WO2010042202A1 - Methods for treatment of podocyte injury - Google Patents

Abstract

The present invention relates to methods and agents for treating, preventing, or reversing 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.

Description

METHODS FOR TREATMENT OF PODOCYTE INJURY

This application claims the benefit of U.S. Provisional Application Serial No 61/195,531, filed on 07 October 2008, and U.S. Provisional Application Senal No. 61/198,728, filed on 06 November 2008, each of which is incorporated by reference herein it its entirety.

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

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 uπnary space and gives πse to long primary processes that extend toward the glomerular basement membrane (GBM) to which they attach by numerous foot processes The foot processes of neighboπng podocytes interdigitate, leaving between them filtration slits bπdged by an extracellular structure, known as the slit diaphragm.

Podocytes are polaπzed cells. Their unique shape is the result of an abundantly πch actin cytoskeleton, which serves as the podocyte's backbone and also serves as a static function to podocytes.

Rearrangement of their actin cytoskeleton also enables podocytes to continually and dynamically alter their shape. The actm cytoskeleton compπses 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. Several actin binding proteins in podocytes, such as synaptopodin and CD2AP, play important roles in maintaining podocyte shape.

In a healthy kidney, podocytes function as a filtration barrier between glomerular capillaries and the uπnary space, preventing proteins from enteπng 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. Additionally, the complex architecture of podocyte specific proteins (e.g , synaptopodin, nephπn) is required for many highly specialized functions of podocytes, which include maintenance of the capillary loop shape; counteracting the intraglomerular pressure; synthesis and maintenance of the GBM; and production and secretion of vascular endothelial growth factor (VEGF) required for glomerular endothelial cell integrity. 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. (1992) Br J Pharmacol 107- 189-195 ) 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; Kπz et al (1994) Kidney Int. 45: 369- 376, Pagtalunan et al (1997) J. Clin. Invest. 99- 342-348.) As a result of these observed changes in podocyte structure, these glomerulopathies are often referred to as podocytopathy (i.e podocyte disease)s (i e podocytopathies) (Koshikawa et al (2005) J Am Soc Nephrol 16:2690-2701 )

There are currently no approved treatments specific for podocyte injury Current treatment strategies typically focus on ameliorating underlying symptoms of podocytopathies (i.e. podocytopathy (i.e. podocyte disease)), such as, for example, proteinuria. Steroid therapy and immunosuppressive agents are often used to treat proteinuric subjects with podocytopathy (e.g., FSGS). ACE inhibitors and angiotensin receptor blockers (ARBs) are commonly administered to hypertensive subjects with podocytopathy (i.e. podocytopathy (i.e. podocyte disease)) and may decrease proteinuria. Although 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.

Therefore, there is a need in the art for methods and agents for effectively preventing or treating podocyte injury, podocyte-associated disorders, and podocytopathy (i.e. podocytopathy (i.e. podocyte disease))

Additionally, there is a need in the art for methods and agents effective at reducing podocyte dysfunction associated with podocyte injury The present invention meets this need by showing for the first time that specific inhibition of connective tissue growth factor (CTGF) reduces podocyte-associated disorders and measurably improves podocyte structure and function following podocyte injury.

SUMMARY OF THE INVENTION

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)) In one aspect, the podocyte injury is due to ischemia, hyperglycemia, diabetes, inflammation, an infection, or a medication.

In certain embodiments, the present invention provides a method for treating or preventing, or reducing a podocyte-associated disorder in a subject in need thereof, the method compπsing administering to the subject a therapeutically effective amount of an agent that inhibits CTGF In certain aspects, the podocyte-associated disorder is podocytopema, podocytuπa, podocyte foot process effacement, a decrease in podocyte slit diaphragm length, or a diminution of podocyte number or density

In other embodiments, the present invention provides a method for treating or preventing a podocytopathy

(i e podocytopathy (i.e. podocyte disease)) in a subject m need thereof, the method compπsing administering to the subject a therapeutically effective amount of an agent that inhibits CTGF. In one aspect, the podocytopathy (i.e. podocytopathy (i e. podocyte disease)) is diabetic nephropathy, minimal change disease, focal segmental glomerulosclerosis (FSGS), membranous nephropathy, collapsing glomerulopathy (CG), or chronic glomerulonephritis.

In yet other embodiments, the present invention provides a method for preventing or reducing podocyte foot process effacement in a subject in need thereof, the method compπsing admmisteπng to the subject a therapeutically effective amount of an agent that inhibits CTGF. In other embodiments, the present invention provides a method for preventing or reducing podocytuπa in a subject in need thereof, the method compπsing admimsteπng to the subject a therapeutically effective amount of an agent that inhibits CTGF

In another embodiment, 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 compπsing admmisteπng 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 compπsing admmisteπng to the subject a therapeutically effective amount of an agent that inhibits CTGF, are specifically provided

In certain embodiments, the invention provides a method for decreasing podocyte actin cytoskeletal rearrangement in a subject m need thereof, the method compπsing admimsteπng to the subject a therapeutically effective amount of an agent that inhibits CTGF. In another embodiment, the invention provides a method for increasing a podocyte marker in a subject in need thereof, the method compπsmg administenng to the subject a therapeutically effective amount of an agent that inhibits CTGF. In another embodiment, 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 compπsing administenng to the subject a therapeutically effective amount of an agent that inhibits CTGF. In certain embodiments, the podocyte marker is podocin, CD2AP, synaptopodin, nephnn, podocalyxin, FAT-I, nephπn, podoplanin, or Neph-1. In other embodiments, the invention provides a method for decreasing kidney desmm levels in a subject in need thereof, the method compπsing administering to the subject a therapeutically effective amount of an agent that inhibits CTGF

In other embodiments, 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 compπsing administering to the subject a therapeutically effective amount of an agent that inhibits CTGF.

In certain embodiments, 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 duπng 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.

