WO2024153636A1 - Vasorin as a biomarker and biotarget in nephrology - Google Patents

Vasorin as a biomarker and biotarget in nephrology Download PDF

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WO2024153636A1
WO2024153636A1 PCT/EP2024/050919 EP2024050919W WO2024153636A1 WO 2024153636 A1 WO2024153636 A1 WO 2024153636A1 EP 2024050919 W EP2024050919 W EP 2024050919W WO 2024153636 A1 WO2024153636 A1 WO 2024153636A1
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vasn
subject
fsgs
expression level
reference value
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PCT/EP2024/050919
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French (fr)
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Pierre-Louis Tharaux
Olivia Lenoir
Julien DANG
Catherine CHAUSSAIN
Nassim MAHTAL
Anja Michaela KRAUTZBERGER
Nicole Endlich
Heinrich SCHREWE
Florian Siegerist
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Institut National de la Santé et de la Recherche Médicale
Université Paris Cité
Assistance Publique-Hôpitaux De Paris (Aphp)
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Publication of WO2024153636A1 publication Critical patent/WO2024153636A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • VASORIN AS A BIOMARKER AND BIOTARGET IN NEPHROLOGY FIELD OF THE INVENTION The invention is in the field of medicine, in particular nephrology.
  • BACKGROUND OF THE INVENTION Chronic kidney diseases (CKD) affect around one out of ten people worldwide and were the 12th leading cause of mortality in 2017 (GBD Chronic Kidney Disease Collaboration. Global, regional, and national burden of chronic kidney disease, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet. 29 févr 2020;395(10225):709 ⁇ 33).
  • Glomerular diseases are the leading cause of CKD. For this reason, glomerular resident cells are important therapeutic targets of current research (Wiggins RC.
  • the spectrum of podocytopathies a unifying view of glomerular diseases. Kidney Int. 2007;71(12):1205 ⁇ 14).
  • the glomerular filtration barrier is composed of three layers: the glomerular basement membrane (GBM), lined by a fenestrated endothelium on the vascular side, and the podocytes, specialized glomerular epithelial cells, on the urinary side.
  • GBM glomerular basement membrane
  • the podocytes specialized glomerular epithelial cells
  • Injured podocytes are considered a common feature of glomerular diseases and invariably respond with foot process effacement and podocyte detachment, leading to focal and segmental glomerulosclerosis (FSGS) and irreversible loss of kidney function (Kriz W, Shirato I, Nagata M, LeHir M, Lemley KV.
  • the podocyte s response to stress: the enigma of foot process effacement. Am J Physiol Renal Physiol. 15 févr 2013;304(4):F333-347).
  • As terminally differentiated cells podocytes are incapable of reentering cell-cycle, limiting their self-renewal capacity.
  • Podocyte depletion is sufficient to cause experimental glomerulosclerosis in rodents (Wharram BL, Goyal M, Wiggins JE, Sanden SK, Hussain S, Filipiak WE, et al. Podocyte depletion causes glomerulosclerosis: diphtheria toxin-induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene. J Am Soc Nephrol.
  • diabetic nephropathy Pagtalunan ME, Miller PL, Jumping-Eagle S, Nelson RG, Myers BD, Rennke HG, et al. Podocyte loss and progressive glomerular injury in type II diabetes. J Clin Invest. 1997;99(2):342 ⁇ 8; Weil EJ, Lemley KV, Mason CC, Yee B, Jones LI, Blouch K, et al. Podocyte detachment and reduced glomerular capillary endothelial fenestration promote kidney disease in type 2 diabetic nephropathy.
  • diabetic nephropathy Pagtalunan ME, Miller PL, Jumping-Eagle S, Nelson RG, Myers BD, Rennke HG, et al. Podocyte loss and progressive glomerular injury in type II diabetes. J Clin Invest. 1997;99(2):342 ⁇ 8; Weil EJ, Lemley KV, Mason CC, Yee B, Jones LI, Blouch K, e
  • the present invention relates to the use of Vasorin (VASN) as a biomarker and a biotarget in nephrology.
  • VASN Vasorin
  • DETAILED DESCRIPTION OF THE INVENTION Aiming to identify factors underlying podocytes loss, the inventors revealed a markedly decreased expression of Vasorin (VASN) using comparative deep RNA sequencing of microdissected glomeruli from controls and patients diagnosed with FSGS or crescentic glomerulonephritis (CGN), that they further confirmed by in situ hybridization and immunohistochemistry.
  • VASN is mainly expressed in podocytes during health. Furthermore, the inventors found that the level of glomerular or podocyte expression of the VASN gene depends on the kind of glomerular disease considered, suggesting this could be a helpful biomarker for differential diagnosis or outcome prediction. Of specific interest, 1/ the expression of VASN is significantly less in kidneys from patients suffering from secondary FSGS or anti-neutrophil cytoplasmic antibodies (ANCA) associated crescentic glomerulonephritis than in control normal kidneys or minimal change disease (MN), suggesting that low VASN abundance would predict poor outcome.2/ Podocyte expression of the VASN gene is significantly less in secondary FSGS than in primary FSGS.
  • ANCA anti-neutrophil cytoplasmic antibodies
  • Podocyte expression of the VASN gene is significantly less in FSGS (primary and secondary) than in MCD cases.4/ Podocyte or glomerular expression of the VASN gene is increased substantially in kidney biopsies diagnosed with membranous nephropathy (MN), minimal change disease (MCD), and lupus nephritis (LN) as compared with controls and FSGS, suggesting that this could be used to discriminate diseases types, causes, outcome, or response to therapy. Global or podocyte-specific, constitutive of inducible gene deletion of Vasn induced dramatic features of podocytopathy and FSGS after 14 days, along with nephrotic syndrome and kidney failure.
  • MN membranous nephropathy
  • MCD minimal change disease
  • LN lupus nephritis
  • VASN is an essential master protein maintaining podocyte quiescence and homeostasis during health and kidney diseases.
  • the first object of the present invention relates to a method for diagnosing a kidney disease in a subject comprising determining in a biological sample obtained from the subject the expression level of Vasorin (VASN) wherein the level indicates whether or not the subject suffers from kidney disease.
  • VASN Vasorin
  • the term “subject” refers to any mammal, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human. In some embodiments, the subject is a human who is susceptible to have a kidney disease, as described below.
  • kidney disease refers to disease caused by kidneys being damaged and kidneys not filtering blood the way they should.
  • kidney disease is a chronic kidney disease (CKD).
  • the kidney disease selected from the group consisting of but not limited to nephrotic syndrome, membranous nephropathy (MN), kidney failure, primary focal segmental glomerulosclerosis (FSGS), secondary FGSG, collapsing FSGS, crescentic glomerulonephritis (CGN), minimal change disease (MCD), diabetic nephropathy (DN), ANCA vasculitis (ANCA) or lupus nephritis (LN).
  • the kidney disease is a chronic kidney disease (CKD).
  • CKD chronic kidney disease
  • CKD chronic kidney disease
  • CKD is used to classify numerous conditions that affect the kidney, destruction of the renal parenchyma and the loss of functional nephrons or glomeruli. It should be further noted that CKD can result from different causes, but the final pathway remains renal fibrosis. CKD is defined as kidney damage or glomerular filtration rate (GFR) ⁇ 60 mL/min/1 .73 m ⁇ 2>for 3 months or more, irrespective of the cause. GFR can be estimated from calibrated serum creatinine and estimating equations, such as the Modification of Diet in Renal Disease (MDRD) Study equation or the Cockcroft-Gault formula. Kidney disease severity is classified into five stages according to the level of GFR.
  • MDRD Modification of Diet in Renal Disease
  • kidney diseases examples include but are not limited to, cardiovascular diseases, hypertension, diabetes, glomerulonephritis, polycystic kidney diseases, and kidney graft rejection.
  • the kidney disease is nephrotic syndrome.
  • nephrotic syndrome refers to a rare disease defined by massive proteinuria (>3g/day, or >lg of urine protein per square meter of body-surface area per day in children) and hypoalbuminemia ( ⁇ 30g/l) and result from loss of integrity of the glomerular filtration barrier. The main causes are genetic and immune.
  • the nephrotic syndrome is idiopathic nephrotic syndrome.
  • INS idiopathic nephrotic syndrome
  • INS is histologically characterized by the absence of lesion on light microscopy and lack of immunoglobulin or complement deposit with sometimes additional focal segmental glomerulosclerosis (FSGS) lesions.
  • FSGS focal segmental glomerulosclerosis
  • the kidney disease is membranous nephropathy (MN).
  • MN membranous nephropathy
  • MN membranous nephropathy
  • MN membranous nephropathy
  • the specific lesion is an apparent thickening of the glomerular capillary walls, which results from immune complex formation on the outer aspect of the basement membrane (Couser, W. G. Primary membranous nephropathy. Clin. J. Am. Soc. Nephrol.12, 983–997 (2017)).
  • the immune deposits consist of immunoglobulin G (IgG), the relevant antigens and complement components, including the membrane attack complex (MAC).
  • IgG immunoglobulin G
  • MAC membrane attack complex
  • the consequence of the immunological conflict is the loss of large amounts of proteins in the urine (proteinuria), which is predominantly mediated by the pathophysiological disturbance of the podocyte structure caused by immune complex deposition and MAC formation. Patients experience decreased serum albumin levels and generalized oedema, which define a condition called nephrotic syndrome.
  • MN nephrotic non-diabetic glomerular diseases by podocytopathy presenting as focal and segmental glomerulosclerosis lesions in some populations (African and Hispanic American individuals).
  • primary MN primary MN
  • sMN secondary MN
  • lupus also referred to as systemic lupus erythematosus (SLE)
  • SLE systemic lupus erythematosus
  • the kidney disease is minimal change disease (MCD).
  • MCD minimal change disease
  • MCNS minimal- change nephrotic syndrome
  • nil disease refers to minimal change nephropathy. It arises from a histopathologic lesion in the glomerulus and is characterized by intense proteinuria, leading to edema and intravascular volume depletion. It is the most common single form of nephrotic syndrome in children, but it can also occur in adults.
  • the kidney disease is focal segmental glomerulosclerosis (FSGS).
  • FSGS focal segmental glomerulosclerosis
  • LM light microscopy
  • IF immunofluorescence
  • EM electron microscopy
  • the lesion of FSGS can be classified into primary, secondary (maladaptive or secondary to infectious agents or to medication or toxin), genetic (including associated with APOL1 risk variants), and unknown form.
  • the kidney disease is crescentic glomerulonephritis (CGN).
  • CGN crescentic glomerulonephritis
  • RPGN rapid progressive glomerulonephritis
  • a flare of CGN indicates recurrence of CGN after a period of time in an individual known to have either prior CGN or a systemic autoimmune disease (such as ANCA vasculitis or lupus) in which one may expect CGN as part of the disease.
  • CGN fluorescentic glomerulonephritis
  • RPGN rapidly progressive glomerulonephritis
  • the kidney disease is diabetic nephropathy (DN).
  • DN diabetic nephropathy
  • DN diabetic nephropathy
  • Diabetic nephropathy is globally the leading cause of chronic kidney disease (CKD) and end-stage renal disease (ESRD).
  • CKD chronic kidney disease
  • ESRD end-stage renal disease
  • the triad of protein leaking into the urine (proteinuria or albuminuria), rising blood pressure with hypertension, and then falling renal function is common to many forms of CKD.
  • Protein loss in the urine due to damage of the glomeruli may become massive and cause low serum albumin with resulting generalized body swelling (edema) so called nephrotic syndrome.
  • the kidney disease is anti-neutrophil cytoplasmic antibodies (ANCA) vasculitis.
  • ANCA anti-neutrophil cytoplasmic antibodies
  • anti-neutrophil cytoplasmic antibodies refers to a group of autoantibodies of the IgG type that react with the cytoplasmic constituents of neutrophils and monocytes.
  • the interaction between primed neutrophils and ANCAs releases factors that activate the alternative complement pathway, initiating an amplification loop that is thought to sustain necrotizing inflammation during flares of ANCA-associated vasculitis (AAV).
  • AAV ANCA-associated vasculitis
  • the kidney disease is proliferative glomerulonephritis caused by systemic lupus erythematosus.
  • systemic lupus erythematosus refers to a systemic autoimmune disease thought to be manifested by a wide range of abnormalities in immune regulation. It is the most common type of lupus.
  • the proliferative glomerulonephritis is lupus nephritis.
  • LN lupus nephritis
  • LN refers to an inflammation of the kidney that is caused by systemic lupus erythematosus (SLE). Up to 60% of lupus patients will develop LN.
  • kidneys When the kidneys are inflamed, they cannot function normally to filter toxins, byproducts, excess salts, excess fluid, and other metabolic and immune products in the blood. If not controlled, LN can lead to kidney failure. Even with treatment, loss of kidney function sometimes progresses. If both kidneys fail, subjects with LN may need dialysis. Ultimately, it may be necessary for the LN subject to receive a kidney transplant. Symptoms of loss of or abnormal kidney function include increased amounts of protein in the urine (proteinuria), foaming in a subject’s urine, and/or a higher level of blood urea nitrogen (BUN).
  • proteinuria protein in the urine
  • BUN blood urea nitrogen
  • a high level of VASN indicates that the subject suffers from a minimal change disease (MCD), diabetic nephropathy (DN), a membranous nephropathy (MN), or a lupus nephritis-LN).
  • MCD minimal change disease
  • DN diabetic nephropathy
  • MN membranous nephropathy
  • FSGS focal segmental glomerulosclerosis
  • the term “high” refers to a measure greater than normal, greater than a standard such as a predetermined reference value or a subgroup measure, or relatively greater than another subgroup measure.
  • a high level of VASN refers to a level of VASN that is greater than a normal VASN level.
  • a normal VASN level may be determined according to any method available to one skilled in the art.
  • a high level of VASN may also refer to a level that is equal to or greater than a predetermined reference value, such as a predetermined cutoff.
  • a high level of VASN may also refer to a level of VASN wherein a high VASN subgroup has relatively greater levels of VASN than another subgroup.
  • two distinct patient subgroups can be created by dividing samples around a mathematically determined point, such as, without limitation, a median, thus creating a subgroup whose measure is high (i.e., higher than the median) and another subgroup whose measure is low.
  • a “high” level may comprise a range of levels that is very high and a range of levels that is “moderately high,” where moderately high is a level that is greater than normal but less than “very high”.
  • a higher level of VASN than a predetermined reference value indicates that the subject suffers from a minimal change disease (MCD), diabetic nephropathy (DN), a membranous nephropathy (MN), or a lupus nephritis (LN).
  • MCD minimal change disease
  • DN diabetic nephropathy
  • MN membranous nephropathy
  • LN lupus nephritis
  • the term “low” refers to a level that is less than normal or less than a standard, such as a predetermined reference value or a subgroup measure that is relatively less than another subgroup level.
  • a low level of VASN means a level of VASN that is less than a normal level of in a particular set of samples of patients.
  • a normal level of VASN measure may be determined according to any method available to one skilled in the art.
  • a low level of VASN may also mean a level that is less than a predetermined reference value, such as a predetermined cutoff.
  • a low level of VASN may also indicate a level wherein a low level VASN subgroup is relatively lower than another subgroup.
  • two distinct patient subgroups can be created by dividing samples around a mathematically determined point, such as, without limitation, a median, thus creating a group whose measure is low (i.e., less than the median) with respect to another group whose measure is high (i.e., greater than the median).
  • a lower level of VASN than a predetermined reference value indicates that the subject suffers from a nephrotic syndrome, a chronic kidney disease, an ANCA-associated vasculitis, crescentic glomerulonephritis (CGN) or a focal segmental glomerulosclerosis (FSGS).
  • predetermined reference value refers to a threshold value or a cut- off value.
  • a “threshold value”, “reference value”, or “cut-off value” can be determined experimentally, empirically, or theoretically.
  • a threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skill in the art.
  • the concentration of the marker of the invention e.g., VASN
  • the predetermined corresponding reference value is the median measured in the population of the subjects for the marker of in the invention (VASN, for example).
  • the threshold value has to be determined to obtain the optimal sensitivity and specificity according to the test function and the benefit/risk balance (clinical consequences of false positive and false negative).
  • the optimal sensitivity and specificity can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data.
  • ROC Receiver Operating Characteristic
  • ROC curve is the receiver operator characteristic curve, also known as the receiver operation characteristic curve. It is mainly used for clinical and biochemical diagnostic tests.
  • ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). It reveals the relationship between sensitivity and specificity with the image composition method.
  • a series of different cut-off values are set as continuous variables to calculate a series of sensitivity and specificity values.
  • sensitivity is used as the vertical coordinate
  • specificity is used as the horizontal coordinate to draw a curve.
  • AUC area under the curve
  • the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values.
  • the AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result improves as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high.
  • This algorithmic method is preferably done with a computer.
  • ROC curve such as MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE- ROC.SAS, GB STAT VI0.0 (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.
  • a further aspect of the invention relates to a method for discriminating a primary focal segmental glomerulosclerosis (FSGS) from a secondary FSGS in a subject comprising i) determining in a biological sample obtained from the subject the expression level of Vasorin (VASN); ii) comparing the expression level determined at step i) with a predetermined reference value and iii) concluding that the subject has or is susceptible to have a primary FSGS when the expression level determined at step i) is higher than its predetermined reference value, or concluding that the subject has or is susceptible to have a secondary FSGS when the expression level determined at step i) is lower than its predetermined reference value.
  • VASN Vasorin
  • a further object of the present invention relates to a method for discriminating minimal change disease (MCD) from FSGS in a subject comprising i) determining in a biological sample obtained from the subject the expression level of Vasorin (VASN); ii) comparing the expression level determined at step i) with a predetermined reference value and iii) concluding that the subject has or is susceptible to have a FSGS when the expression level determined at step i) is lower than its predetermined reference value or concluding that the subject has or is susceptible to have a MCD when the expression level determined at step i) is higher than its predetermined reference value.
  • VASN Vasorin
  • the term “discriminating” refers to identifying, observing a difference, or distinguishing two groups.
  • a further object of the present invention relates to a method for predicting the risk of a subject suffering from a kidney disease progresses to a nephrotic syndrome comprising i) determining in a biological sample obtained from the subject the expression level of Vasorin (VASN), ii) comparing the expression level determined at step i) with a predetermined reference value and iii) concluding that the subject has a significantly higher risk of having a nephrotic syndrome when the expression level determined at step i) is lower than its predetermined reference value, or concluding that the subject a lower risk of having a nephrotic syndrome when the expression level determined at step i) is higher than its predetermined reference value.
  • VASN Vasorin
  • the method is particularly suitable for predicting the occurrence of a nephrotic syndrome, a chronic kidney disease, a kidney failure or death.
  • predicting means that the subject to be analyzed by the method of the invention is allocated either into the group of subjects who will relapse or into a group of subjects who will not relapse after treatment.
  • risk in the context of the present invention, relates to the probability that an event will occur over a specific time period, as in the conversion to relapse, and can mean a subject's "absolute" risk or "relative” risk.
  • Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period.
  • Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low-risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of an event and (1- p) is the probability of no event) to no- conversion.
  • Risk evaluation encompasses predicting the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another, i.e., from a normal condition to relapse or to one at risk of developing relapse.
  • Risk evaluation can also comprise the prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of relapse, either in absolute or relative terms in reference to a previously measured population.
  • the methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion to relapse, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk of having a relapse.
  • the invention can be used to discriminate between normal and other subject cohorts at higher risk of having relapse.
  • the present invention may be used so as to discriminate those at risk of having relapse from normal or those having relapse disease from normal.
  • a further object of the invention relates to a method for determining whether a subject suffering from a kidney disease achieves a response to treatment comprising the following steps i) obtaining a biological sample from said subject, ii) determining the expression level of VASN, iii) comparing the determined level with a predetermined reference level and iii) concluding that the subject achieves a response to the treatment when the level of VASN is higher than the reference value; or concluding that the subject does not achieve a response treatment when the expression level of VASN is lower than the reference value.
  • the subject suffers from FSGS, and the treatment is immunosuppressive.
  • the predetermined level is the level determined in a biological sample obtained from the subject before the treatment.
  • the present invention relates to a method of determining whether a subject suffering from a kidney disease achieves a response to treatment comprising i) determining the level of VASN in a biological sample obtained from the subject before the treatment, ii) determining the level of VASN in a sample obtained from the subject after treatment initiation, iii) comparing the level determined at step i) with the level determined at step ii) and concluding that the subject achieves a response treatment when the level of VASN determined at step ii) is lower than the level determined at step i), or concluding that the subject does not achieves a response treatment when the level of VASN determined at step ii) is lower than the level determined at step i).
  • the terms " achieve a response” or “respond” refer to the response to a treatment of the subject suffering from primary or maladaptive FSGS. Typically, such treatment induces, ameliorates, or otherwise causes an improvement in the pathological symptoms, disease progression, or physiological conditions associated with or resistance to succumbing to a maladaptive FSGS.
  • the term “respond” refers to the ability of corticosteroid or other immunomodulatory treatment to improve the pathological symptoms; thus, the subject presents a clinical improvement compared to the subject who does not receive the treatment. The said subject is considered as a "responder" to the treatment.
  • the term "not respond” refers to a subject who does not present any clinical improvement to the treatment with corticosteroid or other immunomodulatory treatment.
  • This subject is considered a "non-responder” to the treatment.
  • the subject considered a “non-responder” has particular monitoring in the therapeutic regimen.
  • the response to a treatment is determined by Response evaluation criteria in solid tumors (RECIST) criteria. These criteria refer to a set of published rules that define when the disease in the subjects improves ("responds"), stays the same (“stabilizes”), or worsens ("progresses”) during treatment.
  • RECIST Response evaluation criteria in solid tumors
  • immunosuppressive treatment refers to any substance capable of producing an immunosuppressive effect, e.g., the prevention or diminution of the immune response and in particular the prevention or diminution of the production of Ig.
