WO2013041913A1 - Method for monitoring, diagnosis and/or prognosis of acute kidney injury in early stage - Google Patents
Method for monitoring, diagnosis and/or prognosis of acute kidney injury in early stage Download PDFInfo
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- WO2013041913A1 WO2013041913A1 PCT/IB2011/054187 IB2011054187W WO2013041913A1 WO 2013041913 A1 WO2013041913 A1 WO 2013041913A1 IB 2011054187 W IB2011054187 W IB 2011054187W WO 2013041913 A1 WO2013041913 A1 WO 2013041913A1
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Classifications
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- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/54—Interleukins [IL]
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/81—Protease inhibitors
- G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- G01N2333/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
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- 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
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- G01—MEASURING; TESTING
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- 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
- the present invention relates to a method and a kit for monitoring, diagnosis and/or prognosis of acute kidney injury in early stage and determination of treatment in subjects suffering thereof.
- the kidney is an organ that serves multiple functions in the body, among which stand the elimination of body waste substances that are produced during metabolism, and return to the blood of substances that are necessary for the body, so as well as regulating the volume and composition of body fluids.
- the hydroelectrolytic balance of the body is maintained due to the kidney function (Guyton A., Hall J.E. 2001 , Medical Physiology Treaty, tenth edition. Mexico DF, Mexico: McGraw-Hill Interamericana).
- Kidney diseases are one of the most important causes of death in many countries. As early as 1994, more than 15 million of people presented kidney diseases in the United States, causing deterioration in life quality of the patient and death (Guyton A., Hall J.E. 2001 , Medical Physiology Treaty, tenth edition. Mexico DF, Mexico: McGraw-Hill Interamericana).
- kidney disease can be grouped into fairly well defined syndromes. Some are specific to glomerular diseases, and others are present in diseases that affect any of the renal structures. These diseases are among the causes of major morbidity and death in many countries (Guyton A., Hall J.E. 2001 , Medical Physiology Treaty, tenth edition. Mexico DF, Mexico: McGraw-Hill Interamericana).
- Severe kidney disease can be grouped into two main categories: Pathologies
- Kidney failure is a clinical condition in which the kidneys fail to function properly, reducing the glomerular filtration rate. Clinically, this fault is divided into two groups: acute kidney injury (AKI) and chronic renal failure (CRF) (Guyton A., Hall J.E. 2001, Medical Physiology Treaty, tenth edition. Mexico DF, Mexico: McGraw-Hill Interamericana). Renal failure
- oliguria or anuria decrease or absence of urinary excretion
- azotemia which corresponds to the accumulation of nitrogenated products in the blood.
- Metabolic waste products are also accumulated due to water retention, which determines an overload of fluid and salts, which results in edema and hypertension.
- kidneys do not excrete a normal amount of hydrogen ions, resulting in a manifestation of metabolic acidosis.
- the AKI is divided into 3 main categories: pre-renal, intra-renal and post-renal.
- Vasculitis (polyarteritis nodosa)
- Tubular epithelial injury tubular necrosis
- AKI is a pathological condition mainly acquired in hospitals and originated by several factors, such as sepsis, surgical interventions, particularly cardiac surgeries, ischemia, administration of nephrotoxins, therefore, there is a need of modern diagnosis techniques and treatments for preventing and diminish the impact of AKI (Vukusich A., Alvear F., Villanueva P., Gonzalez C, Olivari F., Alvarado N., Zehnder C. 2004, Rev. Med. Chile, 132:1355-1361).
- AKI is a severe problem, therefore, there are a lot of efforts to develop an early intervention of the pathology, especially in high risk patients, such as ICU patients (Vukusich A., Alvear F., Villanueva P., Gonzalez C, Olivari F., Alvarado N., Zehnder C. 2004, Rev. Med. Chile, 132:1355-1361 ; Schrier R. 2010, Nat. Rev. Nephrol, 6:56-59).
- Kidney efficiency allows us to maintain relatively normal blood concentrations of most electrolytes and an appropriate volume of body fluids, while the number of functioning nephrons remains above the 20 to 30% of normal. No serious clinical symptoms are present above this figure (Guyton A., Hall J.E. 2001, Medical Physiology Treaty, tenth edition. Mexico DF, Mexico: McGraw-Hill Inter americana).
- Nephrotoxins analgesics, heavy metals
- ESRD End stage renal disease
- Kidney transplant is the most effective treatment for ESRD. The success of transplant depends on many factors such as type of donor. The donor from which comes the kidney transplant can be either dead or alive. All transplanted organs are submitted to an acute injury due to ischemia and reperfusion process associated with the transplant.
- Living donor transplant is an excellent therapeutic option for the treatment of end stage chronic renal failure, especially in young patients. It allows following a defined pre-transplant protocol to help the better survival of the organ, together with a small ischemic time (less than 30 minutes). Survival becomes greater than 10 years, and higher in 17 to 20% compared to transplants with organs from cadaveric donors.
- the patient enters a waiting list and the selection is made regarding the degree of ABO and HLA compatibility.
- the selection criteria are the age, similarity between the body mass index of donor and recipient, if it corresponds to the first transplant, and if the transplant is of one kidney or both.
- the organ ischemic time could increase several hours (even 1 or 2 days), which increases the deterioration of transplanted organ.
- the renal transplant patient requires careful monitoring, initially in intensive care unit (ICU) or transplant unit and later, hospitalized or as outpatient.
- ICU intensive care unit
- Several surgical or medical complications may occur during monitoring. Although some complications are premature and could be treated while still confined in the ICU or transplant unit, this monitoring should be extended throughout the whole life of the patient, as there are complications that can arise even after a long period of time after receiving transplant (late complications).