In certain embodiments, the methods of the present invention further comprise determining the extent of one or more podocyte-associated disorders (e.g., podocytuπa or podocytopema) 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 compπse determining the extent of one or more podocyte- associated disorders duπng 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 compπse 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. Li particular embodiments, the methods of the present invention further compπse 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 pπmate, or a mammalian subject, e.g , a cat or a dog. In particular embodiments, the subject is a human subject, hi other embodiments, 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 descπbed 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 In particular, 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 In specific embodiments, the agent that inhibits CTGF is a monoclonal antibody or a fragment thereof, wherein the monoclonal antibody or fragment thereof specifically bind to

CTGF. In other specific embodiments, the agent that inhibits CTGF is CLN-I, descπbed in WO 2004/108764, or a fragment thereof. In other specific embodiments, 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.

These and other embodiments of the present invention will readily occur to those of skill in the art in light of the disclosure herein, and all such embodiments are specifically contemplated.

Each of the limitations of the invention can encompass vaπous embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited m its application to the details of construction and the arrangement of components set forth in the following descπption or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being earned out in vaπous ways. Also, the phraseology and terminology used herein is for the purpose of descπption and should not be regarded as limiting. The use of "including,"

"comprising," or "having," "containing", "involving", and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless context clearly dictates otherwise.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawmg(s) will be provided by the U. S Patent and Trademark Office upon request and payment of the necessary fee.

Figures IA, IB, 1C, and ID set forth data showing CTGF induced actin cytoskeletal rearrangement and cell polarization in human podocytes.

Figures 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.

Figures 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.

DESCRIPTION OF THE INVENTION

It is to be understood that the invention is not limited to the particular methodologies, protocols, cell lines, assays, and reagents descπbed herein, as these may vary It is also to be understood that the terminology used herein is intended to describe particular embodiments of the present invention, and is in no way intended to limit the scope of the present invention as set forth in the appended claims

It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless context clearly dictates otherwise Thus, for example, a reference to "a fragment" includes a plurality of such fragments, a reference to an "antibody" is a reference to one or more antibodies and to equivalents thereof known to those skilled m the art, and so forth.

Unless defined otherwise, all technical and scientific terms used herem have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs Although any methods and mateπals similar or equivalent to those descπbed herem can be used in the practice or testing of the present invention, the preferred methods, devices, and mateπals are now descπbed. All publications cited herein are incorporated herein by reference in their entirety for the purpose of descπbing and disclosing the methodologies, reagents, and tools reported in the publications that might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of pπor invention The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, cell biology, genetics, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Gennaro, A.R., ed. (1990) Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co.; Colowick, S. et al., eds., Methods In Enzymology, Academic Press, Inc.; Handbook of Experimental Immunology, VoIs. I-TV

(D.M. Weir and CC. Blackwell, eds., 1986, Blackwell Scientific Publications); Maniatis, T. et al., eds. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, VoIs. I-IIL Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al., eds. (1999) Short Protocols in Molecular Biology, 4th edition, John Wiley & Sons; Ream et al., eds. (1998) Molecular Biology Techniques: An Intensive Laboratory Course, Academic Press); PCR (Introduction to Biotechniques Series), 2nd ed. (Newton & Graham eds., 1997,

Springer Verlag).

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).

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. In some embodiments, 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. Such 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. (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)), wherein 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 section headings are used herein for organizational purposes only, and are not to be construed as in any way limiting the subject matter descπbed herein

Podocyte Injury

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 descπbed including, for example, high blood pressure and ischemia reperfusion injury. (See, e.g., Nagase et al. (2006) Hypertension 47.1084-1093, Kπz et al. (1994) Kidney Lit Suppl. 45.S64-S72.)

Other causes of podocyte injury include toxic substances, endocπnologic disorders, infections, contrast agents, mechanical trauma, cytotoxic agents (e.g., cisplatinum, adπamycin, puromycin), calcineuπn inhibitors, inflammation (e g., due to infection, trauma, anoxia, obstruction, or ischemia), radiation, infections (e.g., bacteπal, 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-bacteπal agent, anti-viral agent, anti-fungal agent, immunosuppressive agent, anti-inflammatory agent, analgestic or anticancer agent), organ failure, organ transplantation, and uropathy. Genetic disorders that cause 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, nephπn 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 (Baπsoni et al. (2003) Am J Nephrol. 23 353-360.) In one embodiment, 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. (Koshikawa et al (2005) J Am Soc Nephrol 16 2690-701 ) In human podocyte injury diseases, the phosphorylation of vaπous 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. (See e.g., Example 7.) Thus, agents that inhibit CTGF are useful for preventing or reducing podocyte cytoskeletal rearrangement As 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.

Additionally, in response to injury, podocytes undergo foot process effacement, or change in podocyte shape, also referred to as fusion, retraction, or simplification Podocyte foot process effacement is characteπzed by gradual simplification of the mter-digitanng foot process pattern, resulting in the formation of a flat and elongated looking cell. (Durvasula et al (2006) Curr Opin Nephrol Hypertens 15 1-7.) Foot process effacement is an active process that is initiated by rearrangement of the podocyte's actm cytoskeleton. Following injury, 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. (Shirato (2002) Microsc Res Tech. 15 241-6 ) Podocyte foot process effacement occurs with different types of podocyte injury and podocytopathy (i.e. podocyte disease)s (i e. podocytopathies).

The present invention shows that CTGF induces cytoskeletal rearrangement in cultured podocytes. (See, e.g., Example 1.) Agents that inhibit CTGF, therefore, are useful for preventing or reducing podocyte cytoskeletal rearrangement. The present invention also shows that 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 restoπng or reducing decreased levels of synaptopodm in podocytes. Additionally, the present invention demonstrates that CTGF increases levels of Arp3 in cultured podocytes; Arp3 is an enzyme responsible for podocyte cytoskeletal rearrangement. (See, e.g., Example 1 ) Thus, agents that inhibit CTGF are useful for preventing or reducing increased levels of Arp3 in podocytes Taken together, 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. As 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.

In addition to cytoskeletal rearrangement and foot process effacement, podocyte injury often leads to disruption of the slit diaphragm (e g , decreased slit diaphragm length) that lies between neighboπng 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. (Somlo et al (2000) Nat Genet 24 333-335; Tryggvason (2001) Curr Opin Nephrol Hypertens. 10:543-549, Endlich et al.

