WO2006096931A1 - Renal repair and regeneration - Google Patents

Abstract

Methods and compositions comprising CSF-1 are provided for regenerating, repairing or otherwise treating renal cells, tissues and/or organs, and more particularly, in prophylactic or therapeutic treatment of diseases or conditions associated with renal damage and/or dysfunction. In particular, CSF-1 may be particularly efficacious in treating acute renal failure. CSF-1 protein or an encoding nucleic acid may be administered to suppress, ameliorate or otherwise treat an existing renal disease or condition or to prevent, inhibit, suppress or otherwise protect against subsequent renal damage and/or renal failure. CSF-1 may also be used to regenerate or repair renal cells, tissue and/or organs ex vivo, the treated cells, tissue and/or organs then being suitable for subsequent transplantation to an animal, such as a human.

Description

TITLE RENAL REPAIR AND REGENERATION

FIELD OF THE INVENTION

THIS INVENTION relates to use of colony stimulating factor 1 (macrophage colony stimulating factor) in relation to the kidney. More particularly, this invention relates to use of colony stimulating factor 1 for treating a renal disease or condition associated with renal damage or dysfunction.

BACKGROUND OF THE INVENTION

Development of the kidney is a process involving branching morphogenesis. The metanephros, or permanent kidney, is first observed at ElO.5 in the mouse. Reciprocal inductive interactions between the ureteric bud (UB), an outgrowth of the Wolffian duct, and the metanephric mesenchyme (MM) result in branching of the UB to form the collecting duct system of the mature kidney and the differentiation of the mesenchyme into the glomeruli and uriniferous tubules (Saxen 1987, Organogenesis of the kidney. Cambridge University Press. Cambridge). In both embryonic and adult kidney, most epithelial structures are surrounded by renal interstitium. Interstitial cells are responsible for the production of extracellular matrix components and development and support of the functional units of the kidney, including feedback control of the glomerular capillary blood flow. The adult (Hume et ah, 1983. J Exp Med. 158 1522-36) and embryonic mammalian renal interstitium contains resident macrophages. The phenotype and potential tissue specific function of renal macrophages, and tissue macrophages in general, is not well defined. A little is known about their growth factor production and receptor profile. The expression of Cxcr4 on renal macrophages allows them to respond to the production of Cxcll2 by the comma and S-shaped bodies of the kidney (Grone et ah, 2002, JASN 13 957-67). Conversely, renal macrophages express CxcllO (IP-IO), allowing them to signal to the Cxcr3 receptor in the developing kidney mesenchyme (Grone et ah, 2002, supra).

The importance of macrophage infiltration in development is mirrored in adult tissue repair. In numerous examples of tissue repair, including models of acute damage to muscle, liver, lung, gastrointestinal tract and peripheral nervous system, infiltration by macrophages and production of macrophage-derived trophic factors appears to be absolutely essential for regeneration (Kluth et al, 2004, Kidney International. 66 542- 57).

But macrophages are the classical two-edged sword. In systems where the damage is severe or progressive and where the damage stimulus remains, including chronic inflammation, macrophages are the dominant cell type in the inflammatory exudates and they are implicated directly in cell death and tissue damage. Indeed, conventional wisdom in both renal disease and allograft rejection has been that macrophages are responsible for damage (Eitner et al, 1998, Transplantation 66 1551-7; Segerer et al, 2003, Curr. Opin. Nephrol. Hypertens. 12 243-9) and many therapeutic strategies have focused on ways in which to reduce macrophage recruitment and activation. A reduction in the production of chemokines involved in macrophage recruitment, proliferation and activation has been shown to be potentially beneficial not only in renal disease classically associated with immune perturbations, such as glomerulonephritis and lupus nephropathy, but also in unilateral ureteric obstruction and diabetes (Naito et al, 1996, MoI. Med. 2 297-312; Utsunomiya et al, 1995, J. Diabetes Complications 9 292-5). However, this is not always the case (Veilhauer et al, 2004, Kidney Blood Press. Res. 27 226-38; Holdsworth et al, 2000, Curr. Opin Neprhol. Hypertens. 9 505-11). Macrophage migration inhibitory factor (MIF), while associated with renal injury in the rat, has been shown to be independent of macrophage recruitment and renal fibrosis in a unilateral ureteral obstruction (UUO) model in the mouse (Rice et al, 2004, Nephrology 9 278-287).

CSF-I (macrophage colony-stimulating factor; M-CSF) is the major growth factor for cells of the macrophage lineage. Increased CSFl levels are associated with renal disease and allograft rejection (Isbel et al, 2001, Nephrol. Dial. Transplant. 16 1638-47; Le Muer et al, 2002, Leukoc. Biol. 72 530-7; Le Muer et al, 2004, Nephrol. Dial. Transplant. 19 1862-5). CSF-I acts on its target cells by binding to colony- stimulating factor 1 receptor (CSF-IR), a cell-surface tyrosine kinase receptor encoded by the c-fins proto-oncogene, which is expressed in macrophage and trophoblast cell lineages (Sasmono et al, 2003, Blood. 101 1155-1163). c-fms is critical for the proliferation, survival and differentiation of macrophages as disruption of the gene results in large depletions of macrophages in most tissues (Dai et ah, 2002, Blood 99 111-20).

Mutation of the CSF-I gene, such as that present in op/op mice, or blockade of CSF-I function with either anti-CSF-1 or anti-c-fms antibodies, greatly reduces renal damage in several models including experimental glomerular nephritis, renal tubular interstitial nephritis, autoimmune nephritis and ureteral ligation (Lenda et αl, 2003, J. Immunology 170 3254-62; Jose et αl, 2003, Am. J. Transplant. 3 294-300). In each of these model systems, CSF-I is produced locally, and probably also systemically (although this is seldom measured), and the interpretation has been that CSF-I acts to recruit and activate macrophages to cause tissue damage.

Administered granulocyte colony stimulating factor (G-CSF) has been shown to protect mouse kidneys from subsequent cisplatin damage. Cisplatin is a widely-used anticancer drug that can induce acute renal failure due to renal tubular injury. The protective effect provided by G-CSF was enhanced by CSF-I (i.e M-CSF; Iwasaki et αl, 2005, JASN 16 658). However, the administration of CSF-I alone prior to the induction of cisplatin damage showed no protective effect.

CSF-I has been reported to impair the progression of lipid-induced nephrotoxiocity in streptozotocin-induced diabetic rats, by modulating the recruitment of macrophages to the glomerulus (Utsunomiya et αl, 1995, supra). However, this contradicts Miyazaki et al., 1997, Clin. Exp. Immunol. 108 318, who showed that increased M-CSF production is associated with an increase in recruitment of macrophages to the glomerulus in lipid-induced nephrotoxicity.