  • Immunosuppressive drugs include, without limitation, thiopurine drugs such as azathioprine (AZA) and metabolites thereof; calcineurin inhibitor immunosuppressants such as cyclosporine and voclosporin; nucleoside triphosphate inhibitors such as mycophenolic acid (Cellcept) and its derivative (Myfortic); derivatives thereof; prodrugs thereof; and combinations thereof.
  • the immunosuppressive drug is a calcineurin inhibitor.
  • calcineurin inhibitor has its general meaning in the art and refers to substances that block calcineurin (i.e., calcium/calmodulin-regulated protein phosphatase involved in intracellular signalling) dephosphorylation of appropriate substrates, by targeting calcineurin phosphatase (PP2B, PP3), a cellular enzyme that is involved in gene regulation.
  • a calcineurin inhibitor of the present invention is typically an immunophilin-binding compound having calcineurin inhibitory activity.
  • Immunophilin-binding calcineurin inhibitors are compounds forming calcineurin inhibiting complexes with immunophilins, e.g. cyclophilin and macrophilin.
  • cyclophilin-binding calcineurin inhibitors are cyclosporines or cyclosporine derivatives (hereinafter cyclosporines) and examples of macrophilin-binding calcineurin inhibitors are ascomycin (FR 520) and ascomycin derivatives (hereinafter ascomycins).
  • ascomycin derivatives are known, which are either naturally occurring among fungal species or are obtainable by manipulation of fermentation procedures or by chemical derivatization.
  • Ascomycin-type macrolides include ascomycin, tacrolimus (FK506), sirolimus and pimecrolimus.
  • Cyclosporine originally extracted from the soil fungus Potypaciadium infilatum, has a cyclic 11-amino acid structure and includes, e.g., Cyclosporines A through I, such as Cyclosporine A, B, C, D and G.
  • Voclosporin is a next-generation calcineurin inhibitor that is a more potent and less toxic semi-synthetic derivative of cyclosporine A.
  • the calcineurin inhibitor of the present invention is the trans-version of voclosporin, trans-ISA247 (Cas number 368455-04-3), which is described in, for example, US Patent Publication No.: 2006/0217309, which is hereby incorporated herein by reference.
  • Tacrolimus (FK506) is another calcineurin inhibitor which is also a fungal product but has a macrolide lactone structure.
  • Sirolimus rapamycin
  • Sirolimus binds to an immunophilin (FK-binding protein 12, FKBP12), forming a complex that inhibits the mammalian target of rapamycin (mTOR) pathway by directly binding the mTOR Complex1 (mTORC1).
  • mTOR mammalian target of rapamycin
  • mTORC1 mTOR Complex1
  • Calcineurin inhibitors such as cyclosporine A, voclosporin, ascomycin, tacrolimus, pimecrolimus, an analog thereof, or a pharmaceutically acceptable salt thereof, can be utilized in a mixed micellar composition of the present disclosure.
  • the immunosuppressive drug is a corticosteroid.
  • corticosteroids has a general meaning in the art and refers to a class of active ingredients having a hydrogenated cyclopentoperhydrophenanthrene ring system endowed with anti- inflammatory activity.
  • Corticosteroid drugs typically include cortisone, cortisol, hydrocortisone (11 ⁇ ,17-dihydroxy, 21-(phosphonooxy)-pregn-4-ene, 3,20-dione disodium), dihydroxycortisone, dexamethasone (21-(acetyloxy)-9-fluoro-1 ⁇ ,17-dihydroxy-16 ⁇ -m- ethylpregna-1,4-diene-3,20-dione), and highly derivatized steroid drugs such as beconase (beclomethasone dipropionate, which is 9-chloro-11- ⁇ , 17,21, trihydroxy-16 ⁇ -methylpregna- 1,4 diene-3,20-dione 17,21-dipropionate).
  • beconase beclomethasone dipropionate, which is 9-chloro-11- ⁇ , 17,21, trihydroxy-16 ⁇ -methylpregna- 1,4 diene-3,20-dione 17,21-dipropionate
  • corticosteroids include prednisone, prednisolone, methylprednisolone, deflazacort and betamethasone, cortisone, hydrocortisone, beclomethasone dipropionate, budesonide, dexamethasone sodium phosphate, flunisolide, fluticasone propionate, triamcinolone acetonide, fluocinolone, fluocinonide, betamethasone dipropionate, betamethasone valerate, desonide, desoximetasone, fluocinolone, triamcinolone, triamcinolone acetonide, clobetasol propionate, and dexamethasone.
  • the immunosuppressive drug is a B cell depleting agent.
  • B cell depleting agent refers to any agent capable of triggering B cells' lymphodepletion.
  • the B cell-depleting agent is an antibody having specificity for CD20. Examples of antibodies having specificity for CD20 include: “C2B8” which is now called “Rituximab” (“RITUXAN®”) (U.S. Pat. No.
  • AME-133 ocaratuzumab; Applied Molecular Evolution
  • IgG1 mAb against CD20 a fully-humanized and optimized IgG1 mAb against CD20
  • A20 antibody or variants thereof such as chimeric or humanized A20 antibody (cA20, hA20, respectively) (U.S. Ser. No.
  • Immunomedics 10/366,709, expressly incorporated herein by reference, Immunomedics); and monoclonal antibodies L27, G28-2, 93-1B3, B-CI or NU-B2 available from the International Leukocyte Typing Workshop (Valentine et al, In: Leukocyte Typing III (McMichael, Ed., p.440, Oxford University Press (1987)).
  • suitable antibodies include, e.g., antibody GA101 (obinutuzumab), a third-generation humanized anti-CD20-antibody of Biogen Idec/Genentech/Roche.
  • BLX-301 of Biolex Therapeutics a humanized anti- CD20 with optimized glycosylation or Veltuzumab (hA20), a 2nd-generation humanized antibody specific for CD20 of Immunomedics or DXL625, derivatives of veltuzumab, such as the bispecific hexavalent antibodies of IBC Pharmaceuticals (Immunomedics) which are comprised of a divalent anti-CD20 IgG of veltuzumab and a pair of stabilized dimers of Fab derived from milatuzumab, an anti-CD20 mAb enhanced with InNexus' Dynamic Cross Linking technology, of Inexus Biotechnology both are humanized anti-CD20 antibodies are suitable.
  • BM-ca a humanized antibody specific for CD20 (Int J. Oncol. 2011 February; 38(2):335-44)), C2H7 (a chimeric antibody specific for CD20 (Mol Immunol. 2008 May; 45(10):2861-8)), PRO131921 (a third generation antibody specific for CD20 developed by Genentech), Reditux (a biosimilar version of rituximab developed by Dr Reddy's), PBO-326 (a biosimilar version of rituximab developed by Probiomed), a biosimilar version of rituximab developed by Zenotech, TL-011 (a biosimilar version of rituximab developed by Teva), CMAB304 (a biosimilar version of rituximab developed by Shanghai CP Guojian), GP-2013 (a biosimilar version of rituximab developed by Sandoz (Novartis)), SAIT- 101 (a biosimilar version of rituximab developed by Samsung BioLogics
  • a sample refers to any substance derived from a living organism.
  • a sample may be derived from blood as a urine sample, a serum sample, a plasma sample, and or a whole blood sample.
  • a sample may be derived from a tissue collected, for example, by a biopsy.
  • a tissue sample may comprise, for example, kidney tissue (e.g., biopsies in glomeruli).
  • the kidney tissue sample comprises podocytes.
  • the tissue sample can, of course, be subjected to a variety of well-known post- collection preparative and storage techniques (e.g., fixation, storage, freezing, etc.).
  • the sample can be fresh, frozen, fixed (e.g., formalin-fixed), or embedded (e.g., paraffin-embedded).
  • the sample has been previously obtained from the subject.
  • the term “Vasorin” or “VASN” refers to a typical type I membrane protein containing tandem arrays of a characteristic leucine-rich repeat motif, an epidermal growth factor-like motif, and a fibronectin type III-like motif at the extracellular domain.
  • VASN is an evolutionarily conserved single-pass type I transmembrane protein, with a high degree of similarity both at the DNA (95%) and protein (83%) levels between rodent and human homologs, suggesting highly conserved functions.
  • the naturally occurring human VASN gene has the following nucleotide sequence as shown in Genbank Accession number NM_138440.3 (SEQ ID NO: 1), and the naturally occurring human VASN protein has the following amino acid sequence as shown in Genbank Accession number NP_612449.2 (SEQ ID NO: 2).
  • SEQ ID NO: 2 MCSRVPLLLPLLLLLALGPGVQGCPSGCQCSQPQTVFCTARQGTTVPRDVPPDTVGLYVFENGITMLDA GSFAGLPGLQLLDLSQNQIASLPSGVFQPLANLSNLDLTANRLHEITNETFRGLRRLERLYLGKNRIRH IQPGAFDTLDRLLELKLQDNELRALPPLRLPRLLLLDLSHNSLLALEPGILDTANVEALRLAGLGLQQL DEGLFSRLRNLHDLDVSDNQLERVPPVIRGLRGLTRLRLAGNTRIAQLRPEDLAGLAALQELDVSNLSL QALPGDLSGLFPRLRLLAAARNPFNCVCPLSWFGPWVRESHVTLASPEETRCHFPPKNAGRLLLELDYA DFGCPATTTTATVPTTRPVVREPTALSSSLAPTWLSPTEPATEAPSPPSTAPPTVGPVPQPQDCPPSTC LNGGTCHLGTRHHLACLCPEGFTGLYCESQMGQGTRPSPTPVTPRPPRSL
  • the expression level of Vasorin is measured at the mRNA or the protein level.
  • Methods to determine the expression level of VASN can be performed by any method known in the art, including, without limitation: deep RNA sequencing, in situ hybridization, direct sequencing or Q-PCR, immunostaining, immunohistochemistry, immunofluorescence, ELISA, flow cytometry, chromatography, proteomics.
  • the level of VASN is determined by immunostaining, immunohistochemistry, immunofluorescence, ELISA, flow cytometry, deep RNA sequencing, in situ hybridization.
  • protein or antibody concentration may be measured, for example, by capillary electrophoresis-mass spectroscopy technique (CE-MS) or ELISA performed on the sample.
  • CE-MS capillary electrophoresis-mass spectroscopy technique
  • ELISA ELISA
  • Such methods comprise contacting a sample with a binding partner capable of selectively interacting with proteins present in the sample.
  • the binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.
  • the presence of the protein or antibody can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich-type assays.
  • Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, capillary electrophoresis- mass spectroscopy technique (CE-MS).etc.
  • the reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.
  • the aforementioned assays generally involve the separation of unbound protein in a liquid phase from solid phase support to which antigen-antibody complexes are bound.
  • Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e.g., in membrane or microtiter well form), polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidene fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like. More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the proteins to be tested.
  • a sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties, and a detectably labeled secondary binding molecule is added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate is washed, and the presence of the secondary binding molecule is detected using methods well-known in the art. In some embodiments, the level of VASN is determined by immunohistochemistry or immunofluorescence.
  • Immunohistochemistry typically includes the following steps i) fixing the human or murine renal tissue sample with formalin, ii) embedding said human or murine renal tissue sample in paraffin, iii) cutting said human or murine renal tissue sample into sections for staining, iv) incubating said sections with the binding partner specific for the marker, v) rinsing said sections, vi) incubating said section with a secondary antibody typically biotinylated and vii) revealing the antigen-antibody complex typically with avidin-biotin-peroxidase complex. Accordingly, the human or murine renal tissue sample is first incubated with binding partners, such as anti- VASN.
  • binding partners such as anti- VASN.
  • the labeled antibodies bound to markers of interest are revealed by the appropriate technique, depending on the label borne by the labeled antibody, e.g., radioactive, fluorescent or enzyme label. Multiple labelling can be performed simultaneously.
  • the method of the present invention may use a secondary antibody coupled to an amplification system (to intensify the staining signal) and enzymatic molecules.
  • Such coupled secondary antibodies are commercially available, e.g. from Dako, EnVision system.
  • Counterstaining may be used, e.g., Hematoxylin & Eosin, DAPI, Hoechst.
  • Other staining methods may be accomplished using any suitable method or system as would be apparent to one of skill in the art, including automated, semi-automated, or manual systems.
  • one or more labels can be attached to the antibody, thereby permitting the detection of the target protein (i.e., the marker).
  • exemplary labels include radioactive isotopes, fluorophores, ligands, chemiluminescent agents, enzymes, and combinations thereof.
  • the label is a quantum dot.
  • Non-limiting examples of labels that can be conjugated to primary and/or secondary affinity ligands include fluorescent dyes or metals (e.g., fluorescein, rhodamine, phycoerythrin, fluorescamine), chromophoric dyes (e.g., rhodopsin), chemiluminescent compounds (e.g.
  • Affinity ligands can also be labeled with enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-lactamase), radioisotopes (e.g., 3H, 14C, 32P, 35S or 125I) and particles (e.g., gold).
  • enzymes e.g., horseradish peroxidase, alkaline phosphatase, beta-lactamase
  • radioisotopes e.g., 3H, 14C, 32P, 35S or 125I
  • particles e.g., gold
  • the different types of labels can be conjugated to an affinity ligand using various chemistries, e.g., the amine reaction or the thiol reaction. However, other reactive groups than amines and thiols can be used, e.g., aldehydes, carboxylic acids and glutamine.
  • Various enzymatic staining methods are known in the art of detecting a protein of interest. For example, enzymatic interactions can be visualized using different enzymes such as peroxidase, alkaline phosphatase, or different chromogens such as DAB, AEC, or Fast Red.
  • the antibody can be conjugated to peptides or proteins that can be detected via a labeled binding partner or antibody.
  • a secondary antibody or second binding partner is necessary to detect the binding of the first binding partner, as it is not labeled.
  • the resulting stained specimens are each imaged using a system for viewing the detectable signal and acquiring an image, such as a digital image of the staining.
  • Methods for image acquisition are well known to one of skill in the art.
  • any optical or non-optical imaging device can be used to detect the stain or biomarker label, such as, for example, upright or inverted optical microscopes, scanning confocal microscopes, cameras, scanning or tunneling electron microscopes, scanning probe microscopes and imaging infrared detectors.
  • the image can be captured digitally.
  • the obtained images can then be used for quantitatively or semi-quantitatively determining the amount of the marker in the sample, or the absolute number of cells positive for the maker of interest, or the surface of cells positive for the marker of interest.
  • Various automated sample processing, scanning and analysis systems suitable for use with IHC are available in the art. Such systems can include automated staining and microscopic scanning, computerized image analysis, serial section comparison (to control for variation in the orientation and size of a sample), digital report generation, and archiving and tracking of samples (such as slides on which tissue sections are placed).
  • Cellular imaging systems are commercially available that combine conventional light microscopes with digital image processing systems to perform quantitative analysis on cells and tissues, including immunostained samples.
  • detection can be made manually or by image processing techniques involving computer processors and software.
  • the images can be configured, calibrated, standardized and/or validated based on factors including, for example, stain quality or stain intensity, using procedures known to one of skill in the art (see, e.g., published U.S. Patent Publication No. US20100136549).
  • the image can be quantitatively or semi-quantitatively analyzed and scored based on the staining intensity of the sample.
  • Quantitative or semi-quantitative histochemistry refers to method of scanning and scoring samples that have undergone histochemistry to identify and quantitate the presence of the specified biomarker (i.e. the marker).
  • Quantitative or semi-quantitative methods can employ imaging software to detect staining densities or amount of staining or methods of detecting staining by the human eye, where a trained operator ranks results numerically.
  • images can be quantitatively analyzed using pixel count algorithms and tissue recognition patterns (e.g., Aperio Spectrum Software, Automated QUantitatative Analysis platform (AQUA® platform), or Tribvn with Ilastic and Calopix software), and other standard methods that measure or quantitate or semi-quantitate the degree of staining; see, e.g., U.S. Pat. No. 8,023,714; U.S. Pat. No.7,257,268; U.S. Pat. No.7,219,016; U.S. Pat. No.7,646,905; published U.S. Patent Publication No. US20100136549 and 20110111435; Camp et al.
  • tissue recognition patterns e.g., Aperio Spectrum Software, Automated QUantitatative Analysis platform (AQUA® platform), or Tribvn with Ilastic and Calopix software
  • a ratio of a strong positive stain (such as a brown stain) to the sum of total stained area can be calculated and scored.
  • the amount of the detected biomarker i.e., the marker
  • the amount is quantified and given as a percentage of positive pixels and/or a score. For example, the amount can be quantified as a percentage of positive pixels. In some examples, the amount is quantified as the percentage of area stained, e.g., the percentage of positive pixels.
  • a sample can have at least or about at least or about 0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more positive pixels as compared to the total staining area.
  • the amount can be quantified as an absolute number of cells positive for the marker of interest.
  • a score is given to the sample that is a numerical representation of the intensity or amount of the histochemical staining of the sample and represents the amount of target biomarker (e.g., the marker) present in the sample.
  • Optical density or percentage area values can be given a scaled score, for example, on an integer scale.
  • the method of the present invention comprises the steps consisting of i) providing one or more immunostained slices of tissue section obtained by an automated slide-staining system by using a binding partner capable of selectively interacting with the marker (e.g., an antibody as above described), ii) proceeding to the digitalization of the slides of step i).by high-resolution scan capture, iii) detecting the slice of a tissue section on the digital picture iv) providing a size reference grid with uniformly distributed units having a same surface, said grid being adapted to the size of the tissue section to be analyzed, and v) detecting, quantifying and measuring the intensity or the absolute number of stained cells in each unit whereby the number or the density of cells stained of each unit is assessed.
  • a binding partner capable of selectively interacting with the marker (e.g., an antibody as above described)
  • the marker e.g., an antibody as above described
  • the level of VASN is determined by tissue proteomics.
  • proteomics refers to a highly sensitive and accurate method for protein identification and expression analysis.
  • proteomics may use liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) or nano-liquid chromatography mass spectrometry (nLC-MS/MS), as described in Chick JM et al., Defining the consequences of genetic variation on a proteome-wide scale. Nature.2016 Jun 23;534(7608):500-5; in Yuka T et al., Proteomic and transcriptomic profiling reveal different aspects of aging in the kidney eLife.
  • the expression level of VASN in the sample is determined at the nucleic acid level.
  • the level of a gene may be determined by assessing the quantity of mRNA encoding for VASN. Methods for determining the amount of mRNA are well-known in the art.
  • the nucleic acid contained in the samples e.g., cell or tissue prepared from the subject
  • the extracted mRNA is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR).
  • amplification e.g., RT-PCR.
  • Other methods of Amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence-based amplification (NASBA).
  • LCR ligase chain reaction
  • TMA transcription-mediated amplification
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence-based amplification
  • the nucleic acid probes include one or more labels, for example, to permit detection of a target nucleic acid molecule using the disclosed probes.
  • a nucleic acid probe includes a label (e.g., a detectable label).
  • a “detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample.
  • a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample.
  • a label associated with one or more nucleic acid molecules can be detected directly or indirectly.
  • a label can be detected by any known or yet-to-be-discovered mechanism, including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons).
  • Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.
  • the methods of the invention comprise the steps of providing total RNAs extracted from cumulus cells and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semi- quantitative RT-PCR.
  • the level of VASN is determined by deep RNA sequencing.
  • deep RNA sequencing refers to a highly sensitive and accurate method for gene expression analysis. This technology has rapidly emerged as the key approach to high- throughput transcriptome profiling to better understand functional biology underlying disease. RNA sequencing data related to other processes (RNA splicing entropy, gene expression, etc.) will provide a signature that can identify subjects with a kidney disease such as FSGS.
  • a further aspect of the present invention relates to a method for preventing the progression to a nephrotic syndrome in a subject suffering from a kidney disease comprising administering to the subject a therapeutically effective amount of an agent that restores the expression of VASN.
  • the method of the present invention is particularly suitable for preventing the progression of chronic kidney disease, kidney failure and/or death.
  • the agent is a vasorin (VASN) polypeptide or a polynucleotide encoding thereof.
  • polynucleotide refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”). It also includes modifications, for example, by alkylation and/or by capping and unmodified forms of the polynucleotide.
  • polynucleotide includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids “PNAs”) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA.
  • PNAs peptide nucleic acids
  • the polynucleotide comprises an mRNA.
  • the mRNA is a synthetic mRNA.
  • the synthetic mRNA comprises at least one unnatural nucleobase.
  • all nucleobases of a certain class have been replaced with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be replaced with an unnatural nucleobase, e.g., 5-methoxyuridine).
  • the polynucleotide (e.g., a synthetic RNA or a synthetic DNA) comprises only natural nucleobases, i.e., A, C, T and G in the case of a synthetic DNA, or A, C, T, and U in the case of a synthetic RNA.
  • the polynucleotide of the present invention is a messenger RNA (mRNA).
  • mRNA messenger RNA
  • the polynucleotide is inserted in a vector, such a viral vector.
  • the term “viral vector” refers to a virion or virus particle that functions as a nucleic acid delivery vehicle and which comprises a vector genome packaged within the virion or virus particle.
  • the vector is a viral vector, an adeno-associated virus (AAV), a retroviral vector, a bovine papilloma virus, an adenovirus vector, a vaccinia virus, or a polyomavirus.
  • the viral vector is a AAV vector.
  • AAV vector means a vector derived from an adeno- associated virus serotype, including, without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and mutated forms thereof.
  • AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking ITR sequences.
  • the viral vector is a retroviral vector.
  • retroviral vector refers to a vector containing structural and functional genetic elements that are primarily derived from a retrovirus.