(2001) Curr Opin Nephrol Hypertens 10:331-340.) Disruptions of the slit diaphragm, evidenced by a decrease in slit diaphragm length or number, has been observed in several glomerular diseases and often results in to protemuπa in patients with minimal change nephritic syndrome (MCNS). (Patrakka et al

(2002) Pediatr Res 52 349-355.) Podocyte-specifϊc proteins, such as, for example, podocin and CD2AP, are responsible for anchoπng 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 nephπn m cultured podocytes (see e.g.,

Example 6); nephπn is a constituent protein of the slit diaphragm. Decreased levels of nephnn result in disruption and loss of slit diaphragm in vivo. Taken together, agents that inhibit CTGF are useful for preventing disruption or loss of slit diaphragm. Further, 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 proteinuπa in several glomerular diseases (Patrakka et al. (2002) Pediatr Res. 52:349- 355; Ruotsalamen et al. (1999) PNAS 96:7962-7967.) Therefore, in some embodiments, 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 hi other embodiments, 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. In yet other embodiments of the present invention, 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 compπses inhibiting CTGF.

A reduction in podocyte markers is a further manifestation of podocyte injury. (See e.g , Shankland et al. (2006) Kidney Int. 69:2132-2147.) Podocyte markers reduced following podocyte injury include¹ Nephπn; GLEPP; Podocin; FAT-I ; CD2AP; Nephl; integrins; mtegπn-lmked kinase; secreted protein acid πch in cysteine; Rho GTPases, β- actinm-4; synaptopodin, cychn-dependent kinase 5; podocalyxm; hic-5; TRPC6; dendπn; snail; notch; HSP27; Iamb4; podocalyxm, podoplanm, NHERF2, Ezπn; a,/3 dystroglycans; ffl3-j8-l integπn collagen type 4; Wnt-4 and Hic-5. Methods for increasing any one or more of these podocyte markers in a subject having podocyte injury, wherein the methods compπse administering to the subject an agent that inhibits CTGF, are specifically provided by the present invention

The present invention shows that CTGF decreases levels of various podocyte markers including, podocin, CD2AP, nephπn, 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)

Nephron 91 416-423 ) Therefore, in one aspect, the present invention provides methods and agents for preventing or reducing loss of podocyte markers in a subject having podocyte injury by admimsteπng an agent that inhibits CTGF In certain aspects, 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.) Thus, methods for decreasing desmin levels in a subject having podocyte injury, wherein the methods compπse administering to the subject an agent that inhibits CTGF, are specifically provided.

Detection of Podocyte Injury

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-α-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-πch 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. 16:1966-76; Asanuma, K. et al (2006) Nat Cell Biol. 8:485-91 ; Pavenstadt et al. (2003) Physiol Rev. 83:253-307 ) On the mRNA and protein level, specific podocyte genes including markers of cellular stress, apoptosis and specific proteins involved in podocyte injury, can be studied as descπbed in Tandon et al. ((2006) Am J Physiol Renal Physiol. 17; Durvasula (2005) Am J Physiol Renal Physiol. 289:F577- 84 )

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.

(2003) Physiol Rev. 83 253-307.) For example, urinary podocyte count (e.g., the presence of podocytuπa) may be used to detect podocyte injury (Yu et al (2005) J Am Soc Nephrol. 16:1733-41) Methods of detection of podocytuπa useful in the present invention are descπbed 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-specifϊc in the uπnary 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 Examples of markers useful for detection of podocyte injury include nephnn, desmin, Glepp-1, and synaptopodin In one aspect, detection of a particular gene can be done using a reverse transcπptase quantitative polymerase chain reaction (RT-PCR), microarrays, Western blots, proteomics, and ln-situ hybridization, and lmmunohistochemistry. Other markers are podocin, FAT-I, CD2AP, Nephl, integπns, integrm-lmked kinase, secreted protein acid πch m cysteine, Rho GTPases, β- actinin-4, podoplanin, cychn-dependent kinase5, podocalyxm, hic-5, TRPC6, dendπn, snail, notch, HSP27, Iamb4, podocalyxm, NHERF2, Ezπn, 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. For example, 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

(1999) PNAS 96:7962-7967). 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).

Additionally, fluorescent microscopy may be used to assess increases or decreases in 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 vaπous 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 descπbed previously by Kim et al. ((2001) Kidney Int. 60:957-968), the contents of which are incorporated herein by reference in their entirety. Accordingly, in one embodiment, the extent of podocyte injury is determined by measuring podocyte response to injury, such as, for example, the degree or level of podocytuπa or podocytopenia.

Podocyte-associated Disorders

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)s, 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 compπse 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. 23.353-360.) In some embodiments, 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. In other embodiments, methods and agents are provided for treating or preventing podocytopenia, wherein the method compnses inhibiting CTGF In yet other embodiments, 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. In yet embodiments, 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. In further embodiments, 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.)

Thus, 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. proteinuria) in an animal model of podocyte injury (See, e g., Example 4 ) As a reduction of protemuna has been shown to be associated with a reduction of podocytuπa, the present invention provides methods and agents useful for decreasing podocytuπa in a subject having podocyte injury, wherein the method compπses 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 uπne (i e podocytuπa), the present invention provides methods and agents useful for decreasing podocytuπa in a subject having podocyte injury, wherein the method compπses 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, podocytuπa. In some embodiments, the present invention provides methods and agents for reducing unnary podocyte count in a subject having podocyte injury, wherein the method compπses inhibiting CTGF. In other embodiments, the present invention provides methods and agents for treating or preventing podocytuπa in a subject having podocyte injury, wherein the method compπses inhibiting CTGF. In yet other embodiments, 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. In further embodiments, 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 compπses inhibiting CTGF.

In certain embodiments, the methods of the present invention further compπse determining the extent or seventy of podocyte injury or damage before, duπng, and/or after admmisteπng to the subject a therapeutically effective amount of an agent that inhibits CTGF. In vaπous aspects, 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 πsk for having podocyte injury is taken pπor to or duπng 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.