In humans, renal disease is a severe and debilitating ailment that is broadly classified as "chronic" or "acute". Chronic renal disease (CRD) refers to the gradual decline in renal function. This ultimately progresses to end stage renal disease (ESRD) when the renal filtration rate falls below 10%. CRD prevalence is rising at 6 — 8% per annum worldwide. Subsequently the incidence of ESRD is also increasing. Currently, the only available treatment options for ESRD are renal transplantation and dialysis. Transplantation extends survival over dialysis, but is associated with surgical morbidity and faces a shortage of viable organs. Dialysis replaces solute clearance but does not replace all renal functions, such as endocrine or metabolic functions. For those receiving dialysis treatment, the quality of life is poor and mortality rates are high (16% pa). Acute renal failure (ARF) is a common outcome in the postoperative patient, due to nephrotoxic or ischaemic insult during treatment for another condition. ARP patients receive dialysis treatment, but the lack of adjunct therapy to dialysis is thought to contribute to the high mortality rate of 50-75%. For both acute and chronic renal conditions, there is an urgent need for more advanced therapeutic approaches.

SUMMARY OF THE INVENTION Notwithstanding the typical association between elevated CSF-I, macrophages and tissue and organ damage, the present inventors have identified CSF-I as having a hitherto unrealized role in supporting and promoting renal tissue repair and regeneration.

The invention is therefore broadly directed to use of CSF-I for regenerating, repairing or otherwise treating renal cells, tissues and/or organs, and more particularly, in prophylactic or therapeutic treatment of diseases or conditions associated with renal damage and/or dysfunction.

In a particular forms, the invention relates to use of CSF-I for treatment of acute renal damage and/or dysfunction.

In a first aspect, the invention provides a method of prophylactically or therapeutically treating a renal disease or condition in an animal including the step of administering a CSF-I protein or an encoding nucleic acid to an animal in need of such treatment.

In one form, the method according to the first aspect may be used to suppress, ameliorate or otherwise treat an existing renal disease or condition. In another form the method according to the first aspect may be used as prophylaxis to prevent, inhibit, suppress or otherwise protect against subsequent renal damage and/or renal failure.

Suitably, in embodiments relating to prophylactic or protective administration of CSF-I to prevent renal damage, CSF-I is administered in the absence of a therapeutically effective amount of G-CSF.

Preferably, the renal disease or condition is acute renal failure. In a second aspect, the invention provides a method of regenerating, repairing or otherwise treating renal tissue in an animal including the step of administering a CSF-I protein or an encoding nucleic acid to an animal in need of such treatment.

In a third aspect, the invention provides a method of regenerating, repairing or otherwise treating renal tissue ex vivo including the step of exposing one or more isolated renal cells, tissues or organs to a CSF-I protein or encoding nucleic acid.

In a fourth aspect, the invention provides a method of renal transplantation, including the step of administering to the animal one or more renal cells, tissues or organs exposed ex vivo to a CSF-I protein or encoding nucleic acid. In a fifth aspect, the invention provides a pharmaceutical composition for use in treating a renal disease or condition, said pharmaceutical composition comprising a CSF-I protein or an encoding nucleic acid and a pharmaceutically acceptable carrier, diluent or excipient.

Suitably, in embodiments relating to prophylactic or protective administration of CSF-I to prevent renal damage, said pharmaceutical composition does not comprise a therapeutically effective amount of G-CSF.

It will be appreciated from the foregoing that the renal cells may be, or may include, isolated renal macrophages as well as kidney cells.

In a sixth aspect, the invention provides use of CSF-I in the manufacture of a medicament for prophylactically or therapeutically treating a renal disease or condition in an animal.

In one embodiment, said medicament is for prophylactically or therapeutically treating acute renal failure in an animal.

In another embodiment, said medicament is for treating an existing renal disease or condition in an animal.

It will be appreciated that the present invention has broad application to animals inclusive of human and non-human mammals. Preferably, the animal is a human.

Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Resident renal macrophages. Left panel: FACS profile of GFP+ cells isolated from embryonic day 15.5 kidneys of c-fms-EGFP transgenic mice represent 2.8% of total cells. Middle panel: RNA section in situ hybridisation of macrophage- specifϊc outlier shows punctate expression within the interstitial macrophages of the kidney. Right panel: Two colour confocal image of a renal macrophage (arrowhead) between renal proximal tubules. Figure 2. Metanephric explants cultured with lOOU/μL (1.25 ng/μL) human recombinant CSF-I for 3 days compared to control explants. Immunofluorescence was performed to reveal the ureteric tree (calbindin 28KD) and the forming nephrons (WTl). 6 day cultures with WTl, calbindin and DAPI (nuclei) merged show the overall increase in size. Figure 3. Representative micrographs demonstrating the histology of mice following 50 minutes of IR injury at 1 week after injury (Panel A and B) compared to mice with IR injury following delayed administered CSF-I at the same time point (Panel C and D). Numerous tubular casts (black arrows) were evident in the renal medulla by 1 week after IR injury (Mag x200) (A). At higher power (Mag x400) interstitial matrix expansion is shown with a prominent inflammatory cell infiltrate (white arrows; B). In IR mice following CSF-I administration starting at 3 days post-renal artery clamping the majority of the tubular epithelium showed normal histology (black arrows) with very few tubular casts present at 1 week white arrows; C Mag x200). At higher power (D; Mag x400) there was widespread tubular epithelial cell replacement (black arrows) with attenuated interstitial matrix expansion.

Figure 4. Measurement of urinary albumin levels (A) and the albumin/creatinine ratio (B) in IR mice with or without the administration of CSF-I delivered day 3-5 after initiation of injury. The administration of CSF-I to IR mice was found to reduce urinary protein levels and the albumin/creatinine ratio comparable to control animals. **P<0.03; Data are means ± SD. Figure 5: Immunofluorescence microscopy of type IV collagen in c-fms-GFV mice following IR receiving vehicle (A and B) or CSF-I treatment (C and D). Panel A demonstrates increased numbers of GFP-positive macrophages in the renal interstitium that was associated with collagen type IV accumulation leading to interstitial expansion (arrows; Mag x400). At higher power (B; Mag x 1,000) arrows show tubular cast formation (white arrows) in the majority of proximal tubules as a result of loss of epithelial cell integrity. Interstitial macrophages (large arrowheads) can be seen associated with type IV collagen accumulation in IR kidneys. Following CSF-I treatment, IR kidneys displayed decreased numbers of interstitial GFP-macrophages, and a normal tubulointerstitium that contained a fine framework of collagen type IV (arrows; C; Mag x400) comparable to normal kidneys. At higher power (D; Mag x 1,000) the CSF-I treated IR kidneys showed normal architecture with an intact proximal tubular epithelial cell lining (arrows) that was surrounded by few GFP-macrophages in the interstitium (large arrowheads) without evidence of fibrosis.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT While CSF-I has previously been observed to assist G-CSF in preventing renal damage (i.e reno-protection) and to modulate recruitment of macrophages to the glomerulus of rats having lipid-induced nephrotoxocity (although its precise role remains controversial), the present invention has arisen, at least in part, from the surprising observation that CSF-I stimulates macrophages to promote growth, regeneration and/or repair of the kidney. By extension, the production of CSF-I in renal disease forms part of a protective/regenerative response that fails only when there is ongoing tissue damage elicited by a separate causal agent. Thus, it proposed that treatment with CSF-I could provide a paradoxical and unexpected approach to therapy for renal diseases and/or conditions.

