WO2000061630A1 - Induction of kidney tubule formation - Google Patents

Abstract

The invention provides a method of stimulating kidney tubule formation in a post-natal mammal by administering to the mammal a substantially pure Wnt polypeptide or Wnt agonist.

Description

INDUCTION OF KIDNEY TUBULE FORMATION Statement as to Federally Sponsored Research This invention was funded in part by the

U.S. Government under grant number HD30249 awarded by the National Institutes of Health. The Government has certain rights in the invention.

Background of the Invention Kidney and urinary tract diseases are major causes of illness and death in the United States resulting in about 50,000 deaths per year. Renal cell carcinoma is the most common type of kidney cancer; this type of cancer affects the lining of the renal tubule and is often metastatic. About one third of the cases diagnosed show metastasis, e.g., to the lung or other organs, at the time of diagnosis. Other types of medical conditions, such as diabetes mellitus and high blood pressure, can lead to chronic kidney failure. Current therapeutic approaches include dialysis and transplantation.

Summary of the Invention The invention provides a method of regenerating kidney tissue and is based on the discovery that Wnt-4 is sufficient to trigger kidney tubulogenesis, whereas Wnt-11 (which is also involved in tubule formation) is not. Kidney tubule formation in a post-natal mammal is stimulated by administering to the mammal a substantially pure Wnt polypeptide or a Wnt agonist. Preferably, the Wnt polypeptide is Wnt-4 or a Wnt-1 class polypeptide such as Wnt-1, Wnt-2, Wnt-3a, Wnt-7a, and Wnt-7b. A Wnt- 1 class polypeptide is a Wnt polypeptide that transforms C57MG cells in culture. More preferably, the polypeptide is Wnt-3a, Wnt-4, Wnt-7a, and Wnt-7b, but not members of the Wnt-5a class of proteins such as Wnt-5a or Wnt-11. For example, the Wnt polypeptide is Wnt-4, and the Wnt agonist is HLDAT86. Wnt-4 ediated-tubulogenesis requires cell contact; accordingly, Wnt compositions are preferably administered to kidney cells in the context of the kidney organ or in a situation in which the cells expressing a Wnt polypeptide or agonist are in close contact with cells involved in tubule formation. In preferred embodiments, sulfated glycosaminoglycans (sGAGs) are co-administered with the Wnt compositions. The mammal to be treated is characterized as suffering from a kidney disorder. Preferably, the mammal is a human, mouse, rat, guinea pig, cow, sheep, horse, pig, rabbit, monkey, dog, or cat. The method is therapeutic or preventative and is administered to a juvenile or adult mammal. Kidney disorders include chronic renal failure, renal cell carcinoma, polycystic kidney disease, chronic obstructive uropathy, and virus- induced nephropathy. For example, the method is used to treat or prevent renal tubule epithelial cell degeneration associated with HIV-1 infection. Administration of the Wnt compositions is local or systemic. For example, the polypeptide or Wnt agonist is administered locally to a renal tissue by, e.g., retrograde perfusion of renal tissue via blood vessels or urine collecting ducts. Wnt compositions are also administered ex vivo to an explanted renal tissue. For example, a kidney is removed from an individual and treated in vi tro with a Wnt composition (e.g, a substantially pure polypeptide or an isolated nucleic acid) and then returned to the body of the same individual or a different individual.

The Wnt composition is a peptide mimetic, e.g., a polypeptide that is more resistant to proteolytic cleavage compared to a naturally-occurring Wnt polypeptide. The Wnt polypeptide is preferably soluble under physiological conditions. Accordingly, the polypeptide is modified to improve its solubility. Alternatively, the Wnt polypeptide is present on the surface of a cell. The method utilizes a Wnt polypeptide that includes an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:l, 2, 3, 4, or 5, a Wnt polypeptide that includes an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:l, 2, 3, 4, or 5, a Wnt polypeptide that includes an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:l, 2, 3, 4, or 5, and a Wnt polypeptide that includes an amino acid sequence that is identical to the amino acid sequence of SEQ ID NO:l, 2, 3, 4, or 5. The Wnt polypeptide preferably has an amino acid sequence at least 85% identical to SEQ ID NO: and functions to stimulate tubulogenesis. For example, the polypeptide may be a fragment of Wnt that stimulates tubulogenesis. A fragment has an amino acid sequence that is identical to part, but not all, of the amino acid sequence of a naturally-occurring Wnt polypeptide. A fragment of a naturally-occurring Wnt polypeptide that stimulates tubulogenesis preferably includes the amino acid sequence of at least the amino-terminal 50% of the naturally- occurring polypeptide. More preferably, the fragment contains the amino acid sequence of at least the amino terminal 75% of a naturally-occurring Wnt polypeptide. For example, the fragment contains at least residues 1- 180 of naturally-occurring Wnt-1 (SEQ ID NO:l). Other fragments of Wnt polypeptides which have been shown to stimulate tubulogenesis, e.g., residues 100-331 of naturally-occurring Wnt-7a (SEQ ID NO:4, highlighted in bold) , are administered. Full-length Wnt polypeptides or fragments thereof are chemically or recombinantly linked to Ig to yield Wnt-Ig fusion proteins. Human or mouse Wnt polypeptides are administered to mammals to stimulate tubulogenesis .

Also within the invention is a method of stimulating kidney tubule formation in a post-natal mammal by administering a substantially pure or isolated nucleic acid encoding a Wnt polypeptide (e.g., a nucleic acid having the nucleotide sequence of SEQ ID NO: 10, 11, or 12) or a Wnt agonist. Nucleic acids that encode a Wnt polypeptide and that have a sequence that is substantially identical to a Wnt-encoding nucleic acid sequence are administered to diseased kidney tissue.

Polypeptides or other compounds of interest are said to be "substantially pure" when they are within preparations that are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest . Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. A polypeptide or nucleic acid molecule which is "substantially identical" to a given reference polypeptide or nucleic acid molecule is a polypeptide or nucleic acid molecule having a sequence that has at least 85%, preferably 90%, and more preferably 95%, 98%, 99% or more identity to the sequence of the given reference polypeptide sequence or nucleic acid molecule. "Identity" has an art-recognized meaning and is calculated using well known published techniques, e.g., Computational Molecular Biology, 1988, Lesk A.M., ed. , Oxford University Press, New York; Biocomputing:

Informatics and Genome Projects, 1993, Smith, D.W., ed. , Academic Press, New York; Computer Analysis of Sequence Data, Part I, 1994, Griffin, A.M. and Griffin, H.G. , eds, Humana Press, New Jersey; Sequence Analysis in Molecular Biology, 1987, Heinje, G. , Academic Press, New York; and Sequence Analysis Primer, 1991, Gribskov, M. and Devereux, J. , eds., Stockton Press, New York). While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans and has a definite meaning with respect to a given specified method. Sequence identity is measured using the Sequence Analysis Software Package of the Genetics Computer Group (GCS) , University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705) , with the default parameters as specified therein.