In other embodiments, the methods of the present invention further compπse determining the extent of one or more podocyte-associated disorders (e.g , podocytuna or podocytopenia) before, duπng, and/or after admmistenng to the subject a therapeutically effective amount of an agent that inhibits CTGF In certain aspects of the present embodiment, 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 pπor 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 vaπous human and experimental glomerular diseases, including minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), collapsing glomerulopathy (CG), membranous nephropathy, and diabetic nephropathy (DN) (Bansoni et al. (2003) Am J Nephrol. 23 353- 360 ) These glomerular diseases are often referred to as podocytopathy (i.e. podocyte disease)s (ι e , podocytopathies) (Koshikawa et al. (2005) J Am Soc Nephrol 16 2690-2701 ) Podocytopathies (i.e. podocytopathy (i e podocyte disease)s) have been classified according to their cause, e.g , congenital, hereditary, and acquired diseases (Shankland (2006) Kidney Int. 69 2131-2147.) Congenital podocytopathy (i e podocyte disease)s include abnormalities in structural podocyte proteins, such as in congenital nephritic syndrome of the Finnish type This disease is characteπzed by mutations in nephπn 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 α>actm-4, podocin and TRPC6. Such mutations may lead to hereditary proteinuria

Acquired podocytopathies (i.e. podocytopathy (i e podocyte disease)s) can be immune and non-immune mediated Examples of immune-mediated podocytopathy (i e. podocyte disease) include membranous nephropathy, minimal change disease, and membranoprohferative glomerulonephritis associated with cryoglobulins Non-immune acquired podocytopathy (i e. podocyte disease)s 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. Other examples of metabolic causes of podocytopathy (i e podocyte disease)s 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 Thus, 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 )

Therefore, 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 compπses 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 In some embodiments, 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 compπses inhibiting CTGF. In other embodiments, 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. In yet other embodiments, the present invention provides methods and agents for delaying progression of a podocytopathy (i e. podocyte disease) in a subject, wherein the method compπses inhibiting CTGF.

Subjects

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 pπmates. In certain embodiments, 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.

In certain embodiments, 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). In one aspect, the subject has or is at πsk of having podocyte injury In another aspect, the subject has or is at πsk of having a podocyte-associated disorder (e g., podocytuπa). In yet another aspect, the subject has or is at πsk of having a podocytopathy (i.e. podocyte disease) (e.g , FSGS).

In other embodiments, the subject of the present methods is identified as a subject having podocyte injury Identification of a subject having podocyte injury can occur before, duπng, or after administration of an agent that inhibits CTGF Detection of podocyte injury is descnbed herein In one aspect, the subject is identified as a subject having podocyte injury by the presence of podocytes m a uπne sample from the subject (i e podocytuna) In another aspect, the subject is identified as a subject having podocyte injury by the abnormal presence in uπne sediment of a gene selectively expressed in the podocyte (e g , the presence of nephπn in a uπne sample) In yet another aspect, 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) In other aspects, the subject is identified as a subject having podocyte injury by detecting podocytopenia in a kidney biopsy sample from the subject. Agents

In any of the methods descπbed above, it is particularly contemplated that 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. In particular, the present invention contemplates that 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. In particular embodiments, 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, π, III, and IV; and fibronectm. Assays for CTGF activity are known in the art, for example the cell migration assay descπbed in Example 5 International Publication No WO 2004/108764, incorporated by reference herein in its entirety.

Exemplary antibodies for use in the methods of the present invention are descπbed, 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 In certain embodiments, 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 compπse 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

2004/108764, incorporated by reference herein in its entirety The exemplary 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 Such antibodies, or fragments thereof, can be administered by vaπous means known to those skilled in the art For example, antibodies to CTGF can be injected intravenously, intrapeπtoneally, or subcutaneously Small molecule inhibitors of CTGF expression and/or activity have also been descπbed, 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

145 A19), and potentially phosphodiesterase IV inhibitors (see, e g , Kohyama et al. (2002) Am J Respir Cell MoI Biol 26.694-701), may be used to modulate CTGF expression. Also, inhibitors of serine/threonine mitogen activated protein kinases, particularly p38, cychn-dependent kinase, e g. CDK2, and glycogen synthase kinase (GSK)-3 have also been implicated in decreased CTGF expression. (See, e.g., Matsuoka et al. (2002) Am J Physiol Lung Cell MoI Physiol 283 L103-L112, Yosimichi et al. (2001)

Eur J Biochem 268:6058-6065; International Publication No. WO 01/38532, and International Publication No. WO 03/092584.) Such compounds can be formulated and administered according to established procedures within the art.

Further, polynucleotides including small interfeπng ribonucleic acids (siRNAs), micro-RNAs (miRNAs), πbozymes, and anti-sense sequences may be used in the present methods to inhibit expression and/or production of CTGF. (See, e.g., Kondo et al. (2000) Biochem Biophys Res Commun. 278:119-124.) Such techniques are well-known to those of skill in the relevant art. Anti-sense constructs that target CTGF expression have been descπbed and utilized to reduce CTGF expression in vaπous cell types. (See, e.g., International Publication No. WO 96/38172, International Publication No. WO 00/27868,

International Publication No. WO 00/35936, International Publication No WO 03/053340; Kothapalh et al (1997) Cell Growth Differ 8(l):61-68; Shimo et al (1998) J Biochem (Tokyo) 124(l):130-140; and Uchio et al. (2004) Wound Repair Regen. 12 60-66 ) 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 appropπate 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

Accordingly, m certain embodiments of the present invention, the agent that inhibits CTGF is an antibody to CTGF In a preferred embodiment, the antibody is a monoclonal antibody to CTGF In another preferred embodiment, the antibody is a human or humanized antibody to CTGF. In a particular embodiment, the antibody is CLN-I, as descπbed m International Publication No WO 2004/108764. In another embodiment, the agent is a small molecule. In another embodiment, the agent is a nucleic acid In a further embodiment, the nucleic acid is selected from the group consisting of a cyclic nucleotide, an oligonucleotide, or a polynucleotide In particular embodiments, 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 In one embodiment, 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. In another embodiment, 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. Current therapeutic approaches to treat kidney complications and disease are known by one of skill in the art, and include, for example, ACE inhibitors, 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

Pharmaceutical Formulations and Routes of Administration 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 compπse 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). In a preferred embodiment, 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 appropπate 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

Pharmacological Basis of Therapeutics, supra )

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-arteπal, mtra-articular, intracardiac, mtracisternal, intradermal, intralesional, intraocular, intrapleural, intrathecal, intrauterine, and intraventricular administration. The indication to be treated, along with the physical, chemical, and biological properties of the agent, dictate the type of formulation and the route of administration to be used, as well as whether local or systemic delivery would be preferred Pharmaceutical dosage forms 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, suppositoπes, ovules, implants, amorphous or crystalline powders, aerosols, and lyophihzed formulanons Depending on route of administration used, 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 system. One or multiple excipients, also referred to as inactive ingredients, can be added to an agent of the invention to improve or facilitate manufacturing, stability, administration, and safety of the agent, and can provide a means to achieve a desired drug release profile Therefore, 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 vaπous pharmacopoeias. (See, e.g., USP, JP, EP, and BP, FDA web page (www.fda gov), Inactive Ingredient Guide 1996, and Handbook of

Pharmaceutical Additives, ed. Ash; Synapse Information Resources, Inc. 2002.)