- immunosuppressive therapy to increase survival of the transplanted organ, survival of the patient, and to improve his/her quality of life. This treatment is adjusted to the clinical and serological condition of each patient.
- Acute Kidney Injury can be classified in different levels depending on the stage corresponding to the deterioration of renal function.
- AKI is classified depending on a variety of parameters, the most common are determination of serum creatinine (SCR) and diuresis (D), which appear late in the development of the disease, and as such, do not allow AKI diagnostic in early stages.
- SCR serum creatinine
- D diuresis
- Creatinine is a metabolic byproduct formed in muscle tissue by degradation of phospho-creatine, which is eliminated from the body through filtration at glomerular level. Total rate of creatinine production will depend on muscular mass, muscular activity, sex, age, and total consumption of proteins. These variables also affect creatinine plasma levels. In spite of these limitations, the most used method for diagnostic of AKI is when serum creatinine levels are above 0.6-1.2 mg/dl.
- Creatinine clearance is the amount of creatinine which is eliminated from the blood flow in a period of time. This parameter is determined to estimate the glomerular filtration. Usual levels of creatinine clearance are between 125-150 ml/min and slightly lower in women.
- Oliguria is defined as a decrease in urine production, with a value below 400 ml per day, considering 400 ml as the minimal amount of urine that should be released in a normal metabolic state to eliminate daily produced solutes.
- Uremia corresponds to elevated levels of urea in blood. Determination of this parameter is through determination of blood urea nitrogen (BUN), which in normal values is between 8 to 18 mg/dl.
- BUN blood urea nitrogen
- RAFLE Risk Injury Failure Loss End-stage kidney disease
- RIFLE is a tool proposed to classify different stages of AKI: Risk, Injury, Failure, Loss, End- stage kidney disease. It is based on serum creatinine (SCR) and diuresis (D) levels of the patient. (Carrillo R., Castro J. "009. RIFLE scale. Journal of Mexican association of critic medicine and intensive therapy, 23(4):241-244).
- AKI 1 , AKI 2, and AKI 3 corresponds to stages R, I and L of RIFLE respectively.
- NAL Neutrophil gelatinase-associated lipocalin
- NGAL is usually expressed in low concentrations, and grown significantly for epithelial damage (Schmidt-Ott K.M., Mori K., Kalandadze A., Li J.Y., Paragas N., Nicholas T., Devarajan P., Barasch J. 2006, Curr Opin Nephrol Hypertens, 15:442-449; Cowland J.B., Borregaard N. 1997, Genomics, 45:17-23). NGAL is a small protein that belongs to the lipocalin family.
- the human NGAL is a single polypeptidic chain, with disulfide bridges, with 178 amino acidic residues, with a mass of 23 KDa, and its dimeric form of 46 KDa (Kjeldsen L., Johnsen A.H., Sengelov H., Borregaard N. 1993, J Biol Chem, 268:10425-10432.)- This protein is expressed in neutrophils of certain epithelia, including the epithelium of proximal renal tubules.
- NGAL is a secreted protein and is characterized by its ability to bind to small and hydrophobic molecules in its form of structurally conserved pocket, through ⁇ -pleated sheets to form macromolecular complexes (Uttenthal L.O. 2005 Clin Lab, 29:39-41).
- NGAL has partially overcome the general obstacles on this issue, and has proven been useful in the diagnosis of acute kidney injury, showing that it is possible to make this diagnosis at early stage (Zappitelli M., Washburn K.K., Arikan A.A., Loftis L., Ma Q., Devarajan P., Parikh C.R., Goldstein S.L. 2007 Crit Care, 11 : R84.).
- NGAL has a significant increase in patients with acute kidney injury, but not in the corresponding controls, this increase been produced within the first 24 to 48 hours, and prior to the increase in creatinine.
- This marker is used in both plasma and urine, but it still requires a complete evaluation in different clinical areas (Mishra J., Dent C, Tarabishi R., Mitsnefes M.M., Ma Q., Kelly C, Ruff S.M., Zahedi K., Shao M., Bean J., Mori K., Barasch J., Devarajan P. 2005, Lancet, 365:1231-1238; Devarajan P. 2007, Contrib Nephrol, 156:203-212).
- Aquaporin 1 is an integral membrane protein and was the first of its kind to be structurally and functionally characterized from human red blood cells.
- the protein has a tetramer structure, and each of its subunits has functionality by itself (Preston G.M., Jung J.S., Guggino W.B., Agre P. 1993, J Biol Chem, 268:17-20.). It has a weight of 28 KDa (Friedman M. 2008, Principles and models of biological transport, second edition, New York, USA: Springer), and its pattern of expression depends on the age and the tissue being examined (kidney, lung, brain and eyes) (Bondy C., Chin E., Smith B.L., Preston G.M., Agre P. 1993, Proc Natl Acad Sci USA, 90:4500-4504).
- This channel is strongly expressed in the renal proximal tubule, in the epithelium of Henle's loop in its descending portion and in the endothelium of the vasa recta.
- the high concentration of this protein in the descending loop of Henle (25% of total protein) suggests an essential role in the renal concentrating mechanism.
- Aquaporin 2 is an integral membrane protein that serves as a channel for water passage. This channel is regulated by vasopressin, and is located in the connecting tubule and collecting duct of the kidney, in its apical region (Fushimi K., Uchida S., Hara Y., Hirata Y., Marumo F., Sasaki S. 1993, Nature, 361 :549-552).