By "isolated nucleic acid molecule" is meant a nucleic acid molecule that is free of the genes which, in the naturally-occurring genome of the organism, flank a gene encoding a Wnt polypeptide. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence such as an immunoglobulin polypeptide. The term excludes large segments of genomic DNA, e.g., such as those present in cosmid clones, which contain a gene of interest flanked by one or more other genes which naturally flank it in a naturally-occurring genome . Nucleic acid molecules include both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. Where single-stranded, the nucleic acid molecule may be a sense strand or an antisense strand. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote at a site other than its natural site; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by polymerase chain reaction (PCR) or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence such as an Ig polypeptide. Wnt nucleic acids (encoding Wnt polypeptides) which hybridize at high stringency to naturally-occurring Wnt-encoding sequences are also administered to stimulate tubulogenesis. Hybridization is carried out using standard techniques such as those described in Ausubel et al . , Current Protocols in Molecular Biology, John Wiley & Sons, (1989) . "High stringency" refers to DNA hybridization and wash conditions characterized by high temperature and low salt concentration, e.g., wash conditions of 65° C at a salt concentration of approximately 0.1 X SSC. "Low" to "moderate" stringency refers to DNA hybridization and wash conditions characterized by low temperature and high salt concentration, e.g. wash conditions of less than 60° C at a salt concentration of at least 1.0 X SSC. For example, high stringency conditions may include hybridization at about 42°C, and about 50% formamide; a first wash at about 65°C, about 2X SSC, and 1% SDS; followed by a second wash at about 65°C and about 0.1% x SSC. Lower stringency conditions suitable for detecting DNA sequences having about 50% sequence identity to csa-1 gene are detected by, for example, hybridization at about 42°C in the absence of formamide; a first wash at about 42 °C, about 6X SSC, and about 1% SDS; and a second wash at about 50°C, about 6X SSC, and about 1% SDS. The invention also includes an ex vivo mammalian kidney containing an exogenous Wnt polypeptide, e.g., having been bathed in or perfused with a solution containing a Wnt polypeptide or agonist. Alternatively, the ex vivo mammalian kidney contains exogenous DNA encoding a Wnt polypeptide. The kidney is bathed or perfused with a solution containing a Wnt-encoding nucleic acid, and cells of the kidney take up the DNA. The cells then express and secrete the recombinant Wnt polypeptide or agonist. For expression of recombinant Wnt polypeptides, Wnt-encoding sequences are operably linked to regulatory sequences, e.g., tissue specific promoters. Kidney-specific promoters are known in the art and include, e.g., the Pax-2 promoter, the cRET promoter, and the Hox b7 promoter. By "operably linked" is meant able to promote transcription of an mRNA corresponding to a polypeptide-encoding sequence located downstream on the same DNA strand.

Description of the Preferred Embodiments A Wnt polypeptide, e.g., Wnt-4, Wnt-1, Wnt-3a, Wnt-7a and Wnt-7b, acts as a trigger to start an intrinsic program in the mesenchymal cells which then proceed to form complex nephron like structures. Wnt-4 is a secreted glycoprotein which is required for kidney tubule formation. Development of the mammalian kidney is initiated by ingrowth of the ureteric bud into the metanephric blastema. In response to signal (s) from the ureter, mesenchymal cells condense, aggregate into pretubular clusters, and undergo epithelialisation to form simple epithelial tubules. Subsequent morphogenesis and differentiation of the tubular epithelium lead to the establishment of a functional nephron. Table 6: Human Wnt-1 amino acid sequence

1 MGLWALLPGW VSATLLLALA ALPAALAANS SGRWWGIVNV ASSTNLLTDS KSLQLVLEPS 61 LQLLSRKQRR IRQNPGILH SVSGGLQSAV RECKWQFRNR R NCPTAPGP HLFGKIVNRG

121 CRETAFIFAI TSAGVTHSVA RSCSEGSIES CTCDYRRRGP GGPDWHWGGC SDNIDFGR F 181 GREFVDSGEK GRDLRFLMNL H NEAGRTTV FSEMRQECKC HG SGSCTVR TCWMRLPTLR

241 AVGDVLRDRF DGASRVLYGN RGSNRASRAE LLRLEPEDPA HKPPSPHD V YFEKSPNFCT

301 YSGRLGTAGT AGRACNSSSP ALDGCE LCC GRGHRTRTQR VTERCNCTFH WCCHVSCRNC

361 THTRVLHECL (SEQ ID NO : 1 )

Table 7: Human Wnt-3a amino acid sequence

CKCHG SGSC EVKTCWWSQP DFRAIGDFLK DKYDSASEMV VEKHRESRGW

VETLRPRYTY F VPTERDLV YYEASPNFCE PNPETGSFGT RDRTCNVSSH GIDGCDL CC GRGHNARAER RREKCRCVFH WCC (SEQ ID NO : 2 )

Table 8: Human Wnt-4 amino acid sequence

CKCH GVSGSCEVKT CWRAVPPFRQ VGHALKEKFD GATEVEPRRV GSSRALVPRN AQFKPHTDED

LVYLEPSPDF CEQD RSGVL GTRGRTCNKT SKAIDGCELL CCGRGFHTAQ VELAERCSCK FHWCLFLSR (SEQ ID NO: 3)

Table 9: Human Wnt-7a amino acid sequence

1 MNRKALRCLG HLFLSLGMVC LRIGGFSSW ALGATIICNK IPGLAPRQRA ICQSRPDAII 61 VIGEGSQMGL DECQFQFRNG RWNCSALGER TVFGKELKVG SRDGAFTYAI IAAGVAHAIT 121 AACTHGNLSD CGCDKEKQGQ YHRDEGWKWG GCSADIRYGI GFAKVFVDAR EIKQNARTLM 181 NLHNNEAGRK ILEENMKLEC KCHGVSGSCT TKTCWTTLPQ FRELGYVLKD KYNEAVHVEP 241 VRASRNKRPT FLKIKKPLSY RKP DTDLVY lEKSPNYCEE DPVTGSVGTQ GRACNKTAPQ 301 ASGCDLMCCG RGYNTHQYAR VWQCNCKFHW CCYVKCNTCS ERTEMYTCK (SEQ ID NO: 4) Table 10: Human Wnt-7b partial amino acid sequence VKC GVSGSCTTKT CWTTLPKFRE VGHLLKEKYN AAVQVEWRA SRLRQPTFLR IKQLRSYQKP METDLVYIEK SPNYCEEDAA TGSVGTQGRI CNRTSPGADG CDTMCCGRGY NTHQYTKVWQ CNCKFHWCCS (SEQ ID NO: 5) Table 11: Human Wnt-1 Nucleotide Sequence

1 atgtatgtat gtatgtatgt atgtatgtat acgtgcgtgc acctgtgtgt gc ggtgtc

61 agtggggctc agacatcacc tgattccctg gaactggagt tacaggtggc tataagccac

121 cacttgggtg ctgagaacag agtccgggcc tctggcagag cagtcagtgc ttttagccac

181 tgagccactc tcatcccccc aattatgttc atcttgagtt gggcaggtac ggtggcggaa 241 taggcctgta atcccagcag tcactggacc atcatgggtt ctacatatta aacctttatg

301 ttaggtaggg tcacacagca agatccggtc acaaaaccag caacaacaaa aaccaaaagg

361 agccagcttc ttcccacaag cattctttcc ctcaggtctt cagctccatc tgacagctac

421 tcggctggtg gtcctatcct ttctgagcct agttgccaga gaaacaagcc cggttcatct

481 tcatgactag cacatctaat gataagcaca ggttgactca aggtgccata gagtgacact 541 aggtacccag agcgacagaa tgacacctat gagtgcacgt cgttaatcac aaacacacac

601 acacacacac acacacacac acacacacac tcatgcaccc acctgcaaac acaattgcag

661 ccttctggac gtctcctgtc acagccccac ctccttcctg atacactgcg ttaagtggtg

721 actgtaacaa aatgacttca tgctctccct gtcctgagcc aaattacaca attatttgga

781 aagggctcaa aatgttcttc gttagaagtt tctggataca ccaatacaca ggagcgtgca 841 ccctcagaac acatgtacac tttgacttaa tctcacgggt gacacaccga cgcttacact

901 ccccctagcc cacagaggca aactgctggg cgcttctgag tttctcactg ccaccagctc

961 ggtttgctca gcctaccccc gcaccccgcg cccgggaatc cctgaccaca gctccaccca

1021 tgctctgtct ccttcttttc cttctctgtc cagccgtcgg ggttcctggg tgaggaagtg

1081 tctccacgga gtcgctggct agaaccacaa ctttcatcct gccattcaga atagggaaga 1141 gaagagacca cagcgtaggg gggacagagg agacggactt cgagaggaca gccccaccgg

1201 cgcgtgtggg ggaggcaatc caggctgcaa acaggttgtc cccagcgcat tgtccccgcg 1261 ccccctggcg gatgctggtc cccgacgggc tccggacgcg cagaagagtg aggccggcgc

