WO2010042202A1 - Procédés pour traiter une lésion podocytaire - Google Patents

Procédés pour traiter une lésion podocytaire Download PDF

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Publication number
WO2010042202A1
WO2010042202A1 PCT/US2009/005529 US2009005529W WO2010042202A1 WO 2010042202 A1 WO2010042202 A1 WO 2010042202A1 US 2009005529 W US2009005529 W US 2009005529W WO 2010042202 A1 WO2010042202 A1 WO 2010042202A1
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podocyte
ctgf
agent
subject
injury
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PCT/US2009/005529
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English (en)
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Noelynn A. Oliver
Roel Goldschmeding
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Fibrogen, Inc.
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Publication of WO2010042202A1 publication Critical patent/WO2010042202A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to methods and agents for treating, preventing, or reducing podocyte injury or podocyte-associated disorders.
  • Methods and agents for treating or preventing podocytopathies i e., podocytopathy (i.e. podocyte disease)s are also provided.
  • Podocytes are highly specialized epithelial cells found in the Bowman's capsule of the kidney. Podocytes have a complex cellular morphology. The podocyte 's cell body bulges into the u ⁇ nary space and gives ⁇ se to long primary processes that extend toward the glomerular basement membrane (GBM) to which they attach by numerous foot processes The foot processes of neighbo ⁇ ng podocytes interdigitate, leaving between them filtration slits b ⁇ dged by an extracellular structure, known as the slit diaphragm.
  • GBM glomerular basement membrane
  • Podocytes are pola ⁇ zed cells. Their unique shape is the result of an abundantly ⁇ ch actin cytoskeleton, which serves as the podocyte's backbone and also serves as a static function to podocytes.
  • actin cytoskeleton also enables podocytes to continually and dynamically alter their shape.
  • the actm cytoskeleton comp ⁇ ses three distinct ultrastructural elements: microfilaments (7-9 nm diameter), intermediate filaments (10 nm), and microtubules (24 nm) Microfilaments are the predominant cytoskeletal constituents of the podocyte foot process, and contain a dense network of F- actm and myosin.
  • actin binding proteins in podocytes such as synaptopodin and CD2AP, play important roles in maintaining podocyte shape.
  • podocytes function as a filtration barrier between glomerular capillaries and the u ⁇ nary space, preventing proteins from ente ⁇ ng the urine
  • the slit diaphragm which consists of a highly organized network of glycoproteins, prevents the passage of larger molecules, such as albumin, from glomerular capillaries into the urinary space.
  • podocyte specific proteins e.g , synaptopodin, neph ⁇ n
  • VEGF vascular endothelial growth factor
  • Podocytes are the target of injury m many glomerular diseases. Podocyte damage results in retraction of their foot processes (i.e. foot process effacement) and is associated with proteinuria (Laurens et al. (1995) Kidney Int. 47: 1078-1086); Pavenstaadt et al.
  • Human glomerulopathies such as minimal change nephropathy (MCN), membranous nephropathy (MN), focal segmental glomerulosclerosis (FSGS), collapsing glomerulopathy (CG), chronic glomerulonephritis (GN), and diabetic nephropathy (DN), typically exhibit foot process effacement of podocytes and loss of slit diaphragm structure (Kerjaschki (1997) Kidney Int 45- 300-313; K ⁇ z et al (1994) Kidney Int. 45: 369- 376, Pagtalunan et al (1997) J. Clin. Invest.
  • MN membranous nephropathy
  • FSGS focal segmental glomerulosclerosis
  • CG collapsing glomerulopathy
  • GN chronic glomerulonephritis
  • DN diabetic nephropathy
  • podocytopathy i.e podocyte disease
  • i e podocytopathies i e podocytopathies
  • podocytopathies i.e. podocytopathy (i.e. podocyte disease)
  • Steroid therapy and immunosuppressive agents are often used to treat proteinuric subjects with podocytopathy (e.g., FSGS).
  • ACE inhibitors and angiotensin receptor blockers are commonly administered to hypertensive subjects with podocytopathy (i.e. podocytopathy (i.e. podocyte disease)) and may decrease proteinuria.
  • these therapies can be useful for reducing symptoms of podocytopathy (i.e podocytopathy (i e.
  • podocyte disease the effect of these compounds on treating podocyte injury and podocyte-associated disorders is limited.
  • the provision of an effective treatment that targets the underlying podocytopathy or injury would therefore allow an entire class of patients to be treated effectively, who were previously only able to obtain symptomatic relief.
  • podocytopathy i.e. podocytopathy (i.e. podocyte disease)
  • CTGF connective tissue growth factor
  • the present invention provides a method for treating, preventing, or reducing podocyte injury in a subject in need thereof (e.g., in a subject having or at risk of having podocyte injury), the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits CTGF, thereby treating, preventing, or reducing podocyte injury in the subject.
  • the present invention also provides an agent that inhibits CTGF for use in a method for treating, preventing, or reducing podocyte injury, a podocyte-associated disorder or a podocytopathy (i.e podocytopathy (i e. podocyte disease))
  • the podocyte injury is due to ischemia, hyperglycemia, diabetes, inflammation, an infection, or a medication.
  • the present invention provides a method for treating or preventing, or reducing a podocyte-associated disorder in a subject in need thereof, the method comp ⁇ sing administering to the subject a therapeutically effective amount of an agent that inhibits CTGF
  • the podocyte-associated disorder is podocytopema, podocytu ⁇ a, podocyte foot process effacement, a decrease in podocyte slit diaphragm length, or a diminution of podocyte number or density
  • the present invention provides a method for treating or preventing a podocytopathy
  • the method comp ⁇ sing administering to the subject a therapeutically effective amount of an agent that inhibits CTGF.
  • the podocytopathy i.e. podocytopathy (i e. podocyte disease)
  • the podocytopathy is diabetic nephropathy, minimal change disease, focal segmental glomerulosclerosis (FSGS), membranous nephropathy, collapsing glomerulopathy (CG), or chronic glomerulonephritis.
  • the present invention provides a method for preventing or reducing podocyte foot process effacement in a subject in need thereof, the method comp ⁇ sing admmiste ⁇ ng to the subject a therapeutically effective amount of an agent that inhibits CTGF.
  • the present invention provides a method for preventing or reducing podocytu ⁇ a in a subject in need thereof, the method comp ⁇ sing admimste ⁇ ng to the subject a therapeutically effective amount of an agent that inhibits CTGF
  • the present invention provides a method for maintaining podocyte number or reducing the extent of podocyte loss in a subject in need thereof, the method comp ⁇ sing admmiste ⁇ ng to the subject a therapeutically effective amount of an agent that inhibits CTGF.
  • Methods for maintaining podocyte slit diaphragm length in a subject in need thereof, the method comp ⁇ sing admmiste ⁇ ng to the subject a therapeutically effective amount of an agent that inhibits CTGF are specifically provided
  • the invention provides a method for decreasing podocyte actin cytoskeletal rearrangement in a subject m need thereof, the method comp ⁇ sing admimste ⁇ ng to the subject a therapeutically effective amount of an agent that inhibits CTGF.
  • the invention provides a method for increasing a podocyte marker in a subject in need thereof, the method comp ⁇ smg administenng to the subject a therapeutically effective amount of an agent that inhibits CTGF.
  • the invention provides a method for maintaining levels of a podocyte marker or reducing the extent of podocyte marker loss in a subject in need thereof, the method comp ⁇ sing administenng to the subject a therapeutically effective amount of an agent that inhibits CTGF.
  • the podocyte marker is podocin, CD2AP, synaptopodin, nephnn, podocalyxin, FAT-I, neph ⁇ n, podoplanin, or Neph-1.
  • the invention provides a method for decreasing kidney desmm levels in a subject in need thereof, the method comp ⁇ sing administering to the subject a therapeutically effective amount of an agent that inhibits CTGF
  • the invention provides a method for decreasing or preventing podocyte detachment from the glomerular basement membrane (GBM) in a subject in need thereof, the method comp ⁇ sing administering to the subject a therapeutically effective amount of an agent that inhibits CTGF.
  • GBM glomerular basement membrane
  • the methods of the present invention further comprise determining the extent of podocyte injury before administering to the subject a therapeutically effective amount of an agent that inhibits CTGF. In other embodiments, the methods of the present invention further comprise determining the extent of podocyte injury du ⁇ ng administration of a therapeutically effective amount of an agent that inhibits CTGF to the subject. In yet other embodiments, the methods of the present invention further comprise determining the extent of podocyte injury after administering to the subject a therapeutically effective amount of an agent that inhibits CTGF. hi particular embodiments, the methods of the present invention further comprise determining the extent of podocyte injury before and after administering to the subject a therapeutically effective amount of an agent that inhibits CTGF.
  • the methods of the present invention further comprise determining the extent of one or more podocyte-associated disorders (e.g., podocytu ⁇ a or podocytopema) before administering to the subject a therapeutically effective amount of an agent that inhibits CTGF.
  • the methods of the present invention further comp ⁇ se determining the extent of one or more podocyte-associated disorders du ⁇ ng administration of a therapeutically effective amount of an agent that inhibits CTGF to the subject, hi yet other embodiments, the methods of the present invention further comp ⁇ se determining the extent of one or more podocyte-associated disorders after admimstenng to the subject a therapeutically effective amount of an agent that inhibits CTGF.