  • the retroviral vector of the present invention derives from a retrovirus selected from the group consisting of alpha retroviruses (e.g., avian leukosis virus), betaretroviruses (e.g., mouse mammary tumor virus), gammaretroviruses (e.g., murine leukemia virus), deltaretroviruses (e.g., bovine leukemia virus), epsilon retroviruses (e.g., Walley dermal sarcoma virus), lentiviruses (e.g., HIV-1, HIV- 2) and spumaviruses (e.g., human spumavirus).
  • alpha retroviruses e.g., avian leukosis virus
  • betaretroviruses e.g., mouse mammary tumor virus
  • gammaretroviruses e.g., murine leukemia virus
  • deltaretroviruses e.g., bovine leukemia virus
  • the retroviral vector of the present invention is a replication-deficient retroviral virus particle, which can transfer a foreign imported RNA of a gene instead of the retroviral mRNA.
  • the structure and composition of the vector genome used to prepare the retroviral vectors of the present invention are in accordance with those described in the art.
  • minimum retroviral gene delivery vectors can be prepared from a vector genome, which only contains, apart from the recombinant nucleic acid molecule of the present invention, the sequences of the retroviral genome which are non-coding regions of said genome, necessary to provide recognition signals for DNA or RNA synthesis and processing.
  • the retroviral vector genome comprises all the elements necessary for the nucleic import and the correct expression of the polynucleotide of interest (i.e. the transgene).
  • elements that can be inserted in the retroviral genome of the retroviral vector of the present invention are at least one (preferably two) long terminal repeats (LTR), such as a LTR5' and a LTR3', a psi sequence involved in the retroviral genome encapsidation, and optionally at least one DNA flap comprising a cPPT and a CTS domains.
  • LTR long terminal repeats
  • the LTR preferably the LTR3'
  • the vector is a Self- INactivating (SIN) vector that contains a non-functional or modified 3' Long Terminal Repeat (LTR) sequence. This sequence is copied to the 5' end of the vector genome during integration, resulting in the inactivation of promoter activity by both LTRs.
  • a vector genome may be a replacement vector in which all the viral coding sequences between the 2 long terminal repeats (LTRs) have been replaced by the recombinant nucleic acid molecule of the present invention.
  • the retroviral vector genome is devoid of functional gag, pol and/or env retroviral genes.
  • functional it is meant a gene that is correctly transcribed and/or correctly expressed.
  • the retroviral vector genome of the present invention in this embodiment contains at least one of the gag, pol and env genes that is either not transcribed or incompletely transcribed; the expression “incompletely transcribed” refers to the alteration in the transcripts gag, gag-pro or gag-pro-pol, one of these or several of these being not transcribed.
  • the retroviral genome is devoid of gag, pol and/or env retroviral genes.
  • the retroviral vector genome is also devoid of the coding sequences for Vif-, Vpr-, Vpu- and Nef-accessory genes (for HIV-1 retroviral vectors) or of their complete or functional genes.
  • the retroviral vector of the present invention is non-replicative i.e., the vector and retroviral vector genome are not able to form new particles budding from the infected host cell. This may be achieved by the absence in the retroviral genome of the gag, pol, or env genes, as indicated in the above paragraph; this can also be achieved by deleting other viral coding sequences and/or cis-acting genetic elements needed for particles formation.
  • the present invention encompasses the use of virus-like particles.
  • virus-like particle refers to a structure resembling a virus particle but devoid of the viral genome, incapable of replication and devoid of pathogenicity.
  • the particle typically comprises at least one type of structural protein from a virus. Preferably only one type of structural protein is present. Most preferably, no other non-structural component of a virus is present.
  • virus-like particles can be spontaneously self-assembled by viral structural proteins under appropriate conditions in vitro while excluding the genetic material and potential replication probability.
  • Virus-like particles with a diameter of approximately 20 to 150 nm, also have the characteristics of nanometer materials, such as large surface area, surface- accessible amino acids with reactive moieties (e.g., lysine and glutamic acid residues), inerratic spatial structure, and good biocompatibility. Therefore, assembled virus-like particles have great potential as a delivery system for specifically carrying a variety of cargos.
  • one or more of the zinc finger motifs of the Gag protein is/are substituted by one or more RNA-binding domain(s).
  • the RNA-binding domain is the Coat protein of the MS2 bacteriophage, of the PP7 phage or of the Q3 phage, the prophage HK022 Nun protein, the U1A protein or the hPum protein. More preferably, the RNA binding domain is the Coat protein of the MS2 bacteriophage or of the PP7 phage. Even more preferably, the RNA-binding domain is the Coat protein of the MS2 bacteriophage. These embodiments are particularly suitable for packaging the mRNA encoding for the VASN polypeptide into the VLP.
  • the mRNA encoding for the VASN polypeptide that is encapsuled in the virus particle of the present invention comprises at least one encapsidation sequence.
  • encapsidation sequence is meant an RNA motif (sequence and three-dimensional structure) recognized specifically by an RNA-binding domain as described above.
  • the encapsidation sequence is a stem-loop motif.
  • the encapsidation sequence of the retroviral particle is the stem-loop motif of the RNA of the MS2 bacteriophage or of the PP7 phage such as.
  • the stem-loop motif and more particularly the stem-loop motif of the RNA of the MS2 bacteriophage or that of the RNA of the PP7 phage may be used alone or repeated several times, preferably from 2 to 25 times, more preferably from 2 to 18 times, for example from 6 to 18 times.
  • the present invention encompasses the use of the LentiFlash® technology that based on non-integrative lentiviral particles constructed using a bacteriophage coat protein and its cognate 19-nt stem-loop, to replace the natural lentiviral Psi packaging sequence, in order to achieve active mRNA packaging into the lentiviral particles (Prel A, Caval V, Gayon R, Ravassard P, Duthoit C, Payen E, Maouche-Chretien L, Creneguy A, Nguyen TH, Martin N, Piver E, Sevrain R, Lamouroux L, Leboulch P, Deschaseaux F, Boutician P, Sensébé L, Pagès JC.
  • the LentiFlash® technology that based on non-integrative lentiviral particles constructed using a bacteriophage coat protein and its cognate 19-nt stem-loop, to replace the natural lentiviral Psi packaging sequence, in order to achieve active mRNA packaging into
  • the retroviral vectors of the present invention can be produced by any well-known method in the art, including by transfection (s) transient (s), in stable cell lines and / or by means of helper virus. The use of stable cell lines may also be preferred for the production of the vectors (Greene, M. R. et al.
  • the retroviral vector of the present invention is obtainable by a transcomplementation system (vector/packaging system) by transfecting in vitro a permissive cell (such as 293T cells) with a plasmid containing the retroviral vector genome of the present invention, and at least one other plasmid providing, in trans, the gag, pol and env sequences encoding the polypeptides GAG, POL and the envelope protein(s), or for a portion of these polypeptides sufficient to enable the formation of retroviral particles.
  • a transcomplementation system vector/packaging system
  • permissive cells are transfected with a) trans-complementation plasmid, lacking packaging signal psi, and the plasmid is optionally deleted of accessory genes vif, nef, vpu and / or vpr, b) a second plasmid (envelope expression plasmid or pseudotyping env plasmid) comprising a gene encoding an envelope protein(s) and c) a plasmid vector comprising a recombinant genome retroviral, optionally deleted from the promoter region of the 3 'LTR or U3 enhancer sequence of the 3' LTR, including, between the LTR sequences 5 'and 3' retroviral, a psi encapsidation sequence, a nuclear export element (preferably RRE element of HIV or other retroviruses equivalent), comprising the nucleic acid molecule of the present invention and optionally a promoter and / or a nuclear import sequence (cPPT sequence e
  • the three plasmids used do not contain homologous sequence sufficient for recombination.
  • Nucleic acids encoding gag, pol and env cDNA can be advantageously prepared according to conventional techniques from viral gene sequences available in the prior art and databases.
  • the trans-complementation plasmid provides a nucleic acid encoding the proteins retroviral gag and pol. These proteins are derived from a lentivirus and, most preferably, from HIV-1.
  • the plasmid is devoid of encapsidation sequence, the sequence coding for an envelope, accessory genes, and advantageously also lacks retroviral LTRs.
  • the sequences coding for gag and pol proteins are advantageously placed under the control of a heterologous promoter, eg, cellular, viral, etc., which can be constitutive or regulated, weak or strong. It is preferably a plasmid containing a sequence transcompl mitant ⁇ psi-CMV-gag- pol-PolyA. This plasmid allows the expression of all the proteins necessary for the formation of empty virions except the envelope glycoproteins.
  • the plasmid transcomplementation may advantageously comprise the TAT and REV genes. Plasmid transcomplementation is advantageously devoid of vif, vpr, vpu and / or nef accessory genes.
  • gag and pol genes and genes TAT and REV can also be carried by different plasmids, possibly separated. In this case, several plasmids are used for trans-complementation, each encoding one or more of said proteins.
  • the promoters used in the plasmid trans-complementation, the envelope plasmid and the plasmid vector, respectively, to promote the expression of gag and pol of the coat protein, the mRNA of the vector genome and the transgene are promoters identical or different, chosen advantageously from ubiquitous promoters or specific, for example, from viral promoters CMV, TK, RSV LTR promoter and the RNA polymerase III promoter such as U6 or H1 or promoters of helper viruses encoding env, gag and pol (i.e., adenoviral, baculoviral, herpes viruses).
  • the plasmids described above can be introduced into competent cells, and viruses produced are harvested.
  • the cells used may be any cell competent, particularly eukaryotic cells, in particular mammalian, eg, human or animal. They can be somatic or embryonic stem or differentiated. Typically the cells include 293T cells, fibroblast cells, hepatocytes, muscle cells (skeletal, cardiac, smooth, blood vessel, etc.)., nerve cells (neurons, glial cells, astrocytes) of epithelial cells, renal, ocular, etc. It may also include insect, plant cells, yeast, or prokaryotic cells. It can also be cells transformed by the SV40 T antigen.
  • the genes gag, pol and env encoded in plasmids or helper viruses can be introduced into cells by any method known in the art, suitable for the cell type considered.
  • the cells and the vector system are contacted in a suitable device (plate, dish, tube, pouch, etc...) for a period of time sufficient to allow the transfer of the vector system or the plasmid in the cells.
  • the vector system or the plasmid is introduced into the cells by calcium phosphate precipitation, electroporation, transduction or by using one of the transfection-facilitating compounds, such as lipids, polymers, liposomes and peptides, etc.. Calcium phosphate precipitation is preferred.
  • the cells are cultured in any suitable medium such as RPMI, DMEM, a specific medium to a culture in the absence of fetal calf serum, etc.
  • the retroviral vectors of the present invention may be purified from the supernatant of the cells. Purification of the retroviral vector to enhance the concentration can be accomplished by any suitable method, such as by density gradient purification (e.g., cesium chloride (CsCl)) or by chromatography techniques (e.g., column or batch chromatography).
  • CsCl cesium chloride
  • chromatography techniques e.g., column or batch chromatography
  • the vector of the present invention can be subjected to two or three CsCl density gradient purification steps.
  • the vector is desirably purified from cells infected using a method that comprises lysing cells infected with adenovirus, applying the lysate to a chromatography resin, eluting the adenovirus from the chromatography resin, and collecting a fraction containing the retroviral vector of the present invention.
  • the vector of the present invention includes "control sequences", which refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription, and translation of a coding sequence in a recipient cell.
  • nucleic acid sequence is a "promoter" sequence, which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene that is capable of binding RNA polymerase and initiating transcription of a downstream (3'- direction) coding sequence.
  • Transcription promoters can include "inducible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), “repressible promoters” (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and “constitutive promoters”.
  • the polypeptide or polynucleotide of the present invention can be conjugated to at least one other molecule.
  • said molecule is selected from the group consisting of polynucleotides, polypeptides, lipids, lectins, carbohydrates, vitamins, cofactors, and drugs.
  • the polypeptide or polynucleotide of the present invention is formulated using one or more lipid-based structures that include but are not limited to liposomes, lipoplexes, or lipid nanoparticles (Paunovska, Kalina, David Loughrey, and James E. Dahlman. "Drug delivery systems for RNA therapeutics.” Nature Reviews Genetics (2022): 1-16).
  • Liposomes are artificially-prepared vesicles that can primarily be composed of a lipid bilayer and can be used as a delivery vehicle for the administration of pharmaceutical formulations.
  • Liposomes can be of different sizes, such as but not limited to, a multilamellar vesicle (MLV) which can be hundreds of nanometers in diameter and can contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which can be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which can be between 50 and 500 nm in diameter.
  • MLV multilamellar vesicle
  • SUV small unicellular vesicle
  • LUV large unilamellar vesicle
  • Liposome design can include but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis.
  • Liposomes can contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.
  • liposomes such as synthetic membrane vesicles are prepared using the methods, apparatus, and devices described in US Patent Publication No. US20130177638, US20130177637, US20130177636, US20130177635, US20130177634, US20130177633, US20130183375, US20130183373 and US20130183372.
  • the liposomes are formed from 1,2-dioleyloxy-N,N- dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and MC3 (as described in US20100324120) and liposomes which can deliver small molecule drugs such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.).
  • DOXIL® 1,2-dioleyloxy-N,N- dimethylaminopropane
  • DiLa2 liposomes from Marina Biotech (Bothell, Wash.)
  • DLin-DMA 1,2-dilinoleyloxy-3-dimethyl
  • polypeptide of polynucleotide of the present invention can be encapsulated by the liposome and/or it can be contained in an aqueous core which can then be encapsulated by the liposome (see International Pub. Nos. WO2012031046, WO2012031043, WO2012030901 and WO2012006378 and US Patent Publication No. US20130189351, US20130195969 and US20130202684).
  • the polynucleotide of the present invention is formulated with stabilized plasmid- lipid particles (SPLP) or stabilized nucleic acid lipid particles (SNALP) that have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (see Wheeler et al. Gene Therapy. 19996:271-281; Zhang et al. Gene Therapy. 19996:1438- 1447; Jeffs et al. Pharm Res.200522:362-372; Morrissey et al., Nat Biotechnol.20052:1002- 1007; Zimmermann et al., Nature.2006441:111-114; Heyes et al.
  • SPLP stabilized plasmid- lipid particles
  • SNALP stabilized nucleic acid lipid particles
  • the term "therapeutically effective amount” refers to a sufficient amount of the polypeptide or the nucleic acid molecule encoding thereof to prevent for use in a method for the treatment of the disease (e.g., a kidney disease) at a reasonable benefit/risk ratio applicable to any medical treatment.
  • the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors, including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
  • the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • the active ingredient of the present invention i.e., the polypeptide or polynucleotide
  • pharmaceutically acceptable excipients and optionally sustained-release matrices such as biodegradable polymers
  • pharmaceutically acceptable excipients such as a pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the invention in another aspect, relates to a kit suitable to perform the diagnosis method as described above, comprising a reagent that specifically reacts with VASN (mRNA or protein), and instructions use.
  • the invention relates to kits for performing the methods of the invention, wherein said kits comprise means for measuring the expression level of the VASN of the invention.
  • the kit according to the invention may include instructional materials containing instructions (e.g., protocols) for the practice of diagnostic methods.
  • the kits may include probes, primers macroarrays or microarrays as above described.
  • the kit may comprise a set of probes as defined above, usually made of DNA, and that may be pre-labelled.
  • probes may be unlabelled and the ingredients for labelling may be included in the kit in separate containers.
  • the kit may further comprise hybridization reagents or other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards.
  • the kit of the invention may comprise amplification primers that may be pre labelled or may contain an affinity purification or attachment moiety.
  • the kit may further comprise amplification reagents and also other suitably packaged reagents and materials needed for the particular amplification protocol.
  • the nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes.
  • a preferred kit also includes the components necessary to determine if amplification has occurred.
  • the kit may also include, for example, PCR buffers and enzymes, positive control sequences, reaction control primers, and instructions for amplifying and detecting the specific sequences.
  • the invention provides diagnostic kits containing the anti- VASN antibodies (monoclonal or polyclonal) including antibody conjugates.
  • the diagnostic kit is a package comprising at least one anti-VASN monoclonal antibody of the disclosure (e.g.
  • kits comprises two anti-VASN antibodies, wherein at least one of the antibodies is an anti-VASN monoclonal antibody.
  • the second antibody is a polyclonal anti- VASN antibody.
  • the kit can include a labeled antibody which binds an anti-VASN monoclonal/polyclonal antibody and is conjugated to an enzyme.
  • the kit can include substrates and cofactors required by the enzyme (e.g., a substrate precursor that provides the detectable chromophore or fluorophore).
  • substrates and cofactors required by the enzyme e.g., a substrate precursor that provides the detectable chromophore or fluorophore.
  • other additives can be included, such as stabilizers, buffers (e.g. , a block buffer or lysis buffer), and the like.
  • Anti- VASN antibodies included in a diagnostic kit can be immobilized on a solid surface, or alternatively, a solid surface (e.g., a slide) on which the antibody can be immobilized is included in the kit.
  • the relative amounts of the various reagents can be varied widely to provide for concentrations in solution of the reagents, which substantially optimizes the sensitivity of the assay.
  • Antibodies and other reagents can be provided (individually or combined) as dry powders, usually lyophilized, including excipients, which on dissolution will provide a reagent solution having the appropriate concentration.
  • FIGURES Figure 1: Podocyte-specific deletion of Vasn induces FSGS in mice
  • c Plasma urea levels in PodoCre-Vasnlox/lox and control mice after 22 days.
  • FIG. 2 Global and post-natal deletion of Vasn induces FSGS in mice
  • FIG. 3 Podocyte-specific and post-natal deletion of Vasn induces FSGS in mice
  • Podocyte count as stained by WT1-positive nuclei in glomerular sections, in kidneys from NEFTA8-Cre-GFP- Vasnlox/lox and control mice at day 22.
  • Podocyte differentiation as stained by NPHS1/nephrin-positive area in glomerular sections, in kidneys from NEFTA8-Cre-GFP- Vasnlox/lox and control mice at day 22.
  • PEC activation as stained by glomerular sections with CD44-positive PEC, in kidneys from NEFTA8-Cre-GFP-Vasnlox/lox and control mice at day 22.
  • Data represent mean +/- s.d. *P ⁇ 0.05, **** P ⁇ 0.0001.
  • VASN is markedly regulated in human kidney diseases
  • mice with podocyte-specific and constitutive deletion of the Vasn gene were generated by crossing Vasnlox/lox mice with Nphs2.Cre (PodoCre) mice on the C57BL6/J background (PodoCre-Vasnlox/lox).
  • Mice with podocyte-specific deletion of the Vasn gene using an inducible Cre/lox system were generated by crossing Vasnlox/lox mice with Nphs2-rtTA (NEFTA8), Tet-O-Cre (Cre) and ROSAmT/mG (GFP) mice on the C57BL6/J background.
  • NEFTA8-Cre-GFP-Vasnlox/lox received doxycycline (ALX-380-273-G005) for 7 days (2 mg/mL in 5% sucrose water) to induce the Cre-recombinase.
  • Age-matched littermates with no Cre-recombinase or no floxed Vasn allele were considered as controls.
  • C57BL6/J mice with overexpression of a fluorescent protein (Venus) under the transcriptional regulation of the Vasn gene (Venus-Vasn) were obtained by transfection of ES cells with a BAC containing the sequence of the Venus framed on both sides with regulatory sequences of the Vasn gene.
  • mice were considered diabetic when fasting glycemia remained above 300 mg/dL two weeks in a row after STZ injection. Mice were killed 8 weeks after the STZ injection.
  • Parabiosis Shared blood circulation between CreERT2-Vasnlox/lox female littermates was made by parabiosis surgery, as previously described (Duyverman AMMJ, Kohno M, Duda DG, Jain RK, Fukumura D. A transient parabiosis skin transplantation model in mice. Nat Protoc.22 mars 2012;7(4):763 ⁇ 70).
  • mice were injected with tamoxifen (TMX) for 5 consecutive days to induce Cre recombinase, underwent parabiosis surgery 7 days after the first TMX injection, and were killed 15 days after the surgery.
  • Biochemical measurements in blood and urine Blood was taken by intracardiac puncture. Urine was either collected using metabolic cages or by puncturing the bladder for newborns. Urinary creatinine and plasma urea concentrations were analyzed by a standard colorimetric method (Olympus AU400) at the Biochemistry Laboratory of Institut Claude Bernard (IFR2, Faculté de Médecine Paris Diderot). Quantitative determination of urinary albumin excretion was measured by a specific ELISA mouse kit (Crystal Chem, #80630).
  • Ultrathin sections were cut with an ultramicrotome and counterstained with uranyl acetate and lead citrate. Samples were examined in the JEM1011 transmission electron microscope (JEOL) with the Orius SC1000 CCD camera (Gatan), operated with Digital Micrograph software (Gatan) for acquisition. Human tissues Acetic acid-formol-alcohol-fixed and paraffin-embedded human renal tissue specimens were obtained from the Pathology department of Hôpital Eurofug Georges Pompidou, Paris, France.
  • Podocyte number was assessed by the number of WT1-positive cells, podocyte differentiation by nephrin- positive area, parietal cells (PECs) activation by CD44-positive glomerular area or the number of CD44-positive glomeruli, per glomerular cross-section, in an average of 30 sections per mice.
  • Tubular injury was assessed by TIM1-positive tubule area per kidney section.
  • In situ apoptotic cells in kidneys were quantified by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) using Click-iT TM Plus TUNEL Assay (Invitrogen, #C10617) according to the manufacturer’s instruction.
  • Immunofluorescence mages were taken with a fluorescent microscope with Apotome module (Zeiss Apotome.2), using Zen 2.6 software (Zeiss).
  • Semi- automatic quantifications were performed using CellProfiler 4.2.1. software (Broad Institute, USA).