Pharmaceutical dosage forms 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 As noted above, 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.

Proper formulation is dependent upon the desired route of administration For intravenous injection, for example, 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 chloπde or dextrose For transmucosal or nasal administration, semisolid, liquid formulations, or patches may be preferred, possibly containing penetration enhancers. Such penetrants are generally known in the art For oral administration, 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 suppositoπes or retention enemas, e g , containing conventional suppository bases such as cocoa butter or other glyceπdes 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, coloπng, and flavoring agents. These excipients can be of synthetic or natural source Examples of such excipients include cellulose deπvatives, 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. dextrose, sucrose, lactose, etc ), talc, tragacanth mucilage, vegetable oils (hydrogenated), and waxes Ethanol and water may serve as granulation aides In certain instances, 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. When a capsule is preferred over a tablet, the agent powder, suspension, or solution thereof can be delivered in a compatible hard or soft shell capsule.

In one embodiment, 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.

For administration by inhalation, or administration to the nose, the agents for use according to the present invention are conveniently delivered in the form of a solution, suspension, emulsion, or semisolid aerosol from pressuπzed 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. For topical aerosols, hydrocarbons like butane, isobutene, and pentane are useful. In the case of a pressuπzed aerosol, the appropπate 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 steπle 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. Depending on the injection site, the vehicle may contain water, a synthetic or vegetable oil, and/or organic co-solvents. In certain instances, such as with a lyophihzed product or a concentrate, the parenteral formulation would be reconstituted or diluted pπor 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 requiπng 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 chloπde as a tonicity agent, for example, the buffer contains phosphate or histidine Co-solvents, such as, for example, polyethylene glycols, may be added. These water-based systems are effective at dissolving agents of the invention and produce low toxicity upon systemic administration. The proportions of the components of a solution system may be varied considerably, without destroying solubility and toxicity characteπstics. Furthermore, the identity of the components may be vaπed. For example, low-toxicity surfactants, such as polysorbates or poloxamers, may be used, as can polyethylene glycol or other co-solvents, biocompatible polymers such as polyvinyl pyrrohdone may be added, and other sugars and polyols may substitute for dextrose.

For agents useful for the present methods of treatment, 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 appropπate 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, veteπnaπan, 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) The MEC will vary for each agent but can be estimated from, for example, in vitro data and animal expeπments 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.

The present agents and compositions may, if desired, be presented in a pack or dispenser device containing one or more unit dosage forms containing the active ingredient Such 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

These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein. EXAMPLES

The invention will be further understood by reference to the following examples, which are intended to be purely exemplary of the invention These examples are provided solely to illustrate the claimed invention. The present invention is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only Any methods that are functionally equivalent are withm the scope of the invention Vaπous modifications of the invention in addition to those descπbed herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall withm the scope of the appended claims.

Example 1: CTGF Induced Podocyte Cytoskeletal Rearrangement In Vitro

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 The effect of CTGF on podocyte actin cytoskeleton (i.e. podocyte shape) was examined using a cell adhesion assay as follows.

Cell adhesion assays were earned out using immortalized human podocyte cells (AB 8/13) as previously descπbed by Saleem et al ((2002) J Am Soc Nephrol 13:630-638). Briefly, proliferation of this cell line takes place by incubation at 33°C while differentiation to the parental podocyte phenotype (e.g., cell spreading) takes place by incubation at 37⁰C. Accordingly, podocytes were cultured to 70% confluence

(i.e. proliferation) m MCDB-131 media supplemented with 10% FBS, L-glutamme and penicillin/streptomycin in a humidified 5% CO2 environment at 33⁰C Following proliferation, podocytes were transferred to an incubator at 37°C for 7 days to induce differentiation. Next, 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.

Following CTGF addition, podocytes were fixed in 3% paraformaldehyde and stained for vaπous markers using standard immunohistochemical techniques. Fluorescent phalloidin staining of filamentous actin was earned out with 1 μg/ml FITC-conjugated phalloidin (Sigma) (Green stam in Figures IA, IB, 1C, and

ID) 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.

As shown in Figures IA, IB, 1C, and ID, addition of CTGF to cultured podocytes resulted in cytoskeletal rearrangement and cell polanzation in cultured podocytes compared to that observed m non-treated controls after 1 or 6 hours of CTGF addition. Specifically, non-treated control podocytes showed well- developed foot processes and contained long bundles of actin filaments that terminated in the peπphery of the podocyte foot processes. Conversely, addition of CTGF resulted in process retraction and podocyte rounding by one hour with a loss of actin filament bundles (see Figures IB and ID)

These results showed that CTGF induced podocyte cytoskeletal rearrangement and cell polarization in cultured podocytes Therefore, these results suggested that 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 As podocyte foot effacement is a result of podocyte cytoskeletal rearrangement, these results further suggested that 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.