As used herein "CSF-I protein" includes and encompasses any CSF-I protein (also known as macrophage colony stimulating factor or M-CSF) of mammalian origin, including any biologically active fragment of a CSF-I protein It will be appreciated that the invention also contemplates use of any of a number of modified and/or fragmentary forms of CSF-I. For example, United States Patent 6,322,779 describes an isolated recombinant, dimeric CSF-I is which is unglycosylated and which can be produced essentially endotoxin and pyrogen-free.

In particular, several C-terminally truncated fragments of CSF-I have been described which retain biological activity.

By way of example, reference is made to United States Patent 6,204,020 and United States Patent 6,146,851 which describe various carboxy-truncated forms of CSF- 1 protein and their encoding nucleic acids.

A biologically active CSF-I dimer is described in United States Patent 5,861,150, wherein at least one of the CSF-I monomers has one or more amino acid substitutions together with a carboxy truncation.

United States Patent 5,672,343 sets forth a CSF-I protein consisting of amino acids 4-522 of the 536 amino acid CSF-I sequence and fragments of CSF-I comprising truncations at various positions C-terminal of residue 149. The invention also contemplates use of any other molecule that has CSF-I agonist activity, including but not limited to any molecule capable of binding, dimerizing and/or activating the cognate CSF-I receptor (CSF-IR or c-fms).

CSF-I protein may be in native form purified from a natural source, including but not limited to human urine. An example of such a product is Mirimostim™ from Mitsubishi Pharma. .

CSF-I may also be in recombinant or chemical synthetic form.

For example, the present invention contemplates chemical synthesis of CSF-I protein, inclusive of solid phase and solution phase synthesis. Such methods are well known in the art, although reference is made to examples of chemical synthesis techniques as provided in Chapter 9 of SYNTHETIC VACCINES Ed. Nicholson (Blackwell Scientific Publications) and Chapter 15 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al, (John Wiley & Sons, Inc. NY USA 1995- 2001).

The invention also contemplates recombinant DNA technology as a means of producing recombinant CSF-I, including but not limited to, standard protocols as for example described in Sambrook et al, MOLECULAR CLONING. A Laboratory Manual (Cold Spring Harbor Press, 1989), in particular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al, (John Wiley & Sons, Inc. NY USA 1995-2001), in particular Chapters 10 and 16; and CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al., (John Wiley & Sons, Inc. NY USA 1995-2001, in particular Chapters 1, 5 and 6).

In one embodiment, the CSF-I protein is bacterially-expressed, non-glycosylated human recombinant CSF-I.

However, use of glycosylated forms of CSF-I (such as produced by mammalian cell expression systems) are also suitable for use according to the invention. Preferably, the CSF-I protein consists of a C-terminal 150 amino acid fragment of CSF-I protein.

The invention also contemplates CSF-I protein "derivatives", which have been altered, for example by addition, conjugation or complexing with other chemical moieties or by post-translational modification techniques, as are well understood in the art.

By way of example only, the invention contemplates derivatives of CSF-I such as, but not limited to, chemical modification of side chains (e.g pegylation of nucleophilic groups such as lysyl ε-amino groups or sulphydryl oxidation by performic acid oxidation to cysteic acid), chemical modification of the C-terminus (eg. carbodiimide activation via O-acylisourea formation followed by subsequent derivitization to a corresponding amide), chemical modification of the N-terminus (eg. acylation with acetic or succinic anhydride), incorporation of non-natural amino acids and/or their derivatives during protein synthesis and the use of crosslinkers, labels (e.g. fluorochromes, radionuclides, biotin) and other adducts. Other CSF-I derivatives may comprise additional amino acid sequences such as fusion partner sequences. Fusion partner sequences, by way of example, assist in protein purification and/or identification. For instance, these include "epitope tags" such as c- myc, FLAG and influenza haemagglutinin tags, polyhistidine (e.g. HIS₆), maltose binding protein, green fluorescent protein (GFP), immunoglobulin heavy chain Fc portion and glutathione S-transferase (GST), although without limitation thereto. For the purposes of fusion polypeptide purification by affinity chromatography, relevant matrices for affinity chromatography are antibody, protein A- or G-, glutathione-, amylose-, and nickel- or cobalt-conjugated resins respectively. Many such matrices are available in "kit" form, such as the QIAexpress™ system (Qiagen) useful with (HIS₆) fusion partners and the Pharmacia GST purification system. Isolated nucleic acids and expression constructs

It will be appreciated from the foregoing and also from renal treatment methods and compositions to be described in more detail hereinafter, that the invention also provides use of an isolated nucleic acid encoding a CSF-I protein. The term "nucleic acid" as used herein designates single-or double-stranded mRNA, RNA, cRNA, RNAi and DNA inclusive of cDNA and genomic DNA and DNA-RNA hybrids. Nucleic acids may also be conjugated with fluorochromes, enzymes and peptides as are well known in the art.

The invention also contemplates variant CSF-I nucleic acids having one or more codon sequences altered by taking advantage of codon sequence redundancy.

A particular example of a variant CSF-I nucleic acid is optimization of a nucleic acid sequence according to codon usage, as is well known in the art. This can effectively "tailor" a nucleic acid for optimal expression in a particular organism, or cells thereof, where preferential codon usage has been established. In certain embodiments, said isolated CSF-I nucleic acid may be present in an expression construct, wherein the said isolated nucleic acid is operably linked or connected to one or more regulatory sequences in an expression vector.

In one particular embodiment, the expression construct is suitable for bacterial expression of CSF-I protein in bacteria such as E. coli. In another particular embodiment, the expression construct is for expression in one or more mammalian cells, tissues or organs in vitro or in vivo.

According to this embodiment, the mammalian cells, tissues or organs include kidney cells, resident renal macrophages and/or bone marrow-derived macrophages.

Accordingly, an "expression vector" may be either a self-replicating extra- chromosomal vector such as a plasmid, or a vector that integrates into a host genome, inclusive of vectors of viral origin such as adenovirus, lentivirus, poxvirus and flavivirus vectors as are well known in the art.

By "operabty linked or connected" is meant that said regulatory nucleotide sequence(s) is/are positioned relative to the recombinant nucleic acid of the invention to initiate, control, regulate or otherwise direct transcription and/or other processes associated with expression of said nucleic acid.

Regulatory nucleotide sequences will generally be appropriate for the host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. Typically, said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, splice donor/acceptor sequences and enhancer or activator sequences.