1321 gcgtgggagg ccatcccaag gggaggggtc ggcggccagt gcagacctgg aggcggggcc 1381 accaggcagg gggcgggggt gagccccgac ggttagcctg tcagctcttt gctcagaccg

1441 gcaagagcca cagcttcgct cgccactcat tgtctgtggc cctgaccagt gcgccctggt

1501 gcttttagtg ccgcccgggc ccggaggggc agcctcttct cactgcagtc agcgccgcaa

1561 ctataagagg cctataagag gcggtgcctc ccgcagtggc tgcttcagcc cagcagccag

1621 gacagcgaac catgctgcct gcggcccgcc tccagactta ttagagccag cctgggaact 1681 cgcatcactg ccctcaccgc tgtgtccagt cccaccgtcg cggacagcaa ccacagtcgt

1741 cagaaccgca gcacagaacc agcaaggcca ggcaggccat ggggctctgg gcgctgctgc

1801 ccagctgggt ttctactacg ttgctactgg cactgaccgc tctgcccgca gccctggctg

1861 ccaacagtag tggccgatgg tggtaagtga gctagtacgg ggtccgccac ttgtcctggg

1921 gcaaagagcc aggcacgggc cttacccagc tcccacgctg tggggatcac caacctacag 1981 acccccctcg tgcattgtga cttcacatcc agggtgctca cacctagaac tagctctgct

2041 gaagtggggc acatcattgg catgcagaag cccagataca ccaggctcag agaccattcc

2101 catttaatac gaccccgttt ctgctgagca acaggtccca acctcgctgt ggtgggtgct

2161 caggtgtccc ttaggtcttg aaccaaaaaa aaaaaaaaaa aaaaaaaaaa accagatatt

2221 agctttgagg tgagggagtg gaattcctaa gtttttcaag gtgggcaagg ctgcaggtgg 2281 ggtttctcct cgggggctga cttgaagaaa ggaagagcta aggtagccat gccttttctg

2341 tccactcact agactctgga gctcagggcc aggcaaggat agggtggtac agcctgtatg

2401 gttaggatgc aggtcccctc ccctggactg aacccttatg catcccgcca ggggcatcgt

2461 gaacatagcc tcctccacga acctgttgac ggattccaag agtctgcagc tggtgctcga

2521 gcccagtctg cagctgctga gccgcaagca gcggcgactg atccgacaga acccggggat 2581 cctgcacagc gtgagtggag ggctccagag cgctgtgcga gagtgcaaat ggcaattccg

2641 aaaccgccgc tggaactgcc ccactgctcc ggggccccac ctcttcggca agatcgtcaa 2701 ccgaggtggg tgcccaggaa agcgacgctt ccgggattaa gggaaaagca gggtcatctc

2761 cagggcatag gcgggcgaag gcagggaaga catcccaggg ttatatgtga tcaaactgag

2821 aatcgcctgg tgccggcagt taccgtaggt cagcaccaga ttctttctag ccttgcgttg

2881 tgagcatgat ctttaacgtt gctggccact ggcccacaga aagggaattc cggatcgtgg

2941 gcgctgggcg acagctgttt ttccctagcc ttcctcaaag gtacctggga agctgatctc 3001 tgagggctag ctagggttgt gcttcgcacc cagcaaagtt tgcactgcca atactagtag

3061 cgatcttggc tatgcagatt tgttctactt gggaatctcc ccttggagct gctctgctag

3121 ggctctggag tctcagtaaa gcttagagag gagggcattc catgcttcgc acacatgact

3181 ccaaggatgt tggactgtag ggtaccaagt cttccaaaca gggtgctgag ttggccccac

3241 gccttctctc aactgatgcg gggtcgcttc acccacaggc tgccgagaaa cagcgttcat 3301 cttcgcaatc acctccgccg gggtcacaca ttccgtggcg cgctcctgct ccgaaggctc

3361 catcgagtcc tgcacctgcg actaccggcg gcgcggccct gggggccccg actggcactg

3421 ggggggctgc agtgacaaca tcgattttgg tcgcctcttt ggccgagagt tcgtggactc

3481 cggggagaag gggcgggacc tacgcttcct catgaacctt cacaacaacg aggcagggcg

3541 aacggtacgt cggtgtgtcc ggaaccaatg gcaggggaga tgtaagacag gtgcacgggg 3601 acagaggcac agggaggggc ttcccgagag agtgggactc taggagggaa gacagagaag

3661 aggtggtggt tgagggcaaa gaggttcctg agctgatgac agaacagaag agattagcag

3721 gctatcaaca cgtgggatgt attgagatgg ctccatggca cacttttgaa agataaaagt

3781 gacttgctgg cgtggagcag agtctggccg aatgtcccta tctcagcggg ccattttgca

3841 cttcctctct cccgagctta gtcacacctg gaccttggct gaagtttcca cagcatcgac 3901 gtgacccggg tggggtgggg gtggggaagt atgggtggtg gttcgtggga tgttggcttt

3961 gaccttttct tccctcctcc cctcgtcccc tcctccccca gaccgtgttc tctgagatgc 4021 gccaagagtg caaatgccac gggatgtccg gctcctgcac ggtgcgcacg tgttggatgc

4081 ggctgcccac gctgcgcgct gtgggcgacg tgctgcgcga ccgcttcgac ggcgcctccc

4141 gcgtccttta cggcaaccga ggcagcaacc gcgcctcgcg ggcggagctg ctgcgcctgg

4201 agcccgaaga ccccgcgcac aagcctccct cccctcacga cctcgtctac ttcgagaaat

4261 cgcccaactt ctgcacgtac agtggccgcc tgggcacagc tggcacagct ggacgagctt 4321 gcaacagctc gtctcccgcg ctggacggct gtgagctgct gtgctgtggc cgaggccacc

4381 gcacgcgcac gcagcgcgtc acggagcgct gcaactgcac cttccactgg tgctgccacg

4441 tcagctgccg caactgcacg cacacgcgcg ttctgcacga gtgtctatga ggtgccgcgc

4501 ctccgggaac gggaacgctc tcttccagtt ctcagacaca ctcgctggtc ctgatgtttg

4561 cccaccctac cgcgtccagc cacagtccca gggttcatag cgatccatct ctcccacctc 4621 ctacctgggg actcctgaaa ccacttgcct gagtcggctc gaaccctttt gccatcctga

4681 gggccctgac ccagcctacc tccctccctc tttgagggag actccttttg cactgccccc

4741 caatttggcc agagggtgag agaaagattc ttcttctggg gtgggggtgg ggaggtcaac

4801 tcttgaaggt gttgcggttc ctgatgtatt ttgcgctgtg acctctttgg gtattatcac

4861 ctttccttgt ctctcgggtc cctataggtc ccttgagttc tctaaccagc acctctgggc 4921 ttcaaggcct ttcccctccc acctgtagct gaagagtttc cgagttgaaa gggcacggaa

4981 agctaagtgg gaaaggaggt tgctggaccc agcagcaaaa ccctacattc tccttgtctc

5041 tgcctcggag ccattgaaca gctgtgaacc atgcctccct cagcctcctc ccaccccttc

5101 ctgtcctgcc tcctcatcac tgtgtaaata atttgcaccg aaatgtggcc gcagagccac

5161 gcgttcggtt atgtaaataa aactatttat tgtgctgggt tccagcctgg gttgcagaga 5221 ccaccctcac cccacctcac tgctcctctg ttctgctcgc cagtcctttt gttatccgac

5281 cttttttctc ttttacccag cttctcatag gcgcccttgc ccaccggatc agtatttcct 5341 tccactgtag ctattagtgg ctcctcgccc ccaccaatgt agtatcttcc tctgaggaat

5401 aaaatatcta tttttatcaa cgactctggt ccttgaatcc agaacacagc atggcttcca

5461 acgtcctctt cccttccaat ggacttgctt ctcttctcat agccaaacaa aagagataga

5521 gttgttgaag atctcttttc cagggcctga gcaaggaccc tgagatcctg acccttggat

5581 gaccctaaat gagaccaact agggatc (SEQ ID NO: 6)