  • FSD Filtration slit density
  • PEMP Podocyte Effacement Measurement Procedure
  • VASN is mainly expressed in podocytes in mice and humans
  • a transgenic mouse with overexpression of a fluorescent protein (Venus) under the transcriptional regulation of the Vasn gene, we were able to locate the expression of Vasn in the kidneys.
  • Vasn mRNA expression was mainly and strongly found in the podocytes and also the vascular smooth muscle cells (data not shown).
  • the same transcription pattern was observed by in situ hybridization on kidney biopsies from humans (data not shown). Milder expression was also found in mesangial cells, PEC and proximal tubular cells. This expression was further confirmed using immunohistochemistry (data not shown).
  • mice with inducible deletion of the Vasn gene either globally after TMX injection (CreERT2-Vasnlox/lox) or podocyte-specific after oral doxycycline administration (NEFTA8-Cre-GFP-Vasnlox/lox). These mice also developed nephrotic syndrome after 14 to 17 days ( Figures 2a-e, and Figures 3a-c). Structurally, we observed severe lesions of FSGS in all these mice, with diffuse foot processes effacement and podocyte partial detachment from the GBM, along with urinary pseudocysts and vesicles accumulation in the cytoplasm ( Figures 1f, 2f, 3g).
  • VASN deficiency does not induce podocyte loss by apoptosis
  • TUNEL assay we did not observe any nuclear staining in podocytes of PodoCre-Vasnlox/lox mice; the only staining we had in glomerular sections was from a few circulating immune cells in the vascular compartment (data not shown).
  • As in situ apoptotic podocyte is theoretically difficult to observe because of the short duration process and rapid detachment in this context, we also studied the viability of immortalized human podocyte cell line in vitro.
  • Podocytes transduced with a lentivirus encoding for an anti-Vasorin small-hairpin RNA did not show increased cell mortality as compared to control cells (scramble) (data not shown). Instead, their metabolic activity was more important, as reflected by a higher aerobic respiration capacity in a resazurin reduction assay (data not shown), and by their oxygen consumption rate, for basal respiration and ATP production. However, their maximal respiration capacity was similar to scramble cells, a reduced spare respiratory capacity that might reflect greater stress sensitivity (data not shown). Our findings suggest that podocyte death is not the primary cause of podocyte detachment.
  • RNA sequencing revealed down- regulation of genes involved in the cell-cycle control, especially in S to G2/M-transition, interferon-mediated immune response, and an up-regulation of genes involved in metabolism, which is consistent with the cell respiration experiments, but also in epithelial-mesenchymal transition, and cell adhesion (data not shown).
  • VASN deficiency increases podocyte focal adhesion dynamics
  • shVASN podocytes had more focal adhesion complexes than scramble cells at baseline, as stained by phopho-paxillin (data not shown).
  • HB-EGF heparin-binding epidermal growth factor
  • shVASN podocytes had some features of epithelial-mesenchymal transition, as characterized by a switch from P-cadherin to N- cadherin expression (data not shown). Altogether, our data suggest that VASN deficiency might lead to podocyte mesenchymal transition and increase their ability to migrate from the GBM. VASN deficiency increases podocyte proliferative capacity
  • One striking feature of shVASN podocytes is the high frequency of multinucleated cells (data not shown). We, therefore, studied the cell cycle using DNA staining by FACS.
  • shVASN podocytes had a higher cell-cycle reentry capacity, as suggested by an increased S- phase with a decreased G0/1-phase cells proportion and an S-phase slow-down, as indicated by a reduced proportion of cells in the G2-M phase (data not shown).
  • cyclins A2, B1, CDK1, and CDK2, proteins involved in S-G2 transition were down-regulated, cyclin B1 decreased expression being further confirmed by IF and WB (data not shown).
  • IF and WB data not shown.
  • podocytes from PodoCre-Vasnlox/lox mice expressed more PCNA, an early-phase proliferative marker, and less p57, a podocyte cell-cycle inhibitor (data not shown).
  • GFP-cell sorting by FACS from NEFTA8-Cre-GFP-Vasnlox/lox mice we also observed an increase in podocyte cell-cycle reentry (data not shown).
  • Our findings suggest that VASN is a gatekeeper of podocytes' cell cycle, ensuring their physiological quiescence.
  • VASN is markedly regulated in human and mice kidney diseases
  • Vasn transcription was markedly regulated. If this expression was strongly upregulated in minimal change diseases, diabetic nephropathy, or lupus proliferative nephritis, it was conversely downregulated in extracapillary diseases such as ANCA vasculitis and secondary FSGS ( Figures 4a).
  • ANCA vasculitis and secondary FSGS Figures 4a.
  • VASN haploinsufficiency increases mice sensitivity to kidney diseases
  • Mice with podocyte-specific heterozygous deletion of Vasn did not show structural and functional renal abnormalities for up to 21 months (data not shown).
  • heterozygous PodoCre-Vasnlox/wt mice had more significant weight loss, podocyte dedifferentiation and loss, and also more severe albuminuria and kidney failure than their wild-type control littermates (data not shown).
  • heterozygous mice with partial Vasn deficiency had significantly accentuated podocyte dedifferentiation and loss, significantly more severe albuminuria, and kidney failure, but also strikingly more PEC activation and crescents formation (data not shown).
  • EGFR but not TGF ⁇
  • signaling pathways contribute to the VASN deficiency phenotype Aiming to determine the signaling pathway of VASN in podocytes
  • TGF ⁇ canonical pathway we studied the TGF ⁇ canonical pathway, as VASN is known to be a soluble TGF ⁇ trap.
  • oral administration of a TGF ⁇ -RI inhibitor did not reduce proteinuria or kidney failure (data not shown).
  • VASN affinity purification and mass spectrometry experiments are ongoing to determine the biological partners of VASN, using human podocyte overexpression of an HA-tagged VASN. Consistently with in vivo data, HA-tagged VASN was mainly found in the cytoplasm, especially in the perinuclear spaces, but also sometimes in the membrane in filopodia-like structures (data not shown). Endocrine substitution of soluble VASN does not prevent glomerular destruction As the expression of VASN in podocytes was mainly nuclear and cytoplasmic, we hypothesized that its membrane and soluble forms did not play a major role in podocyte homeostasis.
  • VASN deficient mice attached to another VASN deficient mice were sick as compared to WT mice (Cre-Cre-).
  • VASN Vasorin
  • VASN podocyte abundance was relatively increased in MN, MCD, primary FSGS and LN which could help differential diagnosis.
  • VASN expression level could be useful for the differential diagnosis between primary and secondary FSGS.
  • VASN appears to be a critical survival factor for the podocyte. Maintening a high level of VASN in podocytes or preventing its downregulation may help protect the glomerular function in patients. VASN is overexpressed in the context of stress, such as hyperfiltration or hyperglycemia. Then its expression would be progressively lost during the development of chronic kidney disease and more rapidly during FSGS and CGN such as ANCA-associated vasculitis-mediated CGN. VASN thus represents a prognostic factor or even a therapeutic target in human renal diseases.
  • REFERENCES Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Abstract

Aiming to identify factors underlying podocytes loss, the inventors revealed a markedly decreased expression of Vasorin (VASN) using comparative deep RNA sequencing of microdissected glomeruli from controls and patients diagnosed with FSGS or crescentic glomerulonephritis (CGN), that they further confirmed by in situ hybridization and immunohistochemistry. In particular, the inventors found that VASN is mainly expressed in podocytes during health. Furthermore, the inventors found that the level of glomerular or podocyte expression of the VASN gene depends on the kind of glomerular disease considered, suggesting this could be a helpful biomarker for differential diagnosis or outcome prediction. Of specific interest, 1/ the expression of VASN is significantly less in kidneys from patients suffering from secondary FSGS or anti-neutrophil cytoplasmic antibodies (ANCA) associated crescentic glomerulonephritis than in control normal kidneys or minimal change disease (MN), suggesting that low VASN abundance would predict poor outcome. 2/ Podocyte expression of the VASN gene is significantly less in secondary FSGS than in primary FSGS. 3/ Podocyte expression of the VASN gene is significantly less in FSGS (primary and secondary) than in MCD cases. 4/ Podocyte or glomerular expression of the VASN gene is increased substantially in kidneys biopsies diagnosed with membranous nephropathy (MN), minimal change disease (MCD), and lupus nephritis (LN) as compared with controls and FSGS, suggesting that this could be used to discriminate disease types, causes, outcome, or response to therapy. Accordingly, the present invention relates to the use of VASN as a biomarker and a biotarget for kidney diseases.

Description

VASORIN AS A BIOMARKER AND BIOTARGET IN NEPHROLOGY FIELD OF THE INVENTION: The invention is in the field of medicine, in particular nephrology. BACKGROUND OF THE INVENTION: Chronic kidney diseases (CKD) affect around one out of ten people worldwide and were the 12th leading cause of mortality in 2017 (GBD Chronic Kidney Disease Collaboration. Global, regional, and national burden of chronic kidney disease, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet. 29 févr 2020;395(10225):709‑33). Glomerular diseases are the leading cause of CKD. For this reason, glomerular resident cells are important therapeutic targets of current research (Wiggins RC. The spectrum of podocytopathies: a unifying view of glomerular diseases. Kidney Int. 2007;71(12):1205‑14). The glomerular filtration barrier is composed of three layers: the glomerular basement membrane (GBM), lined by a fenestrated endothelium on the vascular side, and the podocytes, specialized glomerular epithelial cells, on the urinary side. Recent signs of progress have highlighted the central role of podocytes in kidney function as a key organizer of glomerular development and maintenance. Injured podocytes are considered a common feature of glomerular diseases and invariably respond with foot process effacement and podocyte detachment, leading to focal and segmental glomerulosclerosis (FSGS) and irreversible loss of kidney function (Kriz W, Shirato I, Nagata M, LeHir M, Lemley KV. The podocyte’s response to stress: the enigma of foot process effacement. Am J Physiol Renal Physiol. 15 févr 2013;304(4):F333-347). As terminally differentiated cells, podocytes are incapable of reentering cell-cycle, limiting their self-renewal capacity. Indeed, forced cytokinesis might lead to podocyte aneuploidy, mitotic catastrophe, and loss by detachment (Mulay SR, Thomasova D, Ryu M, Kulkarni OP, Migliorini A, Bruns H, et al. Podocyte loss involves MDM2-driven mitotic catastrophe. J Pathol. juill 2013;230(3):322‑35.; Liapis H, Romagnani P, Anders HJ. New insights into the pathology of podocyte loss: mitotic catastrophe. Am J Pathol.2013;183(5):1364‑74). Podocyte depletion is sufficient to cause experimental glomerulosclerosis in rodents (Wharram BL, Goyal M, Wiggins JE, Sanden SK, Hussain S, Filipiak WE, et al. Podocyte depletion causes glomerulosclerosis: diphtheria toxin-induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene. J Am Soc Nephrol. 2005;16(10):2941‑52), and both clinical and animal model studies have evidenced a correlation between viable podocyte loss and kidney disease activity in humans during various kidney diseases, including diabetic nephropathy (Pagtalunan ME, Miller PL, Jumping-Eagle S, Nelson RG, Myers BD, Rennke HG, et al. Podocyte loss and progressive glomerular injury in type II diabetes. J Clin Invest. 1997;99(2):342‑8; Weil EJ, Lemley KV, Mason CC, Yee B, Jones LI, Blouch K, et al. Podocyte detachment and reduced glomerular capillary endothelial fenestration promote kidney disease in type 2 diabetic nephropathy. Kidney Int.2012;82(9):1010‑7), IgA nephropathy (Lemley KV, Lafayette RA, Safai M, Derby G, Blouch K, Squarer A, et al. Podocytopenia and disease severity in IgA nephropathy. Kidney Int. 2002;61(4):1475‑85), active FSGS (Vogelmann SU, Nelson WJ, Myers BD, Lemley KV. Urinary excretion of viable podocytes in health and renal disease. Am J Physiol Renal Physiol. 2003;285(1):F40-48, Hara M, Yanagihara T, Kihara I. Urinary podocytes in primary focal segmental glomerulosclerosis. Nephron. 2001;89(3):342‑7) and lupus nephritis (Vogelmann SU, Nelson WJ, Myers BD, Lemley KV. Urinary excretion of viable podocytes in health and renal disease. Am J Physiol Renal Physiol. 2003;285(1):F40-48) as well as during the normal ageing process (Puelles VG, Cullen-McEwen LA, Taylor GE, Li J, Hughson MD, Kerr PG, et al. Human podocyte depletion in association with older age and hypertension. Am J Physiol Renal Physiol.2016;310(7):F656‑68). Hence, unravelling mechanisms of podocyte detachment is mandatory for the development of efficient, targeted therapies against glomerular diseases. SUMMARY OF THE INVENTION: The present invention is defined by the claims. In particular, the present invention relates to the use of Vasorin (VASN) as a biomarker and a biotarget in nephrology. DETAILED DESCRIPTION OF THE INVENTION: Aiming to identify factors underlying podocytes loss, the inventors revealed a markedly decreased expression of Vasorin (VASN) using comparative deep RNA sequencing of microdissected glomeruli from controls and patients diagnosed with FSGS or crescentic glomerulonephritis (CGN), that they further confirmed by in situ hybridization and immunohistochemistry. The inventors found that VASN is mainly expressed in podocytes during health. Furthermore, the inventors found that the level of glomerular or podocyte expression of the VASN gene depends on the kind of glomerular disease considered, suggesting this could be a helpful biomarker for differential diagnosis or outcome prediction. Of specific interest, 1/ the expression of VASN is significantly less in kidneys from patients suffering from secondary FSGS or anti-neutrophil cytoplasmic antibodies (ANCA) associated crescentic glomerulonephritis than in control normal kidneys or minimal change disease (MN), suggesting that low VASN abundance would predict poor outcome.2/ Podocyte expression of the VASN gene is significantly less in secondary FSGS than in primary FSGS. 3/ Podocyte expression of the VASN gene is significantly less in FSGS (primary and secondary) than in MCD cases.4/ Podocyte or glomerular expression of the VASN gene is increased substantially in kidney biopsies diagnosed with membranous nephropathy (MN), minimal change disease (MCD), and lupus nephritis (LN) as compared with controls and FSGS, suggesting that this could be used to discriminate diseases types, causes, outcome, or response to therapy. Global or podocyte-specific, constitutive of inducible gene deletion of Vasn induced dramatic features of podocytopathy and FSGS after 14 days, along with nephrotic syndrome and kidney failure. Both in vitro and in vivo, they show that VASN deficiency does not induce podocyte death but rather an epithelial-mesenchymal transition, enhancing their migratory and cell-cycle reentry capacity, which might lead to their secondary detachment from the GBM. Mice with podocyte-specific heterozygous deletion of Vasn did not show any evident baseline renal abnormalities. Still, they had more severe podocyte loss, albuminuria, and kidney failure than control mice when challenged by type 1 diabetic with glomerular hyperfiltration or crescentic glomerulonephritis (CGN) experimental models. These findings suggest that VASN is an essential master protein maintaining podocyte quiescence and homeostasis during health and kidney diseases. Partial loss of VASN in podocytes is expected to aggravate the course of glomerular diseases and may discriminate specific diseases. Diagnosis methods: The first object of the present invention relates to a method for diagnosing a kidney disease in a subject comprising determining in a biological sample obtained from the subject the expression level of Vasorin (VASN) wherein the level indicates whether or not the subject suffers from kidney disease. As used herein, the term “subject” refers to any mammal, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human. In some embodiments, the subject is a human who is susceptible to have a kidney disease, as described below. As used herein, the term “diagnosing” refers to classifying a disease or a symptom, determining the severity of the disease, monitoring disease progression, and forecasting an outcome of a disease and/or prospects of recovery. In the context of the invention, the method according to the invention allows the diagnosis of a kidney disease. As used herein, the term “kidney disease” refers to disease caused by kidneys being damaged and kidneys not filtering blood the way they should. In some embodiments, in the context of the invention, kidney disease is a chronic kidney disease (CKD). In some embodiments, the kidney disease selected from the group consisting of but not limited to nephrotic syndrome, membranous nephropathy (MN), kidney failure, primary focal segmental glomerulosclerosis (FSGS), secondary FGSG, collapsing FSGS, crescentic glomerulonephritis (CGN), minimal change disease (MCD), diabetic nephropathy (DN), ANCA vasculitis (ANCA) or lupus nephritis (LN). In some embodiments, the kidney disease is a chronic kidney disease (CKD). As used herein, the term “chronic kidney disease” or “CKD” has its general meaning in the art and refers to a progressive loss in renal function over a period of months or years. CKD is used to classify numerous conditions that affect the kidney, destruction of the renal parenchyma and the loss of functional nephrons or glomeruli. It should be further noted that CKD can result from different causes, but the final pathway remains renal fibrosis. CKD is defined as kidney damage or glomerular filtration rate (GFR) <60 mL/min/1 .73 m<2>for 3 months or more, irrespective of the cause. GFR can be estimated from calibrated serum creatinine and estimating equations, such as the Modification of Diet in Renal Disease (MDRD) Study equation or the Cockcroft-Gault formula. Kidney disease severity is classified into five stages according to the level of GFR. Examples of etiology of CKD include but are not limited to, cardiovascular diseases, hypertension, diabetes, glomerulonephritis, polycystic kidney diseases, and kidney graft rejection. In some embodiments, the kidney disease is nephrotic syndrome. As used herein, the term “nephrotic syndrome”(NS) refers to a rare disease defined by massive proteinuria (>3g/day, or >lg of urine protein per square meter of body-surface area per day in children) and hypoalbuminemia (< 30g/l) and result from loss of integrity of the glomerular filtration barrier. The main causes are genetic and immune. In some embodiments, the nephrotic syndrome is idiopathic nephrotic syndrome. As used herein, the term "idiopathic nephrotic syndrome" (INS) has its general meaning in the art and represents 80% of the causes of nephrotic syndrome in children and 25% in adults. INS is histologically characterized by the absence of lesion on light microscopy and lack of immunoglobulin or complement deposit with sometimes additional focal segmental glomerulosclerosis (FSGS) lesions. In some embodiments, the kidney disease is membranous nephropathy (MN). As used herein, the term “membranous nephropathy” or “MN” has its general meaning in the art and refers to a pathologically defined disorder of the kidney glomerulus. The specific lesion is an apparent thickening of the glomerular capillary walls, which results from immune complex formation on the outer aspect of the basement membrane (Couser, W. G. Primary membranous nephropathy. Clin. J. Am. Soc. Nephrol.12, 983–997 (2017)). The immune deposits consist of immunoglobulin G (IgG), the relevant antigens and complement components, including the membrane attack complex (MAC). The consequence of the immunological conflict is the loss of large amounts of proteins in the urine (proteinuria), which is predominantly mediated by the pathophysiological disturbance of the podocyte structure caused by immune complex deposition and MAC formation. Patients experience decreased serum albumin levels and generalized oedema, which define a condition called nephrotic syndrome. Most patients report fatigue as an important symptom. MN accounts for ~30% of cases of nephrotic syndrome in adults and is only surpassed in prevalence among nephrotic non-diabetic glomerular diseases by podocytopathy presenting as focal and segmental glomerulosclerosis lesions in some populations (African and Hispanic American individuals). In ~80% of patients, there is no underlying cause of MN (primary MN (pMN)) and 20% are associated with medications, such as NSAIDs, or other diseases (secondary MN (sMN)) such as lupus (also referred to as systemic lupus erythematosus (SLE)), hepatitis B or hepatitis C, and malignancies1. In some embodiments, the kidney disease is minimal change disease (MCD). As used herein, the term "MCD," also known as minimal change disease (MCD), or minimal- change nephrotic syndrome (MCNS), or lipoid nephrosis, or nil disease, refers to minimal change nephropathy. It arises from a histopathologic lesion in the glomerulus and is characterized by intense proteinuria, leading to edema and intravascular volume depletion. It is the most common single form of nephrotic syndrome in children, but it can also occur in adults. In some embodiments, the kidney disease is focal segmental glomerulosclerosis (FSGS). As used herein, the term "focal segmental glomerulosclerosis” (FSGS) refers to a histologic lesion rather than a specific disease entity that is commonly found to underlie the nephrotic syndrome in adults and children. The term "focal" is added because, in FSGS, only some of the glomeruli filters become scarred. The term "Segmental" means that only some sections of the glomerulus become scarred, just parts of them. FSGS is characterized by the presence of sclerosis in parts (segmental) of at least one glomerulus (focal) in the entire kidney biopsy specimen when examined by light microscopy (LM), immunofluorescence (IF), or electron microscopy (EM). The lesion of FSGS can be classified into primary, secondary (maladaptive or secondary to infectious agents or to medication or toxin), genetic (including associated with APOL1 risk variants), and unknown form. In some embodiments, the kidney disease is crescentic glomerulonephritis (CGN). As used herein, the term "crescentic glomerulonephritis” (CGN) or "rapidly progressive glomerulonephritis (RPGN)" should be understood to mean severe glomerular inflammation destroying the glomerular tuft and accumulation of inflammatory cells around the tuft in the shape of a crescent. A flare of CGN indicates recurrence of CGN after a period of time in an individual known to have either prior CGN or a systemic autoimmune disease (such as ANCA vasculitis or lupus) in which one may expect CGN as part of the disease. The terms "crescentic glomerulonephritis (CGN)" and "rapidly progressive glomerulonephritis (RPGN)" may be used interchangeably. In some embodiments, the kidney disease is diabetic nephropathy (DN). As used herein, the term “diabetic nephropathy” (DN) refers to diabetic kidney disease, a chronic loss of kidney function in those with diabetes mellitus. Diabetic nephropathy is globally the leading cause of chronic kidney disease (CKD) and end-stage renal disease (ESRD). The triad of protein leaking into the urine (proteinuria or albuminuria), rising blood pressure with hypertension, and then falling renal function is common to many forms of CKD. Protein loss in the urine due to damage of the glomeruli may become massive and cause low serum albumin with resulting generalized body swelling (edema) so called nephrotic syndrome. In some embodiments, the kidney disease is anti-neutrophil cytoplasmic antibodies (ANCA) vasculitis. As used herein, the term “anti-neutrophil