Constitutive promoters (such as CMV, SV40 and human elongation factor promoters) and inducible/repressible promoters (such as tet-repressible promoters and IPTG-, alcohol-, metallothionine- or ecdysone-inducible promoters) are well known in the art and are contemplated by the invention, as are tissue-specific promoters such as α- crystallin promoters. It will also be appreciated that promoters may be hybrid promoters that combine elements of more than one promoter (such as SRa promoter). The expression construct may also include a fusion partner (typically provided by the expression vector) so that the recombinant CSF-I protein is expressed as a fusion polypeptide with said fusion partner, as hereinbefore described.

Expression constructs may also include a selection marker nucleic acid that confers transformed host cell resistance to a selection agent. Selection markers useful for the purposes of selection of transformed bacteria include bla, kanR and tetR while transformed eukaryotic cells may be selected by markers such as hygromycin, G418 and puromycin, although without limitation thereto.

Expression constructs may be introduced into cells or tissues by any of a number of well known methods typically referred to as "transfection" "transduction", "transformation" and the like. Non-limiting examples of such methods include transformation by heat shock, electroporation, DEAE-Dextran transfection, microinjection, liposome-mediated transfection (e.g. lipofectamine, lipofectin), calcium phosphate precipitated transfection, viral transformation, protoplast fusion, microparticle bombardment and the like.

Pharmaceutical compositions and methods of treatment

A variety of diseases and conditions can damage kidney parenchyma, such as atheroembolic disease, renal vein thrombosis, renal artery embolism, thrombosis, diabetic nephropathy, glomerulonephritis of various etiology, toxic nephrosis, and pyelonephritis. As a result of the damage, renal failure, whether arising from an acute or chronic decline in renal function, is a grave condition that can result in substantial or complete failure of the filtration, reabsorption, endocrine, and homeostatic functions of the kidney.

In one aspect, the invention therefore provides a method of prophylactically or therapeutically treating a renal disease or condition in an animal, such as by regenerating renal tissue in vivo in the animal, by administering a CSF-I protein, or an expression construct encoding a CSF-I protein, to the animal.

It will be appreciated that CSF-I may be administered alone or together with one or more other therapeutic agents that facilitate or assist in treating the renal disease or condition. A non-limiting example of such a therapeutic agent includes immunosuppressive agents {e.g. cyclosporine) and antibiotics (e.g. amoxicillin, cephalosporins, levofloxacin and ciprofloxacin).

In one particular embodiment relating to prophylactic treatment, said one or more other therapeutic agents is not G-CSF. It will also be appreciated that the invention contemplates combination with other treatments such as dialysis, surgery and transplantation.

In a preferred embodiment, the invention provides a method of treating an existing renal disease or condition in an animal.

The term "renal disease or condition" broadly includes and encompasses both acute and chronic renal failure. By "acute renal failure" is meant sudden loss of the ability of the kidneys to excrete wastes, concentrate urine, and/or conserve electrolytes.

Acute renal failure occurs relatively rapidly, such as in the postoperative patient, due to nephrotoxic or ischaemic insult during treatment for another condition. A more comprehensive review and discussion of acute renal failure can be found in Lameire et ah, 2006, JASN 17 923 and Xue et al, 2006, JASN Feb 22.

By "chronic renal disease" is meant a gradual decline in renal function which ultimately progresses to end stage renal disease (ESRD) where the renal filtration rate falls below 10%. In one embodiment, the chronic renal disease is not lipid-induced nephrotoxocity.

In a preferred embodiment, the invention relates to treatment of acute renal failure, such as where rapid renal repair and/or regeneration is required.

However, it will be appreciated that immediate delivery of CSF-I in vivo may also be useful in ongoing treatment of chronic renal disease.

In an alternative, less preferred embodiment, the invention provides use of CSF- 1 for prophylactic administration to an animal to prevent, inhibit, suppress or otherwise protect against subsequent renal damage and/or renal failure.

Suitably, according to such an embodiment CSF-I is administered to the animal in the absence of a therapeutically effective amount of G-CSF.

As used herein, an example of a therapeutically effective amount of G-CSF is an amount which is sufficient to protect against subsequent renal failure.

An example of a therapeutically effective amount of G-CSF is 250 μg/kg, such as described in Iwasaki et ah, 2005, supra. Preferably, CSF-I is administered in the absence of G-CSF.

Thus, a therapeutic agent administered according to the invention may "consist of CSF-I or "consist essentially of CSF-I.

By "consist essentially of is meant that CSF-I is the major, therapeutically active agent administered to said animal. For example, CSF-I provides, accounts for, or constitutes at least 60%, preferably at least 70%, more preferably at least 80% and advantageously at least 85%, 90% or 95-99% of the therapeutic activity administered to the animal.

In particular embodiments, CSF-I is delivered as a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier, diluent or excipient.

In general terms, by "pharmaceutically-acceptable carrier, diluent or excipienf is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers, well known in the art may be used. These carriers may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates and pyrogen-free water. A useful reference describing pharmaceutically acceptable carriers, diluents and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. NJ. USA, 1991) which is incorporated herein by reference.

Any safe route of administration may be employed for providing a patient with the composition of the invention. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed. Intra-muscular and subcutaneous injection is appropriate, for example, for administration of proteinaceous and nucleic acid molcules.

Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like.

These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and/or microspheres.

Pharmaceutical compositions of the present invention suitable for oral or parenteral administration may be presented as discrete units such as capsules, sachets or tablets each containing a pre-determined amount of CSF-I protein or an expression construct encoding same, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the agents of the invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

The above compositions may be administered in a manner compatible with the dosage formulation, and in such amount as is pharmaceutically-effective. The dose administered to a patient, in the context of the present invention, should be sufficient to effect a beneficial response in a patient over an appropriate period of time. The quantity of agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner.

Preferably, pharmaceutical compositions are deliverable directly to the kidney in a manner which avoids or lessens the likelihood of CSF-I -induced side effects that might result from systemic delivery.

In this regard, a CSF-I reservoir may be utilized which is transplantable into an animal, preferably at a site in proximity to the kidney, which delivers a controlled, metered dosage of CSF-I over time.

In one embodiment, CSF-I may be delivered by an implanted osmotic pump that delivers CSF-I to a location proximal to the kidney and/or into the renal blood supply such as via the renal artery. In another embodiment, microsphere-based delivery may be achieved by reconstituting CSF-I in solvent including distilled water and chitosan and combining this solution with polylactic-co-glycolic acid in an aqueous solution so that an emulsion is formed by ultrasonic treatment. The membrane is permeable to CSF-I and biocompatible and biodegradable with human kidney tissue without forming toxic waste products. Release can be controlled by the biodegradation kinetics of the chitosan used. For example, recombinant human colony stimulating factor 1 (rhCSF-1) was delivered in chitosan microcapsules that were injected locally into injured mouse brain so that CSF-I was constitutively released for different lengths of time to enhance survival of neurons in injured brain (Berezovskaya et ah, 1996, Acta Neuropathol. 92 479-86). Another particular, non -limiting example of CSF-I protein delivery technology is provided in United States Patent Application 20040191215.