Table 12: Human Wnt-2 Nucleotide Sequence

1 agcagagcgg acgggcgcgc gggaggcgcg cagagctttc gggctgcagg cgctcgctgc

61 cgctggggaa ttgggctgtg ggcgaggcgg tccgggctgg cctttatcgc tcgctgggcc

121 catcgtttga aactttatca gcgagtcgcc actcgtcgca ggaccgagcg gggggcgggg

181 gcgcggcgag gcggcggccg tgacgaggcg ctcccggagc tgagcgcttc tgctctgggc

241 acgcatggcg cccgcacacg gagtctgacc tgatgcagac gcaagggggt taatatgaac 301 gcccctctcg gtggaatctg gctctggctc cctctgctct tgacctggct cacccccgag

361 gtcaactctt catggtggta catgagagct acaggtggct cctccagggt gatgtgcgat

421 aatgtgccag gcctggtgag cagccagcgg cagctgtgtc accgacatcc agatgtgatg

481 cgtgccatta gccagggcgt ggccgagtgg acagcagaat gccagcacca gttccgccag

541 caccgctgga attgcaacac cctggacagg gatcacagcc tttttggcag ggtcctactc 601 cgaagtagtc gggaatctgc ctttgtttat gccatctcct cagctggagt tgtatttgcc

661 atcaccaggg cctgtagcca aggagaagta aaatcctgtt cctgtgatcc aaagaagatg

721 ggaagcgcca aggacagcaa aggcattttt gattggggtg gctgcagtga taacattgac

781 tatgggatca aatttgcccg cgcatttgtg gatgcaaagg aaaggaaagg aaaggatgcc

841 agagccctga tgaatcttca caacaacaga gctggcagga aggctgtaaa gcggttcttg 901 aaacaagagt gcaagtgcca cggggtgagc ggctcatgta ctctcaggac atgctggctg

961 gccatggccg acttcaggaa aacgggcgat tatctctgga ggaagtacaa tggggccatc

1021 caggtggtca tgaaccagga tggcacaggt ttcactgtgg ctaacgagag gtttaagaag

1081 ccaacgaaaa atgacctcgt gtattttgag aattctccag actactgtat cagggaccga

1141 gaggcaggct ccctgggtac agcaggccgt gtgtgcaacc tgacttcccg gggcatggac 1201 agctgtgaag tcatgtgctg tgggagaggc tacgacacct cccatgtcac ccggatgacc

1261 aagtgtgggt gtaagttcca ctggtgctgc gccgtgcgct gtcaggactg cctggaagct

1321 ctggatgtgc acacatgcaa ggcccccaag aacgctgact ggacaaccgc tacatgaccc

1381 cagcaggcgt caccatccac cttcccttct acaaggactc cattggatct gcaagaacac

1441 tggacctttg ggttctttct ggggggatat ttcctaaggc atgtggcctt tatctcaacg 1501 gaagccccct cttcctccct gggggcccca ggatgggggg ccacacgctg cacctaaagc

1561 ctaccctatt ctatccatct cctggtgttc tgcagtcatc tcccctcctg gcgagttctc 1621 tttggaaata gcatgacagg ctgttcagcc gggagggtgg tgggcccaga ccactgtctc 1681 cacccacctt gacgtttctt ctttctagag cagttggcca agcagaaaaa aaagtgtctc 1741 aaaggagctt tctcaatgtc ttcccacaaa tggtcccaat taagaaattc catacttctc 1801 tcagatggaa cagtaaagaa agcagaatca actgcccctg acttaacttt aacttttgaa 1861 aagaccaaga cttttgtctg tacaagtggt tttacagcta ccacccttag ggtaattggt 1921 aattacctgg agaagaatgg ctttcaatac ccttttaagt ttaaaatgtg tatttttcaa 1981 ggcatttatt gccatattaa aatctgatgt aacaaggtgg ggacgtgtgt cctttggtac 2041 tatggtgtgt tgtatctttg taagagcaaa agcctcagaa agggattgct ttgcattact 2101 gtccccttga tataaaaaat ctttagggaa tgagagttcc ttctcactta gaatctgaag 2161 ggaattaaaa agaagatgaa tggtctggca atattctgta actattgggt gaatatggtg 2221 gaaaataatt tagtggatgg aatatcagaa gtatatctgt acagatcaag aaaaaaagga 2281 agaataaaat tcctatatca t (SEQ ID NO: 7)

Table 13: Murine Wnt-3A Nucleotide Sequence

1 gaattcatgt cttacggtca aggcagaggg cccagcgcca ctgcagccgc gccacctccc

61 agggccgggc cagcccaggc gtccgcgctc tcggggtgga ctccccccgc tgcgcgctca

121 agccggcgat ggctcctctc ggatacctct tagtgctctg cagcctgaag caggctctgg

181 gcagctaccc gatctggtgg tccttggctg tgggacccca gtactcctct ctgagcactc

241 agcccattct ctgtgccagc atcccaggcc tggtaccgaa gcagctgcgc ttctgcagga 301 actacgtgga gatcatgccc agcgtggctg agggtgtcaa agcgggcatc caggagtgcc

361 agcaccagtt ccgaggccgg cgttggaact gcaccaccgt cagcaacagc ctggccatct

421 ttggccctgt tctggacaaa gccacccggg agtcagcctt tgtccatgcc atcgcctccg

481 ctggagtagc tttcgcagtg acacgctcct gtgcagaggg atcagctgct atctgtgggt

541 gcagcagccg cctccagggc tccccaggcg agggctggaa gtggggcggc tgtagtgagg 601 acattgaatt tggaggaatg gtctctcggg agtttgccga tgccagggag aaccggccgg

661 atgcccgctc tgccatgaac cgtcacaaca atgaggctgg gcgccaggcc atcgccagtc

721 acatgcacct caagtgcaaa tgccacgggc tatctggcag ctgtgaagtg aagacctgct

781 ggtggtcgca gccggacttc cgcaccatcg gggatttcct caaggacaag tatgacagtg

841 cctcggagat ggtggtagag aaacaccgag agtctcgtgg ctgggtggag accctgaggc

901 cacgttacac gtacttcaag gtgccgacag aacgcgacct ggtctactac gaggcctcac

961 ccaacttctg cgaacctaac cccgaaaccg gctccttcgg gacgcgtgac cgcacctgca

1021 atgtgagctc gcatggcata gatgggtgcg acctgttgtg ctgcgggcgc gggcataacg

1081 cgcgcactga gcgacggagg gagaaatgcc actgtgtttt ccattggtgc tgctacgtca 1141 gctgccagga gtgcacacgt gtctatgacg tgcacacctg caagtaggag agctcctaac

1201 acgggagcag ggttcattcc gaggggcaag gttcctacct gggggcgggg ttcctacttg

1261 gaggggtctc ttacttgggg actcggttct tacttgaggg cggagatcct acctgtgagg

1321 gtctcatacc taaggacccg gtttctgcct tcagcctggg ctcctatttg ggatctgggt

1381 tcctttttag gggagaagct cctgtctggg atacgggttt ctgcccgagg gtggggctcc 1441 acttggggat ggaattccaa tttgggccgg aagtcctacc tcaatggctt ggactcctct

1501 cttgacccga cagggctcaa atggagacag gtaagctact ccctcaacta ggtggggttc

1561 gtgcggatgg gtgggagggg agagattagg gtccctcctc ccagaggcac tgctctatct

1621 agatacatga gagggtgctt cagggtgggc cctatttggg cttgaggatc ccgtgggggc

1681 ggggcttcac cccgactggg tggaactttt ggagaccccc ttccactggg gcaaggcttc 1741 actgaagact catgggatgg agctccacgg aaggaggagt tcctgagcga gcctgggctc

1801 tgagcaggcc atccagctcc catctggccc ctttccagtc ctggtgtaag gttcaacctg

1861 caagcctcat ctgcgcagag caggatctcc tggcagaatg aggcatggag aagaactcag

1921 gggtgatacc aagacctaac aaaccccgtg cctgggtacc tcttttaaag ctctgcaccc

1981 cttcttcaag ggctttccta gtctccttgg cagagctttc ctgaggaaga tttgcagtcc 2041 cccagagttc aagtgaacac ccatagaaca gaacagactc tatcctgagt agagagggtt