cytoplasmic antibodies” refers to a group of autoantibodies of the IgG type that react with the cytoplasmic constituents of neutrophils and monocytes. The interaction between primed neutrophils and ANCAs releases factors that activate the alternative complement pathway, initiating an amplification loop that is thought to sustain necrotizing inflammation during flares of ANCA-associated vasculitis (AAV). In some embodiments, the kidney disease is proliferative glomerulonephritis caused by systemic lupus erythematosus. As used herein, the term “systemic lupus erythematosus” (SLE) refers to a systemic autoimmune disease thought to be manifested by a wide range of abnormalities in immune regulation. It is the most common type of lupus. In some embodiments, the proliferative glomerulonephritis is lupus nephritis. As used herein, the term “lupus nephritis” (LN) refers to an inflammation of the kidney that is caused by systemic lupus erythematosus (SLE). Up to 60% of lupus patients will develop LN. When the kidneys are inflamed, they cannot function normally to filter toxins, byproducts, excess salts, excess fluid, and other metabolic and immune products in the blood. If not controlled, LN can lead to kidney failure. Even with treatment, loss of kidney function sometimes progresses. If both kidneys fail, subjects with LN may need dialysis. Ultimately, it may be necessary for the LN subject to receive a kidney transplant. Symptoms of loss of or abnormal kidney function include increased amounts of protein in the urine (proteinuria), foaming in a subject’s urine, and/or a higher level of blood urea nitrogen (BUN). Typically, a high level of VASN indicates that the subject suffers from a minimal change disease (MCD), diabetic nephropathy (DN), a membranous nephropathy (MN), or a lupus nephritis-LN). Typically, a low level of VASN indicates that the subject suffers from a nephrotic syndrome, a chronic kidney disease, an ANCA-associated vasculitis, crescentic glomerulonephritis (CGN) or a focal segmental glomerulosclerosis (FSGS). As used herein, the term “high” refers to a measure greater than normal, greater than a standard such as a predetermined reference value or a subgroup measure, or relatively greater than another subgroup measure. For example, a high level of VASN refers to a level of VASN that is greater than a normal VASN level. A normal VASN level may be determined according to any method available to one skilled in the art. A high level of VASN may also refer to a level that is equal to or greater than a predetermined reference value, such as a predetermined cutoff. A high level of VASN may also refer to a level of VASN wherein a high VASN subgroup has relatively greater levels of VASN than another subgroup. For example, without limitation, according to the present specification, two distinct patient subgroups can be created by dividing samples around a mathematically determined point, such as, without limitation, a median, thus creating a subgroup whose measure is high (i.e., higher than the median) and another subgroup whose measure is low. In some cases, a “high” level may comprise a range of levels that is very high and a range of levels that is “moderately high,” where moderately high is a level that is greater than normal but less than “very high”. In particular embodiment, a higher level of VASN than a predetermined reference value indicates that the subject suffers from a minimal change disease (MCD), diabetic nephropathy (DN), a membranous nephropathy (MN), or a lupus nephritis (LN). As used herein, the term “low” refers to a level that is less than normal or less than a standard, such as a predetermined reference value or a subgroup measure that is relatively less than another subgroup level. For example, a low level of VASN means a level of VASN that is less than a normal level of in a particular set of samples of patients. A normal level of VASN measure may be determined according to any method available to one skilled in the art. A low level of VASN may also mean a level that is less than a predetermined reference value, such as a predetermined cutoff. A low level of VASN may also indicate a level wherein a low level VASN subgroup is relatively lower than another subgroup. For example, without limitation, according to the present specification, two distinct patient subgroups can be created by dividing samples around a mathematically determined point, such as, without limitation, a median, thus creating a group whose measure is low (i.e., less than the median) with respect to another group whose measure is high (i.e., greater than the median). In particular embodiment, a lower level of VASN than a predetermined reference value indicates that the subject suffers from a nephrotic syndrome, a chronic kidney disease, an ANCA-associated vasculitis, crescentic glomerulonephritis (CGN) or a focal segmental glomerulosclerosis (FSGS). As used herein, the term “predetermined reference value” refers to a threshold value or a cut- off value. A "threshold value", “reference value”, or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skill in the art. For example, retrospective measurement of the concentration of the marker of the invention (e.g., VASN) in properly banked historical subject samples may be used in establishing the predetermined corresponding reference value. In some embodiments, the predetermined corresponding reference value is the median measured in the population of the subjects for the marker of in the invention (VASN, for example). In some embodiments, the threshold value has to be determined to obtain the optimal sensitivity and specificity according to the test function and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the concentration of the marker of the invention (VASN, for example) in a group of reference, one can use algorithmic analysis for the statistical treatment of the expression levels determined in samples to be tested and thus obtain a classification standard having significance for sample classification. The full name of the ROC curve is the receiver operator characteristic curve, also known as the receiver operation characteristic curve. It is mainly used for clinical and biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal diagnostic test results) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate, and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result improves as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used to draw the ROC curve, such as MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE- ROC.SAS, GB STAT VI0.0 (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc. A further aspect of the invention relates to a method for discriminating a primary focal segmental glomerulosclerosis (FSGS) from a secondary FSGS in a subject comprising i) determining in a biological sample obtained from the subject the expression level of Vasorin (VASN); ii) comparing the expression level determined at step i) with a predetermined reference value and iii) concluding that the subject has or is susceptible to have a primary FSGS when the expression level determined at step i) is higher than its predetermined reference value, or concluding that the subject has or is susceptible to have a secondary FSGS when the expression level determined at step i) is lower than its predetermined reference value. A further object of the present invention relates to a method for discriminating minimal change disease (MCD) from FSGS in a subject comprising i) determining in a biological sample obtained from the subject the expression level of Vasorin (VASN); ii) comparing the expression level determined at step i) with a predetermined reference value and iii) concluding that the subject has or is susceptible to have a FSGS when the expression level determined at step i) is lower than its predetermined reference value or concluding that the subject has or is susceptible to have a MCD when the expression level determined at step i) is higher than its predetermined reference value. As used herein, the term “discriminating” refers to identifying, observing a difference, or distinguishing two groups. A further object of the present invention relates to a method for predicting the risk of a subject suffering from a kidney disease progresses to a nephrotic syndrome comprising i) determining in a biological sample obtained from the subject the expression level of Vasorin (VASN), ii) comparing the expression level determined at step i) with a predetermined reference value and iii) concluding that the subject has a significantly higher risk of having a nephrotic syndrome when the expression level determined at step i) is lower than its predetermined reference value, or concluding that the subject a lower risk of having a nephrotic syndrome when the expression level determined at step i) is higher than its predetermined reference value. In some embodiments, the method is particularly suitable for predicting the occurrence of a nephrotic syndrome, a chronic kidney disease, a kidney failure or death. As used herein, the term “predicting” means that the subject to be analyzed by the method of the invention is allocated either into the group of subjects who will relapse or into a group of subjects who will not relapse after treatment. As used herein, the term "risk", in the context of the present invention, relates to the probability that an event will occur over a specific time period, as in the conversion to relapse, and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low-risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of an event and (1- p) is the probability of no event) to no- conversion. "Risk evaluation," or "evaluation of risk" in the context of the present invention, encompasses predicting the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another, i.e., from a normal condition to relapse or to one at risk of developing relapse. Risk evaluation can also comprise the prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of relapse, either in absolute or relative terms in reference to a previously measured population. The methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion to relapse, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk of having a relapse. In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk of having relapse. In some embodiments, the present invention may be used so as to discriminate those at risk of having relapse from normal or those having relapse disease from normal. A further object of the invention relates to a method for determining whether a subject suffering from a kidney disease achieves a response to treatment comprising the following steps i) obtaining a biological sample from said subject, ii) determining the expression level of VASN, iii) comparing the determined level with a predetermined reference level and iii) concluding that the subject achieves a response to the treatment when the level of VASN is higher than the reference value; or concluding that the subject does not achieve a response treatment when the expression level of VASN is lower than the reference value. In particular, the subject suffers from FSGS, and the treatment is immunosuppressive. In some embodiments, the predetermined level is the level determined in a biological sample obtained from the subject before the treatment. Thus, in some embodiments, the present invention relates to a method of determining whether a subject suffering from a kidney disease achieves a response to treatment comprising i) determining the level of VASN in a biological sample obtained from the subject before the treatment, ii) determining the level of VASN in a sample obtained from the subject after treatment initiation, iii) comparing the level determined at step i) with the level determined at step ii) and concluding that the subject achieves a response treatment when the level of VASN determined at step ii) is lower than the level determined at step i), or concluding that the subject does not achieves a response treatment when the level of VASN determined at step ii) is lower than the level determined at step i). As used herein, the terms " achieve a response" or "respond" refer to the response to a treatment of the subject suffering from primary or maladaptive FSGS. Typically, such treatment induces, ameliorates, or otherwise causes an improvement in the pathological symptoms, disease progression, or physiological conditions associated with or resistance to succumbing to a maladaptive FSGS. In particular, in the context of the invention, the term "respond" refers to the ability of corticosteroid or other immunomodulatory treatment to improve the pathological symptoms; thus, the subject presents a clinical improvement compared to the subject who does not receive the treatment. The said subject is considered as a "responder" to the treatment. The term "not respond" refers to a subject who does not present any clinical improvement to the treatment with corticosteroid or other immunomodulatory treatment. This subject is considered a "non-responder" to the treatment. Accordingly, the subject considered a "non-responder" has particular monitoring in the therapeutic regimen. In some embodiments, the response to a treatment is determined by Response evaluation criteria in solid tumors (RECIST) criteria. These criteria refer to a set of published rules that define when the disease in the subjects improves ("responds"), stays the same ("stabilizes"), or worsens ("progresses") during treatment. In the context of the invention, when the subject is identified as a responder, it means that said subject improves overall and progression-free survival (OS/PFS). As used herein, the term “immunosuppressive treatment” or “immunomodulatory treatment “ refers to any substance capable of producing an immunosuppressive effect, e.g., the prevention or diminution of the immune response and in particular the prevention or diminution of the production of Ig. Immunosuppressive drugs include, without limitation, thiopurine drugs such as azathioprine (AZA) and metabolites thereof; calcineurin inhibitor immunosuppressants such as cyclosporine and voclosporin; nucleoside triphosphate inhibitors such as mycophenolic acid (Cellcept) and its derivative (Myfortic); derivatives thereof; prodrugs thereof; and combinations thereof. Other examples include but are not limited to 6- mercaptopurine ("6-MP"), cyclophosphamide, mycophenolate, prednisolone, sirolimus, dexamethasone, rapamycin, FK506, mizoribine, azathioprine and tacrolimus. In some embodiments, the immunosuppressive drug is a calcineurin inhibitor. As used herein, the term “calcineurin inhibitor” has its general meaning in the art and refers to substances that block calcineurin (i.e., calcium/calmodulin-regulated protein phosphatase involved in intracellular signalling) dephosphorylation of appropriate substrates, by targeting calcineurin phosphatase (PP2B, PP3), a cellular enzyme that is involved in gene regulation. A calcineurin inhibitor of the present invention is typically an immunophilin-binding compound having calcineurin inhibitory activity. Immunophilin-binding calcineurin inhibitors are compounds forming calcineurin inhibiting complexes with immunophilins, e.g. cyclophilin and macrophilin. Examples of cyclophilin-binding calcineurin inhibitors are cyclosporines or cyclosporine derivatives (hereinafter cyclosporines) and examples of macrophilin-binding calcineurin inhibitors are ascomycin (FR 520) and ascomycin derivatives (hereinafter ascomycins). A wide range of ascomycin derivatives are known, which are either naturally occurring among fungal species or are obtainable by manipulation of fermentation procedures or by chemical derivatization. Ascomycin-type macrolides include ascomycin, tacrolimus (FK506), sirolimus and pimecrolimus. Cyclosporine, originally extracted from the soil fungus Potypaciadium infilatum, has a cyclic 11-amino acid structure and includes, e.g., Cyclosporines A through I, such as Cyclosporine A, B, C, D and G. Voclosporin is a next-generation calcineurin inhibitor that is a more potent and less toxic semi-synthetic derivative of cyclosporine A. In some embodiments, the calcineurin inhibitor of the present invention is the trans-version of voclosporin, trans-ISA247 (Cas number 368455-04-3), which is described in, for example, US Patent Publication No.: 2006/0217309, which is hereby incorporated herein by reference. Further compositions of voclosporin are described, for example, in U.S. Pat. No. 7,060,672, which is hereby incorporated herein by reference. Tacrolimus (FK506) is another calcineurin inhibitor which is also a fungal product but has a macrolide lactone structure. Sirolimus (rapamycin) is a microbial product isolated from the actinomycete Streptomyces hygroscopicus. Sirolimus binds to an immunophilin (FK-binding protein 12, FKBP12), forming a complex that inhibits the mammalian target of rapamycin (mTOR) pathway by directly binding the mTOR Complex1 (mTORC1). Pimecrolimus is also a calcineurin inhibitor. Calcineurin inhibitors such as cyclosporine A, voclosporin, ascomycin, tacrolimus, pimecrolimus, an analog thereof, or a pharmaceutically acceptable salt thereof, can be utilized in a mixed micellar composition of the present disclosure. In some embodiments, the immunosuppressive drug is a corticosteroid. As used, the term “corticosteroids” has a general meaning in the art and refers to a class of active ingredients having a hydrogenated cyclopentoperhydrophenanthrene ring system endowed with anti- inflammatory activity. Corticosteroid drugs typically include cortisone, cortisol, hydrocortisone (11β,17-dihydroxy, 21-(phosphonooxy)-pregn-4-ene, 3,20-dione disodium), dihydroxycortisone, dexamethasone (21-(acetyloxy)-9-fluoro-1β,17-dihydroxy-16α-m- ethylpregna-1,4-diene-3,20-dione), and highly derivatized steroid drugs such as beconase (beclomethasone dipropionate, which is 9-chloro-11-β, 17,21, trihydroxy-16β-methylpregna- 1,4 diene-3,20-dione 17,21-dipropionate). Other examples of corticosteroids include prednisone, prednisolone, methylprednisolone, deflazacort and betamethasone, cortisone, hydrocortisone, beclomethasone dipropionate, budesonide, dexamethasone sodium phosphate, flunisolide, fluticasone propionate, triamcinolone acetonide, fluocinolone, fluocinonide, betamethasone dipropionate, betamethasone valerate, desonide, desoximetasone, fluocinolone, triamcinolone, triamcinolone acetonide, clobetasol propionate, and dexamethasone. In some embodiments, the immunosuppressive drug is a B cell depleting agent. As used herein, the term “B cell depleting agent” refers to any agent capable of triggering B cells' lymphodepletion. In some embodiments, the B cell-depleting agent is an antibody having specificity for CD20. Examples of antibodies having specificity for CD20 include: “C2B8” which is now called “Rituximab” (“RITUXAN®”) (U.S. Pat. No. 5,736,137, expressly incorporated herein by reference), a chimaeric pan-B antibody targeting CD20; the yttrium- [90]-labeled 2B8 murine antibody designated “Y2B8” or “Ibritumomab Tiuxetan” ZEVALIN® (U.S. Pat. No. 5,736,137, expressly incorporated herein by reference), a murine IgG1 kappa mAb covalently linked to MX-DTPA for chelating to yttrium-[90]; murine IgG2a “BI,” also called “Tositumomab,” optionally labeled with radioactive 131I to generate the “1311-B1” antibody (iodine 131 tositumomab, BEXXAR™) (U.S. Pat. No. 5,595,721, expressly incorporated herein by reference); murine monoclonal antibody “1F5” (Press et al. Blood 69 (2):584-591 (1987) and variants thereof including “framework patched” or humanized 1F5 (WO03/002607, Leung, S.; ATCC deposit HB-96450); murine 2H7 and chimeric 2H7 antibody (U.S. Pat. No. 5,677,180, expressly incorporated herein by reference); humanized 2H7, also known as ocrelizumab (PRO-70769); Ofatumumab (Arzerra), a fully human IgG1 against a novel epitope on CD20 huMax-CD20 (Genmab, Denmark; WO2004/035607 (U.S. Ser. No. 10/687,799, expressly incorporated herein by reference)); AME-133 (ocaratuzumab; Applied Molecular Evolution), a a fully-humanized and optimized IgG1 mAb against CD20; A20 antibody or variants thereof such as chimeric or humanized A20 antibody (cA20, hA20, respectively) (U.S. Ser. No. 10/366,709, expressly incorporated herein by reference, Immunomedics); and monoclonal antibodies L27, G28-2, 93-1B3, B-CI or NU-B2 available from the International Leukocyte Typing Workshop (Valentine et al, In: Leukocyte Typing III (McMichael, Ed., p.440, Oxford University Press (1987)). Further, suitable antibodies include, e.g., antibody GA101 (obinutuzumab), a third-generation humanized anti-CD20-antibody of Biogen Idec/Genentech/Roche. Moreover, BLX-301 of Biolex Therapeutics, a humanized anti- CD20 with optimized glycosylation or Veltuzumab (hA20), a 2nd-generation humanized antibody specific for CD20 of Immunomedics or DXL625, derivatives of veltuzumab, such as the bispecific hexavalent antibodies of IBC Pharmaceuticals (Immunomedics) which are comprised of a divalent anti-CD20 IgG of veltuzumab and a pair of stabilized dimers of Fab derived from milatuzumab, an anti-CD20 mAb enhanced with InNexus' Dynamic Cross Linking technology, of Inexus Biotechnology both are humanized anti-CD20 antibodies are suitable. Further suitable antibodies are BM-ca (a humanized antibody specific for CD20 (Int J. Oncol. 2011 February; 38(2):335-44)), C2H7 (a chimeric antibody specific for CD20 (Mol Immunol. 