In one embodiment, CSF-I may be provided in a macroporous reservoir comprising CSF-I in a biologically and chemically inert particle having interconnected pores. The pores are open to the particle surface for communication between the exterior of the particle and the internal pore spaces. Examples of particles for formation of such macroporous reservoirs are described, for example, in U.S. Pat. No.5, 135,740.

In another embodiment, CSF-I reservoir may be provided by way of a microcapsule and/or microparticle, having CSF-I contained or dispersed therein. Both microcapsules and microparticles are well known in the pharmaceutical and drug delivery industries (see, for example, Baker, R. W., CONTROLLED RELEASE OF BIOLOGICALLY ACTIVE AGENTS, John Wiley & Sons, NY, 1987; Ranade V. and Hollinger, M., DRUG DELIVERY SYSTEMS, CRC Press, 1996).

A microcapsule would typically comprise a reservoir or bolus of CSF-I contained within a polymer membrane shell. A microparticle would typically be a monolithic system where CSF-I is dispersed throughout the particle.

Specific procedures for encapsulation of biologically active agents which may be relevant to CSF-I are disclosed in U.S. Pat. No. 4,675,189 and U.S. patent application No. 20010033868. In yet another embodiment, the invention contemplates a polymer gel formulation comprising CSF-I. An example of a polymer for use in such a gel formulation is a polyoxyethylene-polyoxypropylene block copolymer (Pluronic.RTM.). These copolymers exhibit reverse thermal gelation behavior, have good drug release characteristics, and have a low toxicity. The copolymers gel as a function of temperature and polymer concentration, where an aqueous solution gels as the solution is warmed. The gel has a low viscosity at room temperature, but at a typical body temperature the viscosity increases.

Other suitable polymers for preparation of CSF-I delivery reservoirs include, but are not limited to collagen (Pieper et al, 2000, Biomaterials 21 1689-1699); fibrin (Grassl et. al, 2002, J. Biomed. Mater. Res. 60 607-612); yaupon gels (Ramamurthi et al, 2002, J. Biomed. Mater. Res. 60 196-205); derivatized dextrans (Letourneur et al, 2002, J. Biomed. Mater. Res. 60 94-100); heparin alginate (Laham et al, 1999, Circulation 100 1865-1871); alginate (U.S. Pat. Nos. 6,238,705 & 6,096,344); and chochleates (U.S. Pat. No. 6,403,056).

In yet another embodiment, CSF-I may be delivered by way of a liposome. Liposomes are typically, although not exclusively, spherical lipid vesicles, ranging in size from 0.01 to 10 microns, and consist of one or more lipid bilayer encapsulating an aqueous space. A variety of amphipathic lipids are used to form the bilayer, such as phospholipids, as for example described in U.S. Pat. No. 5,013,556. The lipid molecules are generally arranged with their polar head groups toward the water phase and the hydrophobic hydrocarbon tails adjacent to one another in the bilayer, thus forming closed, concentric bimolecular lipid leaflets separating aqueous compartment.

As previously described, the invention also contemplates delivery of an expression construct that comprises an isolated nucleic acid encoding CSF-I protein.

For example, the invention contemplates intravenous injection of a plasmid DNA expression construct comprising a CMV promoter operably linked or connected to a CSF-I nucleotide sequence using a procedure such as described for delivery of hepatocyte growth factor to renal glomeruli of mice (Dai et al, 2004, J Am Soc

Nephrol. 15 2637-47)

In another example, the invention contemplates transduction of primary cultures of isolated macrophages with a CSF-I expression construct (for increased expression).

Macrophages are then injected systemically or into the renal artery and will localize to the damaged kidney, as has been demonstrated with respect to IL-IO in rats with nephritis (Wilson et ah, 2002, MoI Ther. 6 710-7).

In yet another example, the invention contemplates therapeutic delivery of a recombinant viral vector encoding CSF-I injected intravenously into renal disease patients

Delivery of CSF-I expression constructs may be facilitated by use of appropriate delivery agents.

Biodegradable hydrogels may be formulated from cationized gelatin prepared through aminization containing plasmid DNA including a human CSF-I nucleotide sequence operably linked or connected to a promoter operable in a mammalian cell (eg a CMV promoter).

Similar approaches using microspheres and hydrogels (containing DNA constructs encoding recombinant matrix metalloproteinases) have shown that injection into the renal subcapsule of C57BL/6 mice which have had streptozotocin-induced diabetes, showed promise as a prophylactic treatment of kidney fibrolysis and dysfunction in the STZ-induced diabetic mouse model. (Aoyama et ah, 2003, Tissue Eng. 9 1289).

The invention also provides a method of regenerating, repairing or otherwise treating renal tissue ex vivo for transplantation into an animal, by administering a CSF-I protein to one or more kidney cells, tissues or organs in vitro prior to transplantation.

As used herein, "transplantation" includes and encompasses transplantation of autologous and heterologous cells, tissues and organs, as understood in the art.

With improved surgical techniques and medical management of rejection, renal transplantation has become the treatment of choice for chronic and end-stage renal disease (ESRD).

The use of immunosuppressive agents such as cyclosporine, OKT3, and FK506 has resulted in a 1-year survival rate for mismatched renal grafts of 80%. A 90% 1-year graft survival rate has been reported with non-identical grafts from living related donors and a 95% 1-year success rate for grafts with identical human lymphocyte antigen. The half-life of grafts from living related donors varies from 13-24 years. Other medical managements have further extended the functional life of renal transplants while ensuring a better quality of life for the transplant recipient.

Surgical techniques for transplantation were recently advanced with the use of laparoscopic surgical techniques. The frequency of left kidney harvesting via a laparoscopic approach has resulted in more frequent transplantation of kidneys with multiple renal arteries.

The invention therefore contemplates treatment of whole kidney or isolated kidney tissue in vitro, such as by soaking or perfusing with CSF-I, to thereby facilitate the effectiveness of transplantation to a recipient. The CSF-I treatment may further comprise other agents such as immunosuppressants (eg. OKT3, cyclosporine or FK506), growth factors and/or cytokines other than CSF-I that suppress rejection and/or assist renal regeneration and/or repair (eg. CcI and Cxcl).

In a particular embodiment, the invention contemplates enhancing growth of renal progenitor or stem cells (once committed to a renal fate) by addition of CSF-I to culture before injection into the renal capsule.

It will also be appreciated that resident, renal macrophages may be used therapeutically.

Although not wishing to be bound by any particular theory, it is possible that CSF-I acts to induce renal macrophages to produce soluble factors that promote renal cell growth and development.