  • the methods of the present invention further comp ⁇ se determining the extent of one or more podocyte-associated disorders before and after admimstenng to the subject a therapeutically effective amount of an agent that inhibits CTGF.
  • the subject of the present methods may be a podocyte, a population of podocytes, a tissue, an organ, an organism, an animal, a p ⁇ mate, or a mammalian subject, e.g , a cat or a dog.
  • the subject is a human subject
  • the subject of the present methods is identified as a subject having podocyte injury Identification of a subject having podocyte injury can occur before, during, or after administration of an agent that inhibits CTGF. Detection of podocyte injury is desc ⁇ bed herein.
  • the agent that inhibits CTGF used in the methods of the present invention may, for example, be a polypeptide, a polynucleotide, or a small molecule
  • the agent that inhibits CTGF may be an annbody that binds to CTGF, or binds to a fragment thereof; an antisense molecule; an siRNA, or a small molecule chemical compound
  • the agent that inhibits CTGF is a monoclonal antibody or a fragment thereof, wherein the monoclonal antibody or fragment thereof specifically bind to
  • the agent that inhibits CTGF is CLN-I, desc ⁇ bed in WO 2004/108764, or a fragment thereof.
  • the agent that inhibits CTGF is the antibody produced by the cell line deposited with the American Type Culture Collection (ATCC) on 20th May 2004 with Deposit Number PTA-6006, or a fragment thereof.
  • Figures IA, IB, 1C, and ID set forth data showing CTGF induced actin cytoskeletal rearrangement and cell polarization in human podocytes.
  • FIGS 2A, 2B, 2C, and 2D set forth data showing CTGF increased Arp3 protein levels in human podocytes.
  • Figure 3 sets forth data showing CTGF increased Arp3 expression in human podocytes
  • Figures 4A, 4B, 4C, and 4D set forth data showing CTGF decreased podocm protein levels in human podocytes
  • Figures 5A, 5B, 5C and 5D set forth data showing CTGF decreased CD2AP protein levels in human podocytes.
  • FIGS. 6A and 6B set forth data showmg CTGF decreased podocm and CD2AP expression in human podocytes.
  • Figure 7 sets forth data showmg CTGF decreased synaptopodm expression in human podocytes.
  • Figures 8 A and 8B set forth data showing CTGF inhibition decreased activation of p42/44 MAPK in human podocytes
  • Figure 9 sets forth data showing CTGF inhibition reduced migration of cultured podocytes.
  • the present invention relates, in part, to the discovery that connective tissue growth factor (CTGF) plays a key role in specific aspects of podocyte injury and podocyte-associated disease, including podocyte cytoskeletal rearrangement (i.e., podocyte foot process effacement) and reduced glomerular podocyte count (i.e., podocytopenia).
  • CGF connective tissue growth factor
  • the present invention is based, in part, on the discovery of unexpected benefits of inhibition of CTGF in treatment of podocyte injury, podocyte-associated disorders, and podocytopathy (i.e. podocytopathy (i.e. podocyte disease)).
  • the present invention demonstrates that inhibition of CTGF reduced various pathological aspects of podocyte injury and podocytopathy (i.e. podocytopathy (i.e. podocyte disease)) not previously associated with CTGF.
  • the present invention shows that inhibition of CTGF provides a therapeutic approach to treat or ameliorate specific physiological and pathological aspects of podocyte injury and podocytopathy (i.e. podocytopathy (i.e. podocyte disease)).
  • the present invention provides methods and agents for treating, reducing, ameliorating, or reversing pathological processes (e.g., podocyturia, podocytopenia, foot process effacement) associated with podocyte injury and impaired podocyte function in a subject having or at risk of having podocyte injury.
  • pathological processes e.g., podocyturia, podocytopenia, foot process effacement
  • the subject is an animal, more preferably a mammal, and most preferably a human.
  • the present invention also provides agents that inhibit CTGF for use in the methods described herein.
  • agents may include small molecule compounds; peptides and proteins including antibodies or functionally active fragments thereof; and polynucleotides including small interfering ribonucleic acids (siRNAs), micro-RNAs (miRNAs), ribozymes, and anti-sense sequences.
  • siRNAs small interfering ribonucleic acids
  • miRNAs micro-RNAs
  • ribozymes ribozymes
  • anti-sense sequences See, e.g., Zeng (2003) Proc Natl Acad Sci USA 100:9779-9784; and Kurreck (2003) Eur J Biochem 270: 1628-1644.
  • the present invention further provides agents for use in manufacturing a medicament for treating, preventing, or reducing podocyte injury, podocyte-associated disorders, and podocytopathy (i.e.
  • podocytopathy i.e. podocyte disease
  • the agent inhibits CTGF.
  • the present invention further provides agents for use in methods of treating, preventing or reducing podocyte injury, podocyte-associated disorders, and podocytopathy (i e podocytopathy (i e podocyte disease)), wherein the agent inhibits CTGF
  • the present invention provides methods and agents useful for the treatment, prevention, or reduction of podocyte injury.
  • Podocyte injury is damage to a podocyte-associated with insult or challenge. Numerous causes of podocyte injury have been desc ⁇ bed including, for example, high blood pressure and ischemia reperfusion injury. (See, e.g., Nagase et al. (2006) Hypertension 47.1084-1093, K ⁇ z et al. (1994) Kidney Lit Suppl. 45.S64-S72.)
  • podocyte injury causes include toxic substances, endoc ⁇ nologic disorders, infections, contrast agents, mechanical trauma, cytotoxic agents (e.g., cisplatinum, ad ⁇ amycin, puromycin), calcineu ⁇ n inhibitors, inflammation (e g., due to infection, trauma, anoxia, obstruction, or ischemia), radiation, infections (e.g., bacte ⁇ al, fungal, or viral), dysfunction of the immune system (e g., an autoimmune disease, a systemic disease, or IgA nephropathy), genetic disorders, medications (e.g , anti-bacte ⁇ al agent, anti-viral agent, anti-fungal agent, immunosuppressive agent, anti-inflammatory agent, analgestic or anticancer agent), organ failure, organ transplantation, and uropathy.
  • cytotoxic agents e.g., cisplatinum, ad ⁇ amycin, puromycin
  • calcineu ⁇ n inhibitors e.g., due to infection, trauma,
  • podocyte injury may include, for example, congenital nephritic syndrome of the Finnish type, fetal membranous nephropathy or mutations in one or more podocyte specific proteins, such as, for example, a- actin-4, podocin, neph ⁇ n or CD2AP
  • the methods and agents of the present invention are useful for the treatment of podocyte injury of any cause or of any etiology
  • podocytes respond to injury in several ways. For example, early manifestations of podocyte injury include vacuolization, pseudocyst formation, and detachment from the GBM, resulting in podocyte loss (Ba ⁇ soni et al. (2003) Am J Nephrol. 23 353-360.)
  • the extent of podocyte injury is determined by measuring podocyte response to injury, such as, for example, the degree of podocyte foot process effacement
  • Activation of the family of mitogen-activated protein kinases (MAPK) in podocytes increases cytoskeletal rearrangement and decreases barrier function often resulting in proteinuria.
  • MAPK mitogen-activated protein kinases
  • the phosphorylation of va ⁇ ous MAPK enzymes in podocytes has been observed. Decreased activation of these MAPK enzymes results in decreased podocyte cytoskeletal rearrangement.
  • the present invention shows that inhibition of CTGF decreases activation of p42/44 MAPK in podocytes.
  • agents that inhibit CTGF are useful for preventing or reducing podocyte cytoskeletal rearrangement
  • podocyte foot process effacement is a result of podocyte cytoskeletal rearrangement
  • use of the agents and methods of the present invention to treat, prevent, or reduce podocyte foot process effacement is also provided.
  • podocytes undergo foot process effacement, or change in podocyte shape, also referred to as fusion, retraction, or simplification
  • Podocyte foot process effacement is characte ⁇ zed by gradual simplification of the mter-digitanng foot process pattern, resulting in the formation of a flat and elongated looking cell.
  • Foot process effacement is an active process that is initiated by rearrangement of the podocyte's actm cytoskeleton.
  • foot process length may be reduced by up to 70% and foot process width may decrease up to 60% compared to that of a non-mjured podocyte, resulting in a flattened and spread out cell.
  • Podocyte foot process effacement occurs with different types of podocyte injury and podocytopathy (i.e. podocyte disease)s (i e. podocytopathies).
  • CTGF induces cytoskeletal rearrangement in cultured podocytes.
  • Agents that inhibit CTGF therefore, are useful for preventing or reducing podocyte cytoskeletal rearrangement.
  • CTGF decreases levels of synaptopodin in cultured podocytes; synaptopodm is a protein responsible for podocyte actm cytoskeleton integrity. (See e.g., Example 5.) Therefore, agents that inhibit CTGF are useful for resto ⁇ ng or reducing decreased levels of synaptopodm in podocytes.
  • CTGF increases levels of Arp3 in cultured podocytes; Arp3 is an enzyme responsible for podocyte cytoskeletal rearrangement.
  • agents that inhibit CTGF are useful for preventing or reducing increased levels of Arp3 in podocytes
  • the present invention provides methods and agents for reducing or preventing podocyte cytoskeletal rearrangement in a subject having podocyte injury, wherein the method comprises inhibiting CTGF.
  • the agents and methods of the present invention to treat, prevent, or reduce podocyte foot process effacement is also provided.