- mice have been modified to selectively express this aquaporin in the connecting tubule and not in the collecting duct. Also mice completely deficient in this protein have been developed. It has been observed that deficient mice die postnatally (5-12 days), while mice, which only the expression in the collecting duct was blocked, grow to adulthood, showing decreased body weight, 10 times of increase in urine production and decreased urine osmolarity. When deprived of water for 3 hours, there is no significant change in urine osmolarity, demonstrating that there are not compensatory mechanisms (Rojek A., Fiichtbauer E.M., Kwon T.H., Fr0kiaer J., Nielsen S. 2006, Proc Natl Acad Sci USA, 103:6037-6042).
- Aquaporin 3 is an integral membrane protein, with a weight of 30 kDa (Wakayama Y., Jimi T., Inoue M., Kojima H., Shibuya S., Murahashi M., Hara H., Oniki H. 2002, Histochem J, 34:331-337) and is expressed in the basolateral membrane of epithelial cells of renal collecting duct. Unlike other aquaporins, this protein can also transport glycerol (Ma T., Frigeri A., Hasegawa H., Verkman A.S. 1994, J Biol Chem, 269:21845-21849.).
- NKCCC2 in the Henle's loop
- NHE-3 and NaPill in the proximal tubule
- the group of Dr. Mark Knneper has developed proteomic studies in healthy human urine. These studies have analyzed the cellular and exosomal urinary fractions, obtained by differential centrifugation (Knepper M.A., Pisitkun T., Shen R.F. 2004, Proc Natl Acad Sci USA, 101 :13368-13373). The exosomal fraction has received particular interest because it is believed to be a rich source of representative proteins of the renal epithelium.
- Exosomes are produced from the endocytosis of apical membrane proteins. After this, the endosome fuses with the multivesicular body (MVB). Consequently, apical membrane proteins are segregated in the MVB outer membrane and internalized by invagination of the membrane. Finally, the outer membrane of the MVB fuses with the apical membrane, releasing their internal vesicles, called exosomes, in the urinary space (Knepper M.A., Pisitkun T., Shen R.F. 2004, Proc Natl Acad Sci USA, 101 :13368-13373).
- the proteins currently identified in urinary exosomes correspond to plasma membrane proteins (NKCC2, CD24, etc..) cytoplasm proteins (GAPDH, etc.) and nuclear proteins (AFT3 and WT-1) (Zhou H., Cheruvanky A., Hu X., Matsumoto T., Hiramatsu N., Cho M.E., Berger A., Leelahavanichkul A., Doi K., Chawla L.S., Illei G.G., Kopp J.B., Balow J.E., Austin H.A. 3rd, Yuen P.S., Star R.A. 2008, Kidney Int, 74:613-621).
- US2010203529 describes exosomes that can be used for detecting biomarkers in diagnostic, for example the stage or progression of a disease, describing also that biomarkers from cell-of-origin can be used to further determine treatment regimes for diseases and establishing the treatment efficacy.
- EP2191276 the document describes a prenatal diagnosis method by isolating exosomes from a fluid, wherein the exosomes are identified by a specific biomarker, in particular CD24.
- GB2463401 describes a method for characterising phenotype, diagnosing a disease by determining a bio-signature of an exosome in a sample from the subject.
- the markers mentioned comprise miRNA profiles, or antigens including CD63, CD9, CD81 , B7H3, EpCam, PSCA, TNFR, MFG-E8, Rab, SETAP, PCMA or 5T4. Further, the method mentions that it can be used to determine the cell of origin of the exosomes for profiling physiological states or determining phenotypes.
- WO2009115561 describes a polypeptide for identification of membrane vesicles or exosomes. Furthermore, the invention describes immunogenic preparations for preventing and/or treating an infection due to a pathogen or a tumoral antigen.
- KR20070058441 describes methods and compositions for use in immunosupression reactions.
- the compositions comprise exosomes with immunosupressive activity, wherein the exosomes can be derived from different cell types, mainly from the immune system. Furthermore, the exosomes can be exposed to molecules to enhance the immunosupressive activity.
- the exosomes are used in the treatment of diseases or disorders associated with immune system malfunction.
- US 2007254351 describes a method for isolation of hepatitis C virus, comprising separation of exosomes from blood plasma from an individual infected with the virus.
- AU2004203482 describes membrane vesicles comprising molecules (exosomes), wherein the molecules are from the major histocompatibility complex, and wherein the exosomes are used as immunogen or for diagnostic purposes.
- US 2004197314 describes compositions and methods to express a polypeptide in the membrane of a vesicle (exosome), focused mainly in the synthetic production of exosomes.
- CA2453198 describes exosomes which can be used in identification and quantification of immune suppressive factors in biological fluids of cancer patients. These exosomes can be used alone or in combination with other immunological assays as a prognosis indicator for cancer patient.
- EP1523990 describes exosomes derived from tumor cells. These exosomes have tumor specific antigens and molecules for stimulation of lymphocytes.
- Devarajan (2007) proposes a panel of acute kidney injury (AKI) markers for early detection of injury, as well as predicting the outcome of kidney injury in a patient. (Proteomics for biomarker discovery in acute kidney injury. Devarajan P., Williams L.M. 2007, Semin. Nephrol. 27(6):637- 651).
- NGAL as a potential biomarker indicator for organ recovery predictor in cases of kidney transplantation (NGAL in urinary exosomes as a source of kidney dysfunction biomarker in renal transplantation.
- Boltansky A. Alvarez S., Vukusich A., Hurtado M., Ursu M., Innocenti G., Carvajal D., Suazo C, Villanueva S., Carreno J., Altuzarra R., Yen C, Tapia D., Irarrazabal C.E. 2010, Renal Week, Denver, CO, J Am Soc Nephrol 21 :959).