Examples of such factors include the chemokines within the CcI and Cxcl families. The expression of receptors for CcI and Cxcl chemokines on renal cells, including podocytes and collecting duct cells (Huber et al., 2002, J Immunol. 168 6244-

52.) suggests that these ligands can signal to the kidney itself rather than simply playing a role in monocyte attraction

Therefore, the invention contemplates delivery of isolated renal macrophages to renal tissue to thereby promote CSF-I -mediated repair and regeneration of renal tissue.

Resident renal macrophages may be readily isolated by way of surface markers such as c-fins, class II MHC, CD83, CD 14 and/or CD86 by cell isolation methods well known in the art (e.g by FACS sorting or by magnetic bead enrichment). While in preferred forms the invention provides methods of treatment of renal diseases or conditions in humans, the invention also contemplates veterinary treatments of non-human animals such as poultry, livestock (e.g. cattle, horses, goats and sheep), performance animals (e.g. racehorses including sires and broodmares) and domestic animals, although without limitation thereto.

So that preferred forms of the invention may be better understood and put into practical effect, reference is made to the following non-limiting examples .

EXAMPLES

Examplel Characterizing the location and arrival of resident renal macrophages

Materials and Methods CSF-I

All experiments described herein used human recombinant CSF-I. Isolation of renal embryonic tissue and macrophages Outbred CDl female mice were mated and sacrificed by cervical dislocation for the collection of control kidney tissue at El 5.5. Embryonic kidneys were dissected in ice-cold PBS and stored at -70°C in preparation for RNA extraction.

Male c-fms transgenic mice (Sasmono et al, 2003, supra) were mated to CDl outbred females. Pregnant females were sacrificed at E15.5 and transgenic offspring were determined by visualization of c-fins EGFP expression in the placenta of the embryos. The kidneys of both transgenic and non-transgenic embryos were dissected separately in ice-cold PBS. 10-20 kidneys were incubated in ImI Dissociation Media

(lmg/ml CoIIagenase B. 1.2U/ml Dispase, 5U/ml DNase II, in HANKS media) at 37°C for 20min. Kidneys were then dissociated with a P-1000 and incubated for a further 5min at 37⁰C. This step was repeated before the kidneys were dissociated with a 23- gauge syringe and passed through a 4OuM cell strainer (BD Bioscience). An equal volume of ice-cold PBS was washed through the strainer and the cells were centrifuged at 3000xrpm for 5min. The supernatant was discarded and the cells resuspended in 2-

3ml of ice-cold PBS. The cells were passed again through a 4OuM cell strainer, checked under a microscope to ensure they were a single-cell suspension, and stored on ice ready for fluorescence activated cell sorting (FACS). Isolation of EGFP positive macrophages was carried out on a FACS Vantage SE DiVa flow cytometer (BD Biosciences).

Approximately 200 transgenic kidneys were subjected to FACS analysis with non- transgenic littermate kidneys used as a reference. All animal experimentation was covered by Animal Ethics Committee number IMB/479/03/NIH. RNA in situ hybridisation

Section RNA in situ hybridisation was performed as previously described

(Roche DIG Application Manual) with minor modifications (Holmes GP et al. Mech.

Dev. 79:57-72, 1998). Sections were dehydrated through an ethanol series prior to hybridization overnight at 65°C. Posthybridisation washes consisted of 6xSSC (5min, 65°C), 2xSSC/50% formamide/lOmM EDTA (30min, 65°C), 2xSSC (2x30min, 65°C) and 0.2xSSC (2x30min, 65°C).

Isolation and preparation of tissue for confocal analysis

Male c-fms transgenic mice (Sasmono et al, 2003, supra) were mated to CDl outbred females. Pregnant females were sacrificed at El 1.5, E12.5, E15 and newborn and transgenic offspring were determined by visualization of c-fms EGFP expression in the placenta of the embryos. The kidneys of transgenic embryos were dissected separately in ice-cold PBS. c-fms kidneys were fixed in 4% paraformaldehyde for 3 hrs at room temperature. The kidneys were subsequently equilibrated in 30% sucrose overnight at 4°C before being mounted in Tissue-Tek OCT medium in isopentane cooled over dry ice.

Results

To facilitate the isolation and characterisation of tissue macrophages, we have generated transgenic mice in which the control elements of the c-fms gene direct expression of a green fluorescent protein (EGFP) reporter. In the so-called MacGreen mice, all tissue macrophages, including interstitial macrophages in the kidney and phagocytes in the embryo from the earliest appearance in the yolk sac, express high levels of EGFP fluorescence (Sasmono et al, 2003, supra).

As shown in Figure 1 (left), GFP⁺ cells isolated from embryonic day 15.5 kidneys of c-fms-EGFP transgenic mice represented 2.8% of total cells. Confocal analysis of kidneys from these mice revealed that these cells appear within the kidney from 12 days post coitum (dpc) and are spread throughout the renal interstitium. As the tubules of the developing nephrons arise and the interstitial space contracts, the renal macrophages become intimately associated with the basement membranes of the adjacent proximal and distal tubules. Their cellular processes wrap around adjacent tubules (Figure 1, right) facilitating an intimate relationship with the cells of these tubules.

Example 2

Human recombinant CSF-I has a growth-promoting effect on the developing kidney. Materials and Methods Metanephric organ culture Metanephric organ culture was used to test the effect of recombinant CSF-I on the developing metanephros. Metanephroi from El 1.5 mice were grown for 1-6 days on Poretics 13mm polycarbonate inserts (Osmonics Inc) with a membrane pore size of l.Oμm at 37⁰C with 5% CO2 in 300μl of DMEM/Hams F12 media (Invitrogen) supplemented with 50μg/ml transferrin and 2OmM glutamine. Metanephroi were either grown in media alone or media supplemented with CSFl to a final concentration of 100U/μL (1.25ng/μ L). Immunofluorescence

Co-immunofluorescence for calbindin-D28K and WTl was performed at the end of the culture period to visualise growth and differentiation of the ureteric epithelium and formation of early nephron structures in explanted metanephroi as previously described (Piper et ah, 2002, Int. J. Dev. Biol. 46 545). Metanephroi were fixed in 100% methanol at -2O⁰C for 20 minutes. Monoclonal anti-calbindin-D28K (Sigma Chemical Company) was used at a dilution of 1:100 and C-terminal WTl polyclonal antibody C19 (Santa Cruz, SC- 192) was used at a dilution of 1:100. Secondary antibodies used were Cy3-conjugated anti-rabbit IgG (Sigma) at a dilution of 1:500, and Alexa Fluor 488 conjugated goat anti-mouse (Molecular Probes) at at a dilution of 1 :200. Explants were also treated with DAPI for visualisation of individual nuclei. Digital images were captured using a Dage "MTI" peltier cooled charge coupled device digital camera attached to an AX70 Olympus microscope, and artificially coloured and overlayed using Adobe Photoshop 7 software. Statistical analysis To semi-quantitatively assess the effects of CSF-I conditioned media on in vitro metanephric development, branch tips, branch points and WTl -positive bodies (forming nephrons) present in each explanted metanephros were counted. A one-way ANOVA followed by a Tukey's post-hoc test was used to determine if there was a significant difference in the number of ureteric tip, branch and/or WTl positive bodies in CSF-I- treated metanephroi in comparison to untreated metanephroi. Results

At 11.5dpc, mouse embryonic kidneys (metanephroi) are comprised of a T- shaped UB surrounded by MM. These can be isolated via microdissection and cultured as explants for up to 6 days. During culture, the ureteric epithelium undergoes branching morphogenesis and a mesenchyme-to-epithelial transition occurs, generating immature nephrons. Metanephric explant culture is an excellent model system for examining kidney development.