  • podocyte injury often leads to disruption of the slit diaphragm (e g , decreased slit diaphragm length) that lies between neighbo ⁇ ng podocytes in the glomerulus.
  • the slit diaphragm is a complex of proteins located in the extracellular space, bridging adjacent podocyte foot processes and measuring 30-40 nm in length.
  • Podocyte-specif ⁇ c proteins such as, for example, podocin and CD2AP, are responsible for ancho ⁇ ng podocyte foot processes to the slit diaphragm in vivo.
  • the present invention shows that CTGF decreases levels of podocin and CD2AP in cultured podocytes (See, e g., Example 2.) Decreased levels of podocin and CD2AP are associated with disruption and loss of slit diaphragm in vivo. Additionally, the present invention shows that CTGF decreases extracellular level of neph ⁇ n m cultured podocytes (see e.g.,
  • neph ⁇ n is a constituent protein of the slit diaphragm. Decreased levels of nephnn result in disruption and loss of slit diaphragm in vivo.
  • agents that inhibit CTGF are useful for preventing disruption or loss of slit diaphragm.
  • the present invention provides methods and agents useful for increasing podocyte slit diaphragm length or number in a subject having podocyte injury, the methods comprising administering to the subject an agent that inhibits CTGF.
  • Slit diaphragm loss is associated with proteinu ⁇ a in several glomerular diseases (Patrakka et al. (2002) Pediatr Res.
  • the present invention provides methods and agents for increasing podocyte slit diaphragm length or number in a subject having podocyte injury, wherein the method comprises inhibiting CTGF
  • the present invention provides methods and agents for treating or preventing disruption of the slit diaphragm in a subject with podocyte injury, wherein the method comprises inhibiting CTGF.
  • methods and agents are provided for increasing the length of the slit diaphragm in a subject with podocyte injury, podocyte-associated disorder, or podocytopathy (i.e. podocytopathy (i.e. podocyte disease)), wherein the method comp ⁇ ses inhibiting CTGF.
  • podocyte markers reduced following podocyte injury include 1 Neph ⁇ n; GLEPP; Podocin; FAT-I ; CD2AP; Nephl; integrins; mteg ⁇ n-lmked kinase; secreted protein acid ⁇ ch in cysteine; Rho GTPases, ⁇ - actinm-4; synaptopodin, cychn-dependent kinase 5; podocalyxm; hic-5; TRPC6; dend ⁇ n; snail; notch; HSP27; Iamb4; podocalyxm, podoplanm, NHERF2, Ez ⁇ n; a,/3 dystroglycans; ffl3-j8-l integ ⁇ n collagen type 4; Wnt-4 and Hic-5.
  • CTGF decreases levels of various podocyte markers including, podocin, CD2AP, neph ⁇ n, and synaptopodin, in cultured podocytes (See, e g , Example 2 ) Therefore, agents that inhibit CTGF are useful for preventing or decreasing a reduction in podocyte markers in injured podocytes Extensive loss of podocyte markers is observed in idiopathic collapsing glomerulopathy (ICG) and is associated with nephrotic syndrome and progression to end stage renal disease. (Yang et al. (2002)
  • the present invention provides methods and agents for preventing or reducing loss of podocyte markers in a subject having podocyte injury by admimste ⁇ ng an agent that inhibits CTGF
  • the podocyte marker is podocm, CD2AP, nephnn, or synaptopodin
  • Increased kidney desmin levels are an early marker of podocyte injury (Nishiyama (2008) J Hypertens 26 1849-59, Kuhlmann et al (2004) Am J Physiol Renal Physiol 286 F526-F533.)
  • the present invention shows that CTGF inhibition reduces kidney desmin levels in subjects with podocyte injury. (See e.g , Example 9.)
  • methods for decreasing desmin levels in a subject having podocyte injury wherein the methods comp ⁇ se administering to the subject an agent that inhibits CTGF, are specifically provided.
  • Disturbances in cultured podocyte function following injury can be studied by the use of activation, adherence, migration, and proliferation assays.
  • One indication of early podocyte damage is disruption in the PINCH- 1-ILK- ⁇ -parvm complex, resulting in reduced podocyte matrix adhesion, foot process formation, or increase in apoptosis of podocytes.
  • Another indicator of early podocyte damage is a disrupt on of function of synaptopodin, a member of a class of proline- ⁇ ch actin associated proteins that are expressed in podocyte foot processes.
  • synaptopodin is essential for the integrity of the podocyte actm cytoskeleton and for the regulation of podocyte cell migration (See Yang et al (2005) J Am Soc Nephrol.
  • Podocyte injury can also be assessed in blood and urine samples. (See e.g., Hara et al. (2005) J Am Soc Nephrol. 16:408-16, Vogelmann et al. (2003) Am J Physiol Renal Physiol. 285:F40-8; Pavenstadt et al.
  • urinary podocyte count e.g., the presence of podocytu ⁇ a
  • urinary podocyte count may be used to detect podocyte injury (Yu et al (2005) J Am Soc Nephrol. 16:1733-41)
  • Methods of detection of podocytu ⁇ a useful in the present invention are desc ⁇ bed in more detail in U S. Application Publication No. 03/0198959, the contents of which are incorporated herein by reference in their entirety.
  • Podocyte injury and loss can be detected with a high degree of sensitivity by the abnormal presence in unne sediment of a gene selectively expressed in the podocyte so as to be podocyte-specif ⁇ c in the u ⁇ nary tract.
  • Such methods of detection useful in the instant invention are descnbed m more detail in International Publication No WO 03/082202, the contents of which are incorporated herein by reference m their entirety
  • markers useful for detection of podocyte injury include nephnn, desmin, Glepp-1, and synaptopodin
  • detection of a particular gene can be done using a reverse transc ⁇ ptase quantitative polymerase chain reaction (RT-PCR), microarrays, Western blots, proteomics, and ln-situ hybridization, and lmmunohistochemistry.
  • RT-PCR reverse transc ⁇ ptase quantitative polymerase chain reaction
  • markers are podocin, FAT-I, CD2AP, Nephl, integ ⁇ ns, integrm-lmked kinase, secreted protein acid ⁇ ch m cysteine, Rho GTPases, ⁇ - actinin-4, podoplanin, cychn-dependent kinase5, podocalyxm, hic-5, TRPC6, dend ⁇ n, snail, notch, HSP27, Iamb4, podocalyxm, NHERF2, Ez ⁇ n, a,/3 dystroglycans, ffl3-/3-l integnn collagen type 4 , Wnt-4 and Hic-5, which can be detected from biopsy specimen, unne or blood analysis
  • podocyte injury and loss can be detected using various methods available and known to one of skill in the art.
  • podocyte injury and podocyte abnormalities or pathologies may be detected by electron microscopy of kidney biopsy.
  • Electron microscopy provides information about the degree of injury to glomerular cells, and the consistency of the basement membrane.
  • Electron microscopy also detects fibrils and provides information on alterations of the ultrastrucrure of podocytes, such as podocyte foot process effacement and flattening Podocyte abnormalities, including vacuolization, microcystic or pseudocystic changes, the presence of cytoplasmic inclusion bodies, and detachment from the GBM, can be detected by electron microscopy (See, e.g , Shankland et al (2006) Kidney Int. 69: 2131-2147.) Electron microscopy may also be used to assess the slit diaphragm of neighboring podocytes (Ruotsalamen et al).
  • Podocyte injury often results in disruption of the slit diaphragm, such as, for example, a reduction in the length of the slit diaphragm
  • Light and fluorescent microscopy provides another useful method for measuring podocyte injury.
  • Light microscopy can be used for example, to evaluate loss of podocytes following injury (i e. podocytopenia).
  • podocyte specific markers e g., Schwarz et al (2001) J. CIm. Invest. 108:1621-1629) or podocyte number in the glomerulus (Dandapani et al. (2007) J. Biol. Chem. 282 467-477)
  • Injury to podocytes often results in decreases in va ⁇ ous podocyte markers and decreases in podocyte count (i e. podocytopenia).
  • Immunohistochemical techniques may be employed to detect podocytes and podocyte markers, such techniques have been desc ⁇ bed previously by Kim et al. ((2001) Kidney Int. 60:957-968), the contents of which are incorporated herein by reference in their entirety.
  • the extent of podocyte injury is determined by measuring podocyte response to injury, such as, for example, the degree or level of podocytu ⁇ a or podocytopenia.
  • podocyte injury is associated with several podocyte-associated disorders, such as, for example, podocytopenia
  • the etiology of podocytopenia a decrease in podocyte number, includes apoptosis, detachment from the GBM, and the inability or lack of podocytes to proliferate or hypertrophy (Shankland (2006) Kidney Int 69. 2131-2147).
  • Podocyte number can be reduced by either a decrease in proliferation due to lack of DNA synthesis, DNA damage or hypertrophy, or an increase in podocyte loss due to detachment from the GBM, and apoptosis.
  • Decreased podocyte number i.e podocytopenia
  • contributes to the progression of podocytopathy i e.