2101 ctctaggaat ctctatgggg actgctagga aggatcctgg gcatgacagc ctcgtatgat

2161 agcctgcatc cgctctgaca cttaatactc agatctcccg ggaaacccag ctcatccggt

2221 ccgtgatgtc catgccccaa atgcctcaga gatgttgcct cactttgagt tgtatgaact

2281 tcggagacat ggggacacag tcaagccgca gagccagggt tgtttcagga cccatctgat

2341 tccccagagc ctgctgttga ggcaatggtc accagatccg ttggccacca ccctgtcccg

2401 agcttctcta gtgtctgtct ggcctggaag tgaggtgcta catacagccc atctgccaca 2461 agagcttcct gattggtacc actgtgaacc gtccctcccc ctccagacag gggaggggat

2521 gtggccatac aggagtgtgc ccggagagcg cggaaagagg aagagaggct gcacacgcgt

2581 ggtgactgac tgtcttctgc ctggaacttt gcgttcgcgc ttgtaacttt attttcaatg

2641 ctgctatatc cacccaccac tggatttaga caaaagtgat tttctttttt tttttttctt

2701 ttctttctat gaaagaaatt attttagttt atagtatgtt tgtttcaaat aatggggaaa 2761 gtaaaaagag agaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa (SEQ ID NO: 8)

Table 14: Human Wnt-3a nucleotide sequence tgtaagtgcc acgggctgtc gggcagctgc gaggtgaaga catgctggtg gtcgcaaccc gacttccgcg ccatcggtga cttcctcaag gacaagtacg acagcgcctc ggagatggtg gtggagaagc accgggagtc ccgcggctgg gtggagaccc tgcggccgcg ctacacctac ttcaaggtgc ccacggagcg cgacctggtc tactacgagg cctcgcccaa cttctgcgag cccaaccctg agacgggctc cttcggcacg cgcgaccgca cctgcaacgt cagctcgcac ggcatcgacg gctgcgacct gctgtgctgc ggccgcggcc acaacgcgcg agcggagcgg cgccgggaga agtgccgctg cgtgtttcac tggtgctgt (SEQ ID NO: 9)

Human nucleic acid sequences which encode Wnt-4, Wnt-7a, and Wnt-7b are shown Tables 15, 16, and 17, respectively. Human and mouse Wnt polypeptides function similarly in transformation assays. Accordingly, human or mouse Wnt polypeptides or nucleic acids are administered to mammals to therapeutically stimulate tubulogenesis. The amino acid and nucleotide sequences of Wnt polypeptides are known in the art, e.g., human Wnt-1 (GENBANK^® X03072) , human Wnt-2 (GENBANK^® X07876) , human Wnt-4 (GENBANK^® AAB30677) , human Wnt-7a (GENBANK^® 000755) , mouse Wnt-1 (GENBANK^® P04426) , mouse Wnt-2

(GENBANK^® P21552), mouse Wnt-3a (GENBANK^® P27467) , mouse Wnt-4 (GENBANK^® P22724 and M89787) , mouse Wnt-7a (GENBANK^® M89802) , and mouse Wnt-7a (GENBANK^® M89801) .

Kidney tubulogenesis is a multi-step process with a hierarchy of signaling systems. A permissive signal from the ureter to the mesenchyme triggers survival and tubulogenesis in the mesenchyme, signals from the mesenchyme to the ureter are required for proliferation and branching morphogenesis of the ureter. Other signaling systems within the ureter are required for local adhesion and proliferation, changes which may mediate branching morphogenesis, and within the mesenchyme, for tubulogenesis as evidenced by the role of Wnt-4. The data described herein indicate that Wnt-4 is sufficient to trigger tubulogenesis in isolated metanephric mesenchyme, whereas Wnt-11 which is expressed in the tip of the growing ureter is not. Wnt-4 signaling depends on cell contact and sulphated glycosaminoglycans . Wnt-4 is required for triggering tubulogenesis but not for later developmental events. The Wnt-4 signal can be replaced by other members of the Wnt gene family including Wnt-1, Wnt-3a, Wnt-7a and Wnt-7b. Further, dorsal spinal cord, which has been thought to mimic ureteric signaling in tubule induction, induces Wnt-4 mutant as well as wild-type mesenchyme suggesting that spinal cord derived signal (s) likely act by mimicking the normal mesenchymal action of Wnt-4. These results indicate that Wnt-4 is a key auto-regulator of the mesenchymal to epithelial transformation that leads to tubulogenesis and nephrogenesis .

Therapeutic administration of a Wnt polypeptide or agonist

Wnt polypeptides or agonists are useful to treat kidney disorders such as chronic renal insufficiency, end-stage chronic renal failure, glomerulonephritis, glomerulosclerosis, interstitial nephritis, pyelonephritis, kidney failure due to viral disease, kidney failure after transplantation. Wnt polypeptides are at least about 10 amino acids, usually about 20 contiguous amino acids, preferably at least 40 contiguous amino acids, more preferably at least 50 contiguous amino acids, and most preferably at least about 60 to 80 contiguous amino acids in length and have the biological activity of triggering tubulogenesis. For example, a Wnt polypeptide is at least 50% of the length of the corresponding naturally- occurring Wnt polypeptide and has the amino acid sequences of the amino-terminal half of the naturally- occurring polypeptide. Such peptides are generated by methods known to those skilled in the art, including proteolytic cleavage of the protein, de novo synthesis of the fragment, or genetic engineering, e.g., cloning and expression of a fragment of Wnt-encoding cDNA. Therapeutic compositions are administered in a pharmaceutically acceptable carrier (e.g., physiological saline) . Carriers are selected on the basis of mode and route of administration and standard pharmaceutical practice. A therapeutically effective amount of a composition (e.g., Wnt polypeptide or agonist) is an amount which is capable of producing a medically desirable result, e.g., tubulogenesis, in a treated animal. As is well known in the medical arts, dosage for any one animal depends on many factors, including the animal's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently (e.g., other Wnt polypeptides) is 0.1 to 100 mg/kg body weight. Administration is generally be parenterally, e.g., intravenously, subcutaneously, intramuscularly, or intraperitoneally. The compositions of the invention can be administered locally i.e., at the site of organ damage or systemically. For example, the route of delivery is by intravenous infusion, localized injection or implants. The polypeptides or agonists may be formulated so as to have a continual presence in the tissue during the course of treatment, e.g., by being covalently attached to a polymer such as polyethylene glycol (PEG) . Such continuous release formulations are administered at weekly intervals or at multiples of weekly intervals. Examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the polypeptide or agonist, which matrices are in the form of shaped films, or microcapsules . Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) as described by Langer et al . , 1981, J. Biomed. Mater. Res., 15: 167-277 and Langer, 1982, Chem. Tech., 12: 98-105 or poly (vinylalcohol) ) , polylactides (U.S. Pat. No.

3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al . , 1983, Biopolymers, 22: 547-556), non-degradable ethylene-vinyl acetate (Langer et al . , supra) , degradable lactic acid-glycolic acid copolymers, polylactate polyglycolate (PLGA) , and poly-D- (-) -3-hydroxybutyric acid (EP 133,988). While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid provide release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. Sustained-release Wnt compositions also include liposomally entrapped Wnt polypeptides or agonists. Liposomes containing Wnt compositions are prepared by methods known in the art, e.g., Epstein et al . , 1985, Proc. Natl. Acad. Sci. USA, 82: 3688-3692; Hwang et al . , 1980, Proc. Natl. Acad. Sci. USA, 77: 4030-4034; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. The compositions may also be administered directly to a tissue site, e.g., by biolistic delivery to an internal or external target site or by catheter into a body lumen. Therapeutic compositions are administered by retrograde perfusion of kidney via the ureter or other urine collecting lumens, e.g., using a catheter or perfusion apparatus, such as that described in U.S. Pat. No. 5,871,464.