We have established kidney explant culture to screen secreted factors for their ability to perturb or promote kidney development. One of the proteins that we have added to explant cultures is human recombinant CSF-I. The C-terminal 150 amino acids of this protein is bioactive and contains 4 helix bundles similar to those of other members of this cytokine family (G-CSF, GM-CSF). It can be produced in bacteria and correctly fold to form a bioactive protein. Addition of recombinant CSFl to kidney explants resulted in a dramatic and statistically significant enhancement of renal development (Figure 2). Explants grew with the same morphology as normal, but at a much greater rate and to a greater overall size. This was evident after only 24 hours and detectable at doses as low as lOOU/μL. This implies that renal CSFl signalling via c-fms (CSFRl) on resident macrophages plays a positive role in kidney development.

Example 3 Human recombinant CSF-I has a growth-promoting effect on the damaged adult kidney

Materials and Methods Mouse surgery Male c-fms transgenic mice (20-25g, Monash University Animal House, Australia), carrying a green fluorescent protein (GFP) driven by the c-fms (CSF-I R) promoter, were divided into 3 groups. The first group (n=4) were anaesthetized with 2% inhaled isofluorane (Abbott Australasia Pty Ltd, Kurnell, Australia) and ischemia/reperfusion (IR) injury was induced via 50 minutes of left renal artery clamping. A vascular clamp (0.4- 1.0mm; S&T Fine Science Tools, CA) was used for this procedure via a flank incision. Each mouse in this group received three intraperitoneal injections of CSF-I (20μg/timepoint) at day 3, 4, and 5 after initiation of IR injury. The right contralateral kidney served as a control for CSF-I treatment. The second group of mice (n=4) underwent 50 minutes of left renal artery clamping and vehicle injections (phosphate buffered saline; PBS) were administered at days 3, 4, and 5. The third group (n=5) of mice served as a sham-operated control group where the animals were anaesthetized and a flank incision was performed without renal artery clamping. All experiments were approved by a Monash University Animal Ethics Committee which adheres to the "Australian Code of Practice for the Care and Use of Animals for Scientific Purposes". Preparation of tissue for microscopy

At 1 week after IR injury, mice were perfusion-fixed with 4% paraformaldehyde (PFA) under 2% inhaled isofluorane anesthesia. A midline incision was made to expose both the heart and the inferior vena cava. A 27" gauge needle was injected into the left ventricle of mice and flushed for 3 minutes with PBS containing heparin and NaNitropruside. At the same time, the inferior vena cava was cut to provide an outlet for the perfusate. Mice were perfusion-fixed with preheated 4% PFA at lOOmmHg for 10 minutes. Mid-coronal kidney sections were immersion fixed in 4% PFA (Sigma- Aldrich), embedded in paraffin wax and cut at 4μm. Sections were stained with haematoxylin and eosin and Periodic Acid Schiff (PAS) for histolopathological analysis.

For fluorescence visualization of <>/77Z,y-EGFP-macrophages, following perfusion-fixation kidney tissue was fixed in 4% PFA for 8 hours, transferred to PBS containing 30% sucrose for overnight incubation at 4 ⁰C, embedded in O.C.T. (TissueTek^® Japan) and stored at -80 ⁰C. Frozen sections were cut (5 μm) using a cryostat (Leica, Germany) and visualized under an Olympus Provis AX70 fluorescent microscope.

For determination of collagen type IV localisation, sections were incubated with 1% bovine serum albumin (BSA). A goat anti-human collagen type IV primary antibody (Southern Biotech, Birmingham, AL; 1:100 dilution) was added for 1 hour followed by a chicken anti-goat Alexa Fluor 647 conjugate (1:1000; Molecular Probes). Sections were mounted with Fluorescent Mounting Media (DakoCytomation) before visualization under an Olympus Provis AX70 fluorescent microscope.

Measurement of proteinuria and urine creatinine

Mice were housed in metabolic cages, with free access to food and water on the days of urine collection. Albumin and creatinine levels, and the albumin/creatinine ratio were measured in 24 hour urine samples using an Albuwell murine microalbuminuria ELISA assay and creatinine companion kit (both from Exocell Inc.), respectively. Results

Promotion of renal repair in IR kidneys with CSF-I treatment

At lweek widespread damage was evident in IR kidneys of mice receiving vehicle treatment. Characteristic of the renal damage was extensive loss of tubular epithelium and tubular cast formation particularly in the outer medullary region where numerous tubular casts were observed (Figure 3A & B). In these IR kidneys, interstitial matrix expansion was associated with the accumulation of extracellular matrix proteins resulting in the development of interstitial fibrosis (Figure 3 A & B). 50 minutes of IR injury led to a severe inflammatory response and the extensive loss of the tubular epithelium, without the necrotic insult demonstrated with longer durations of renal artery clamping.

In comparison, mice with IR injury receiving CSF-I treatment starting at 3 days after initiation of injury, showed normal renal histology in the cortical and medullary regions (Figure 3C). In the outer medullary region the tubules appeared intact with complete re-epithelialisation evident (Figure 3D). There were very few tubular casts apparent in these kidneys (Figure 3 C & D). Furthermore, in IR kidneys with CSF-I treatment there was a marked attenuation of interstitial matrix expansion as a result of diminished interstitial fibrosis. Functional recovery of IR mice with CSF-I treatment

Urine protein levels were measured in 24 hour urine samples obtained from sham-operated control mice and IR mice with/without CSF-I delayed administration (Figure 4). There was a significant reduction in the urine protein levels in IR kidneys with CSF-I treatment compared to IR kidneys without CSF-I administration (324.4+250.1 vs. 23.84+15.7; P<0.03). Although creatinine was not found to be significantly different between the mice with IR injury compared to control or IR + CSF-I treatment, the albumin/creatinine ratio was found to be significantly reduced following CSF-I treatment (42.24±25.60 vs. 604.22+496.20; P<0.03). The fact that urinary creatinine levels were not significantly different between groups is probably reflective of right kidney compensation following the left unilateral renal artery clamping. However, the albumin/creatinine ratio is a good indicator of kidney function.