- the present invention is based on the immunopurification of exosomes containing AQP-1 , AQP-2, AQP-3 (AQPs), NKCC2, NHE-3, and/or NaPill.
- the examples of application show that the procedure is accurate in determining renal injury in a patient.
- the present invention relates to a method for monitoring, diagnosis and/or prognosis of acute kidney injury in early stage and determination of treatment in subjects suffering thereof, the method comprising the steps of a) providing a urine sample; b) enriching the urine sample in exosomes present in the urine sample using at least one step of immunopurification; c) detecting an acute kidney injury (AKI) marker in the exosome.
- the invention further comprises a diagnostic kit for determining the presence and/or level of a specific kidney injury marker, for simple and early determination of the onset of AKI in a subject, the kit comprising means for enriching the urine sample in exosomes, using at least one step of immunopurification, and means for detecting a predetermined kidney injury marker of a condition.
- the present invention relates to a method for monitoring, diagnosis and/or prognosis of acute kidney injury in early stage and determination of treatment in subjects suffering thereof, the method comprising the steps of a) providing a urine sample; b) enriching the urine sample in exosomes present in the urine sample using at least one step of immunopurification; c) detecting an acute kidney injury (AKI) marker in the exosome.
- the urine sample is obtained from the first urination in the morning for catheterized patients, and from the second urination for other patients and is maintained at -80°C until it is analyzed.
- the present invention considers as means for enriching the urine sample in exosomes at least one step of immunopurification.
- by using antibodies directed to the intracellular, extracellular or any domain of particular proteins that are preferably and differentially expressed in specific structures of the kidney allows enrichment of exosomes.
- the method of the present invention considers antibodies directed to intracellular, extracellular or any domain of proteins that are preferably and differentially expressed in specific structures of the kidney, and suitable buffer to allow the antibodies to interact with the domains of proteins present in the external surface of exosomes.
- the method of the present invention can include at least one further mean for enrichment of exosomes, previous to the immunopurification.
- the means for enriching the urine sample in exosomes are laboratory methods and devices useful in separating larger elements from the urine sample, which could interfere in the detection phase later.
- the interfering elements can be cells from the patient.
- Centrifuge tubes can be considered enriching means, since the processing in a laboratory centrifuge allows enrichment of exosomes, eliminating larger particles, such as for example cells.
- the sample is centrifuged for 5 to 30 minutes at 5000 to 10000 rpm.
- the sample is ultracentifuged for 30 to 120 minutes at 30000 to 45000 rpm.
- Microfilter cartridges, microfilter columns, or other microfilter media, up to 0.22 micrometers are also considered as exosome enriching means, since micro filtration allows passing of exosomes through microfilters, thus enriching the urine sample in exosomes.
- the method considers, as means for detecting and/or quantifying a predetermined kidney injury marker, a primary antibody directed to a predetermined kidney injury marker, and a secondary antibody conjugated with a label, directed to the primary antibody.
- the label of the secondary antibody can be a fluorescent marker, an enzyme, a radioactive marker, a chemical compound, an infrared compound.
- the primary antibody can be conjugated directly with a label, in which case, the secondary antibody is not needed.
- the primary antibody can be also conjugated with a fluorescent marker, an enzyme, a radioactive marker, a chemical compound, an infrared compound.
- the immunopurification of a specific exosomal fraction from a specific kidney structure is carried out with an antibody selected, but not limited to: anti-aquoporin-1 (anti-AQP-1), anti-aquoporin-2 (anti-AQP-2), anti-aquoporin-3 (anti-AQP-3), anti- NKCC2, anti-NHE-3 and/or anti-NaPill or a combination thereof.
- an antibody selected, but not limited to: anti-aquoporin-1 (anti-AQP-1), anti-aquoporin-2 (anti-AQP-2), anti-aquoporin-3 (anti-AQP-3), anti- NKCC2, anti-NHE-3 and/or anti-NaPill or a combination thereof.
- the specific kidney injury marker is selected, but not limited to NGAL, Cystatin-3, KIM-1 , IL-lbeta, and/or IL-18, or a combination thereof.
- the immunopurification of a specific exosomal fraction from a specific kidney structure is carried out with antibodies directed to any domain of anti-AQPl , anti-AQP2, anti-AQP3, anti-NKCC2, anti-NHE-3, anti-NaPill, or a combination thereof as means for enriching the urine sample in exosomes.
- the specific kidney injury marker is selected from NGAL, Cystatin-3, KIM-1 , IL-lbeta, IL-18 or a combination thereof.
- the kidney injury marker which presence and/or level is determined is NGAL and/or Cystatin-3
- the kidney injury marker which presence and/or level is determined is NGAL
- anti-AQP-3 is used for immunopurifying a specific exosome fraction from a specific kidney structure
- the kidney injury marker which presence and/or level is determined is KIM-1, IL-lbeta and/or Cystatin-3.
- the exosomal fraction comes from the proximal tubule, and the presence and/or level of a specific marker indicates injury in said structure.
- the exosomal fraction comes from the distal tubule, and the presence and/or level of a specific marker indicates injury in said structure.
- the exosomal fraction comes from the collecting duct, and the presence and/or level of a specific marker indicates injury in said structure.
- the exosomal fraction comes from the Henle's loop, and the presence and/or level of a specific marker indicates injury in said structure.
- the exosomal fraction comes from the proximal tubule, and the presence and/or level of a specific marker indicates injury in said structure.
- the exosomal fraction comes from the proximal tubule, and the presence and/or level of a specific marker indicates injury in said structure.
- the method optionally includes a step for evaluating the acute kidney injury in a subject for monitoring, diagnosis, prognosis and/or determining a treatment in the subject based on the presence and/or level of the specific kidney injury marker.