Several enzymes regulate the podocyte's actin cytoskeletal body For example, the Arp2/3 complex is a known mediator of actin nucleation in podocytes. (Welsch et al. (2001) Am J Physiol Renal Physiol 281. F769-F777.) Increased Arp3 levels are associated with greater cytoskeletal rearrangement in the podocyte's actin cytoskeleton. Thus, the effect of CTGF on Arp3 levels in podocytes was examined using a cell adhesion assay as descπbed 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 vaπous markers using standard immunohistochemical techniques. Immunofluorescence was performed using rabbit anti-Arp3 (4 μg/ml;

Boehπnger Mannheim Biochemicals, Indianapolis, IN) primary antibody and Texas red-conjugated goat anti-rabbit secondary antibody (Jackson Immunoresearch Laboratoπes, Inc , West Grove, PA). Fluorescent phalloidm staining of filamentous actin was earned out with 1 μg/ml FITC-conjugated phalloidm (Sigma) (Texas Red stam in Figures 2A, 2B, 2C, and 2D) Nuclei were counter-stained with Hoechst 33342 (DAPI) (DAPI/Blue stain m Figures 2A, 2B, 2C, and 2D) Immunostained podocytes were visualized using a Zeiss Axioscope with Axiovision 4

As shown in Figures 2A and 2C, Arp3 was present in non-treated 7 and 14 day differentiated podocytes. Addition of CTGF, however, increased Arp3 levels in both 7 and 14 day differentiated podocytes compared to that observed in non-treated controls (see increased Texas Red staining in Figures 2B and 2D).

These results showed that CTGF increased Arp3 levels m cultured podocytes Therefore, these results suggested that 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. Thus, these results further suggested that 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.

In another seπes of expeπments, the effect of CTGF on expression of Arp3 was examined using a cell adhesion assay as described above with the following modifications, podocytes were differentiated for 7 or 14 days and time of incubation of podocytes with CTGF was 15 or 180 minutes. Following addition of CTGF, total protein was extracted from the differentiated podocytes and separated by SDS-PAGE. Proteins were then transferred to Immobilon-P and probed with specific antibodies to Arp3 and β-Actin. Next, proteins were visualized with HRP-conjugated secondary antibodies using luminol.

As shown in Figure 3, little Arp3 expression was observed in non-treated control podocytes. Podocytes incubated with CTGF, however, showed increased Arp3 expression in both 7 and 14 day differentiated podocytes compared to that observed in non-treated controls (see Figure 3)

These results showed that CTGF increased Arp3 expression in cultured podocytes. Therefore, these results suggested that 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 Thus, these results further suggested that 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.

Therefore, the effect of CTGF on the expression of vaπous podocyte and slit diaphragm proteins was examined using a cell adhesion assay as descπbed above in Example 1 with the following modifications: podocytes were differentiated for either 7 or 14 days and the amount of time of podocyte incubation with CTGF was 3 hours

Following incubation with CTGF, podocytes were fixed in 3% paraformaldehyde and stamed for vaπous markers using standard immunohistochemical techniques Immunofluorescence was performed using rabbit antt-podocin and anti-CD2AP primary antibodies, and Texas red-conjugated goat anti-rabbit secondary antibody (Jackson Immunoresearch Laboratoπes, Inc , West Grove, PA) (Texas Red stain in Figures 4A, 4B, 4C, 4D, 5 A, 5B, 5C, and 5D) Fluorescent phalloidin staining of filamentous actin was carried out with 1 μg/ml FITC-conjugated phalloidin (Sigma) (Green stain in Figures 4A, 4B, 4C, 4D, 5 A, 5B, 5C, and 5D) Nuclei were counter-stained with Hoechst 33342 (DAPI) (DAPI/Blue stain in Figures 4A, 4B, 4C, 4D, 5A, 5B, 5C, and 5D). Immunostained podocytes were visualized using a Zeiss Axioscope with Axiovision 4.

As shown in Figures 4A, 4B, 4C, and 4D, 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).

These results showed that CTGF reduced CD2AP and podocin levels in cultured podocytes. Thus, these results suggested that use of CTGF inhibitors of the present invention is effective for decreasing the reduction of podocyte marker proteins following podocyte injury. These results further suggested that 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.

In another series of experiments, the effect of CTGF on expression of podocyte proteins CD2AP and podocin was examined using a cell adhesion assay as descπbed above in Example 1 with the following modifications: podocytes were differentiated for 7 or 14 days and time of podocyte incubation with CTGF was 15 or 180 minutes. Following CTGF addition, 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 CD2AP, podocin, and β-Actin. Next, proteins were visualized with HRP- conjugated secondary antibodies using luminol.

As shown in Figure 6A, 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).

These results showed that CTGF reduced expression of podocyte specific proteins, CD2AP and podocin, in cultured podocytes. Since these proteins are responsible for anchoring podocyte foot processes to the slit diaphragm in cultured podocytes, these results suggested that CTGF mediates loss of slit diaphragm at least in part by reduction in expression of CD2AP and podocin. 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. These results further suggested that methods and compounds of the present invention would be effective at treating, preventing, or reducing podocyte injury and podocyte dysfunction following injury, such as, reduced podocyte markers. Example 3: In Vitro Model of Podocyte Injury

The effectiveness CTGF inhibition on podocyte injury using methods and agents of the present invennon is examined using the cell adhesion assay as descπbed above in Example 1 except that differentiated podocytes are treated for 1-24 hours with 10 μg/mL puromycin m the presence or absence of an agent that inhibits CTGF (e.g , an anti-CTGF antibody)

Following treatment, podocytes are fixed in 3% paraformaldehyde and stained using standard immunohistochemical techniques. Podocyte shape and actm cytoskeleton are evaluated as descπbed 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.

Example 4: CTGF Inhibition Reduced Podocyte Dysfunction in an Animal Model of Podocyte Injury

Puromycin is a podocyte toxin which induces podocyte foot process effacement and podocyte shedding in the urine (i.e. podocytuπa) 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

Puromycin ammonucleoside (PA) (50 mg/kg) (Sigma-Aldπch Co, St. Louis, MO) was injected intravenously into male Sprague-Dawley rats (Charles River Co) weighing 290 to 320 g. Following the development of proteinuria, as determined by increasing urinary protein concentration, 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

Uπne 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 Uπne, 15th Ed , Philadelphia, WB Saunders, 1974).

In this podocyte injury model, proteinuπa serves as a marker for podocyte injury and podocyte dysfunction (Ricardo et al (1994) J Am Soc Nephrol 4 1974-1986). As shown below in Table 1, a single injection of PA increased uπnary 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).

TABLE l

Mean ± SE; *p<0 05 compared to Control+IgG (one-way ANOVA)

These results showed that an agent and methods of the present invention were effective at reducing proteinuria in an animal model of podocyte injury Since proteinuria is a marker of podocyte injury, these results showed that agents and methods of the present invention are effective at treating podocyte injury in vivo. Further, these results showed that agents and methods of the present invention were effective at treating podocytopathy (i e. podocyte disease) m a subject having podocyte injury.