  • podocyte disease such as, for example, diabetic nephropathy (Pagtalunan (1997) J Clin Invest. 99 342-348 )
  • the present invention provides methods for treating, preventing, or reducing podocytopenia in a subject with podocyte injury, wherein the methods comp ⁇ se administering to the subject an agent that inhibits CTGF
  • the present invention shows that CTGF inhibition reduces decreased podocyte count associated with podocyte injury in an animal model of podocyte injury (See, e g , Example 10 ) Therefore, agents that inhibit CTGF are effective at preventing or reducing loss of podocytes (i e podocytopenia) in a subject
  • Podocyte loss is an early feature of podocytopathies (i e podocytopathy (i e podocyte disease)s) that leaves the GBM denuded Since podocytes lack proliferative capacity under normal circumstances, they cannot repopulate denuded areas Parietal epithelial cells contact the denuded GBM, forming a synechia resulting in the development of scar tissue (i.e tuft adhesions), considered a first sign of glomerular injury (Bansonia, (2003) Am J Nephrol.
  • the present invention provides methods and agents for maintaining podocyte number or reducing the loss of podocytes in a subject having podocyte injury, wherein the method comprising inhibiting CTGF.
  • methods and agents are provided for treating or preventing podocytopenia, wherein the method compnses inhibiting CTGF
  • the present invention provides methods and agents for treating or preventing podocyte-associated disorders in a subject having podocyte injury, wherein the method compnses inhibiting CTGF.
  • the present invention provides methods and agents for treating or preventing glomerular scarring in a subject having podocyte injury, wherein the method compnses inhibiting CTGF.
  • the present invention provides methods and agents for delaying progression of a podocytopathy (i e. podocytopathy (i e. podocyte disease)) in a subject, wherein the method compnses inhibiting CTGF.
  • Podocytuna the presence of podocytes in the unne, is podocyte-associated disorder that results from podocyte injury Podocytes reside in the unnary space of the bowman's capsule, foot process effacement is associated with podocyte detachment from the GBM and results in the shedding of podocytes in the unne Podocyte excretion in the unne is associated with kidney disorders; and the rate of podocyte excretion in the unne has been shown to correlate with both kidney disease activity and kidney disease type (See, e g , Yu et al (2005) J Am Soc Nephrol 16.1733-41 ) Studies have shown that podocytuna can decrease despite persistent protemuna (Petermann et al (2007) Nephron Clin Pract 106 c61-c66.)
  • podocytuna may serve as the first non-invasive marker of active glomerular damage Accordingly, use of agents and methods of the present invention to treat, prevent, or reduce podocytuna by inhibiting CTGF are provided
  • Podocytuna is observed in animal models of podocyte injury In these animal models, podocytuna closely parallels the onset of marked proteinuria (See Yu et al (2005) J Am Soc Nephrol 16: 1733-41 ) Methods and agents of the present invention reduced unnary protein concentration (i e.
  • the present invention provides methods and agents useful for decreasing podocytu ⁇ a in a subject having podocyte injury, wherein the method comp ⁇ ses inhibiting CTGF Additionally, the present invention shows that inhibition of CTGF reduces loss of podocytes in animals with podocyte injury (See, e.g.
  • Example 10 As a reduction of podocyte loss would result in a reduction in podocyte shedding in the u ⁇ ne (i e podocytu ⁇ a), the present invention provides methods and agents useful for decreasing podocytu ⁇ a in a subject having podocyte injury, wherein the method comp ⁇ ses inhibiting CTGF.
  • the present invention further demonstrates that inhibition of CTGF (e.g., by administration of an antibody to CTGF) treats or prevents podocyte-associated disorders, and m particular, podocytu ⁇ a.
  • the present invention provides methods and agents for reducing unnary podocyte count in a subject having podocyte injury, wherein the method comp ⁇ ses inhibiting CTGF.
  • the present invention provides methods and agents for treating or preventing podocytu ⁇ a in a subject having podocyte injury, wherein the method comp ⁇ ses inhibiting CTGF.
  • the present invention provides methods and agents for treating or preventing podocyte-associated disorders m a subject having podocyte injury, wherein the method compnses inhibiting CTGF.
  • the present invention provides methods and agents for delaying progression of a podocytopathy (i.e podocytopathy (i e. podocyte disease)) m a subject, wherein the method comp ⁇ ses inhibiting CTGF.
  • the methods of the present invention further comp ⁇ se determining the extent or seventy of podocyte injury or damage before, du ⁇ ng, and/or after admmiste ⁇ ng to the subject a therapeutically effective amount of an agent that inhibits CTGF.
  • an improvement or a reduction in the extent or seventy of podocyte injury following administration of the agent that inhibits CTGF can be determined as follows.
  • a first measurement of a podocyte marker level (e.g., podocin) in a subject having or at ⁇ sk for having podocyte injury is taken p ⁇ or to or du ⁇ ng a course of treatment with an agent of the invention.
  • a subsequent measurement of podocyte marker levels is taken dunng or after the course of treatment.
  • the first and subsequent measurements are compared and a difference showing maintenance of podocyte marker levels in the subject, or an increase in such levels in the subject, is indicative of an improvement or reduction in the extent or seventy of the podocyte injury or damage.
  • the methods of the present invention further comp ⁇ se determining the extent of one or more podocyte-associated disorders (e.g , podocytuna or podocytopenia) before, du ⁇ ng, and/or after admmistenng to the subject a therapeutically effective amount of an agent that inhibits CTGF
  • an improvement or a reduction in the extent or seventy of one or more podocyte-associated disorders following administration of the agent that inhibits CTGF can be determined as follows A first measurement of unnary podocyte count is taken p ⁇ or to or dunng the course of a treatment with an agent of the invention.
  • a subsequent measurement of unnary podocyte count is taken dunng or after the course of treatment
  • the first and subsequent measurements are compared and a difference showing a decrease in urinary podocyte count is indicative of an improvement or reduction in the extent or seventy of the podocyte-associated disorder
  • Podocytopathy i.e. podocyte disease
  • Podocytes are injured in va ⁇ ous human and experimental glomerular diseases, including minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), collapsing glomerulopathy (CG), membranous nephropathy, and diabetic nephropathy (DN)
  • MCD minimal change disease
  • FGS focal segmental glomerulosclerosis
  • CG collapsing glomerulopathy
  • DN diabetic nephropathy
  • podocytopathy i.e. podocyte disease
  • ⁇ e podocytopathies
  • Podocytopathies i.e.
  • podocytopathy i e podocyte disease
  • Congenital podocytopathy i e podocyte disease
  • Congenital podocytopathy include abnormalities in structural podocyte proteins, such as in congenital nephritic syndrome of the Finnish type This disease is characte ⁇ zed by mutations in neph ⁇ n leading to a loss of normal podocyte function, resulting in the onset of fetal proteinuria
  • Another congenital podocytopathy i.e.
  • podocyte disease associated with podocyte injury is the development of maternal antibodies to neutral endopeptidase and metallomembrane endopeptidase in mothers who are deficient in this enzyme, resulting in fetal membranous nephropathy.
  • Hereditary diseases of podocyte injury typically include mutations in podocyte-specific proteins, such as ⁇ >actm-4, podocin and TRPC6. Such mutations may lead to hereditary proteinuria
  • Acquired podocytopathies can be immune and non-immune mediated
  • immune-mediated podocytopathy include membranous nephropathy, minimal change disease, and membranoprohferative glomerulonephritis associated with cryoglobulins
  • Non-immune acquired podocytopathy include infectious diseases such as HIV-associated nephropathy due to the local infection of podocytes by the HIV virus. It has been speculated that Parvo Bl-9 virus may induce collapsing glomerulopathy in HIV-negative patients.
  • podocytopathy i e podocyte disease
  • Other examples of metabolic causes of podocytopathy include diabetes, metabolic syndrome, systemic hypertension, any cause of a reduced nephron number, such as reflux nephropathy or chronic glomerulopathies, as well as infiltrative diseases of podocytes, such as amyloid, where individual amyloid spicules "project" through the GBM, penetrating into the overlying podocytes
  • podocyte injury may be associated with both immune and non-immune mediated diseases, resulting in damage to the glomerular filtration barrier
  • Proteinuria is a marker of podocyte injury m animal models of podocyte injury. Methods and agents of the present invention reduced proteinuria m an animal model of podocyte injury (See, e g , Example 4 )
  • the present invention provides methods and agents useful for treating podocytopathy (i e podocytopathy (i e podocyte disease)) in a subject having podocyte injury, wherein the method comp ⁇ ses inhibiting CTGF
  • the present invention demonstrates that inhibition of CTGF (e g , by administration of an antibody to CTGF) is effective for treating or preventing podocytopathy (i e. podocytopathy (i e podocyte disease)), and in particular, puromycm-mduced nephrosis
  • the present invention provides methods and agents for treating or preventing podocytopathy (i e. podocyte disease) in a subject having podocyte injury, wherein the method comp ⁇ ses inhibiting CTGF.
  • the podocytopathy (i.e podocyte disease) treated or prevented by the methods and agents of the present invention is diabetic nephropathy, minimal change disease, focal segmental glomerulosclerosis (FSGS), membranous nephropathy, collapsing glomerulopathy, and chronic glomerulonephritis.
  • the present invention provides methods and agents for delaying progression of a podocytopathy (i e. podocyte disease) in a subject, wherein the method comp ⁇ ses inhibiting CTGF.
  • the present invention provides methods for treating podocyte injury, podocyte-associated disorders, and podocytopathy (i e. podocyte disease) in a subject
  • the subject can be, e g., a podocyte, a population of podocytes, a tissue, an organ, or an organism
  • the invention is applicable to a variety of different organisms, including, for example, vertebrates, large animals, and p ⁇ mates.