- the invention further comprises a diagnostic kit for determining the presence and/or level of a specific kidney injury marker, for simple and early determination of the onset of AKI in a subject, the kit comprising means for enriching the urine sample in exosomes, using at least one step of immunopurification, and means for detecting a predetermined kidney injury marker of a condition.
- the diagnostic kit comprises means for obtaining a urine sample from a patient.
- the means for obtaining the urine sample are selected among a urinary probe, in the case the patient is unable to provide a urine sample by him/her self; or a container to receive a urine sample from a patient.
- the present invention considers as means for enriching the urine sample in exosomes at least one step of immunopurification. In particular, by using antibodies directed to the intracellular, extracellular or any domain of particular proteins that are preferably and differentially expressed in specific structures of the kidney, allows enrichment of exosomes.
- kit of the present invention considers antibodies directed to intracellular, extracellular or any domain of proteins that are preferably and differentially expressed in specific structures of the kidney, and suitable reaction buffer to allow the antibodies to interact with the domains of proteins present in the external surface of exosomes.
- the kit also comprises blocking agents or solutions and stock solutions of the specific kidney injury markers.
- the kit comprises a 96 well plate wherein the wells are covered with the antibodies directed to the intracellular, extracellular or any domain of the particular proteins that are preferably and differentially expressed in specific structures of the kidney, allowing enrichment of exosomes.
- the kit of the present invention can include at least one further mean for enrichment of exosomes, previous to the immunopurification.
- the means for enriching the urine sample in exosomes considered in the invention are laboratory methods and devices useful in separating larger elements from the urine sample, which could interfere in the detection phase later.
- the interfering elements can be cells from the patient.
- Centrifuge tubes can be considered enriching means, since the processing in a laboratory centrifuge allows enrichment of exosomes, eliminating larger particles, such as for example cells.
- Microfilter cartridges, microfilter columns, or other microfilter media, up to 0.22 micrometers are also considered as exosome enriching means, since microfiltration allows passing of exosomes through micro filters, thus enriching the urine sample in exosomes.
- the kit considers, as means for detecting and/or quantifying a predetermined kidney injury marker, a primary antibody directed to a predetermined kidney injury marker, and a secondary antibody conjugated with a label, directed to the primary antibody.
- the label of the secondary antibody can be a fluorescent marker, an enzyme, a radioactive marker, a chemical compound, an infrared compound.
- the primary antibody can be conjugated directly with a label, in which case, the secondary antibody is not needed.
- the primary antibody can be also conjugated with a fluorescent marker, an enzyme, a radioactive marker, a chemical compound, an infrared compound.
- the kit comprises antibodies selected, but not limited to: anti-aquoporin-1 (anti-AQP-1), anti-aquoporin-2 (anti-AQP-2), anti-aquoporin-3 (anti-AQP-3), anti- NKCC2, anti-NHE-3 and/or anti-NaPill or a combination thereof.
- the kit comprises primary antibodies, either conjugated with a label or not for detection of a marker selected, but not limited to NGAL, Cystatin-3, KIM-1 , IL-lbeta, and/or IL-18 or a combination thereof.
- the kit comprises antibodies directed to any domain of anti-AQPl , anti-AQP2, anti-AQP3, anti-NKCC2, anti-NHE-3, anti-NaPill, or a combination thereof as means for enriching the urine sample in exosomes, when using immunopurification as a method for enrichment.
- the kit comprises primary antibodies, either conjugated with a label or not, for detection of NGAL, Cystatin-3, KIM-1 , IL-lbeta, IL-18.
- the kidney injury marker which presence and/or level is determined is NGAL and/or Cystatin-3
- the kidney injury marker which presence and/or level is determined is NGAL
- anti-AQP-3 is used for immunopurifying a specific exosome fraction from a specific kidney structure
- the kidney injury marker which presence and/or level is determined is KIM-1, IL-lbeta and/or Cystatin-3.
- the exosomal fraction comes from the proximal tubule, and the presence and/or level of a specific marker indicates injury in said structure.
- the exosomal fraction comes from the distal tubule, and the presence and/or level of a specific marker indicates injury in said structure.
- the exosomal fraction comes from the collecting duct, and the presence and/or level of a specific marker indicates injury in said structure.
- anti-NKCC2 is used for immunopurifying a specific fraction from a specific kidney structure
- the exosomal fraction comes from the Henle's loop, and the presence and/or level of a specific marker indicates injury in said structure.
- the exosomal fraction comes from the proximal tubule, and the presence and/or level of a specific marker indicates injury in said structure.
- the exosomal fraction comes from the proximal tubule, and the presence and/or level of a specific marker indicates injury in said structure.
- the kit of the invention optionally includes instructions for using the kit.
- the method and the kit of the invention comprise a step of immunopurification of urinary exosomes. Therefore, the invention also describes a method for inmunopurifying urinary exosomes and the use of antibodies for purifying urinary exosomes.
- the invention describes a method for purifying urinary exosomes, the method comprising the steps of (a) incubating a urine sample, or optionally, a decellularized urine sample with an antibody directed to the intracellular, extracellular, or any domain of proteins preferably and differentially expressed in the surface of different kidney structures, thus forming an exosome- antibody complex; (b) incubating the exosome-antibody complex resulting from (a) with a tag that recognizes any region of the antibody and is bound to an insoluble agent, thus forming an exosome-antibody-tag-insoluble agent complex; (c) separating the exosome-antibody-tag- insoluble agent complex from the supernatant; (d) washing of the exosome-antibody-tag- insoluble agent complex with an adequate buffer.