Example 5: CTGF Decreased Expression of Synaptopodin in Podocytes In Vitro

Synaptopodin is essential for the integrity of the podocyte actin cytoskeleton and for the regulation of podocyte cell migration. (See Yang et al. (2005) J Am Soc Nephrol. 16:1966-76; Asanuma, K. et al. (2006) Nat Cell Biol. 8:485-91; Pavenstadt et al. (2003) Physiol Rev. 83:253-307.) 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. At each time point, following CTGF addition, 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 β-Actin. Next, proteins were visualized with HRP-conjugated secondary antibodies using luminol.

As shown in Figure 7, 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 descπbed 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.

Following incubation with CTGF, podocytes were fixed m 3% paraformaldehyde and stained for vaπous 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 lμg/ml FITC -conjugated phalloidin (Sigma).

Nuclei were counter-stained with Hoechst 33342 (DAPI) Immunostained podocytes were visualized using a Zeiss Axioscope with Axiovision 4

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. These results suggested that methods and agents of the present invention are useful for treating or preventing disruption or loss of the slit diaphragm in a subject with podocyte injury 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 These results further suggested that methods and agents of the present invention would be effective at treating, preventing, or reducing podocyte injury and podocyte dysfunction following injury, such as, reduced podocyte markers Example 7: CTGF Inhibition Reduced Activation of p42/44 MAPK In Vitro

Mitogen-activated protein kinase (MAPK) activation is associated with podocyte injury in proteinuric glomerulopathies. (Koshikawa et al (2005) J Am Soc Nephrol 16-2690-701.) The effect of CTGF inhibition on activation of p42/44 MAPK in cultured podocytes was examined using a cell adhesion assay substantially as descπbed above in Example 1 with the following modifϊcanons. podocytes were differentiated for 14 days, the amount of time of podocyte incubation with CTGF was 10, 30, and 180 minutes, and certain podocyte cultures were treated with CLN-I pπor to CTGF addition (i.e., pretreatment).

At each time point following CTGF addition, total protein was extracted from the differentiated podocyte cells and separated by SDS-PAGE. Proteins were then transferred to Immobilon-P and probed with specific primary antibodies to phosphorylated and non-phosphorylated p42/44 MAPK. Next, proteins were visualized with HRP-conjugated secondary antibodies using luminol. Phosphorylated p42/44 and total p42/44 content was quantified by densitometry using Scion Image software. Figure 8B shows the ratio of phosphorylated p42/44 MAPK to total p42/44 MAPK content in cultured podocytes.

As shown in Figures 8 A and 8B, 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. In particular, pretreatment with CLN-I completely prevented CTGF -induced increases in phosphorylation of p42/44 MAPK 10 minutes after

CTGF addition (see Figures 8A and 8B).

These results showed that CTGF inhibition reduced phosphorylation of p42/44 MAPK in cultured podocytes. MAPK activation is necessary for podocyte injury; therefore, methods and agents of the present invention are useful for treating, preventing, or reducing podocyte injury in a subject.

Example 8: CTGF Inhibition Ameliorates Podocyte Injury in an Animal Model of Podocyte Injury

The effects of agents and methods of the present invention on podocyte injury were determined in a rodent model of podocyte injury as follows. 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 pπor to puromycm injection). On day 0 (after all groups had received three doses of antibodies), 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. Uπne 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.

In this podocyte injury model, albuminuria serves as a marker for podocyte injury and podocyte dysfunction (Ricardo et al (1994) J Am Soc Nephrol 4 1974-1986) As shown below in Table 2, a single injection of puromycm increased urinary albumin concentration in all groups at day 3 At day 4 however, 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). By day 7, animals administered an anti-CTGF antibody had urinary albumin levels restored to normal levels of healthy animals.

TABLE 2

Urinary Albumin Concentration (mg/L)

Group

Day 2 Day 3 Day 4 Day 7

Healthy Control + CLN-I

5. 00 ± 0. 71 n/a ND 6.00 ± 0.00 (lOmg/kg)

PA -t- IgG (10mg/kg) 8. 00 ± 2. 36 44.50 ± 8.94 67 83 ± 4 55 57.88 ± 7.66

PA + CLN-I (10mg/kg) 6. 00 ± 0. 00 45.13 ± 7.87 54 14 ± 9 70 4.00 ± 0.00

Mean ± SE, ND = not detected

These results showed that methods and agents of the present invention were effective at reducing albuminuria in an animal model of podocyte injury. Since albuminuria is a marker of podocyte injury, these results showed that agents and methods of the present invention are effective at treating or reducing podocyte injury in a subject.

In this podocyte injury model, podocytuπa closely parallels the onset of marked albuminuria such that a reduction in albuminuria is associated with a reduction in podocytuπa. As shown in this seπes of expenments, methods and agents of the present invention reduced albuminuria; therefore, these results suggested that methods and agents of the present invention are useful for reducing podocytuπa m a subject with podocyte injury. Example 9: CTGF Inhibition Reduced Kidney Desmin Levels in an Animal Model of Podocyte Injury

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 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 descπbed above in Example 8 Segments of kidney were placed in 10% zinc formalin (0.1 M) for 24 hours, dehydrated m a graded seπes 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. Briefly, 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 below 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)

TABLE 3

Mean ± SE

These results showed that methods and agents of the present invention were effective at reducing glomerular desmin levels in an animal model of podocyte injury Desmm is a sensitive early marker of podocyte injury, therefore, these results showed that methods and agents are useful for reducing, treating, or preventing podocyte injury in a subject.