2008 May; 45(10):2861-8)), PRO131921 (a third generation antibody specific for CD20 developed by Genentech), Reditux (a biosimilar version of rituximab developed by Dr Reddy's), PBO-326 (a biosimilar version of rituximab developed by Probiomed), a biosimilar version of rituximab developed by Zenotech, TL-011 (a biosimilar version of rituximab developed by Teva), CMAB304 (a biosimilar version of rituximab developed by Shanghai CP Guojian), GP-2013 (a biosimilar version of rituximab developed by Sandoz (Novartis)), SAIT- 101 (a biosimilar version of rituximab developed by Samsung BioLogics), a biosimilar version of rituximab developed by Intas Biopharmaceuticals, CT-P10), a biosimilar version of rituximab developed by Celltrion), a biosimilar version of rituximab developed by Biocad, Ublituximab (LFB-R603, a transgenically produced mAb targeting CD20 developed by GTC Biotherapeutics (LFB Biotechnologies)), PF-05280586 (presumed to be a biosimilar version of rituximab developed by Pfizer), Lymphomun (Bi-20, a trifunctional anti-CD20 and anti-CD3 antibody, developed by Trion Pharma), a biosimilar version of rituximab developed by Natco Pharma, a biosimilar version of rituximab developed by iBio, a biosimilar version of rituximab developed by Gedeon Richter/Stada, a biosimilar version of rituximab developed by Curaxys, a biosimilar version of rituximab developed by Coherus Biosciences/Daiichi Sankyo, a biosimilar version of rituximab developed by BioXpress, BT-D004 (a biosimilar version of rituximab developed by Protheon), AP-052 (a biosimilar version of rituximab developed by Aprogen), a biosimilar version of ofatumumab developed by BioXpress, MG-1106 (a biosimilar version of rituximab developed by Green Cross), IBI-301 (a humanized monoclonal antibody against CD20 developed by Innovent Biologics), BVX-20 (a humanized mAb against the CD20 developed by Vaccinex), 20-C2-2b (a bispecific mAb-IFNalpha that targets CD20 and human leukocyte antigen-DR (HLA-DR) developed by Immunomedics), MEDI-552 (developed by MedImmune/AstraZeneca), the anti-CD20/streptavidin conjugates developed by NeoRx (now Poniard Pharmaceuticals), the 2nd generation anti-CD20 human antibodies developed by Favrille (now MMRGlobal), TRU-015, an antibody specific for CD20 fragment developed by Trubion/Emergent BioSolutions, as well as other precloinical approaches by various companies and entities. All aforementioned publications, references, patents and patent applications are incorporated by reference in their entirety. All antibodies disclosed therein may be used within the present invention. As used herein, the term "biological sample" refers to any substance derived from a living organism. For example, a sample may be derived from blood as a urine sample, a serum sample, a plasma sample, and or a whole blood sample. Alternatively, a sample may be derived from a tissue collected, for example, by a biopsy. Such a tissue sample may comprise, for example, kidney tissue (e.g., biopsies in glomeruli). In particular, the kidney tissue sample comprises podocytes. The tissue sample can, of course, be subjected to a variety of well-known post- collection preparative and storage techniques (e.g., fixation, storage, freezing, etc.). The sample can be fresh, frozen, fixed (e.g., formalin-fixed), or embedded (e.g., paraffin-embedded). In a particular embodiment, the sample has been previously obtained from the subject. As used herein, the term “Vasorin” or “VASN” refers to a typical type I membrane protein containing tandem arrays of a characteristic leucine-rich repeat motif, an epidermal growth factor-like motif, and a fibronectin type III-like motif at the extracellular domain. VASN is an evolutionarily conserved single-pass type I transmembrane protein, with a high degree of similarity both at the DNA (95%) and protein (83%) levels between rodent and human homologs, suggesting highly conserved functions. The naturally occurring human VASN gene has the following nucleotide sequence as shown in Genbank Accession number NM_138440.3 (SEQ ID NO: 1), and the naturally occurring human VASN protein has the following amino acid sequence as shown in Genbank Accession number NP_612449.2 (SEQ ID NO: 2). SEQ ID NO: 1: 1 gactccggag cccgagcccg gggcgggtgg acgcggactc gaacgcagtt gcttcgggac 61 ccaggacccc ctcgggcccg acccgccagg aaagactgag gccgcggcct gccccgcccg 121 gctccctgcg ccgccgccgc ctcccgggac agaagatgtg ctccagggtc cctctgctgc 181 tgccgctgct cctgctactg gccctggggc ctggggtgca gggctgccca tccggctgcc 241 agtgcagcca gccacagaca gtcttctgca ctgcccgcca ggggaccacg gtgccccgag 301 acgtgccacc cgacacggtg gggctgtacg tctttgagaa cggcatcacc atgctcgacg 361 caggcagctt tgccggcctg ccgggcctgc agctcctgga cctgtcacag aaccagatcg 421 ccagcctgcc cagcggggtc ttccagccac tcgccaacct cagcaacctg gacctgacag 481 ccaacaggct gcatgaaatc accaatgaga ccttccgtgg cctgcggcgc ctcgagcgcc 541 tctacctggg caagaaccgc atccgccaca tccagcctgg tgccttcgac acgctcgacc 601 gcctcctgga gctcaagctg caggacaacg agctgcgggc actgcccccg ctgcgcctgc 661 cccgcctgct gctgctggac ctcagccaca acagcctcct ggccctggag cccggcatcc 721 tggacactgc caacgtggag gcgctgcggc tggctggtct ggggctgcag cagctggacg 781 aggggctctt cagccgcttg cgcaacctcc acgacctgga tgtgtccgac aaccagctgg 841 agcgagtgcc acctgtgatc cgaggcctcc ggggcctgac gcgcctgcgg ctggccggca 901 acacccgcat tgcccagctg cggcccgagg acctggccgg cctggctgcc ctgcaggagc 961 tggatgtgag caacctaagc ctgcaggccc tgcctggcga cctctcgggc ctcttccccc 1021 gcctgcggct gctggcagct gcccgcaacc ccttcaactg cgtgtgcccc ctgagctggt 1081 ttggcccctg ggtgcgcgag agccacgtca cactggccag ccctgaggag acgcgctgcc 1141 acttcccgcc caagaacgct ggccggctgc tcctggagct tgactacgcc gactttggct 1201 gcccagccac caccaccaca gccacagtgc ccaccacgag gcccgtggtg cgggagccca 1261 cagccttgtc ttctagcttg gctcctacct ggcttagccc cacagagccg gccactgagg 1321 cccccagccc gccctccact gccccaccga ctgtagggcc tgtcccccag ccccaggact 1381 gcccaccgtc cacctgcctc aatgggggca catgccacct ggggacacgg caccacctgg 1441 cgtgcttgtg ccccgaaggc ttcacgggcc tgtactgtga gagccagatg gggcagggga 1501 cacggcccag ccctacacca gtcacgccga ggccaccacg gtccctgacc ctgggcatcg 1561 agccggtgag ccccacctcc ctgcgcgtgg ggctgcagcg ctacctccag gggagctccg 1621 tgcagctcag gagcctccgt ctcacctatc gcaacctatc gggccctgat aagcggctgg 1681 tgacgctgcg actgcctgcc tcgctcgctg agtacacggt cacccagctg cggcccaacg 1741 ccacttactc cgtctgtgtc atgcctttgg ggcccgggcg ggtgccggag ggcgaggagg 1801 cctgcgggga ggcccataca cccccagccg tccactccaa ccacgcccca gtcacccagg 1861 cccgcgaggg caacctgccg ctcctcattg cgcccgccct ggccgcggtg ctcctggccg 1921 cgctggctgc ggtgggggca gcctactgtg tgcggcgggg gcgggccatg gcagcagcgg 1981 ctcaggacaa agggcaggtg gggccagggg ctgggcccct ggaactggag ggagtgaagg 2041 tccccttgga gccaggcccg aaggcaacag agggcggtgg agaggccctg cccagcgggt 2101 ctgagtgtga ggtgccactc atgggcttcc cagggcctgg cctccagtca cccctccacg 2161 caaagcccta catctaagcc agagagagac agggcagctg gggccgggct ctcagccagt 2221 gagatggcca gccccctcct gctgccacac cacgtaagtt ctcagtccca acctcgggga 2281 tgtgtgcaga cagggctgtg tgaccacagc tgggccctgt tccctctgga cctcggtctc 2341 ctcatctgtg agatgctgtg gcccagctga cgagccctaa cgtccccaga accgagtgcc 2401 tatgaggaca gtgtccgccc tgccctccgc aacgtgcagt ccctgggcac ggcgggccct 2461 gccatgtgct ggtaacgcat gcctgggccc tgctgggctc tcccactcca ggcggaccct 2521 gggggccagt gaaggaagct cccggaaaga gcagagggag agcgggtagg cggctgtgtg 2581 actctagtct tggccccagg aagcgaagga acaaaagaaa ctggaaagga agatgcttta 2641 ggaacatgtt ttgctttttt aaaatatata tatatttata agagatcctt tcccatttat 2701 tctgggaaga tgtttttcaa actcagagac aaggactttg gtttttgtaa gacaaacgat 2761 gatatgaagg ccttttgtaa gaaaaaataa aagatgaagt gtgtttcttg ggctca. SEQ ID NO: 2: MCSRVPLLLPLLLLLALGPGVQGCPSGCQCSQPQTVFCTARQGTTVPRDVPPDTVGLYVFENGITMLDA GSFAGLPGLQLLDLSQNQIASLPSGVFQPLANLSNLDLTANRLHEITNETFRGLRRLERLYLGKNRIRH IQPGAFDTLDRLLELKLQDNELRALPPLRLPRLLLLDLSHNSLLALEPGILDTANVEALRLAGLGLQQL DEGLFSRLRNLHDLDVSDNQLERVPPVIRGLRGLTRLRLAGNTRIAQLRPEDLAGLAALQELDVSNLSL QALPGDLSGLFPRLRLLAAARNPFNCVCPLSWFGPWVRESHVTLASPEETRCHFPPKNAGRLLLELDYA DFGCPATTTTATVPTTRPVVREPTALSSSLAPTWLSPTEPATEAPSPPSTAPPTVGPVPQPQDCPPSTC LNGGTCHLGTRHHLACLCPEGFTGLYCESQMGQGTRPSPTPVTPRPPRSLTLGIEPVSPTSLRVGLQRY LQGSSVQLRSLRLTYRNLSGPDKRLVTLRLPASLAEYTVTQLRPNATYSVCVMPLGPGRVPEGEEACGE AHTPPAVHSNHAPVTQAREGNLPLLIAPALAAVLLAALAAVGAAYCVRRGRAMAAAAQDKGQVGPGAGP LELEGVKVPLEPGPKATEGGGEALPSGSECEVPLMGFPGPGLQSPLHAKPYI As used herein, the terms “expression level”, “level”, “concentration” and “quantity” can be used equivalently. In some embodiments, the expression level of Vasorin (VASN) is measured at the mRNA or the protein level. Methods to determine the expression level of VASN can be performed by any method known in the art, including, without limitation: deep RNA sequencing, in situ hybridization, direct sequencing or Q-PCR, immunostaining, immunohistochemistry, immunofluorescence, ELISA, flow cytometry, chromatography, proteomics. Thus,in some embodiments, the level of VASN is determined by immunostaining, immunohistochemistry, immunofluorescence, ELISA, flow cytometry, deep RNA sequencing, in situ hybridization. Typically, protein or antibody concentration may be measured, for example, by capillary electrophoresis-mass spectroscopy technique (CE-MS) or ELISA performed on the sample. Such methods comprise contacting a sample with a binding partner capable of selectively interacting with proteins present in the sample. The binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal. The presence of the protein or antibody can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich-type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, capillary electrophoresis- mass spectroscopy technique (CE-MS).etc. The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith. The aforementioned assays generally involve the separation of unbound protein in a liquid phase from solid phase support to which antigen-antibody complexes are bound. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e.g., in membrane or microtiter well form), polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidene fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like. More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the proteins to be tested. A sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties, and a detectably labeled secondary binding molecule is added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate is washed, and the presence of the secondary binding molecule is detected using methods well-known in the art. In some embodiments, the level of VASN is determined by immunohistochemistry or immunofluorescence. Immunohistochemistry typically includes the following steps i) fixing the human or murine renal tissue sample with formalin, ii) embedding said human or murine renal tissue sample in paraffin, iii) cutting said human or murine renal tissue sample into sections for staining, iv) incubating said sections with the binding partner specific for the marker, v) rinsing said sections, vi) incubating said section with a secondary antibody typically biotinylated and vii) revealing the antigen-antibody complex typically with avidin-biotin-peroxidase complex. Accordingly, the human or murine renal tissue sample is first incubated with binding partners, such as anti- VASN. After washing, the labeled antibodies bound to markers of interest are revealed by the appropriate technique, depending on the label borne by the labeled antibody, e.g., radioactive, fluorescent or enzyme label. Multiple labelling can be performed simultaneously. Alternatively, the method of the present invention may use a secondary antibody coupled to an amplification system (to intensify the staining signal) and enzymatic molecules. Such coupled secondary antibodies are commercially available, e.g. from Dako, EnVision system. Counterstaining may be used, e.g., Hematoxylin & Eosin, DAPI, Hoechst. Other staining methods may be accomplished using any suitable method or system as would be apparent to one of skill in the art, including automated, semi-automated, or manual systems. For example, one or more labels can be attached to the antibody, thereby permitting the detection of the target protein (i.e., the marker). Exemplary labels include radioactive isotopes, fluorophores, ligands, chemiluminescent agents, enzymes, and combinations thereof. In some embodiments, the label is a quantum dot. Non-limiting examples of labels that can be conjugated to primary and/or secondary affinity ligands include fluorescent dyes or metals (e.g., fluorescein, rhodamine, phycoerythrin, fluorescamine), chromophoric dyes (e.g., rhodopsin), chemiluminescent compounds (e.g. luminal, imidazole) and bioluminescent proteins (e.g. luciferin, luciferase), haptens (e.g. biotin). A variety of other useful fluorescers and chromophores are described in Stryer L (1968) Science 162:526-533 and Brand L and Gohlke J R (1972) Annu. Rev. Biochem. 41:843-868. Affinity ligands can also be labeled with enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-lactamase), radioisotopes (e.g., 3H, 14C, 32P, 35S or 125I) and particles (e.g., gold). The different types of labels can be conjugated to an affinity ligand using various chemistries, e.g., the amine reaction or the thiol reaction. However, other reactive groups than amines and thiols can be used, e.g., aldehydes, carboxylic acids and glutamine. Various enzymatic staining methods are known in the art of detecting a protein of interest. For example, enzymatic interactions can be visualized using different enzymes such as peroxidase, alkaline phosphatase, or different chromogens such as DAB, AEC, or Fast Red. In other examples, the antibody can be conjugated to peptides or proteins that can be detected via a labeled binding partner or antibody. In an indirect IHC assay, a secondary antibody or second binding partner is necessary to detect the binding of the first binding partner, as it is not labeled. The resulting stained specimens are each imaged using a system for viewing the detectable signal and acquiring an image, such as a digital image of the staining. Methods for image acquisition are well known to one of skill in the art. For example, once the sample has been stained, any optical or non-optical imaging device can be used to detect the stain or biomarker label, such as, for example, upright or inverted optical microscopes, scanning confocal microscopes, cameras, scanning or tunneling electron microscopes, scanning probe microscopes and imaging infrared detectors. In some examples, the image can be captured digitally. The obtained images can then be used for quantitatively or semi-quantitatively determining the amount of the marker in the sample, or the absolute number of cells positive for the maker of interest, or the surface of cells positive for the marker of interest. Various automated sample processing, scanning and analysis systems suitable for use with IHC are available in the art. Such systems can include automated staining and microscopic scanning, computerized image analysis, serial section comparison (to control for variation in the orientation and size of a sample), digital report generation, and archiving and tracking of samples (such as slides on which tissue sections are placed). Cellular imaging systems are commercially available that combine conventional light microscopes with digital image processing systems to perform quantitative analysis on cells and tissues, including immunostained samples. See, e.g., the CAS-200 system (Becton, Dickinson & Co.). In particular, detection can be made manually or by image processing techniques involving computer processors and software. Using such software, for example, the images can be configured, calibrated, standardized and/or validated based on factors including, for example, stain quality or stain intensity, using procedures known to one of skill in the art (see, e.g., published U.S. Patent Publication No. US20100136549). The image can be quantitatively or semi-quantitatively analyzed and scored based on the staining intensity of the sample. Quantitative or semi-quantitative histochemistry refers to method of scanning and scoring samples that have undergone histochemistry to identify and quantitate the presence of the specified biomarker (i.e. the marker). Quantitative or semi-quantitative methods can employ imaging software to detect staining densities or amount of staining or methods of detecting staining by the human eye, where a trained operator ranks results numerically. For example, images can be quantitatively analyzed using pixel count algorithms and tissue recognition patterns (e.g., Aperio Spectrum Software, Automated QUantitatative Analysis platform (AQUA® platform), or Tribvn with Ilastic and Calopix software), and other standard methods that measure or quantitate or semi-quantitate the degree of staining; see, e.g., U.S. Pat. No. 8,023,714; U.S. Pat. No.7,257,268; U.S. Pat. No.7,219,016; U.S. Pat. No.7,646,905; published U.S. Patent Publication No. US20100136549 and 20110111435; Camp et al. (2002) Nature Medicine, 8:1323-1327; Bacus et al. (1997) Analyt Quant Cytol Histol, 19:316-328). A ratio of a strong positive stain (such as a brown stain) to the sum of total stained area can be calculated and scored. The amount of the detected biomarker (i.e., the marker) is quantified and given as a percentage of positive pixels and/or a score. For example, the amount can be quantified as a percentage of positive pixels. In some examples, the amount is quantified as the percentage of area stained, e.g., the percentage of positive pixels. For example, a sample can have at least or about at least or about 0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more positive pixels as compared to the total staining area. For example, the amount can be quantified as an absolute number of cells positive for the marker of interest. In some embodiments, a score is given to the sample that is a numerical representation of the intensity or amount of the histochemical staining of the sample and represents the amount of target biomarker (e.g., the marker) present in the sample. Optical density or percentage area values can be given a scaled score, for example, on an integer scale. Thus, in some embodiments, the method of the present invention comprises the steps consisting of i) providing one or more immunostained slices of tissue section obtained by an automated slide-staining system by using a binding partner capable of selectively interacting with the marker (e.g., an antibody as above described), ii) proceeding to the digitalization of the slides of step i).by high-resolution scan capture, iii) detecting the slice of a tissue section on the digital picture iv) providing a size reference grid with uniformly distributed units having a same surface, said grid being adapted to the size of the tissue section to be analyzed, and v) detecting, quantifying and measuring the intensity or the absolute number of stained cells in each unit whereby the number or the density of cells stained of each unit is assessed. In some embodiments, the level of VASN is determined by tissue proteomics. As used herein, the term “proteomics” refers to a highly sensitive and accurate method for protein identification and expression analysis. For example, proteomics may use liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) or nano-liquid chromatography mass spectrometry (nLC-MS/MS), as described in Chick JM et al., Defining the consequences of genetic variation on a proteome-wide scale. Nature.2016 Jun 23;534(7608):500-5; in Yuka T et al., Proteomic and transcriptomic profiling reveal different aspects of aging in the kidney eLife. 2021 10:e62585; or in Lasse M et al., An integrated organoid omics map extends modeling potential of kidney disease. Nat Commun 14, 4903 (2023). In some embodiments, the expression level of VASN in the sample is determined at the nucleic acid level. Typically, the level of a gene may be determined by assessing the quantity of mRNA encoding for VASN. Methods for determining the amount of mRNA are well-known in the art. For example, the nucleic acid contained in the samples (e.g., cell or tissue prepared from the subject) is first extracted according to standard methods, for example, using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR). Other methods of Amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence-based amplification (NASBA). Typically, the nucleic acid probes include one or more labels, for example, to permit detection of a target nucleic acid molecule using the disclosed probes. In various applications, such as in situ hybridization procedures, a nucleic acid probe includes a label (e.g., a detectable label). A “detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample. Thus, a labeled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) (to which the labeled uniquely specific nucleic acid molecule is bound or hybridized) in a sample. A label associated with one or more nucleic acid molecules (such as a probe generated by the disclosed methods) can be detected directly or indirectly. A label can be detected by any known or yet-to-be-discovered mechanism, including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons). Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colorless substance into a colored substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials. In some embodiments, the methods of the invention comprise the steps of providing total RNAs extracted from cumulus cells and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semi- quantitative RT-PCR. In some embodiments, the level of VASN is determined by deep RNA sequencing. As used herein, the term “deep RNA sequencing” refers to a highly sensitive and accurate method for gene expression analysis. This technology has rapidly emerged as the key approach to high- throughput transcriptome profiling to better understand functional biology underlying disease. RNA sequencing data related to other processes (RNA splicing entropy, gene expression, etc.) will provide a signature that can identify subjects with a kidney disease such as FSGS. In some embodiments, the method of the present invention is performed in vitro or ex vivo. Therapeutic methods: A further aspect of the present invention relates to a method for preventing the progression to a nephrotic syndrome in a subject suffering from a kidney disease comprising administering to the subject a therapeutically effective amount of an agent that restores the expression of VASN. In some embodiments, the method of the present invention is particularly suitable for preventing the progression of chronic kidney disease, kidney failure and/or death. In some embodiments, the agent is a vasorin (VASN) polypeptide or a polynucleotide encoding thereof. As used herein, the term “polynucleotide” refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”). It also includes modifications, for example, by alkylation and/or by capping and unmodified forms of the polynucleotide. More particularly, the term “polynucleotide” includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids “PNAs”) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. In some embodiments, the polynucleotide comprises an mRNA. In another aspect, the mRNA is a synthetic mRNA. In some embodiments, the synthetic mRNA comprises at least one unnatural nucleobase. In some embodiments, all nucleobases of a certain class have been replaced with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be replaced with an unnatural nucleobase, e.g., 5-methoxyuridine). In some embodiments, the polynucleotide (e.g., a synthetic RNA or a synthetic DNA) comprises only natural nucleobases, i.e., A, C, T and G in the case of a synthetic DNA, or A, C, T, and U in the case of a synthetic RNA. In some embodiments, the polynucleotide of the present invention is a messenger RNA (mRNA). In some embodiments, the polynucleotide is inserted in a vector, such a viral vector. As used herein, the term “viral vector” refers to a virion or virus particle that functions as a nucleic acid delivery vehicle and which comprises a vector genome packaged within the virion or virus particle. Typically, the vector is a viral vector, an adeno-associated virus (AAV), a retroviral vector, a bovine papilloma virus, an adenovirus vector, a vaccinia virus, or a polyomavirus. In some embodiments, the viral vector is a AAV vector. As used herein, the term "AAV vector" means a vector derived from an adeno- associated virus serotype, including, without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and mutated forms thereof. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking ITR sequences. In some embodiments, the viral vector is a retroviral vector. As used herein, the term “retroviral vector” refers to a vector containing structural and functional genetic elements that are primarily derived from a retrovirus. In some embodiments, the retroviral vector of the present invention derives from a retrovirus selected from the group consisting of alpha retroviruses (e.g., avian leukosis virus), betaretroviruses (e.g., mouse mammary tumor virus), gammaretroviruses (e.g., murine leukemia virus), deltaretroviruses (e.g., bovine leukemia virus), epsilon retroviruses (e.g., Walley dermal sarcoma virus), lentiviruses (e.g., HIV-1, HIV- 2) and spumaviruses (e.g., human spumavirus). In some embodiments, the retroviral vector of the present invention is a replication-deficient retroviral virus particle, which can transfer a foreign imported RNA of a gene instead of the retroviral mRNA. The structure and composition of the vector genome used to prepare the retroviral vectors of the present invention are in accordance with those described in the art. Especially, minimum retroviral gene delivery vectors can be prepared from a vector genome, which only contains, apart from the recombinant nucleic acid molecule of the present invention, the sequences of the retroviral genome which are non-coding regions of said genome, necessary to provide recognition signals for DNA or RNA synthesis and processing. In some embodiment, the retroviral vector genome comprises all the elements necessary for the nucleic import and the correct expression of the polynucleotide of interest (i.e. the transgene). As examples of elements that can be inserted in the retroviral genome of the retroviral vector of the present invention are at least one (preferably two) long terminal repeats (LTR), such as a LTR5' and a LTR3', a psi sequence involved in the retroviral genome encapsidation, and optionally at least one DNA flap comprising a cPPT and a CTS domains. In some embodiments of the present invention, the LTR, preferably the LTR3', is deleted for the promoter and the enhancer of U3 and is replaced by a minimal promoter, allowing transcription during vector production while an internal promoter is added to allow expression of the transgene. In particular, the vector is a Self- INactivating (SIN) vector that contains a non-functional or modified 3' Long Terminal Repeat (LTR) sequence. This sequence is copied to the 5' end of the vector genome during integration, resulting in the inactivation of promoter activity by both LTRs. Hence, a vector genome may be a replacement vector in which all the viral coding sequences between the 2 long terminal repeats (LTRs) have been replaced by the recombinant nucleic acid molecule of the present invention. In some embodiments, the retroviral vector genome is devoid of functional gag, pol and/or env retroviral genes. By "functional" it is meant a gene that is correctly transcribed and/or correctly expressed. Thus, the retroviral vector genome of the present invention in this embodiment contains at least one of the gag, pol and env genes that is either not transcribed or incompletely transcribed; the expression "incompletely transcribed" refers to the alteration in the transcripts gag, gag-pro or gag-pro-pol, one of these or several of these being not transcribed. In some embodiments, the retroviral genome is devoid of gag, pol and/or env retroviral genes. In some embodiments, the retroviral vector genome is also devoid of the coding sequences for Vif-, Vpr-, Vpu- and Nef-accessory genes (for HIV-1 retroviral vectors) or of their complete or functional genes. Typically, the retroviral vector of the present invention is non-replicative i.e., the vector and retroviral vector genome are not able to form new particles budding from the infected host cell. This may be achieved by the absence in the retroviral genome of the gag, pol, or env genes, as indicated in the above paragraph; this can also be achieved by deleting other viral coding sequences and/or cis-acting genetic elements needed for particles formation. Thus the present invention encompasses the use of virus-like particles. As used herein, the term “virus-like particle” or “VLP” refers to a structure resembling a virus particle but devoid of the viral genome, incapable of replication and devoid of pathogenicity. The particle typically comprises at least one type of structural protein from a virus. Preferably only one type of structural protein is present. Most preferably, no other non-structural component of a virus is present. Thus, virus-like particles can be spontaneously self-assembled by viral structural proteins under appropriate conditions in vitro while excluding the genetic material and potential replication probability. Virus-like particles, with a diameter of approximately 20 to 150 nm, also have the characteristics of nanometer materials, such as large surface area, surface- accessible amino acids with reactive moieties (e.g., lysine and glutamic acid residues), inerratic spatial structure, and good biocompatibility. Therefore, assembled virus-like particles have great potential as a delivery system for specifically carrying a variety of cargos. In some embodiments, one or more of the zinc finger motifs of the Gag protein is/are substituted by one or more RNA-binding domain(s). In some embodiments, the RNA-binding domain is the Coat protein of the MS2 bacteriophage, of the PP7 phage or of the Q3 phage, the prophage HK022 Nun protein, the U1A protein or the hPum protein. More preferably, the RNA binding domain is the Coat protein of the MS2 bacteriophage or of the PP7 phage. Even more preferably, the RNA-binding domain is the Coat protein of the MS2 bacteriophage. These embodiments are particularly suitable for packaging the mRNA encoding for the VASN polypeptide into the VLP. Thus, in some embodiments, the mRNA encoding for the VASN polypeptide that is encapsuled in the virus particle of the present invention comprises at least one encapsidation sequence. By “encapsidation sequence” is meant an RNA motif (sequence and three-dimensional structure) recognized specifically by an RNA-binding domain as described above. Preferably, the encapsidation sequence is a stem-loop motif. Even more preferably, the encapsidation sequence of the retroviral particle is the stem-loop motif of the RNA of the MS2 bacteriophage or of the PP7 phage such as. The stem-loop motif and more particularly the stem-loop motif of the RNA of the MS2 bacteriophage or that of the RNA of the PP7 phage may be used alone or repeated several times, preferably from 2 to 25 times, more preferably from 2 to 18 times, for example from 6 to 18 times. In some embodiments, the present invention encompasses the use of the LentiFlash® technology that based on non-integrative lentiviral particles constructed using a bacteriophage coat protein and its cognate 19-nt stem-loop, to replace the natural lentiviral Psi packaging sequence, in order to achieve active mRNA packaging into the lentiviral particles (Prel A, Caval V, Gayon R, Ravassard P, Duthoit C, Payen E, Maouche-Chretien L, Creneguy A, Nguyen TH, Martin N, Piver E, Sevrain R, Lamouroux L, Leboulch P, Deschaseaux F, Bouillé P, Sensébé L, Pagès JC. Highly efficient in vitro and in vivo delivery of functional RNAs using new versatile MS2-chimeric retrovirus-like particles. Mol Ther Methods Clin Dev.2015 Oct 21;2:15039. doi: 10.1038/mtm.2015.39. PMID: 26528487; PMCID: PMC4613645). The retroviral vectors of the present invention can be produced by any well-known method in the art, including by transfection (s) transient (s), in stable cell lines and / or by means of helper virus. The use of stable cell lines may also be preferred for the production of the vectors (Greene, M. R. et al. Transduction of Human CD34 + Repopulating Cells with a Self-Inactivating Lentiviral Vector for SCID-X1 Produced at Clinical Scale by a Stable Cell Line. Hum. Gene Ther. Methods 23, 297–308 (2012).) For instance, the retroviral vector of the present invention is obtainable by a transcomplementation system (vector/packaging system) by transfecting in vitro a permissive cell (such as 293T cells) with a plasmid containing the retroviral vector genome of the present invention, and at least one other plasmid providing, in trans, the gag, pol and env sequences encoding the polypeptides GAG, POL and the envelope protein(s), or for a portion of these polypeptides sufficient to enable the formation of retroviral particles. As an example, permissive cells are transfected with a) trans-complementation plasmid, lacking packaging signal psi, and the plasmid is optionally deleted of accessory genes vif, nef, vpu and / or vpr, b) a second plasmid (envelope expression plasmid or pseudotyping env plasmid) comprising a gene encoding an envelope protein(s) and c) a plasmid vector comprising a recombinant genome retroviral, optionally deleted from the promoter region of the 3 'LTR or U3 enhancer sequence of the 3' LTR, including, between the LTR sequences 5 'and 3' retroviral, a psi encapsidation sequence, a nuclear export element (preferably RRE element of HIV or other retroviruses equivalent), comprising the nucleic acid molecule of the present invention and optionally a promoter and / or a nuclear import sequence (cPPT sequence eg CTS ) of the RNA. Advantageously, the three plasmids used do not contain homologous sequence sufficient for recombination. Nucleic acids encoding gag, pol and env cDNA can be advantageously prepared according to conventional techniques from viral gene sequences available in the prior art and databases. The trans-complementation plasmid provides a nucleic acid encoding the proteins retroviral gag and pol. These proteins are derived from a lentivirus and, most preferably, from HIV-1. The plasmid is devoid of encapsidation sequence, the sequence coding for an envelope, accessory genes, and advantageously also lacks retroviral LTRs. Therefore, the sequences coding for gag and pol proteins are advantageously placed under the control of a heterologous promoter, eg, cellular, viral, etc., which can be constitutive or regulated, weak or strong. It is preferably a plasmid containing a sequence transcomplémentant Δpsi-CMV-gag- pol-PolyA. This plasmid allows the expression of all the proteins necessary for the formation of empty virions except the envelope glycoproteins. The plasmid transcomplementation may advantageously comprise the TAT and REV genes. Plasmid transcomplementation is advantageously devoid of vif, vpr, vpu and / or nef accessory genes. It is understood that the gag and pol genes and genes TAT and REV can also be carried by different plasmids, possibly separated. In this case, several plasmids are used for trans-complementation, each encoding one or more of said proteins. The promoters used in the plasmid trans-complementation, the envelope plasmid and the plasmid vector, respectively, to promote the expression of gag and pol of the coat protein, the mRNA of the vector genome and the transgene are promoters identical or different, chosen advantageously from ubiquitous promoters or specific, for example, from viral promoters CMV, TK, RSV LTR promoter and the RNA polymerase III promoter such as U6 or H1 or promoters of helper viruses encoding env, gag and pol (i.e., adenoviral, baculoviral, herpes viruses). For the production of the retroviral vector of the present invention, the plasmids described above can be introduced into competent cells, and viruses produced are harvested. The cells used may be any cell competent, particularly eukaryotic cells, in particular mammalian, eg, human or animal. They can be somatic or embryonic stem or differentiated. Typically the cells include 293T cells, fibroblast cells, hepatocytes, muscle cells (skeletal, cardiac, smooth, blood vessel, etc.)., nerve cells (neurons, glial cells, astrocytes) of epithelial cells, renal, ocular, etc. It may also include insect, plant cells, yeast, or prokaryotic cells. It can also be cells transformed by the SV40 T antigen. The genes gag, pol and env encoded in plasmids or helper viruses can be introduced into cells by any method known in the art, suitable for the cell type considered. Usually, the cells and the vector system are contacted in a suitable device (plate, dish, tube, pouch, etc...) for a period of time sufficient to allow the transfer of the vector system or the plasmid in the cells. Typically, the vector system or the plasmid is introduced into the cells by calcium phosphate precipitation, electroporation, transduction or by using one of the transfection-facilitating compounds, such as lipids, polymers, liposomes and peptides, etc.. Calcium phosphate precipitation is preferred. The cells are cultured in any suitable medium such as RPMI, DMEM, a specific medium to a culture in the absence of fetal calf serum, etc. Once transfected, the retroviral vectors of the present invention may be purified from the supernatant of the cells. Purification of the retroviral vector to enhance the concentration can be accomplished by any suitable method, such as by density gradient purification (e.g., cesium chloride (CsCl)) or by chromatography techniques (e.g., column or batch chromatography). For example, the vector of the present invention can be subjected to two or three CsCl density gradient purification steps. The vector, is desirably purified from cells infected using a method that comprises lysing cells infected with adenovirus, applying the lysate to a chromatography resin, eluting the adenovirus from the chromatography resin, and collecting a fraction containing the retroviral vector of the present invention. In some embodiments, the vector of the present invention includes "control sequences", which refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription, and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell. Another nucleic acid sequence, is a "promoter" sequence, which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene that is capable of binding RNA polymerase and initiating transcription of a downstream (3'- direction) coding sequence. Transcription promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters”. In some embodiments, the polypeptide or polynucleotide of the present invention can be conjugated to at least one other molecule. Typically, said molecule is selected from the group consisting of polynucleotides, polypeptides, lipids, lectins, carbohydrates, vitamins, cofactors, and drugs. In some embodiments, the polypeptide or polynucleotide of the present invention is formulated using one or more lipid-based structures that include but are not limited to liposomes, lipoplexes, or lipid nanoparticles (Paunovska, Kalina, David Loughrey, and James E. Dahlman. "Drug delivery systems for RNA therapeutics." Nature Reviews Genetics (2022): 1-16). Liposomes are artificially-prepared vesicles that can primarily be composed of a lipid bilayer and can be used as a delivery vehicle for the administration of pharmaceutical formulations. Liposomes can be of different sizes, such as but not limited to, a multilamellar vesicle (MLV) which can be hundreds of nanometers in diameter and can contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which can be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which can be between 50 and 500 nm in diameter. Liposome design can include but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes can contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations. As a non-limiting example, liposomes such as synthetic membrane vesicles are prepared using the methods, apparatus, and devices described in US Patent Publication No. US20130177638, US20130177637, US20130177636, US20130177635, US20130177634, US20130177633, US20130183375, US20130183373 and US20130183372. In some embodiments, the liposomes are formed from 1,2-dioleyloxy-N,N- dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and MC3 (as described in US20100324120) and liposomes which can deliver small molecule drugs such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.). The polypeptide of polynucleotide of the present invention can be encapsulated by the liposome and/or it can be contained in an aqueous core which can then be encapsulated by the liposome (see International Pub. Nos. WO2012031046, WO2012031043, WO2012030901 and WO2012006378 and US Patent Publication No. US20130189351, US20130195969 and US20130202684). In some embodiments, the polynucleotide of the present invention is formulated with stabilized plasmid- lipid particles (SPLP) or stabilized nucleic acid lipid particles (SNALP) that have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (see Wheeler et al. Gene Therapy. 19996:271-281; Zhang et al. Gene Therapy. 19996:1438- 1447; Jeffs et al. Pharm Res.200522:362-372; Morrissey et al., Nat Biotechnol.20052:1002- 1007; Zimmermann et al., Nature.2006441:111-114; Heyes et al. J Contr Rel.2005107:276- 287; Semple et al. Nature Biotech.201028:172-176; Judge et al. J Clin Invest.2009119:661- 673; deFougerolles Hum Gene Ther. 2008 19:125-132; U.S. Patent Publication No US20130122104). As used herein, the term "therapeutically effective amount" refers to a sufficient amount of the polypeptide or the nucleic acid molecule encoding thereof to prevent for use in a method for the treatment of the disease (e.g., a kidney disease) at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors, including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day. Typically the active ingredient of the present invention (i.e., the polypeptide or polynucleotide) is combined with pharmaceutically acceptable excipients and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. The term "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In another aspect, the invention relates to a kit suitable to perform the diagnosis method as described above, comprising a reagent that specifically reacts with VASN (mRNA or protein), and instructions use. In a particular embodiment, the invention relates to kits for performing the methods of the invention, wherein said kits comprise means for measuring the expression level of the VASN of the invention. In a particular embodiment, the kit according to the invention may include instructional materials containing instructions (e.g., protocols) for the practice of diagnostic methods. The kits may include probes, primers macroarrays or microarrays as above described. For example, the kit may comprise a set of probes as defined above, usually made of DNA, and that may be pre-labelled. Alternatively, probes may be unlabelled and the ingredients for labelling may be included in the kit in separate containers. The kit may further comprise hybridization reagents or other suitably packaged reagents and materials needed for the particular hybridization protocol, including solid-phase matrices, if applicable, and standards. Alternatively, the kit of the invention may comprise amplification primers that may be pre labelled or may contain an affinity purification or attachment moiety. The kit may further comprise amplification reagents and also other suitably packaged reagents and materials needed for the particular amplification protocol. The nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes. A preferred kit also includes the components necessary to determine if amplification has occurred. The kit may also include, for example, PCR buffers and enzymes, positive control sequences, reaction control primers, and instructions for amplifying and detecting the specific sequences. In a particular embodiment, the invention provides diagnostic kits containing the anti- VASN antibodies (monoclonal or polyclonal) including antibody conjugates. The diagnostic kit is a package comprising at least one anti-VASN monoclonal antibody of the disclosure (e.g. , either in lyophilized form or as an aqueous solution) and one or more reagents useful for performing a diagnostic assay (e.g., diluents, a labeled antibody that binds to an anti-VASN antibody, an appropriate substrate for the labeled antibody, VASN in a form appropriate for use as a positive control and reference standard standard, a negative control). In specific embodiments, a kit comprises two anti-VASN antibodies, wherein at least one of the antibodies is an anti-VASN monoclonal antibody. Optionally, the second antibody is a polyclonal anti- VASN antibody. Alternatively, the kit can include a labeled antibody which binds an anti-VASN monoclonal/polyclonal antibody and is conjugated to an enzyme. Where the anti-VANS monoclonal antibody or other antibody is conjugated to an enzyme for detection, the kit can include substrates and cofactors required by the enzyme (e.g., a substrate precursor that provides the detectable chromophore or fluorophore). In addition, other additives can be included, such as stabilizers, buffers (e.g. , a block buffer or lysis buffer), and the like. Anti- VASN antibodies included in a diagnostic kit can be immobilized on a solid surface, or alternatively, a solid surface (e.g., a slide) on which the antibody can be immobilized is included in the kit. The relative amounts of the various reagents can be varied widely to provide for concentrations in solution of the reagents, which substantially optimizes the sensitivity of the assay. Antibodies and other reagents can be provided (individually or combined) as dry powders, usually lyophilized, including excipients, which on dissolution will provide a reagent solution having the appropriate concentration. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES: Figure 1: Podocyte-specific deletion of Vasn induces FSGS in mice (a) Schematic protocol to evaluate renal histology and function over time in mice with constitutive and specific deletion of the Vasn gene in the podocyte. (b) Survival rate of heterozygous (PodoCre-Vasnlox/wt) or homozygous (PodoCre-Vasnlox/lox) podocyte-specific Vasn deficient and control mice (c) Plasma urea levels in PodoCre-Vasnlox/lox and control mice after 22 days. (d) Urine albumin-to-creatinine ratio in PodoCre-Vasnlox/lox and control mice from day 4 to day 22. (e) Plasma albumin levels in PodoCre-Vasnlox/lox and control mice after 22 days. (f) Filtration slit density as evaluated by 3D-SIM/PEMP from kidneys of PodoCre-Vasnlox/lox and control mice from day 4 to day 22. (c-f) Data represent mean +/- s.d. *P<0.05, ** P<0.01, **** P<0.0001. Figure 2: Global and post-natal deletion of Vasn induces FSGS in mice (a) Schematic protocol to evaluate renal histology and function over time in mice with global and post-natal deletion of the Vasn gene induced by tamoxifen intraperitoneal injection (2mg/day, in Corn Oil, 5 days) (CreERT2-Vasnlox/lox). (b) Representative pictures of CreERT2- Vasnlox/lox and control mice upon peritoneal incision. (c) Plasma urea levels in CreERT2- Vasnlox/lox and control mice from day 10 to day 29. (d) Plasma albumin levels in CreERT2- Vasnlox/lox and control mice from day 10 to day 29. (e) Urine albumin-to-creatinine ratio in CreERT2-Vasnlox/lox and control mice from day 0 to day 21. (f) Filtration slit density as evaluated by 3D-SIM/PEMP from kidneys of CreERT2-Vasnlox/lox and control mice from day 10 to day 22. (c-f) Data represent mean +/- s.d. *P<0.05, **** P<0.0001. Figure 3: Podocyte-specific and post-natal deletion of Vasn induces FSGS in mice (a) Schematic protocol to evaluate renal histology and function over time in mice with post- natal deletion of the Vasn gene in podocytes induced by oral administration of doxycycline (2% in sucrose water, 7 days) (NEFTA8-Cre-GFP-Vasnlox/lox). (b) Body weight of NEFTA8-Cre- GFP-Vasnlox/lox and control mice from day 0 to day 21. (c) Urine albumin-to-creatinine ratio in NEFTA8-Cre-GFP-Vasnlox/lox and control mice from day 0 to day 21. (d) Podocyte count, as stained by WT1-positive nuclei in glomerular sections, in kidneys from NEFTA8-Cre-GFP- Vasnlox/lox and control mice at day 22. (e) Podocyte differentiation, as stained by NPHS1/nephrin-positive area in glomerular sections, in kidneys from NEFTA8-Cre-GFP- Vasnlox/lox and control mice at day 22. (f) PEC activation, as stained by glomerular sections with CD44-positive PEC, in kidneys from NEFTA8-Cre-GFP-Vasnlox/lox and control mice at day 22. (b-f) Data represent mean +/- s.d. *P<0.05, **** P<0.0001. Figure 4: VASN is markedly regulated in human kidney diseases (a) Quantification of VASN transcripts by glomerular sections in kidney biopsies from controls or patients with MCD, DN, IgA nephropathy (IgAN), ANCA vasculitis, proliferative LN, membranous nephropathy (MN), and primary, secondary FSGS, either pooled by glomeruli or by biopsies. (b) Relative quantification of VASN mRNA expression using comparative deep RNA sequencing of laser capture microdissected glomeruli from patients with FSGS or ANCA- associated CGN versus healthy controls. Data represent mean +/- s.d. Individual values are shown in dots. *** P<0.001, **** P<0.0001. EXAMPLE: Material & Methods Animals Mice with global deletion of a floxed Vasn gene (Vasnlox/lox) using an inducible Cre/lox system (CreERT2) were developed under a C57BL6/J strain and kindly provided by our collaborators (H. Schrewe, Max Planck Institute, Berlin). CreERT2-Vasnlox/lox mice received intraperitoneal injection of tamoxifen (TMX) (Combi-Blocks, #10540-29-1) during 5 consecutive days (10 mg/mL in corn oil, 200 µL/day) to induce the Cre-recombinase. Mice with podocyte-specific and constitutive deletion of the Vasn gene were generated by crossing Vasnlox/lox mice with Nphs2.Cre (PodoCre) mice on the C57BL6/J background (PodoCre-Vasnlox/lox). Mice with podocyte-specific deletion of the Vasn gene using an inducible Cre/lox system were generated by crossing Vasnlox/lox mice with Nphs2-rtTA (NEFTA8), Tet-O-Cre (Cre) and ROSAmT/mG (GFP) mice on the C57BL6/J background. NEFTA8-Cre-GFP-Vasnlox/lox received doxycycline (ALX-380-273-G005) for 7 days (2 mg/mL in 5% sucrose water) to induce the Cre-recombinase. Age-matched littermates with no Cre-recombinase or no floxed Vasn allele were considered as controls. C57BL6/J mice with overexpression of a fluorescent protein (Venus) under the transcriptional regulation of the Vasn gene (Venus-Vasn) were obtained by transfection of ES cells with a BAC containing the sequence of the Venus framed on both sides with regulatory sequences of the Vasn gene. Experiments were conducted according to the French veterinary guidelines and those formulated by the European Community for experimental animal use (L358-86/609EEC) and were approved by the French Ministry of Research and local university research ethics committee (APAFIS- reference n°2019062016505841). Induction of crescentic glomerulonephritis 6- to 8-week-old mice received a retroorbital intravenous injection of sheep anti-mouse glomerular basement membrane nephrotoxic serum (NTS) to induce the CGN model, as previously described (Henique C, Bollée G, Loyer X, Grahammer F, Dhaun N, Camus M, et al. Genetic and pharmacological inhibition of microRNA-92a maintains podocyte cell cycle quiescence and limits crescentic glomerulonephritis. Nat Commun.282017;8(1):1829).15 µL of NTS was diluted with 85 µL of sterile PBS on day 1, and then pure NTS was injected (2.5 µL/g) on days 2 and 3. Mice were killed at day 10. Induction of type 1 diabetes mellitus with streptozotocin (STZ) 10- to 12-week-old males were rendered diabetic by STZ (Sigma-Aldrich) intraperitoneal injection on two consecutive days (100 mg/kg in sodium citrate buffer, pH=4.5). Mice were considered diabetic when fasting glycemia remained above 300 mg/dL two weeks in a row after STZ injection. Mice were killed 8 weeks after the STZ injection. Parabiosis Shared blood circulation between CreERT2-Vasnlox/lox female