- the urine sample, or decellularized urine sample is incubated from 20 to 60 minutes with the antibody, in an adequate buffer, at room temperature.
- the antibodies used in the purification method for urinary exosomes are selected, but not limited to: anti-aquoporin-1 (anti-AQP-1), anti-aquoporin-2 (anti-AQP-2), anti- aquoporin-3 (anti-AQP-3), anti- NKCC2, anti-NHE-3 and/or anti-NaPiII or a combination thereof.
- the antibodies used in the purification method for urinary exosomes are directed to any domain of anti-AQPl , anti-AQP2, anti-AQP3, anti-NKCC2, anti-NHE-3, anti-NaPiII, or a combination thereof.
- the exosomal fraction comes from the proximal tubule, and the presence and/or level of a specific marker indicates injury in said structure.
- the exosomal fraction comes from the distal tubule, and the presence and/or level of a specific marker indicates injury in said structure.
- the exosomal fraction comes from the collecting duct, and the presence and/or level of a specific marker indicates injury in said structure.
- the exosomal fraction comes from the Henle's loop, and the presence and/or level of a specific marker indicates injury in said structure.
- the exosomal fraction comes from the proximal tubule, and the presence and/or level of a specific marker indicates injury in said structure.
- the exosomal fraction comes from the proximal tubule, and the presence and/or level of a specific marker indicates injury in said structure.
- the tag that recognizes any region of the antibody is protein A or protein G or fractions thereof, bound to agarose or sepharose beads.
- the tag is biotin or a fraction thereof, and is bound to the antibody.
- the complex exosome-antibody-biotin is formed.
- the insoluble agent are magnetic beads bound to a compound with affinity to biotin or a fraction thereof, such as, but not limited to an antibody.
- the separation of the exosome-antibody-tag-insoluble agent from the supernatant is performed by centrifugation or sedimentation.
- the invention also discloses the use of an antibody or a combination of antibodies directed to the intracellular, extracellular, or any domain of proteins preferably and differentially expressed in the surface of different kidney structures for immunopurification of urinary exosomes.
- the antibodies used for immunopurification of urinary exosomes are selected, but not limited to: anti-aquoporin-1 (anti-AQP-1), anti-aquoporin-2 (anti-AQP-2), anti- aquoporin-3 (anti-AQP-3), anti- NKCC2, anti-NHE-3 and/or anti-NaPiII or a combination thereof.
- the antibodies used for immunopurification of urinary exosomes are directed to any domain of anti-AQPl , anti-AQP2, anti-AQP3, anti-NKCC2, anti-NHE-3, anti- NaPill, or a combination thereof.
- the exosomal fraction comes from the proximal tubule, and the presence and/or level of a specific marker indicates injury in said structure.
- the exosomal fraction comes from the distal tubule, and the presence and/or level of a specific marker indicates injury in said structure.
- the exosomal fraction comes from the collecting duct, and the presence and/or level of a specific marker indicates injury in said structure.
- the exosomal fraction comes from the Henle' s loop, and the presence and/or level of a specific marker indicates injury in said structure.
- the exosomal fraction comes from the proximal tubule, and the presence and/or level of a specific marker indicates injury in said structure.
- the exosomal fraction comes from the proximal tubule, and the presence and/or level of a specific marker indicates injury in said structure.
- the method and kit of the invention have the advantage to provide a specific, simple and early detection of kidney injury markers that is no possible with the standard methods applied until now.
- the method and kit of the invention also provide the advantage of identifying the specific structure that is injured in the kidney, since it uses specific antibodies for specific proteins present in specific structures of the kidney.
- the method and kit of the present invention can be used for monitoring, diagnosis, prognosis and/or determination of treatment in any subject.
- the method and kit of the invention is useful for monitoring the development of renal injury in patients that underwent kidney transplant, due to the ischemia-reperfusion process associated with the transplant.
- the method and kit of the invention is also useful for monitoring, diagnosis, prognosis and/or determination of treatment in patients in intensive care unit (ICU) that could develop AKI and need an early detection.
- ICU intensive care unit
- Figure 1 Representative Western blot comparing the abundance of NGAL in the cellular fraction and exosomal fraction from a urine sample of a kidney transplant patient at post-transplant days 1 , 2, 3, and 4.
- Figure 2 Representative Western blot showing abundance of NGAL-24 and NGAL-46 in complete urine (U) and exosomal fraction (E) from a patient on day 1 post-transplant.
- Figure 3 Graphical representation of NGAL in the total exosomal fraction of urine in patients day 1 post-transplant.
- AU Arbitrary units.
- Figure 5 Comparative analysis between two levels of SCR and NGAL in individuals with kidney transplant.
- Figure 6 Relative abundance of NGAL (Arbitrary Units) in exosome-free urine (white bars, EF- U) and total exosome fraction (black bars, TE) in ICU patients classified as AKI1 (5 patients), AKI2 (3 patients), and AKI 3 (5 patients).
- NGAL Extracellular NGAL
- Figure 7 TE/EF-U ratio of NGAL abundance in arbitrary units in patients from ICU classified as AKI1 (5 patients), AKI2 (3 patients) and AKI3 (5 patients).
- Figure 8 Relative NGAL abundance in exosome-free urine (EF-U), total exosomes (TE) and immunopurified exosomes (IP-E) in ICU patients classified as AKI1 (5 patients).
- the method and products of the present invention were tested by analysing a group of 12 human patients, plus healthy individuals acting as a control group.
- the inclusion criteria used for healthy controls were the absence of renal symptomatology and the inclusion criteria for patients were all individuals who underwent kidney transplant during the period of duration of the present study. Patients were enrolled considering an informed consent approval.