CSF-I reduces type IV collagen accumulation and the number of interstitial macrophages in the IR kidney

In c-fms-GΕF IR mice, increased numbers of GFP-positive macrophages in the renal interstitium were associated with collagen type IV accumulation and interstitial expansion (Figure 5A & B). Tubular cast formation in the majority of proximal tubules was observed (Figure 5B) as a result of loss of epithelial cell integrity. Following CSF-I treatment, IR mouse kidneys displayed decreased numbers of interstitial GFP- macrophages, and a normal tubulointerstitium that contained a fine framework of collagen type IV comparable to normal kidneys (Figure 5C & D). Furthermore, the CSF-I treated IR kidneys showed normal architecture with an intact proximal tubular epithelial cell lining that was surrounded by few GFP -macrophages in the interstitium without evidence of fibrosis (Figure 5D). Conclusions

CSF-I administration to mice with IR injury resulted in the promotion of renal repair by accelerated tubular epithelial cell replacement and attenuation of interstitial fibrosis. IR is a model of acute tubular necrosis that is characterized pathologically by tubule cell damage resulting from prolonged renal ischemia. The accumulation of macrophages was distinctly observed in the tubulointerstitium of IR kidneys at 1 week after the initiation of injury. This was associated with numerous tubular casts formed as a result of the complete loss of the loss of functioning tubular epithelial cells leading to diffuse effacement and loss of the proximal tubule cell brush border. In IR kidneys large numbers of macrophages were also evident in the interstitium due to inflammation induced from hypoxic insult. Elevated urine protein levels were also observed in IR mice subsequent to the loss of renal function.

CSF-I was found to promote both a structural and functional recovery of the kidneys from IR mice. Importantly, CSF-I treatment was initiated at 3 days after IR injury, a time when renal damage and inflammation is already evident. CSF-I treatment of IR mice resulted in a restoration of the tubular epithelium, attenuation of interstitial matrix expansion and recovery of renal function, comparable to control kidneys.

Markedly reduced numbers of interstitial macrophages were observed in the in CSF-I- treated IR kidneys, compared to IR kidneys without treatment. The population of macrophages observed in the IR mice with CSF-I -treatment appeared to surround re- epithelialised renal tubules and were present without evidence of extracellular matrix accumulation.

The invention described herein has been supported by a research grant from the National Institutes of Health (USA) .

Throughout this specification, the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. Various changes and modifications may be made to the embodiments described and illustrated herein without departing from the broad spirit and scope of the invention.

All computer programs, algorithms, patent and scientific literature referred to in this specification are incorporated herein by reference in their entirety.

Claims

1. A method of treating an existing renal disease or condition in an animal including the step of administering CSF-I protein, or an encoding nucleic acid, to an animal in need of such treatment, to thereby suppress, ameliorate or otherwise treat said existing renal disease or condition.

2. The method of Claim 1, wherein the CSF-I protein is bacterially-expressed, non- glycosylated human recombinant CSF-I.

3. The method of Claim 2, wherein the CSF-I protein is a C-terminal 150 amino acid fragment of CSF-I protein.

4. The method of Claim 1, wherein the animal is a human.

5. The method of Claim 1, wherein the existing renal disease or condition is acute renal failure.

6. A method of prophylactically treating a renal disease or condition in an animal including the step of administering CSF-I protein, or an encoding nucleic acid, to an animal in need of such treatment, in the absence of a therapeutically-effective amount of G-CSF, to thereby prevent, inhibit, suppress or otherwise protect against subsequent renal damage and/or renal failure.

7. The method of Claim 6, wherein the CSF-I protein is bacterially-expressed, non- glycosylated human recombinant CSF-I.

8. The method of Claim 7, wherein the CSF-I protein is a C-terminal 150 amino acid fragment of CSF-I protein.

9. The method of Claim 6, wherein the animal is a human.

10. The method of Claim 6, wherein the renal disease or condition is acute renal failure.

11. A method of regenerating or repairing renal tissue in an animal including the step of administering a CSF-I protein, or an encoding nucleic acid, to an animal in need of such treatment, to thereby regenerate or repair renal tissue in said animal.

12. The method of Claim 11, wherein the CSF-I protein is bacterially-expressed, non-glycosylated human recombinant CSF-I.

13. The method of Claim 12, wherein the CSF-I protein is a C-terminal 150 amino acid fragment of CSF-I protein.

14. The method of Claim 11 , wherein the animal is a human.

15. A method of regenerating, repairing or otherwise treating renal tissue ex vivo including the step of exposing one or more isolated renal cells, tissues or organs to a CSF-I protein to thereby effect proliferation and/or regeneration of said cells, tissues or organs.

16. The method of Claim 15, wherein the CSF-I protein is bacterially-expressed, non-glycosylated human recombinant CSF-I.

17. The method of Claim 15, wherein the CSF-I protein is a C-terminal 150 amino acid fragment of CSF-I protein.

18. The method of Claim 15, wherein the renal tissue is human, renal tissue.

19. A method of renal transplantation, including the step of administering to an animal one or more renal cells, tissues or organs exposed ex vivo to a CSF-I protein or encoding nucleic acid according to Claim 15.

20. The method of Claim 19, wherein the CSF-I protein is bacterially-expressed, non-glycosylated human recombinant CSF- 1.

21. The method of Claim 20, wherein the CSF-I protein is a C-terminal 150 amino acid fragment of CSF-I protein.

22 The method of Claim 19, wherein the animal is a human.

23. A pharmaceutical composition for use in treating an existing renal disease or condition, said pharmaceutical composition comprising a CSF-I protein or an encoding nucleic acid and a pharmaceutically acceptable carrier, diluent or excipient.

24. A pharmaceutical composition for use in regenerating or repairing human renal tissue, said pharmaceutical composition comprising a CSF-I protein or an encoding nucleic acid and a pharmaceutically acceptable carrier, diluent or excipient.

25. A pharmaceutical composition for use in prophylactically treating a renal disease or condition, said pharmaceutical composition comprising a CSF-I protein or an encoding nucleic acid and a pharmaceutically acceptable carrier, diluent or excipient, in the absence of a therapeutically effective amount of G-CSF.

26. The pharmaceutical composition of any one of Claims 23-25, wherein the CSF-I protein is bacterially-expressed, non-glycosylated human recombinant CSF-I .

27. The pharmaceutical composition of Claim 26, wherein the CSF-I protein is a C- terminal 150 amino acid fragment of CSF-I protein.

28. The pharmaceutical composition of Claim 24, for regenerating or repairing human renal tissue in vivo.

29. The pharmaceutical composition of Claim 24, for regenerating or repairing human renal tissue ex vivo.

30. Use of CSF-I in the manufacture of a medicament for treating an existing renal disease or condition in an animal.

31. Use of CSF-I in the manufacture of a medicament for prophylactically or therapeutically treating acute renal failure in an animal.

32. Use according to Claim 30 or Claim 31, wherein the animal is a human.

33. Use according to Claim 32, wherein CSF-I is native CSF-I obtained from human urine.