  • the subject is a mammalian subject, and m particular embodiments, the subject is a human subject. Although applications with humans are clearly foreseen, veterinary applications are also envisaged herein.
  • the present methods of treatment involve administration of an effective amount of an agent to a subject, wherein the agent inhibits CTGF, and wherein the subject would benefit from treatment of: podocyte injury, a podocyte-associated disorder; or a podocytopathy (i.e. podocyte disease).
  • the subject has or is at ⁇ sk of having podocyte injury
  • the subject has or is at ⁇ sk of having a podocyte-associated disorder (e g., podocytu ⁇ a).
  • the subject has or is at ⁇ sk of having a podocytopathy (i.e. podocyte disease) (e.g , FSGS).
  • the subject of the present methods is identified as a subject having podocyte injury Identification of a subject having podocyte injury can occur before, du ⁇ ng, or after administration of an agent that inhibits CTGF Detection of podocyte injury is descnbed herein
  • the subject is identified as a subject having podocyte injury by the presence of podocytes m a u ⁇ ne sample from the subject (i e podocytuna)
  • the subject is identified as a subject having podocyte injury by the abnormal presence in u ⁇ ne sediment of a gene selectively expressed in the podocyte (e g , the presence of neph ⁇ n in a u ⁇ ne sample)
  • the subject is identified as a subject having podocyte injury by analysis of kidney biopsy from the subject (e g , detection of podocyte foot effacement)
  • the subject is identified as a subject having podocyte injury by detecting podocytopenia in a kidney biopsy sample from the subject.
  • the agent that inhibits CTGF may be a polypeptide, polynucleotide, or small molecule includes; for example, an antibody that binds to CTGF, an antisense molecule, siRNAs, small molecule chemical compounds, etc.
  • inhibiting CTGF can be accomplished by any of the means well- known in the art for modulating the expression and activity of CTGF.
  • Use of an agent that inhibits CTGF, such as, for example, a human monoclonal antibody directed against CTGF is preferred, although any agent or method of inhibiting expression of the gene encoding CTGF, inhibiting production of CTGF, or inhibiting activity of CTGF is provided by the present invention
  • the present invention involves an agent that inhibits CTGF.
  • CTGF sequences from a large number of species are known in the art and an exemplary CTGF is human CTGF, which has SWISSPROT Accession No P29729.
  • the agent may decrease one or more of the biological activities of CTGF Activities associated with CTGF include, but are not limited to, stimulation of cell migration, production of extracellular matrix by a cell in vivo or ex vivo, and/or reduction m fibrosis m a subject.
  • the biological activity is selected from the group consisting of cell growth, differentiation of fibroblasts and/or endothelial cells, and induction of expression of proteins involved in extracellular matrix formation and remodeling including, e.g., collagens including, but not limited to, types I, ⁇ , III, and IV; and fibronectm.
  • Assays for CTGF activity are known in the art, for example the cell migration assay desc ⁇ bed in Example 5 International Publication No WO 2004/108764, incorporated by reference herein in its entirety.
  • antibodies for use in the methods of the present invention are desc ⁇ bed, e g., in U S. Patent No 5,408,040; International Publication No WO 99/07407; International Publication No. WO 99/33878; and International Publication No. WO 00/35936
  • antibodies for use in the methods of the present invention specifically bind to a region in the N-terminal portion of CTGF, the N- terminal portion may be considered to comp ⁇ se ammo acid residues 1 to 198 of human CTGF (SWISSPROT Accession No P029729).
  • An example of a suitable region in the N-termmal portion of CTGF is from L143 to V154 of human CTGF (SWISSPROT Accession No. P029729).
  • An exemplary antibody for use in the methods of the present invention is descnbed in International Publication No WO
  • CLN-I antibody is produced by the cell line deposited with the American Type Culture Collection (ATCC) on 20th May 2004 with Deposit Number PTA-6006
  • ATCC American Type Culture Collection
  • Such antibodies, or fragments thereof, can be administered by va ⁇ ous means known to those skilled in the art
  • antibodies to CTGF can be injected intravenously, intrape ⁇ toneally, or subcutaneously Small molecule inhibitors of CTGF expression and/or activity have also been desc ⁇ bed, for example, International Publication No.
  • WO 96/38172 identifies modulators of cAMP such as cholera toxin and 8Br-cAMP as inhibitors of CTGF expression Therefore, compounds identified as, e.g , prostaglandin and/or prostacyclin analogs such as Iloprost (see, e g , International Publication No. WO 00/02450, Ricupero et al. (1999) Am J Physiol 277 Ll 165-1171, also, see Ertl et al (1992) Am Rev Respir Dis
  • polynucleotides including small interfe ⁇ ng ribonucleic acids (siRNAs), micro-RNAs (miRNAs), ⁇ bozymes, and anti-sense sequences may be used in the present methods to inhibit expression and/or production of CTGF.
  • siRNAs small interfe ⁇ ng ribonucleic acids
  • miRNAs micro-RNAs
  • ⁇ bozymes and anti-sense sequences
  • anti-sense sequences may be used in the present methods to inhibit expression and/or production of CTGF.
  • anti-sense constructs that target CTGF expression have been desc ⁇ bed and utilized to reduce CTGF expression in va ⁇ ous cell types.
  • Such antisense constructs can be used to reduce expression of CTGF and thereby ameliorate or prevent the pathological processes induced by CTGF.
  • Such constructs can be designed using approp ⁇ ate vectors and expressional regulators for cell- or tissue- specific expression and constitutive or inducible expression.
  • Such genetic constructs can be formulated and administered according to established procedures within the art
  • the agent that inhibits CTGF is an antibody to CTGF
  • the antibody is a monoclonal antibody to CTGF
  • the antibody is a human or humanized antibody to CTGF.
  • the antibody is CLN-I, as desc ⁇ bed m International Publication No WO 2004/108764.
  • the agent is a small molecule.
  • the agent is a nucleic acid
  • the nucleic acid is selected from the group consisting of a cyclic nucleotide, an oligonucleotide, or a polynucleotide
  • the agent is an antisense oligonucleotide or an siRNA
  • the present invention also provides the use of the present methods m combination with other therapies
  • the method of the present invention is used m combination with another therapy, e.g., to further augment the therapeutic effect on podocyte injury, etc.
  • the two treatments may be administered at the same time or consecutively, e.g., during a treatment time course or following disease progression and remission.
  • the method is used in combination with another therapeutic method having a similar or different mode of action, e.g., an ACE inhibitor, ARBs, statin, advanced glycation endproduct (AGE) inhibitor, etc.
  • ACE inhibitor angiotensin receptor blockers
  • statins advanced glycation endproduct inhibitors
  • calcium channel blockers etc.
  • Use of any of these therapeutic agents in combination with the use of methods and agents of the present invention for treating podocyte injury, podocyte-associated disorder, or podocytopathy (i e. podocyte disease) is specifically provided
  • the agents of the present invention can be delivered directly or in pharmaceutical compositions containing excipients, as is well known in the art.
  • Present methods of treatment can comp ⁇ se administration of an effective amount of an agent of the present invention to a subject having or at nsk for having podocyte injury, a podocyte-associated disorder or a podocytopathy (i.e podocyte disease).
  • the subject is a mammalian subject, and m a most preferred embodiment, the subject is a human subject.
  • An effective amount, e.g., dose, of agent or drug can readily be determined by routine experimentation, as can an effective and convenient route of administration and an approp ⁇ ate formulation Vanous formulations and drug delivery systems are available in the art. (See, e.g., Gennaro, ed. (2000) Remington's Pharmaceutical Sciences, supra; and Hardman, Limbird, and Gilman, eds (2001) The
  • Suitable routes of administration may, for example, include oral, rectal, topical, nasal, pulmonary, ocular, intestinal, and parenteral administration
  • Primary routes for parenteral administration include intravenous, intramuscular, and subcutaneous administration
  • Secondary routes of administration include intraperitoneal, lntra-arte ⁇ al, mtra-articular, intracardiac, mtracisternal, intradermal, intralesional, intraocular, intrapleural, intrathecal, intrauterine, and intraventricular administration.
  • compositions of an agent of the invention may be provided in an instant release, controlled release, sustained release, or target drug-delivery system
  • Commonly used dosage forms include, for example, solutions and suspensions, (micro-) emulsions, ointments, gels and patches, liposomes, tablets, dragees, soft or hard shell capsules, supposito ⁇ es, ovules, implants, amorphous or crystalline powders, aerosols, and lyophihzed formulanons
  • special devices may be required for application or administration of the agent, such as, for example, syringes and needles, inhalers, pumps, injection pens, applicators, or special flasks
  • Pharmaceutical dosage forms are often composed of the agent, an excipient(s), and a container/closure
  • excipients also referred to as inactive ingredients
  • the type of excipient(s) to be added to the agent can depend on various factors, such as, for example, the physical and chemical properties of the agent, the route of administration, and the manufacturing procedure
  • Pharmaceutically acceptable excipients are available in the art, and include those listed in va ⁇ ous pharmacopoeias. (See, e.g., USP, JP, EP, and BP, FDA web page (www.fda gov), Inactive Ingredient Guide 1996, and Handbook of
  • compositions of an agent of the present invention may be manufactured by any of the methods well-known in the art, such as, for example, by conventional mixing, sieving, dissolving, melting, granulating, dragee-makmg, tablettmg, suspending, extruding, spray-drymg, levigating, emulsifying, (nano/micro-) encapsulating, entrapping, or lyophihzation processes
  • the agents of the present invention can include one or more physiologically acceptable inactive ingredients that facilitate processing of active molecules into preparations for pharmaceutical use.