Example 10: CTGF Inhibition Reduced Podocytopenia in an Animal Model of Podocyte Injury

WT-I (Wilms' tumor 1) is a useful marker for measuπng 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 measuπng WT-I staining as follows. Kidney sections were obtained on day 7 from the PA animals descnbed above in Example 8. Segments of kidney were placed in 10% zinc formalin (0 1 M) for 24 hours, dehydrated in a graded seπes 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 WT-I using standard immunohistochemical techniques Briefly, immunostainmg was performed using rabbit anti-WT-1 primary antibody (Santa Cruz Biotechnology) and HRP-conjugated anti-rabbit IgG secondary antibody (DAKO) Immunoreactivity was detected using DAB substrate (Sigma) and sections were counterstamed with hematoxylin Kidney sections were examined under light microscope; WT-I positive cells (i e podocytes) and hematoxylin positive cells were counted in 20 glomeruli for each kidney section. Podocyte count was reported as a ratio of WT-I positive cells to total glomerular cells

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)

TABLE 4

Mean ± SE

These results showed that methods and agents of the present invention were effective at reducing decreased podocyte count (i e podocytopenia) in an animal model of podocyte injury. Therefore, these results showed that agents and methods of the present invention are effective at treating, preventing, or reducing podocytopenia in a subject with podocyte injury. In this animal model of podocyte injury, podocyte loss results in podocyte shedding in the urine (i e podocytuπa) Taken together, these results showed that agents and methods of the present invention are effective at reducing podocyte loss in an animal model of podocyte injury. Therefore methods and agents of the present invention are effective at treating, preventing, or reducing podocytuπa in a subject with podocyte injury Further, these results showed that agents and methods of the present invention were effective at treating podocytopathy (i e. podocyte disease) in a subject having podocyte injury. Example 11: CTGF Inhibition Reduced Podocyte Migration In Vitro

The effect of CTGF inhibition on cell migration in cultured podocytes was examined using a scratch wound cell migration assay as follows. Human AB 8/13 podocytes were used in the cell migration assays. Podocytes were cultured to 80% confluence under permissive temperature conditions (33⁰C) in 12-well plates and then differentiated at a non-permissive temperature (37°C) for 14 days. Next, podocytes were serum starved for 24 hours and two parallel scratches were made with a 200 μl plastic pipette tip. Podocytes were treated with CLN-I (lOμg/ml) or a control IgG antibody (lOμg/ml) and then incubated in the presence of CTGF (25ng/ml).

Following incubation with CTGF, podocytes were fixed in 3% paraformaldehyde, permeabilized with

0.01% Triton X-100, and stained with Hoechst 33342. The number of migrated podocytes was determined by measuring the fluorescence signal in the scraped area. Figure 9 shows the number of migrated podocytes following incubation with CTGF.

As shown in Figure 9, CTGF addition increased cell migration of differentiated podocytes in culture.

Pretrearment of podocytes with CLN-I, however, completely prevented CTGF-mduced migration of differentiated podocytes. (See Figure 9.)

These results showed that CTGF inhibition prevented CTGF-mduced migration of cultured podocytes. Podocyte motility and migration can result in podocyte detachment from the glomerular basement membrane. Thus, methods and agents of the present invention are useful for decreasing or preventing podocyte detachment from the glomerular basement membrane. Further, podocyte motility and migration can lead to podocyte shedding in the urine (i.e., podocytuπa). Therefore, methods and agents of the present invention are useful for treating, preventing, or reducing podocytuπa.

Vaπous modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing descπption. Such modifications are intended to fall within the scope of the appended claims.

All references cited herein are hereby incorporated by reference herein in their entirety.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method for treating or reducing podocyte injury in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits CTGF.

2. A method for treating or reducing a podocyte-associated disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits CTGF.

3. A method for treating a podocytopathy in a subject having podocyte injury, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits CTGF.

4. An agent that inhibits CTGF for use in a method for treating or reducing podocyte injury, a podocyte- associated disorder, or a podocytopathy.

5. The method of claim 1 or an agent of claim 4, wherein the podocyte injury is due to ischemia, hyperglycemia, diabetes, inflammation, an infection, or a medication.

6. The method of claim 2 or an agent of claim 4, wherein the podocyte-associated disorder is selected from the group consisting of podocytopenia, podocyturia, podocyte foot process effacement, a decrease in podocyte slit diaphragm length, and a diminution of podocyte number or density.

7. The method of claim 3 or an agent of claim 4, wherein the podocytopathy is selected from the group consisting of minimal change disease, focal segmental glomerulosclerosis (FSGS), membranous nephropathy, collapsing glomerulopathy (CG), diabetic nephropathy, and chronic glomerulonephritis.

8. 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 comprising administering to the subject a therapeutically effective amount of an agent that inhibits CTGF.

9. An agent according to any preceding claim wherein the agent maintains levels of a podocyte marker or reduces the extent of podocyte marker loss.

10. An agent according to any preceding claim wherein the agent decreases podocyte actin cytoskeletal rearrangement.

11. The method of claim 8 or an agent of claim 9, wherein the podocyte marker is selected from the group consisting of podocin, CD2AP, synaptopodin, nephπn, podocalyxin, FAT-I, nephrin, podoplanm, and Neph-1.

12. A method for decreasing kidney desmin levels in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits CTGF.

13. An agent according to any preceding claim, wherein the agent decreases kidney desmin levels.

14. A method for decreasing or preventing podocyte detachment from the glomerular basement membrane (GBM) in a subject m need thereof, the method comprising admimsteπng to the subject a therapeutically effective amount of an agent that inhibits CTGF.

15. An agent according to any preceding claim, wherein the agent decreases or prevents podocyte detachment from the glomerular basement membrane (GBM).

16. The method according to any one of the preceding claims, wherein the subject is a mammalian subject.

17. An agent according to any one of the preceding claims, wherein the method is for treating, preventing, or reducing podocyte injury, a podocyte-associated disorder, or a podocytopathy in a mammalian subject.

18. The method according to any one of the preceding claims, wherein the subject is a human subject.

19. An agent according to any one of the preceding claims, wherein the method is for treating, preventing, or reducing podocyte injury, a podocyte-associated disorder, or a podocytopathy in a human subject.

20. The method or an agent according to any one of the preceding claims, wherein the agent that inhibits CTGF is a polypeptide, a polynucleotide, or a small molecule.

21. The method or an agent according to claim 20, wherein the agent that inhibits CTGF is an antibody that binds to CTGF or to a fragment thereof; an antisense molecule; an siRNA; or a small molecule chemical compound.

22. The method or an agent according to claim 21, wherein the agent that inhibits CTGF is a monoclonal antibody or a fragment thereof, and wherein the monoclonal antibody or fragment thereof specifically bind to CTGF

3. The method or an agent according to claim 21, wherein the agent that inhibits CTGF is CLN-I, or a fragment thereof.