littermates was made by parabiosis surgery, as previously described (Duyverman AMMJ, Kohno M, Duda DG, Jain RK, Fukumura D. A transient parabiosis skin transplantation model in mice. Nat Protoc.22 mars 2012;7(4):763‑70). Mice were injected with tamoxifen (TMX) for 5 consecutive days to induce Cre recombinase, underwent parabiosis surgery 7 days after the first TMX injection, and were killed 15 days after the surgery. Biochemical measurements in blood and urine Blood was taken by intracardiac puncture. Urine was either collected using metabolic cages or by puncturing the bladder for newborns. Urinary creatinine and plasma urea concentrations were analyzed by a standard colorimetric method (Olympus AU400) at the Biochemistry Laboratory of Institut Claude Bernard (IFR2, Faculté de Médecine Paris Diderot). Quantitative determination of urinary albumin excretion was measured by a specific ELISA mouse kit (Crystal Chem, #80630). Cytospin Fresh urines were collected from mice in metabolic cages and centrifugated at 2000 rpm for 10 minutes, then resuspended in 200 µL of sterile PBS before being put into cytospin funnels (Shandon Cytospin 4, Thermo Scientific). Urinary Cells were then collected in SuperFrost+ slides at 2000 rpm for 5 minutes, dried for 10 minutes, fixed in 4% paraformaldehyde (PFA), and permeabilized by Tris-buffer-saline (TBS) with 0.1% Triton before immunostaining. Electron microscopy Small pieces of the renal cortex (1 mm3) were fixed in Trump fixative (Electron Microscopy Science) and embedded in Araldite M (Sigma Aldrich). Ultrathin sections were cut with an ultramicrotome and counterstained with uranyl acetate and lead citrate. Samples were examined in the JEM1011 transmission electron microscope (JEOL) with the Orius SC1000 CCD camera (Gatan), operated with Digital Micrograph software (Gatan) for acquisition. Human tissues Acetic acid-formol-alcohol-fixed and paraffin-embedded human renal tissue specimens were obtained from the Pathology department of Hôpital Européen Georges Pompidou, Paris, France. Human tissue was used after informed consent by all the patients and approval form, and kidney biopsies collection was approved by the Inserm Ethics Committee (IRB00003888, FWA00005831 NIH OHRP, Office of Human Research Protection) and the local Ethics Committee (Comité de Protection des Personnes Ile de France IV, IRB: 00003835). Histology Mice kidneys were fixed in 10% formalin, paraffin-embedded, then 4-µm thick sections were processed for Masson’s trichrome and immunostaining. Cryosections were used for Oil Red O staining. For immunofluorescence, after heat antigen retrieval using citrate buffer (pH=6), sections were incubated overnight (4°C) with the following primary antibodies: rabbit anti- WT1 (Abcam, ab89901), guinea pig anti-nephrin (Progen, GP-N2), rat anti-CD44 (Abcam, ab119348), goat anti-podocalyxin (R&D Systems, BAF1556), rat anti-CD31 (Dianova, DIA- 310), goat anti-TIM1 (R&D Systems, AF1817), rabbit anti-fibronectin (Merck, AB2033), rabbit anti-collagen IV (Abcam, ab19808), rabbit anti-GFP (Abcam, ab290), mouse anti-PCNA (Abcam, ab29), anti-p57 (Abcam, ab75974), rabbit anti-phospho-S6 (Ser235/236) (Cell Signaling Technology, #4858), rat anti-LAMP1 (Abcam, ab25245), rabbit anti-phospho- SMAD3 (S423+S425) (Abcam, ab52903). Secondary antibodies were Alexa 488- and Alexa 568-conjugated antibodies from Invitrogen. Nuclei were stained in blue using Hoechst. For immunohistochemistry, kidney tissues were stained with primary rabbit anti-Vasorin antibody (Abcam, ab233181) and rabbit phospho-EGFR (Y1068) (Abcam, ab40815), then incubated with polymer-based peroxidase-conjugated antibody (Histofine®, Nichirei Biosciences). Staining was revealed with 3,39-diaminobenzidine (DAB) reagent (Sigma-Aldrich) and counterstained with hematoxylin. For quantification of histological lesions, an average of 50 glomerular cross-sections per mouse was blindly counted for crescents in NTS model. Images were obtained with a Nikon Eclipse E600 microscope and Axiocam 208 camera (Zeiss), using Zen 2.6 software (Zeiss). Podocyte number was assessed by the number of WT1-positive cells, podocyte differentiation by nephrin- positive area, parietal cells (PECs) activation by CD44-positive glomerular area or the number of CD44-positive glomeruli, per glomerular cross-section, in an average of 30 sections per mice. Tubular injury was assessed by TIM1-positive tubule area per kidney section. In situ apoptotic cells in kidneys were quantified by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) using Click-iT TM Plus TUNEL Assay (Invitrogen, #C10617) according to the manufacturer’s instruction. Immunofluorescence mages were taken with a fluorescent microscope with Apotome module (Zeiss Apotome.2), using Zen 2.6 software (Zeiss). Semi- automatic quantifications were performed using CellProfiler 4.2.1. software (Broad Institute, USA). Filtration slit density (FSD) was measured with Podocyte Effacement Measurement Procedure (PEMP) as published before (Siegerist F, Ribback S, Dombrowski F, Amann K, Zimmermann U, Endlich K, et al. Structured illumination microscopy and automatized image processing as a rapid diagnostic tool for podocyte effacement. Sci Rep.132017;7(1):11473). Statistical analyses Data are expressed as means ± SEM. Statistical analyses were calculated by using GraphPad Prism software (GraphPad Software, La Jolla, CA). Non-parametric tests were used for quantitative variables. All tests were 2-tailed, and a P-value < 0.05 was considered significant. *P < 0.05, **P < 0.01, ***P < 0.001. In case of multiple groups comparisons, ANOVA was carried out followed by Tukey’s multiple comparison test. Results VASN is mainly expressed in podocytes in mice and humans By using a transgenic mouse with overexpression of a fluorescent protein (Venus) under the transcriptional regulation of the Vasn gene, we were able to locate the expression of Vasn in the kidneys. Vasn mRNA expression was mainly and strongly found in the podocytes and also the vascular smooth muscle cells (data not shown). The same transcription pattern was observed by in situ hybridization on kidney biopsies from humans (data not shown). Milder expression was also found in mesangial cells, PEC and proximal tubular cells. This expression was further confirmed using immunohistochemistry (data not shown). Constitutive or post-natal deletion of Vasn in podocytes induced severe features of FSGS Mice with constitutive and global deletion of the Vasn gene had no apparent clinical abnormalities at birth compared to their wild-type littermates but subsequently developed nephrotic syndrome, kidney failure and died within one month after birth. To determine if VASN deficiency in podocytes was responsible of this lethal phenotype, we generated podocyte-specific VASN-deficient mice (PodoCre-Vasn lox/lox). Strikingly, PodoCre-Vasnlox/lox mice totally mimicked global VASN deficient mice and developed nephrotic syndrome, kidney failure around day 14 after birth, and died within one month (Figures 1a-e). To further assess if this phenotype was due to developmental defect, we also generated mice with inducible deletion of the Vasn gene, either globally after TMX injection (CreERT2-Vasnlox/lox) or podocyte-specific after oral doxycycline administration (NEFTA8-Cre-GFP-Vasnlox/lox). These mice also developed nephrotic syndrome after 14 to 17 days (Figures 2a-e, and Figures 3a-c). Structurally, we observed severe lesions of FSGS in all these mice, with diffuse foot processes effacement and podocyte partial detachment from the GBM, along with urinary pseudocysts and vesicles accumulation in the cytoplasm (Figures 1f, 2f, 3g). These vesicles were partly composed of lipid droplets, as suggested by the Oil Red O staining, but also lysosomes, as stained by LAMP1 (data not shown). By immunofluorescence, we found profound podocyte dedifferentiation, hypertrophy, and loss, as stained by nephrin, phospho-S6 and WT1 markers. This is associated with local PEC activation, as stained by CD44, and tubular injury, as stained by TIM-1 (data not shown). Altogether, these data suggest that VASN has a fundamental but no or little developmental role in podocyte and glomerular homeostasis. VASN deficiency does not induce podocyte loss by apoptosis We sought to determine if VASN deficiency led to podocyte loss by apoptosis. Using TUNEL assay, we did not observe any nuclear staining in podocytes of PodoCre-Vasnlox/lox mice; the only staining we had in glomerular sections was from a few circulating immune cells in the vascular compartment (data not shown). As in situ apoptotic podocyte is theoretically difficult to observe because of the short duration process and rapid detachment in this context, we also studied the viability of immortalized human podocyte cell line in vitro. Podocytes transduced with a lentivirus encoding for an anti-Vasorin small-hairpin RNA (shVASN) did not show increased cell mortality as compared to control cells (scramble) (data not shown). Instead, their metabolic activity was more important, as reflected by a higher aerobic respiration capacity in a resazurin reduction assay (data not shown), and by their oxygen consumption rate, for basal respiration and ATP production. However, their maximal respiration capacity was similar to scramble cells, a reduced spare respiratory capacity that might reflect greater stress sensitivity (data not shown). Our findings suggest that podocyte death is not the primary cause of podocyte detachment. Subsequently, we aimed to decipher the mechanisms of podocyte loss by transcriptomic analysis on shVASN and scramble podocytes. RNA sequencing revealed down- regulation of genes involved in the cell-cycle control, especially in S to G2/M-transition, interferon-mediated immune response, and an up-regulation of genes involved in metabolism, which is consistent with the cell respiration experiments, but also in epithelial-mesenchymal transition, and cell adhesion (data not shown). We then aimed to assess these different cell functions in vitro. VASN deficiency increases podocyte focal adhesion dynamics By immunofluorescence and western blot analysis, we observed that shVASN podocytes had more focal adhesion complexes than scramble cells at baseline, as stained by phopho-paxillin (data not shown). This could be further increased by the addition of heparin-binding epidermal growth factor (HB-EGF), a potent inducer of podocyte in vitro migration and proliferation, in the culture medium after initial serum starvation (FCS 0.1%), at one hour (data not shown). We then tested the resistance of shVASN podocytes to mechanical stretches using an oscillating pressure culture chamber, which mimics the physiological glomerular hydrostatic pressure. Surprisingly, shVASN podocytes had greater resistance to mechanical stretches than scramble cells (data not shown). Our findings suggest that VASN deficiency does not induce primary podocyte adhesion defect. VASN deficiency induces podocyte EMT and increases podocyte migration capacity By transmission electronic microscopy, we observed in vivo podocytes' total detachment from the GBM, a feature that we confirmed by cytospin of freshly collected urine from VASN- deficient mice (data not shown). These podocytes did not show any nuclear abnormalities, suggesting again that cell death was not the primary cause of their detachment, but we could not affirm that these cells shedding is not due to secondary detachment caused by increased filtration stretch, a phenomenon commonly described in secondary FSGS. We then culture glomeruli isolated from VASN-deficient mice, and found that podocytes spreading from these glomeruli was more important than in control mice, suggesting either increased migratory or proliferative capacity (data not shown). Importantly, even if we seeded the same number of glomeruli on culture plates, we found more glomeruli attached on day 5. Therefore we could not exclude the increased outgrowth due to increased glomeruli density and matrix attachment, thereby leading to more paracrine survival and proliferation factors secretion. Next, we studied the migration ability of podocytes in vitro by a wound-healing assay. shVASN podocytes showed greater mobility, and strikingly, HB-EGF failed to increase their migration capacity as compared to scramble cells (data not shown). Finally, shVASN podocytes had some features of epithelial-mesenchymal transition, as characterized by a switch from P-cadherin to N- cadherin expression (data not shown). Altogether, our data suggest that VASN deficiency might lead to podocyte mesenchymal transition and increase their ability to migrate from the GBM. VASN deficiency increases podocyte proliferative capacity One striking feature of shVASN podocytes is the high frequency of multinucleated cells (data not shown). We, therefore, studied the cell cycle using DNA staining by FACS. We found that shVASN podocytes had a higher cell-cycle reentry capacity, as suggested by an increased S- phase with a decreased G0/1-phase cells proportion and an S-phase slow-down, as indicated by a reduced proportion of cells in the G2-M phase (data not shown). We sought to determine if the different cell-cycle phases proteins were accordingly regulated using qPCR, WB and IF. Indeed, we found that cyclins D1/2 transcription, proteins involved in G1-S transition, were upregulated, cyclin D1 increased expression being further confirmed by IF and WB (data not shown). Conversely, cyclins A2, B1, CDK1, and CDK2, proteins involved in S-G2 transition, were down-regulated, cyclin B1 decreased expression being further confirmed by IF and WB (data not shown). In vivo, we found that podocytes from PodoCre-Vasnlox/lox mice expressed more PCNA, an early-phase proliferative marker, and less p57, a podocyte cell-cycle inhibitor (data not shown). Finally, using GFP-cell sorting by FACS from NEFTA8-Cre-GFP-Vasnlox/lox mice, we also observed an increase in podocyte cell-cycle reentry (data not shown). Our findings suggest that VASN is a gatekeeper of podocytes' cell cycle, ensuring their physiological quiescence. VASN is markedly regulated in human and mice kidney diseases We wanted to determine the expression of VASN during various kidney diseases. By in situ hybridization, we found that Vasn transcription was markedly regulated. If this expression was strongly upregulated in minimal change diseases, diabetic nephropathy, or lupus proliferative nephritis, it was conversely downregulated in extracapillary diseases such as ANCA vasculitis and secondary FSGS (Figures 4a). Using comparative deep RNA sequencing of microdissected glomeruli from controls and patients diagnosed with FSGS or CGN, we were able to observe the same downregulation (Figures 4b). We then confirmed that mice challenged with a type 1 diabetic/hyperfiltration model induced by STZ, or a CGN model induced by NTS, also have a downregulation of Vasn transcription in podocytes (data not shown). VASN haploinsufficiency increases mice sensitivity to kidney diseases Mice with podocyte-specific heterozygous deletion of Vasn (PodoCre-Vasnlox/wt) did not show structural and functional renal abnormalities for up to 21 months (data not shown). We challenged these mice with streptozotocin (STZ) to induce an early-stage of type 1 diabetic/hyperfiltration model. We found that heterozygous PodoCre-Vasnlox/wt mice had more significant weight loss, podocyte dedifferentiation and loss, and also more severe albuminuria and kidney failure than their wild-type control littermates (data not shown). We then challenged these mice with NTS to induce an experimental CGN. Again, we found that heterozygous mice with partial Vasn deficiency had significantly accentuated podocyte dedifferentiation and loss, significantly more severe albuminuria, and kidney failure, but also strikingly more PEC activation and crescents formation (data not shown). These results indicate that even partial alteration of VASN expression in podocytes is linked with aggravated glomerular injury. EGFR, but not TGFβ, signaling pathways contribute to the VASN deficiency phenotype Aiming to determine the signaling pathway of VASN in podocytes, we studied the TGFβ canonical pathway, as VASN is known to be a soluble TGFβ trap. Unfortunately, we did not find evidence for kidney TGFβ activation in situ, as stained by phospho-SMAD3 Ab in VASN deficient mice (data not shown). Also, oral administration of a TGFβ-RI inhibitor did not reduce proteinuria or kidney failure (data not shown). As we and others have previously shown that the EGFR pathway is involved in the pathophysiology of CGN and FSGS (Bollée G, Flamant M, Schordan S, Fligny C, Rumpel E, Milon M, et al. Epidermal growth factor receptor promotes glomerular injury and renal failure in rapidly progressive crescentic glomerulonephritis. Nature Medicine.2011;17(10):1242‑50), and that the HB-EGF/EGFR axis increased murine podocyte migration and proliferation capacity in vitro (Bollée G, Flamant M, Schordan S, Fligny C, Rumpel E, Milon M, et al. Epidermal growth factor receptor promotes glomerular injury and renal failure in rapidly progressive crescentic glomerulonephritis. Nature Medicine.2011;17(10):1242‑50), we next studied if EGFR signaling contributes to the phenotype of VASN deficiency. Indeed, we already showed that HB-EGF could not further increase podocytes mobility. In vitro addition of EGFR inhibitor resulted in reduced migration capacity and also proliferation capacity, as suggested by BrdU incorporation (data not shown). In vivo, we found increased phosphorylation of EGFR in podocytes from PodoCre-Vasnlox/lox mice (data not shown). These data highlight a probably downstream but partial contribution of EGFR in the pathophysiology of VASN deficiency. To gain further insights into the precise mechanisms of VASN in podocytes, affinity purification and mass spectrometry experiments are ongoing to determine the biological partners of VASN, using human podocyte overexpression of an HA-tagged VASN. Consistently with in vivo data, HA-tagged VASN was mainly found in the cytoplasm, especially in the perinuclear spaces, but also sometimes in the membrane in filopodia-like structures (data not shown). Endocrine substitution of soluble VASN does not prevent glomerular destruction As the expression of VASN in podocytes was mainly nuclear and cytoplasmic, we hypothesized that its membrane and soluble forms did not play a major role in podocyte homeostasis. We created a shared blood circulation between mice, as previously described, to test whether the circulation of soluble VASN could rescue the VASN deficient mice phenotype (Duyverman AMMJ, Kohno M, Duda DG, Jain RK, Fukumura D. A transient parabiosis skin transplantation model in mice. Nat Protoc.22 mars 2012;7(4):763‑70) (data not shown). As expected, VASN deficient mice attached to another VASN deficient mice (Cre+ Cre+) were sick as compared to WT mice (Cre-Cre-). FSGS features, albuminuria, and kidney function were not reduced in VASN-deficient mice attached to WT mice (Cre+ Cre-), suggesting that endocrine soluble VASN did not play a major role in podocyte physiology (data not shown). Conclusion: Here we demonstrated that podocyte expression of Vasorin (VASN) orchestrates glomerular homeostasis by maintaining podocyte and PEC quiescence in a physiological context and during renal diseases such as FSGS and CGN. We showed significant regulation of VASN in different glomerular diseases (Figure 4). In particular, we showed a strong decrease in VASN mRNA abundance in kidney biopsies from FSGS or CGN cases as compared with histologically normal controls. Conversely, VASN podocyte abundance was relatively increased in MN, MCD, primary FSGS and LN which could help differential diagnosis. In particular, VASN expression level could be useful for the differential diagnosis between primary and secondary FSGS. VASN appears to be a critical survival factor for the podocyte. Maintening a high level of VASN in podocytes or preventing its downregulation may help protect the glomerular function in patients. VASN is overexpressed in the context of stress, such as hyperfiltration or hyperglycemia. Then its expression would be progressively lost during the development of chronic kidney disease and more rapidly during FSGS and CGN such as ANCA-associated vasculitis-mediated CGN. VASN thus represents a prognostic factor or even a therapeutic target in human renal diseases. REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS: 1. A method for diagnosing a kidney disease in a subject comprising determining in a biological sample obtained from the subject the expression level of Vasorin (VASN), wherein the level indicates whether or not the subject suffers from a kidney disease. 2. The method of claim 1, wherein a high level of VASN indicates that the subject suffers from a minimal change disease (MCD), diabetic nephropathy (DN), a membranous nephropathy (MN) or a lupus nephritis (LN). 3. The method of claim 1 wherein a low level of VASN indicates that the subject suffers from a nephrotic syndrome, a chronic kidney disease, an ANCA-associated vasculitis, crescentic glomerulonephritis (CGN), or a focal segmental glomerulosclerosis (FSGS). 4. A method for discriminating a primary focal segmental glomerulosclerosis (FSGS) from a secondary FSGS in a subject comprising i) determining in a biological sample obtained from the subject the expression level of Vasorin (VASN); ii) comparing the expression level determined at step i) with a predetermined reference value and iii) concluding that the subject has or is susceptible to have a a primary FSGS when the expression level determined at step i) is higher than its predetermined reference value, or concluding that the subject has or is susceptible to have a secondary FSGS when the expression level determined at step i) is lower than its predetermined reference value. 5. A method for discriminating minimal change disease (MCD) from FSGS in a subject comprising i) determining in a biological sample obtained from the subject the expression level of Vasorin (VASN); ii) comparing the expression level determined at step i) with a predetermined reference value and iii) concluding that the subject has or is susceptible to have a FSGS when the expression level determined at step i) is lower than its predetermined reference value or concluding that the subject has or is susceptible to have a MCD when the expression level determined at step i) is higher than its predetermined reference value. 6. A method for predicting the risk of a subject suffering from a kidney disease progresses to chronic kidney failure comprising i) determining in a biological sample obtained from the subject the expression level of Vasorin (VASN), ii) comparing the expression level determined at step i) with a predetermined reference value and iii) concluding that the subject has a significantly higher risk of having a chronic kidney failure when the expression level determined at step i) is lower than its predetermined reference value, or concluding that the subject a lower risk of having a chronic kidney failure when the expression level determined at step i) is higher than its predetermined reference value. 7. The method of claim 6 for predicting the occurrence of nephrotic syndrome, chronic kidney disease, kidney failure, or death. 8. A method for determining whether a subject suffering from a kidney disease achieves a response to a treatment comprising the following steps i) obtaining a biological sample from said subject, ii) determining the expression level of VASN, iii) ii) comparing the expression level determined at step i) with a predetermined reference value and iv) concluding that the subject achieves a response to the treatment when the level of VASN is higher than the reference value or concluding that the subject does not achieve a response treatment when the expression level of VASN is lower than the reference value. 9. The method of claim 8 wherein the subject suffers from a FSGS, and the treatment is an immunosuppressive treatment. 10. A method for preventing the progression to a nephrotic syndrome in a subject suffering from a kidney disease comprises administering a therapeutically effective amount of an agent that restores the expression of VASN. 11. The of claim 10 for preventing progression to chronic kidney disease, kidney failure, and/or death. 12. The method of claim 10 wherein the agent is a vasorin (VASN) polypeptide or a polynucleotide encoding thereof.
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