  • the agent may be formulated in aqueous solution, if necessary using physiologically compatible buffers, including, for example, phosphate, histidme, or citrate for adjustment of the formulation pH, and a tonicity agent, such as, for example, sodium chlo ⁇ de or dextrose
  • physiologically compatible buffers including, for example, phosphate, histidme, or citrate for adjustment of the formulation pH
  • a tonicity agent such as, for example, sodium chlo ⁇ de or dextrose
  • semisolid, liquid formulations, or patches may be preferred, possibly containing penetration enhancers.
  • the agents can be formulated in liquid or solid dosage forms and as instant or controlled/sustained release formulations Suitable dosage forms for oral ingestion by a subject include tablets, pills, dragees, hard and soft shell capsules, liquids, gels, syrups, slurries, suspensions, and emulsions.
  • the agents may also be formulated in rectal compositions, such as supposito ⁇ es or retention enemas, e g , containing conventional suppository bases such as cocoa butter or other glyce ⁇ des
  • Solid oral dosage forms can be obtained using excipients, which may include, fillers, disintegrants, binders (dry and wet), dissolution retardants, lubricants, ghdants, antiadherants, canonic exchange resins, wetting agents, antioxidants, preservatives, colo ⁇ ng, and flavoring agents.
  • excipients can be of synthetic or natural source
  • excipients include cellulose de ⁇ vatives, citric acid, dicalcium phosphate, gelatine, magnesium carbonate, magnesium/sodium lauryl sulfate, mannitol, polyethylene glycol, polyvinyl pyrrohdone, silicates, silicium dioxide, sodium benzoate, sorbitol, starches, stearic acid or a salt thereof, sugars (i.e.
  • Ethanol and water may serve as granulation aides
  • coating of tablets with, for example, a taste-masking film, a stomach acid resistant film, or a release-retarding film is desirable
  • Natural and synthetic polymers, in combination with colorants, sugars, and organic solvents or water, are often used to coat tablets, resulting in dragees.
  • the agent powder, suspension, or solution thereof can be delivered in a compatible hard or soft shell capsule.
  • the agents of the present invention can be administered topically, such as through a skin patch, a semi-solid or a liquid formulation, for example a gel, a (micro-) emulsion, an ointment, a solution, a (nano/micro)-suspension, or a foam.
  • the penetration of the agent into the skin and underlying tissues can be regulated, for example, using penetration enhancers; the appropriate choice and combination of lipophilic, hydrophilic, and amphiphilic excipients, including water, organic solvents, waxes, oils, synthetic and natural polymers, surfactants, emulsifiers, by pH adjustment; and use of complexing agents.
  • Other techniques, such as iontophoresis may be used to regulate skm penetration of an agent of the invention. Transdermal or topical administration would be preferred, for example, in situations in which local delivery with minimal systemic exposure is desired.
  • the agents for use according to the present invention are conveniently delivered in the form of a solution, suspension, emulsion, or semisolid aerosol from pressu ⁇ zed packs, or a nebuliser, usually with the use of a propellant, e.g., halogenated carbons dervided from methan and ethan, carbon dioxide, or any other suitable gas.
  • a propellant e.g., halogenated carbons dervided from methan and ethan, carbon dioxide, or any other suitable gas.
  • hydrocarbons like butane, isobutene, and pentane are useful.
  • the approp ⁇ ate dosage unit may be determined by providing a valve to deliver a metered amount
  • Capsules and cartridges of, for example, gelatin, for use in an inhaler or insufflator may be formulated These typically contain a powder mix of the agent and a suitable powder base such as lactose or starch.
  • Agents formulated for parenteral administration by injection are usually ste ⁇ le and, can be presented in unit dosage forms, e g., m ampoules, syringes, injection pens, or in multi-dose containers, the latter usually containing a preservative.
  • the agents may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents, such as buffers, tonicity agents, viscosity enhancing agents, surfactants, suspending and dispersing agents, antioxidants, biocompatible polymers, chelating agents, and preservatives.
  • the vehicle may contain water, a synthetic or vegetable oil, and/or organic co-solvents.
  • the parenteral formulation would be reconstituted or diluted p ⁇ or to admimstranon Depot formulations, providing controlled or sustained release of an agent of the invention, may include injectable suspensions of nano/micro particles or nano/micro or non-micronized crystals
  • Polymers such as poly(lactic acid), poly(glycohc acid), or copolymers thereof, can serve as controlled/sustained release matrices, in addition to others well known in the art
  • Other depot delivery systems may be presented in form of implants and pumps requi ⁇ ng incision
  • Suitable earners for intravenous injection for the agents of the invention are well-known in the art and include water-based solutions containing a base, such as, for example, sodium hydroxide, to form an ionized compound, sucrose or sodium chlo ⁇ de as a tonicity agent, for example, the buffer contains phosphate or histidine Co-solvents, such as, for example, polyethylene glycols, may be added.
  • a base such as, for example, sodium hydroxide
  • sucrose or sodium chlo ⁇ de as a tonicity agent
  • the buffer contains phosphate or histidine Co-solvents, such as, for example, polyethylene glycols, may be added.
  • Co-solvents such as, for example, polyethylene glycols
  • low-toxicity surfactants such as polysorbates or poloxamers
  • polyethylene glycol or other co-solvents polyethylene glycol or other co-solvents
  • biocompatible polymers such as polyvinyl pyrrohdone may be added, and other sugars and polyols may substitute for dextrose.
  • a therapeutically effective dose can be estimated initially using a variety of techniques well-known in the art. Initial doses used in animal studies may be based on effective concentrations established in cell culture assays. Dosage ranges approp ⁇ ate for human subjects can be determined, for example, using data obtained from animal studies and cell culture assays.
  • a therapeutically effective dose or amount of a compound, agent, or drug of the present invention refers to an amount or dose of the compound, agent, or drug that results in amelioration of symptoms or a prolongation of survival in a subject Toxicity and therapeutic efficacy of such molecules can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e g , by determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population)
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50/ ED50 Agents that exhibit high therapeutic indices are preferred
  • the effective amount or therapeutically effective amount is the amount of the agent or pharmaceutical composition that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought by the researcher, vete ⁇ na ⁇ an, medical doctor, or other clinician, e.g., improved kidney function, reduced podocytopenia, etc
  • Dosages preferably fall withm a range of circulating concentrations that includes the ED50 with little or no toxicity. Dosages may vary withm this range depending upon the dosage form employed and/or the route of administration utilized. The exact formulation, route of administration, dosage, and dosage interval should be chosen according to methods known in the art, m view of the specifics of a subject's condition
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety that are sufficient to achieve the desired effects, e g , improved vascular function, improved cardiac function, etc, i e , minimal effective concentration (MEC)
  • MEC minimal effective concentration
  • the MEC will vary for each agent but can be estimated from, for example, in vitro data and animal expe ⁇ ments Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration
  • the amount of agent or composition administered may be dependent on a variety of factors, including the sex, age, and weight of the subject being treated, the seventy of the affliction, the manner of administration, and the judgment of the prescribing physician.
  • compositions may, if desired, be presented in a pack or dispenser device containing one or more unit dosage forms containing the active ingredient
  • a pack or device may, for example, comprise metal or plastic foil, such as a blister pack, or glass and rubber stoppers such as in vials.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • Compositions comprising an agent of the invention formulated in a compatible pharmaceutical earner may also be prepared, placed in an appropnate container, and labeled for treatment of an indicated condition
  • Example 1 CTGF Induced Podocyte Cytoskeletal Rearrangement In Vitro
  • podocytes In response to injury, podocytes undergo foot process effacement, or change in podocyte shape.
  • Podocyte foot process effacement is an active process that is initiated by changes in the podocyte 's actm cytoskeleton and can result in podocyte detachment from the glomerular basement membrane, podocyte shedding in the urine, and proteinuria
  • CTGF a cell adhesion assay
  • m MCDB-131 media supplemented with 10% FBS, L-glutamme and penicillin/streptomycin in a humidified 5% CO2 environment at 33 0 C
  • podocytes were transferred to an incubator at 37°C for 7 days to induce differentiation.
  • differentiated podocytes were stimulated with CTGF by transferring podocytes to 96-well culture plates that had been pre-coated with recombinant human CTGF (10ng/ml). Podocytes were cultured with CTGF for either 1 or 6 hours.
  • FITC-conjugated phalloidin Green stam in Figures IA, IB, 1C, and
  • Nuclei were counter-stamed with Hoechst 33342 (Blue stain in Figures IA, IB, 1C, and ID). Immunostained podocytes were visualized using a Zeiss Axioscope with Axiovision 4.
  • CTGF inhibition provided for by the methods and agents of the present invention is an effective means for reducing podocyte cytoskeletal rearrangement and cell polarization (i e shape change) following podocyte injury
  • CTGF inhibitton provided for by methods and agents of the present invention is effective at treating, preventing, or reducing podocyte injury and podocyte dysfunction following injury, such as, podocyte foot effacement.
  • the Arp2/3 complex is a known mediator of actin nucleation in podocytes.
  • the Arp2/3 complex is a known mediator of actin nucleation in podocytes.
  • Increased Arp3 levels are associated with greater cytoskeletal rearrangement in the podocyte's actin cytoskeleton.