Analogs, homologs, or mimetics of the above peptides may also be used to induce and promote kidney tubule formation in a post-natal mammal. Analogs can differ from the naturally-occurring Wnt polypeptides by amino acid sequence, or by modifications which do not affect the sequence, or both. Modifications (which do not normally alter primary sequence) include in vivo or in vi tro chemical derivitization of polypeptides, e.g., acetylation or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps, e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. To improve the solubility and therapeutic half-life of Wnt polypeptides, Wnt-Ig fusion proteins are produced. Methods of making Ig fusion proteins is well known in the art (e.g., as described in Current Protocols of Immunology, 1994, Coligan et al . , eds . , John Wiley & Sons, Inc., p. 10 . 19 . 1 - 10 . 19 . 11 ) .

To render the therapeutic peptides less susceptible to cleavage by peptidases, the peptide bonds of a peptide may be replaced with an alternative type of covalent bond (a "peptide mimetic"). Where proteolytic degradation of the peptides following injection into the subject is a problem, replacement of a particularly sensitive peptide bond with a noncleavable peptide mimetic renders the resulting peptide more stable, and thus more useful as a therapeutic. Such mimetics, and methods of incorporating them into polypeptides, are well known in the art. Similarly, the replacement of an L- amino acid residue with a D-amino acid is a standard way of rendering the polypeptide less sensitive to proteolysis. Also useful are amino-terminal blocking groups such as t-butyloxycarbonyl, acetyl, theyl, succinyl, methoxysuccinyl , suberyl, adipyl, azelayl, dansyl, benzyloxycarbonyl , fluorenylmethoxycarbonyl , methoxyazelayl , methoxyadipyl , methoxysuberyl , and 2,4,- dinitrophenyl . Peptides may be administered to a subject intravenously in a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are biologically compatible vehicles which are suitable for administration to an animal: e.g., physiological saline. Wnt polypeptides are generally administered in vivo to allow regeneration of kidney tissue in the context of the autologous organ. However, kidney tissue or dissociated cells (derived from kidney tissue or embryonic tissue) may be treated outside the body (i.e., ex vivo) and then transplanted back into the body from which it was derived or into a different mammal. In the case of ex vivo therapy, a damaged or diseased kidney is removed from an individual , treated with a Wnt polypeptide (or DNA encoding a Wnt polypeptide) and then transplanted into the same individual or a different individual .

Therapeutic administration of DNA encoding a Wnt polypeptide or agonist

Gene therapy for regeneration of kidney tissue is carried out by directly administering the claimed DNA to a mammal or by transfecting kidney cells, e.g., renal mesenchymal cells or endothelial cells, with Wnt-encoding DNA in vivo or ex vivo. Gene transfer into kidney tissue is carried out using known methods, e.g., bathing the tissue or cells in a solution containing Wnt-encoding

DNA. Alternatively, kidney tissue is perfused in vivo or explanted kidney tissue is perfused ex vivo, using a perfusion apparatus, such as that described in U.S. Pat. No. 5,871,464. After the cells are contacted with DNA, the cells or organ is transplanted into a recipient (or returned to the host from which it was removed) . If the cells in suspension, the cells are infused into the mammal to be treated.

To express a Wnt polypeptide in a kidney cell, a Wnt-encoding DNA is introduced into a target cell, e.g., a mesenchymal or epithelial kidney cell, of the mammal by standard vectors and/or gene delivery systems. For example, expression of exogenous Wnt DNA in an epithelial cell induces production and secretion of a Wnt polypeptide, which in turn, leads to tubulogenesis and kidney regeneration. Suitable gene delivery systems may include liposomes, receptor-mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses, adenovirus, and adeno-associated virus, among others. A therapeutically effective amount is an amount of the nucleic acid of the invention which is capable of producing a medically desirable result in a treated animal, e.g., tubulogenesis.

DNA or transfected cells may be administered in a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are biologically compatible vehicles which are suitable for administration to a mammal, e.g., physiological saline. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Dosages will vary, but a preferred dosage for intravenous administration of DNA is from approximately 10⁶ to 10²² copies of the DNA molecule. The compositions of the invention may be administered locally or systemically. As with other therapeutic compositions such as peptides, administration of a nucleic acid composition is generally be parenterally, e.g., intravenously. DNA is also administered by retrograde perfusion of kidney tissue using, e.g., a catheter. DNA may also be administered directly to the target site, e.g., by biolistic delivery to a kidney tissue or by an implantable device. Methods of delivering nucleic acids to kidney tissue are known in the art, e.g, those described by Sukhatme et al . in U.S. Pat. No. 5,869,230. Nucleic acids are expressed under the control of tissue-specific, e.g., kidney-specific, promoters such as the Pax-2 promoter, the cRET promoter, and the Hox b7 promoter. Promoter constructs for inducible and constitutive expression of heterologous sequences are well known in the art and commercially-available. For example, nucleic acids are expressed under the control of the cytomegalovirus (CMV) β-actin promoter for general constitutive expression.

Method of screening for compounds which increase Wnt expression

A screening assay to identify compounds which are capable of inducing or increasing Wnt polypeptide expression in kidney tissue of a post-natal mammal (i.e., non-embryonic cells) is carried out as follows. For example, a sample of kidney cells, e.g., cultured mesenchymal or epithelial cells, is incubated in the presence of a candidate compound. A sample of control cells is incubated in the absence of the compound. Each sample of cells is evaluated for the expression of a Wnt polypeptide, e.g, Wnt-4. To test for presence of the Wnt gene product, each sample of cells can be incubated with a Wnt-specific antibody and the cells evaluated for binding of the antibody by methods well known in the art, e.g., immunofluorescent staining. The amount of antibody binding correlates with the level of expression of the Wnt polypeptide. Wnt expression is also measured at the level of gene transcription. For example, Wnt transcripts can be measured by Northern blotting techniques using Wnt-specific DNA probes or by PCR using Wnt-specific DNA primers. A increase in the amount of Wnt gene expression in cells contacted with a candidate compound compared to the amount in untreated cells indicates that the candidate compound is capable of inducing or increasing the expression of a Wnt polypeptide in kidney cells (and inducing tubulogenesis) . The compound is tested in tissue or organ culture systems as described below to determine whether the compound triggers tubulogenesis. Mouse model of renal development

Mouse renal development is characterized by the continuous interaction of epithelial and mesenchymal compartments both of which are derived from the intermediate mesenchyme. These compartments are the nephric duct and its derivative, the ureter, and the nephrogenic mesenchyme which lies adjacent to these ducts. As a consequence of these interactions, three embryonic kidneys are laid down from anterior to posterior in time and space. While the initial organ, the pronephros is only a very transient structure established at 8-8.5 days post coitum (d.p.c), the mesonephros extends by posterior elongation of the nephric duct and subsequent tubule induction in the adjacent mesonephrogenic mesenchyme between 9 and 11 d p.c. Although forming elaborate tubules, the mesonephros of the male never becomes a functional organ but contributes to the ductal network of the rete testis. Metanephric development is initiated when a bud emerges from the nephric duct at the level of the hind limbs around 10.5 d.p.c. The ureteric duct subsequently invades the metanephric blastema which lies at the posterior end of the intermediate mesoderm. In a process repeated many times, mesenchymal cells condense around the tip of the ureter, i.e., bud, aggregate, epithelialize and undergo morphogenetic movements . Cellular differentiation occurs to form a major part of the nephron, the functional unit of the vertebrate kidney. The ureter continues to grow and to branch forming the collecting duct system of the mature organ. 7-10 days post partum, nephron formation ceases as the mesenchymal stem cells in the periphery of the kidney are exhausted. The role of Wnt-11, Wnt-4 and other Wnt family members in tubule induction was studied. Wnt-4, but not Wnt-11 was found to be able to induce tubule formation, suggesting that spinal cord mediated tubulogenesis reflects the normal mesenchymal function of Wnt-4 rather than that of a ureteric bud derived signal.