- Exosomes were obtained considering two different approaches.
- the first one corresponded to a centrifuge separation in two steps, the first using centrifugation, and the second step using ultracentrifugation.
- the second method the main feature of the present invention, considered a first stage of centrifugation, and a second step of immunopurification.
- a fraction of SI obtained as described in the first step for the method with 2 centrifugation steps, was used for immunopurification of exosomes using AQP1 , AQP2, or AQP3 antibodies for obtaining exosomes from different regions of the kidney.
- Samples were resuspended in a load buffer (100 mM Tris-HCl, pH 6.8, 200 mM dithiotreitol (DTT), 4% SDS, 0.2% bromophenol blue, 20% glycerin), so as to load 100 micrograms of total proteins of each sample in a gel.
- a load buffer 100 mM Tris-HCl, pH 6.8, 200 mM dithiotreitol (DTT), 4% SDS, 0.2% bromophenol blue, 20% glycerin
- the membranes were washed 3 times with 1 % PBS- Tween 20 (Winkler), to proceed with a secondary antibody for 2 hours at room temperature with agitation. Finally, the membranes were washed 3 times with PBS-Tween 20 1 % for further processing.
- Abundance of proteins was determined by a semi-quantitative analysis, using densitometric determination of bands obtained by Western Blot, using Adobe Photoshop CS4 software, with 2 measurements (total band and a fixed size band for all samples) for each band obtained as to standardize the determination.
- Total protein concentration was determined in both cellular fractions, using a commercially available kit (BCA Protein Assay Reagent, Pierce).
- Example 5 Results of the analysis according to the method of the present invention Characteristics of the group study
- Serum creatinine is an important parameter to test the viability of the transplanted organ. The first day after transplant, all of the patients had values for serum creatinine higher than normal (0.8 to 1.4 mg/dl), showing higher values the patients whom received the organ from a cadaver donor (Table 4).
- ischemic time to which the organ was subjected to Previous to the transplant.
- the times range from 27 to 1182 minutes, being in general the higher times for those patients whom received the organ form a cadaver donor, with times ranging from 300 to 1182 minutes, compared to the times from patients receiving the organ from a living donor, from 27 to 360 minutes (Table 4).
- NGAL-23 and NGAL-466 All NGAL isoform abundance was analysed (NGAL-23 and NGAL-46) from total urinary exosomes from the first day post-transplant, using Western blot techniques, in 12 patients included in the study, obtaining information for 11 of them. These results show that NGAL was expressed in almost all of the cases. The range of expression was between 7.3 and 170.3 AU, i.e. about 20 times of difference between the extreme cases (Figure 3).
- Pisitkun in 2004 (Pisitkun T., Shen R.F., Knepper M.A. 2004, Proc Natl Acad Sci USA, 101 :13368-13373) described the presence of aquaporins (AQPs) in urinary exosomes from normal patients.
- AQPs aquaporins
- the present study looked for AQPs (AQP1, AQP2, AQP3) expression in immunopurified exosomes from patients whom underwent renal transplant. The election of each of these components is because its expression is associated with specific zones in the nephron.
- AQP1 is usually associated with proximal tubules (Knepper M.A.
- AQP2 is associated with distal tubules and collecting ducts (Ma T., Yang B., Gillespie A., Carlson E.J., Epstein C.J., Verkman A.S. 1998, J Biol Chem, 273 :4296-4299)
- AQP3 is associated with collecting ducts from the nephron (Sasaki S., Ishibashi K., Marumo F. 1998, Annu Rev Physiol, 60:199-220).
- This analysis allows determining the abundance of each biomarker present in a particular type of exosome.
- the approach is to establish a relationship between the potential origin of the biomarker of renal injury analysed.
- Figure 4 shows the normalization of NGAL-46 isoform by the level of AQPs in urinary exosomes of patients with positive information.
- NGAL-46 was chosen since the expression of NGAL-46 is higher than NGAL-23 ( Figure 1).
- the results show that NGAL abundance in relationship to the abundance of AQPl is statistically higher than other AQPs ( Figure 4, p ⁇ 0.05).
- Figure 4 p ⁇ 0.05
- SCR glomerular filtration
- NGAL markers of renal injury
- NGAL is an excellent marker for renal injury.
- Ischemic time of an organ is a determinant factor of the damage the organ suffers before transplant, since the longer this time, the lower the function it will have. This is observed in patients 1 and 7, which have a high expression of NGAL related with ischemic values of 360 and 1182 minutes. The contrary is observed in patients 2 and 6, with ischemic values of 27 and 90 minutes. This relationship can be established in most of the cases, with exception of patient 11, with a low value of ischemic time and high levels of NGAL (Table 4, Figure 3). Patient 5 shows the higher level of NGAL of the group, but there is no available data of ischemic time.
- Pisitkun in 2004 (Pisitkun T., Shen R.F., Knepper M.A. 2004, Proc Natl Acad Sci USA, 101 : 13368-13373) showed that AQPs were present in urinary exosomes of normal patients.
- AQP1 is associated with proximal tubules in nephrons ((Knepper M.A. 1997, Am J Physiol, 272:F3-F12), and AQP2 is associated with distal tubules, and AQP3 is associated with collecting ducts
- the abundance of NGAL was analysed in exosomes immunopurified with AQP1 , AQP2, and AQP3.
- NGAL in each of the immunopurified exosomes suggests a significantly higher abundance of NGAL in exosomes with AQP1 in their membranes (p ⁇ 0.05, Figure 4), followed by exosomes with AQP2 and AQP3, with no significant difference among the latter two.
- the data suggests that exosomes come from the proximal tubule of the kidney have a higher abundance of renal injury marker NGAL.