  • CTGF the effect of CTGF on Arp3 levels in podocytes was examined using a cell adhesion assay as desc ⁇ bed above with the following modifications podocytes were differentiated for either 7 or 14 days and time of podocyte incubation with CTGF was 6 hours. Following addition of CTGF, podocytes were fixed in 3% paraformaldehyde and stained for va ⁇ ous markers using standard immunohistochemical techniques. Immunofluorescence was performed using rabbit anti-Arp3 (4 ⁇ g/ml;
  • CTGF inhibition provided for by the methods and agents of the present invention is an effective means for reducing or preventing increased Arp3 levels in podocytes following injury.
  • Arp3 mediates podocyte cytoskeletal rearrangement which results in podocyte foot process effacement.
  • CTGF inhibition provided for by methods and agents of the present invention is effective at treating, preventing, or reducing podocyte injury and podocyte dysfunction following injury, such as, podocyte foot effacement.
  • CTGF inhibition provided for by the methods and agents of the present invention would be effective at reducing or preventing increased Arp3 expression in podocytes following injury Arp3 mediates podocyte cytoskeletal rearrangement which results in podocyte foot process effacement
  • CTGF inhibition provided for by methods and agents of the present invention would be effective at treating, preventing, or reducing podocyte injury and podocyte dysfunction following injury, such as, podocyte foot effacement
  • Example 2 CTGF Decreased Expression of Podocyte Specific Proteins Podocin and CD2AP In Vitro A reduction in podocyte and slit diaphragm proteins is commonly observed following podocyte injury.
  • CTGF addition decreased podocin levels in both 7 and 14 day differentiated podocytes compared to that observed in non-treated controls (see decreased Texas red staining in Figures 4A, 4B, 4C, and 4D). CTGF addition also decreased CD2AP levels in both 7 and 14 day differentiated podocytes compared to that observed in non-treated controls (see decreased Texas Red staining in Figures 5A, 5B, 5C, and 5D).
  • CTGF reduced CD2AP and podocin levels in cultured podocytes.
  • use of CTGF inhibitors of the present invention is effective for decreasing the reduction of podocyte marker proteins following podocyte injury.
  • methods and compounds of the present invention would be effective at treating, preventing, or reducing podocyte injury and podocyte dysfunction following injury, such as, for example, reduced podocyte markers.
  • CTGF addition decreased podocin expression in both 7 and 14 day differentiated podocytes compared to that observed in non-treated controls.
  • CTGF addition also decreased CD2AP expression in both 7 and 14 day differentiated podocytes compared to that observed in non-treated controls (see Figure 6B).
  • CTGF inhibition on podocyte injury using methods and agents of the present invennon is examined using the cell adhesion assay as desc ⁇ bed above in Example 1 except that differentiated podocytes are treated for 1-24 hours with 10 ⁇ g/mL puromycin m the presence or absence of an agent that inhibits CTGF (e.g , an anti-CTGF antibody)
  • an agent that inhibits CTGF e.g , an anti-CTGF antibody
  • podocytes are fixed in 3% paraformaldehyde and stained using standard immunohistochemical techniques. Podocyte shape and actm cytoskeleton are evaluated as desc ⁇ bed above in Example 1 Alternatively, total protein is extracted from treated podocyte cells and separated by SDS-PAGE. Proteins are then transferred to Immobilon-P and probed with specific antibodies to various podocyte proteins (e g , CD2AP, podocm). Next, proteins are visualized with HRP-conjugated secondary antibodies using luminol.
  • podocyte proteins e g , CD2AP, podocm
  • Puromycin is a podocyte toxin which induces podocyte foot process effacement and podocyte shedding in the urine (i.e. podocytu ⁇ a) as observed, for example, in human minimal change nephropathy (Inokuchi et al. (1996) Kidney Lit 50. 1278-1287, Ryan et al (1975) Kidney Int. 8 219-232).
  • Rodent models of puromycin aminonucleoside-induced nephrosis are accepted models of podocyte injury (Ricardo et al. (1994) J Am Soc Nephrol 4:1974-1986). The effect of agents and methods of the present invention on podocyte injury in vivo was examined using a rodent model of podocyte injury as follows
  • PA Puromycin ammonucleoside
  • 50 mg/kg (Sigma-Ald ⁇ ch Co, St. Louis, MO) was injected intravenously into male Sprague-Dawley rats (Charles River Co) weighing 290 to 320 g.
  • rats were randomized into three treatment groups on day 21 and treated as follows.
  • Group 1 was treated with anti- CTGF antibody (CLN-I, 3 mg/kg; described in WO 2004/108764)
  • Group 2 was treated with anti-CTGF antibody (CLN-I, 10 mg/kg)
  • Group 3 was treated with an isotype-matched irrelevant human IgG (IgG, 10 mg/kg)
  • Antibodies were administered i.p. at doses of 3 mg/kg or 10 mg/kg (approximate injection volume of 0.5 ml) three times weekly for three weeks
  • U ⁇ ne samples were collected at days 14 and 28, and urine protein levels (i.e. urinary protein concentrations) were determined for each sample by the sulfosalicylic method as previously reported (Bradley GM, Benson ES Examination of the U ⁇ ne, 15th Ed , Philadelphia, WB Saunders, 1974).
  • proteinu ⁇ a serves as a marker for podocyte injury and podocyte dysfunction (Ricardo et al (1994) J Am Soc Nephrol 4 1974-1986).
  • a single injection of PA increased u ⁇ nary protein concentration in all groups at day 14 After 7 days of treatment, however, animals administered an anti-CTGF antibody of the present invention had reduced urinary protein levels (i e reduced proteinuria) compared to that observed in animals administered control IgG (see Table 1).
  • Synaptopodin is essential for the integrity of the podocyte actin cytoskeleton and for the regulation of podocyte cell migration.
  • the effect of CTGF on podocyte expression of synaptopodin was examined using a cell adhesion assay substantially as described above in Example 1 with the following modifications: podocytes were differentiated for 14 days and the amount of time of podocyte incubation with CTGF was 0.5, 1, 6, and 12 hours.
  • total protein was extracted from differentiated podocyte cells and separated by SDS-PAGE. Proteins were then transferred to Immobilon-P and probed with specific antibodies to synaptopodin and ⁇ -Actin. Next, proteins were visualized with HRP-conjugated secondary antibodies using luminol.
  • CTGF addition decreased synaptopodin expression in podocytes compared to that observed in non-treated controls. Specifically, synaptopodin protein levels were decreased as early as 30 minutes after CTGF addition to cultured podocytes (see Figure 7). These results showed that CTGF reduced expression of the podocyte specific protein synaptopodin in cultured podocytes.
  • Synaptopodin is responsible for the integrity of the podocyte actin cytoskeleton; therefore, these results suggested that CTGF mediates foot process effacement at least in part by reduction in expression of synaptopodin These results further suggested that CTGF inhibition would be useful for treating, preventing, or reducing podocyte foot process effacement These results also suggested that methods and agents of the present invention would be effective at decreasing the reduction of podocyte marker protein following podocyte injury.
  • Example 6 CTGF Decreased Extracellular Levels of Nephrin in Podocytes In Vitro Nephrm, a constituent protein of the slit diaphragm, is associated with maintaining the normal function of the glomerular filtration barrier. Extracellular levels of nephrin in podocytes are decreased following injury. (Nagase et al. (2006) Hypertension 47:1084-1093.) The effect of CTGF on extracellular levels of nephrin was examined using a cell adhesion assay substantially as desc ⁇ bed above in Example 1 with the following modifications: podocytes were differentiated for 14 days and the amount of time of podocyte incubation with CTGF was 1 hour.
  • podocytes were fixed m 3% paraformaldehyde and stained for va ⁇ ous markers using standard immunohistochemical techniques. Immunofluorescence was performed using anti-extracellular nephrin primary antibody, and Texas red-conjugated secondary antibody. Fluorescent phalloidin staining of filamentous actin was earned out with l ⁇ g/ml FITC -conjugated phalloidin (Sigma).
  • CTGF addition decreased extracellular levels of nephrin m differentiated podocytes compared to that observed in non-treated controls (data not shown). These results showed that CTGF reduced extracellular levels of nephrin m cultured podocytes Nephrm is responsible for slit diaphragm integrity in cultured podocytes; therefore, these results suggested that CTGF mediates disruption or loss of slit diaphragm at least in part by reduction in extracellular levels of nephrm.
  • Mitogen-activated protein kinase (MAPK) activation is associated with podocyte injury in proteinuric glomerulopathies.
  • MAPK Mitogen-activated protein kinase
  • CTGF addition increased phosphorylation of p42/44 MAPK in differentiated podocytes at all timepomts studied.
  • Pretreatment of podocytes with CLN-I however, abrogated CTGF-mduced phosphorylation of p42/44 MAPK.
  • pretreatment with CLN-I completely prevented CTGF -induced increases in phosphorylation of p42/44 MAPK 10 minutes after
  • Example 8 CTGF Inhibition Ameliorates Podocyte Injury in an Animal Model of Podocyte Injury
  • mice Male Sprague-Dawley rats (Charles River Co) weighing 290 to 320 g were randomized mto three treatment groups. Group 1 and Group 2 were treated with anti-CTGF antibody (CLN-I, 10 mg/kg), and Group 3 was treated with an isotype matched irrelevant human IgG (IgG, 10 mg/kg). Antibodies were administered i.p at a dose of 10 mg/kg (approximate injection volume of 0 5 ml) three times weekly beginning on day -4 (i.e. 4 days p ⁇ or to puromycm injection).