The following reagents and procedures were used to evaluate Wnt signalling in the developing kidney. Mice

Wnt-4 heterozygotes were derived and genotyped using known methods, e.g., that described by Stark et al., 1994, Nature 372:679-683. Embryos for kidney dissections were derived from matings of Swiss Webster (SW) wild-type animals or Wnt-4 heterozygotes . For timed pregnancies, plugs were checked in the morning after mating, noon was taken as 0.5 d.p.c. Cell lines

Cell lines which stably express various Wnt genes or LacZ were prepared using standard methods, e.g, that described by Pear et al . , 1993, Proc. Natl. Acad. Sci. USA 90: 8392-8396. For Wnt polypeptide expression, full- length cDNAs encoding Wnt-1 (van Ooyen and Nusse, 1984, Cell 39: 233-240), Wnt-3a (Roelink and Nusse, 1991, Genes Dev. 5: 381-388), Wnt-4, Wnt-5a, Wnt-7a, Wnt-7b (Gavin et al., 1990, Genes Dev. 4: 2319-2332), Wnt-11 (Kispert et al., 1996, Development 122:3627-3637) and lacZ were cloned into an expression vector, e.g, the retroviral expression vector pLNCX which confers expression of foreign genes under the control of the CMV promotor. Bosc23 packaging cells were transfected with recombinant DNA constructs. Viral supernatants were collected 48-72 h later and used to infect standard NIH3T3 cells. After 10 d of selection in G418, pools of cells were used for recombination experiments. 50,000 cells were plated in 50 μl of medium on polycarbonate filter and grown for 18-24 h at 37°C in 5% C0₂. Organ culture techniques

Metanephric kidneys from SW or Wnt-4 intercrosses were dissected in phosphate buffered saline (PBS) . To generate a preparation of dissociated kidney cells from embryonic or mature tissue, the tissue is dissected and enzymatically digested. For example, metanephric mesenchyme was dissected manually from the ureter (bud stage [10.75 d.p.c] to early T stage [11.5 d p.c.]), following a 2 min. incubation in 3% pancreatin/trypsin (GibcoBRL) in Tyrode's solution. In recombination experiments with wild-type mesenchymes, samples were pooled before being distributed to individual experiments. In experiments with Wnt-4 mutant embryos, metanephric mesenchyme from each kidney of the embryo was kept separate. The remainder of an embryo was used for genotyping by Southern analysis. In recombination experiments with dorsal spinal cord, metanephric mesenchyme from two kidneys was surrounded by two dissected pieces of dorsal spinal cord from the same embryo on a 1 μm polycarbonate filter (Costar) . For direct recombination experiments with Wnt-expressing cells, two mesenchymes were placed on top of modified NIH3T3 cells. For transfilter experiments, 50,000 cells in 50 μl medium were seeded on a 1 μm filter 18-24 h prior to the recombination. Cells were then covered with a 1 μm filter and two mesenchymes placed on this filter. Filters (4-6 mm in size) were supported by stainless steel grids on the surface of the culture medium (Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine,

1 x penicillin/streptomycin) . Medium was changed every 2 d. For studies of glycosaminoglycan dependence of tubule induction, the medium was supplemented with 30 mM NaCl0₃ after O h, 24 h and 48 h, respectively. In experiments concerning pore size dependence of induction, the pore size of the upper filter in the transfilter set-up was varied from 0.05 μm, 0.1 μm, 0.4 μm, 0.8 μm to 1 μm. For marker experiments, at least 6 specimens were processed. For in si tu hybridization analysis, filters were submerged in cold methanol for 10 seconds and then fixed in 4% paraformaldehyde in PBS overnight prior to stepwise transfer into methanol and storage at -20°C. For histological analysis, filters were fixed in Bouin's solution and stored in 70% ethanol at 4°C. Jn situ hybridization analysis In si tu hybridization analysis on whole mount cultures were performed using standard methods. Full- length cDNAs for WT-1 (Pritchard-Jones et al . , 1990, Nature 346:194-197), Pax-2 (Dressier et al . , 1990, Development 109:787-795), Pax-8 (Plachov et al . , 1990, Development 110:643-651), Wnt-4 (Gavin et al . , 1990, Genes Dev. 4:2319-2332) and E-cadherin (Ringwald et al . , 1987, EMBO J. 6:3647-3653) were labeled with Digoxigenin for whole mount detection. Histological analysis and documentation

Samples were dehydrated, embedded in wax and sectioned at 5 μm. Sections were dewaxed, rehydrated and stained with haematoxylin and eosin. Brightfield images of cultures and marker stainings were taken with a binocular on Kodak 64T slide film. Histological sections were photographed on the same film on a Leitz Axiophot. Slides were scanned and documented in Adobe Photoshop 4.0. Spinal cord mimics a mesenchymal signal for tubule induction

The identification of Wnt-4 as a mesenchymal signal essential for tubule formation provides a strategy for evaluating the role of spinal cord explants as heterologous inducers of kidney tubulogenesis. If the spinal cord mimics a ureteric signal upstream of Wnt-4, this signal would not rescue the mesenchymal requirement for Wnt-4 in tubulogenesis. To test this possibility, isolated metanephric mesenchyme from individual embryos derived from intercrosses between mice heterozygous for a likely null allele of Wnt-4 were cultured on a polycarbonate filter in direct contact with dorsal spinal cord from the same embryo . In the absence of spinal cord, all mesenchyme cultures rapidly degenerated as expected. Surprisingly, when cultured in the presence of spinal cord, mesenchyme from Wnt-4 mutant embryos developed as well as that of wild-type or heterozygous siblings (Table 1) .

Table 1: Induction of tubulogenesis in Wnt-4/Wnt-4 mutant metanephric mesenchyme by dorsal spinal cord

Exp # # Recombinants #induced/#total

+/+ Wnt-4/+ Wnt-4/Wnt-4

1 8 2/2 5/5 1/1

2 7 1/1 3/3 3/3

3 7 3/3 3/3 1/1 4 5 1/1 3/3 1/1

5 9 3/3 4/4 2/2

6 11 7/7 4/4 -

7 11 3/3 4/4 4/4 Total 58 20/20 26/26 12/12

Isolated metanephric mesenchyme was recombined with dorsal spinal cord from the same embryo on a nucleopore filter. Induction was monitored by bright field microscopy. Embryos of a total of seven litters were analyzed. Induction of tubulogenesis in wild-type and Wnt-4 mutant metanephric mesenchyme by dorsal spinal cord was analyzed as follows. Isolated metanephric mesenchyme and dorsal spinal cord from the same 11.5 d embryo were recombined on a nucleopore filter. After 48 h and 96 h, cultures were monitored as whole mounts using bright field microscopy; after 144 h, they were analyzed as histological sections. Induction of tubulogenesis in wild-type and Wnt-4/Wnt-4 mutant metanephric mesenchyme were indistinguishable. After 48 h, induction was visible as bright round zones of condensing mesenchyme. After 96 h, the zones of condensing mesenchyme had undergone epithelialization to form complex tubules. At 144 h, epithelial tubular structures and glomeruli indicated that full differentiation of induced tubules occurred in all recombinants.

The induction of tubulogenesis in Wnt-4 mutant mesenchyme indicates that spinal cord signaling acts by either mimicking the action of Wnt-4 itself, or a factor downstream of Wnt-4. Further, although Wnt-4 is expressed in the spinal cord, the observation that spinal cord from Wnt-4 mutants is capable of induction indicates that Wnt-4 expression in the spinal cord is not essential for this process, suggesting the involvement of other Wnts expressed in this tissue. Wnt polypeptides which are sufficient to trigger tubulogenesis

In order to investigate whether Wnt-4 is sufficient for tubulogenesis, and if this property is shared by other Wnts normally expressed in the spinal cord, NIH3T3 cell lines which stably express various Wnt genes were established. Direct recombinations were performed between Wnt-expressing cells and isolated wild- type metanephric mesenchyme .