- NGAL is present in the exosomal fraction of urine samples taken the first day post-transplant of the kidney in all patients analysed, suggesting that the expression of molecular factors in the exosomes is due to development of renal injury in the transplant, associated to the transplant procedure.
- patients 1, 4, and 7 showed together high levels of NGAL.
- 1 and 7 showed a retarded function of the transplant, requiring dialysis therapy during the first week after transplant.
- NGAL is mainly expressed in urinary exosomes obtained from proximal tubules.
- the study group comprised 28 individuals admitted to the ICU, with ages ranging from 21 to 95 years, 19 male and 9 female. 46% (13/28) developed AKI according to RIFLE classification.
- Example 7 Analysis of biomarkers of kidney injury in total exosomes, exosome-free urine, and immunopurified exosomes.
- AKI biomarker NGAL was analysed by Western blot techniques in Exosome-Free urine (EF-U), total exosome fraction (TE) and immunopurified exosomes (IP-E). From 28 patients in the study, 17 showed detectable levels of NGAL using Western blot technique.
- the Table 6 below shows the results for the detection of biomarkers in the patients of the study.
- NGAL abundance was higher in the exosomal fraction in 85% of cases, compared to EF-U fraction.
- 5 patients, with AKI diagnose showed preferential expression of NGAL in the exosomal fraction (patients 5, 8, 9, 12, and 16), while their levels of NGAL in EF-U fraction were extremely low.
- Only 2 patients with positive AKI diagnose and NGAL showed equivalent abundance of NGAL in both fractions (patients 2 and 7).
- NGAL levels in the urine of patients admitted to the ICU and diagnosed with AKI1 , AKI2, and AKI3 were determined separately. Results clearly show that with an increasing level of injury, NGAL abundance also increases, although NGAL abundance is higher in TE compared to EF-U in the case of AKIl and AKI2 ( Figure 6).
- the data shows that immunopurifying exosomes from the proximal tubule section of the nephron increases the abundance of NGAL and thus the sensitivity in early detection of AKI in critical patients.
- the analysis to the group of 28 patients of this example allows establishing the determination of NGAL in urine is a good method for AKI diagnosis, in a period of 24 hours before the usual method of creatinine levels in serum allows.
- NGAL abundance in the exosomal fraction is higher than in exosome-free urine fraction, thus showing a higher sensitivity method for early AKI diagnose, in particular for stages AKIl and AKI2.
- Immunopurification of exosomes allows increasing the sensitivity of AKI detection due to higher abundance of NGAL in immunopurified exosomes with AQP1.
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RU2014115999/15A RU2014115999A (en) | 2011-09-22 | 2011-09-22 | METHOD FOR MONITORING, DIAGNOSTIC AND / OR FORECASTING OF ACUTE KIDNEY DAMAGE AT EARLY STAGE |
BR112014006741A BR112014006741A2 (en) | 2011-09-22 | 2011-09-22 | method for monitoring, diagnosis and / or prognosis of early acute kidney injury |
CA2849577A CA2849577A1 (en) | 2011-09-22 | 2011-09-22 | Method for monitoring, diagnosis and/or prognosis of acute kidney injury in early stage |
EP20110872834 EP2758774A4 (en) | 2011-09-22 | 2011-09-22 | Method for monitoring, diagnosis and/or prognosis of acute kidney injury in early stage |
JP2014531321A JP5893742B2 (en) | 2011-09-22 | 2011-09-22 | Method for monitoring, diagnosing and / or prognosing acute kidney injury in the early stage |
CN201180074742.3A CN103917869A (en) | 2011-09-22 | 2011-09-22 | Method for monitoring, diagnosis and/or prognosis of acute kidney injury in early stage |
AU2011377379A AU2011377379A1 (en) | 2011-09-22 | 2011-09-22 | Method for monitoring, diagnosis and/or prognosis of acute kidney injury in early stage |
US14/346,679 US20140329334A1 (en) | 2011-09-22 | 2011-09-22 | Method for monitoring, diagnosis and/or prognosis of acute kidney injury in early stage |
PCT/IB2011/054187 WO2013041913A1 (en) | 2011-09-22 | 2011-09-22 | Method for monitoring, diagnosis and/or prognosis of acute kidney injury in early stage |
KR1020147009382A KR20140076571A (en) | 2011-09-22 | 2011-09-22 | Method for monitoring, diagnosis and/or prognosis of acute kidney injury in early stage |
MX2014003422A MX2014003422A (en) | 2011-09-22 | 2011-09-22 | Method for monitoring, diagnosis and/or prognosis of acute kidney injury in early stage. |
IL231626A IL231626A0 (en) | 2011-09-22 | 2014-03-20 | Method for monitoring, diagnosis and/or prognosis of acute kidney injury in early stage |
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Also Published As
Publication number | Publication date |
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HK1199754A1 (en) | 2015-07-17 |
IL231626A0 (en) | 2014-05-28 |
JP2014528076A (en) | 2014-10-23 |
AU2011377379A1 (en) | 2014-04-10 |
MX2014003422A (en) | 2014-10-13 |
US20140329334A1 (en) | 2014-11-06 |
KR20140076571A (en) | 2014-06-20 |
IN2014DN03194A (en) | 2015-05-22 |
CA2849577A1 (en) | 2013-03-28 |
EP2758774A1 (en) | 2014-07-30 |
CN103917869A (en) | 2014-07-09 |
JP5893742B2 (en) | 2016-03-23 |
RU2014115999A (en) | 2015-10-27 |
BR112014006741A2 (en) | 2017-03-28 |
EP2758774A4 (en) | 2015-04-29 |
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