  • puromycm aminonucleoside 50 mg/kg was injected intravenously into rats in Group 2 and Group 3 Group 1 served as a healthy control Antibody treatment was continued in all groups through day 7.
  • U ⁇ ne samples were collected at days 2, 3, 4, and 7, and urine albumin levels (i e urinary albumin concentrations) were determined for each sample by an immunoturbidimetnc assay (Roche Diagnostics). Briefly, anti-albumm antibodies react with the albumin in the sample to form albumm/anhbody complexes which, following agglutination, are measured turbidimetncally.
  • albuminuria serves as a marker for podocyte injury and podocyte dysfunction (Ricardo et al (1994) J Am Soc Nephrol 4 1974-1986)
  • a single injection of puromycm increased urinary albumin concentration in all groups at day 3
  • animals administered an anti-CTGF antibody had reduced urinary albumin levels (i e reduced albuminuria) compared to that observed in animals administered control IgG (see Table 2).
  • animals administered an anti-CTGF antibody had urinary albumin levels restored to normal levels of healthy animals.
  • PA -t- IgG (10mg/kg) 8. 00 ⁇ 2. 36 44.50 ⁇ 8.94 67 83 ⁇ 4 55 57.88 ⁇ 7.66
  • kidney desmin levels are an early marker of podocyte injury.
  • the effect of CTGF inhibition on kidney desmin levels m an animal model of podocyte injury was examined as follows. Kidney sections were obtained on day 3 from the PA animals desc ⁇ bed above in Example 8 Segments of kidney were placed in 10% zinc formalin (0.1 M) for 24 hours, dehydrated m a graded se ⁇ es of ethanol solutions, and then embedded in paraffin. Three-micrometer thick sections of the kidney tissue were cut, deparaffinized, hydrated in distilled water, and then stained for desmm using standard immunohistochemical techniques.
  • immunostainmg was performed using mouse anti-human desmin primary antibody (BioGemx) and HRP-conjugated anti-mouse IgG secondary antibody (DAKO). Immunoreactivity was detected usmg DAB substrate (Sigma).
  • the specimens were analyzed using morphometnc techniques Briefly, images of kidney sections were captured with a digital camera-light microscope system and the surface area of glomerular desmin staining was determined by image analysis software (ImagePro Plus, MediaCybernetics).
  • Table 3 shows the percent of glomerular area that stained positive for desmin (i.e. kidney desmin levels) at day 3 following treatment with anti-CTGF antibody in PA animals As shown below in Table 3, PA animals administered an anti-CTGF antibody showed a reduction in kidney desmm levels at day 3 compared to that observed in animals administered control IgG (see Table 3)
  • Example 10 CTGF Inhibition Reduced Podocytopenia in an Animal Model of Podocyte Injury
  • WT-I (Wilms' tumor 1) is a useful marker for measu ⁇ ng podocyte number and density in kidney sections (Kim et al (2001) Kidney Int 60 957 968.) The effect of CTGF inhibition on kidney podocyte count following podocyte injury was examined by measu ⁇ ng WT-I staining as follows. Kidney sections were obtained on day 7 from the PA animals descnbed above in Example 8.
  • Table 4 below shows changes in kidney podocyte count (as determined by WT-I staining) following treatment with anti-CTGF antibody in PA animals As shown below m Table 4, PA animals treated with anti-CTGF antibody showed greater podocyte counts at day 3 and day 7 as compared to those observed in PA animals treated with IgG antibody. Additionally, CTGF inhibition with CLN-I reduced the decrease in podocyte count at day 7 compared to that observed in PA animals treated with IgG antibody (see Table 4)
  • podocytes were fixed in 3% paraformaldehyde, permeabilized with
  • CTGF addition increased cell migration of differentiated podocytes in culture.
  • CTGF inhibition prevented CTGF-mduced migration of cultured podocytes.
  • Podocyte motility and migration can result in podocyte detachment from the glomerular basement membrane.
  • methods and agents of the present invention are useful for decreasing or preventing podocyte detachment from the glomerular basement membrane.
  • podocyte motility and migration can lead to podocyte shedding in the urine (i.e., podocytu ⁇ a). Therefore, methods and agents of the present invention are useful for treating, preventing, or reducing podocytu ⁇ a.

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Abstract

La présente invention concerne des procédés et des agents permettant de traiter, prévenir ou faire régresser une lésion podocytaire ou des troubles associés aux podocytes. L'invention concerne également des procédés et des agents permettant de traiter ou prévenir des podocytopathies, c'est-à-dire des maladies podocytaires.
PCT/US2009/005529 2008-10-07 2009-10-07 Procédés pour traiter une lésion podocytaire WO2010042202A1 (fr)

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WO2012054321A2 (fr) * 2010-10-19 2012-04-26 University Of Miami Essais, procédés et trousses pour la prédiction d'une maladie rénale, et stratégies personnalisées de traitement
US8252762B2 (en) 2008-08-25 2012-08-28 Excaliard Pharmaceuticals, Inc. Antisense oligonucleotides directed against connective tissue growth factor and uses thereof
US8946172B2 (en) 2008-08-25 2015-02-03 Excaliard Pharmaceuticals, Inc. Method for reducing scarring during wound healing using antisense compounds directed to CTGF
US9173894B2 (en) 2011-02-02 2015-11-03 Excaliard Pharamaceuticals, Inc. Method of treating keloids or hypertrophic scars using antisense compounds targeting connective tissue growth factor (CTGF)
EP3143131A4 (fr) * 2014-05-13 2017-10-04 Rush University Medical Center Dosages et composés pour traiter les maladies rénales
EP3396380A1 (fr) * 2017-04-27 2018-10-31 Ernst-Moritz-Arndt-Universität Greifswald Outil de diagnostic pour déterminer l'effacement des processus de pied de podocyte
US10195227B2 (en) 2012-04-13 2019-02-05 L&F Research Llc Method of using cyclodextrin

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US8252762B2 (en) 2008-08-25 2012-08-28 Excaliard Pharmaceuticals, Inc. Antisense oligonucleotides directed against connective tissue growth factor and uses thereof
US8772260B2 (en) 2008-08-25 2014-07-08 Isis Pharmaceuticals, Inc Methods for inhibiting expression of connective tissue growth factor
US8946172B2 (en) 2008-08-25 2015-02-03 Excaliard Pharmaceuticals, Inc. Method for reducing scarring during wound healing using antisense compounds directed to CTGF
US9096851B2 (en) 2008-08-25 2015-08-04 Excaliard Pharmaceuticals, Inc. Antisense oligonucleotides directed against connective tissue growth factor and uses thereof
US9688987B2 (en) 2008-08-25 2017-06-27 Excaliard Pharmaceuticals, Inc. Antisense oligonucleotides directed against connective tissue growth factor and uses thereof
WO2012054321A2 (fr) * 2010-10-19 2012-04-26 University Of Miami Essais, procédés et trousses pour la prédiction d'une maladie rénale, et stratégies personnalisées de traitement
WO2012054321A3 (fr) * 2010-10-19 2012-08-02 University Of Miami Essais, procédés et trousses pour la prédiction d'une maladie rénale, et stratégies personnalisées de traitement
US10765697B2 (en) 2010-10-19 2020-09-08 L & F Research LLC Method and treating renal disease
US10183038B2 (en) 2010-10-19 2019-01-22 L&F Research Llc Method for preventing and treating renal disease
US9173894B2 (en) 2011-02-02 2015-11-03 Excaliard Pharamaceuticals, Inc. Method of treating keloids or hypertrophic scars using antisense compounds targeting connective tissue growth factor (CTGF)
US10980828B2 (en) 2012-04-13 2021-04-20 L & F Research LLC Method of using cyclodextrin
US10195227B2 (en) 2012-04-13 2019-02-05 L&F Research Llc Method of using cyclodextrin
EP3143131A4 (fr) * 2014-05-13 2017-10-04 Rush University Medical Center Dosages et composés pour traiter les maladies rénales
WO2018197633A1 (fr) * 2017-04-27 2018-11-01 Ernst-Moritz-Arndt-Universität Greifswald Outil de diagnostic pour déterminer l'effacement de pédicelles de podocytes
KR20190139997A (ko) * 2017-04-27 2019-12-18 에른스트-모리츠-아른츠 유니버시태트 그레이프스발트 족세포 족돌기 소실을 결정하는 진단 도구
JP2020537739A (ja) * 2017-04-27 2020-12-24 ウニベルジテート グライフスヴァルト ポドサイトの足突起消失を決定するための診断ツール
EP3396380A1 (fr) * 2017-04-27 2018-10-31 Ernst-Moritz-Arndt-Universität Greifswald Outil de diagnostic pour déterminer l'effacement des processus de pied de podocyte
KR102510833B1 (ko) 2017-04-27 2023-03-15 니포카 게엠베하 족세포 족돌기 소실을 결정하는 진단 도구
US11747326B2 (en) 2017-04-27 2023-09-05 Nipoka Gmbh Diagnostic tool to determine podocyte foot process effacement
JP7442083B2 (ja) 2017-04-27 2024-03-04 ニポカ ゲーエムベーハー ポドサイトの足突起消失を決定するための診断ツール

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