Isolated metanephric mesenchyme from 2-3 11.5 d kidneys was placed on top of NIH3T3 cells expressing various Wnt genes. As a control, mesenchymes were placed on NIH3T3 cells expressing LacZ and were placed onto a filter without an underlying cell layer. Induction was scored after 6 d using the morphological appearance of the culture (as documented by brightfield microscopy) , and histological analysis of selected samples. For each cell type 2-3 independent experiments were performed.

Induction of tubulogenesis in isolated metanephric mesenchyme by NIH3T3 cells expressing various Wnt genes was evaluated as follows. Brightfield microscopy (24 h, 88 h) and histological analysis (144 h) of direct recombinations between NIH3T3 cells expressing Wnt genes and isolated metanephric mesenchyme. After 24 h, bright zones indicating induction were visible in recombinants between wild-type mesenchyme and Wnt-1, Wnt-3a, Wnt-4, Wnt-7a and Wnt-7b expressing cells. These condensing mesenchymal cells had epithelialized and formed tubular structures after 88 h. After 144 h highly elaborate tubular structures were apparent. In contrast, cells expressing Wnt-5a, Wnt-11, or as a control lacZ, respectively, did not support survival and differentiation of metanephric mesenchyme.

Co-cultures with Wnt-1, Wnt-3a, Wnt-4, Wnt-7a and Wnt-7b expressing cells developed on schedule with those induced by spinal cord and formed complex epithelial tubules with differentiated glomeruli at 144 h (Table 2) . In contrast, cells expressing Wnt-5a, Wnt-11 or a lacZ control did not support survival and differentiation of metanephric mesenchyme (Table 2) . Table 2 : Induction of tubulogenesis in isolated metanephric mesenchyme by NIH3T3 cells expressing various Wnt genes

Cell line #induced/#

Wnt-1 16/16

Wnt-3a 14/14

Wnt-4 14/14

Wnt-5a 0/12

Wnt-7a 12/12

Wnt-7b 11/12

Wnt-11 0/12

LacZ 1/14 mesenchyme 1/12

Wnt mRNA expression was comparable amongst the various lines. These data indicate that a subset of Wnt genes, which includes Wnt-4 and not Wnt-11, induces tubule formation. As all of these are expressed in the spinal cord at the time of assay, it is likely that these signals account for the robust inducing activity of the spinal cord. However, of these Wnt-4 is the only member which is actually expressed in and which is also required for mesenchymal aggregation. Wnt-4 triggers the complete program of tubular differentiation

In order to investigate whether Wnt-4 is sufficient to induce fully developed tubules in isolated metanephric mesenchyme, the induction properties of NIH3T3 cells expressing Wnt-4 were analyzed more carefully by assessing the differentiation state of the mesenchyme by histological and molecular criteria.

Histological analysis of tubule induction in isolated metanephric mesenchyme by NIH3T3 cells expressing Wnt-4 was evaluated as follows. NIH3T3 cells expressing Wnt-4 were recombined with isolated metanephric mesenchyme directly and in a transfilter setup. Cultures were analyzed by sectioning and histological staining after 24 h, 48 h, 96 h and 192 h of culture. Tubule induction in transfilter assays appeared slightly delayed compared to direct recombinations.

After 48 h, zones of condensed and aggregated mesenchyme were detected, and after 96 h, epithelial tubules were apparent. After 8 d in culture, fully differentiated tubular structures including glomeruli were detected. Tubule induction by spinal cord was demonstrated in the art-recognized system in which cells are cultured with polycarbonate filters of a certain pore size (e.g., the method described by Grobstein, 1956, Science 118:52- 55) . Wnt-4-expressing cells were seeded on one filter; these cells were separated from isolated mesenchyme by another filter of 1 μm pore size. Induction took place transfilter, though with a delay when compared with direct recombinants .

Transfilter cultures appeared less compact and flatter. Zones of condensed mesenchyme formed after 24 h, and aggregating mesenchyme and simple epithelial bodies appeared after 48 h. Epithelial tubules were seen after 96 h, and glomeruli were detected by 8 days. To verify that these morphological features reflected an underlying differentiation of the mesenchyme in response to Wnt-4, the temporal and spatial expression of a number of molecular markers was examined. Marker analysis of tubule induction in isolated metanephric mesenchyme by NIH3T3 cells expressing Wnt-4 were analyzed as follows. NIH3T3 cells expressing Wnt-4 were recombined with isolated metanephric mesenchyme in a transfilter set-up and scored for marker expression by in si tu analysis after 24 h, 48 h, 96 h and 192 h of culture, respectively. Expression of WT-1, Pax-2, Pax-8, Wnt-4 and E-cadherin, respectively, were in accordance with expression data from in vivo and in vi tro studies of tubular differentiation.

WT-1 was broadly expressed after 1 d refining to small intensely labeled foci by 8 days of culture. This expression profile parallels the expression of this gene during metanephric development which is first expressed in condensing mesenchyme, then in simple epithelial bodies before it is restricted to podocytes in the glomeruli. In the recombinants, WT-1 expression was detected in glomeruli after 8 d in agreement with the histological analysis. Like WT-1, Pax-2 is also broadly expressed after 1 d, but becomes restricted to epithelial bodies and is lost after 4 d reflecting initial expression in condensing metanephric mesenchyme, continuing expression in simple epithelial bodies and subsequent down-regulation as glomeruli start to differentiate. Wnt-4 is expressed in aggregating mesenchyme, in the epithelial bodies which they generate and is subsequently down-regulated as these mature into S-shaped bodies. Pax-8, a paired-box transcription factor, has a similar early expression to Wnt-4 which has been shown to depend on Wnt-4 activity. In cultures, Wnt-4 was transiently expressed between 24 h and 96 h, peaking at 48 h. Pax-8 expression extended longer in S- shaped bodies. E-cadherin, which is expressed in the proximal tubules in vivo, was present after 24 h and was maintained, consistent with the differentiation of epithelial tubules along the proximal distal axis.

These data indicate that tubulogenesis in isolated metanephric mesenchyme induced by Wnt-4 follows a similar progression to that observed in the metanephric kidney in vivo. At the stage at which the metanephric mesenchyme (T-stage of the ureter) was isolated, initial ureteric signaling had occurred, as evidenced by the condensation of mesenchyme around the tip of the ureteric bud. However, this alone is insufficient to support mesenchymal survival and tubulogenesis. In contrast, Wnt-4 expressing cells were sufficient to support these processes. In order to exclude that Wnt-4 only maintains Wnt-4 expression in the isolated mesenchyme, mesenchyme derived from 10.75 d.p.c. embryos was also analyzed. At this stage, the ureter bud had just emerged and the metanephric mesenchyme can first be identified. Wnt-4 expressing cells triggered the complete differentiation program as judged by brightfield observation (12 out of 12 cases) and by molecular criteria (Pax-8 induction in 8 out of 8 cases after 4 d of culture) . Wnt-4 signaling requires cell contact

Tubule induction in isolated metanephric mesenchyme was analyzed with respect to filter pore size. Experiments using the spinal cord as a heterologous inducer suggest a requirement for cell -cell contact as pore sizes below 0.1 μm, which prevent the extension of cytoplasmic processes, block induction. Pore size dependence of tubule induction by Wnt-4 expressing cells was tested as follows. NIH3T3 cells expressing Wnt-4 were recombined with isolated metanephric mesenchyme in a transfilter set-up with various pore sizes of the nucleopore filter. Induction was scored after 4 d by Pax-8 expression in whole mount in si tu analysis. Pore sizes of 0.1 μm and bigger supported full induction of metanephric mesenchyme, whereas 0.05 μm pore size reduced or abolished induction (Table 3) . Table 3 : Induction of tubulogenesis in isolated metanephric mesenchyme by NIH3T3 cells expressing Wnt-4 in transfilter assays with increasing pore size

Pore size # induced/# total

0.05 μm 3*/l3 0.1 μm 14/16

0.4 μm 14/14

0.8 μm 6/6 1 μm 3/3

^* In each of the specimen scored as induced, only 1-4 spots of Pax-8 expression were seen in contrast to 15-30 with all the other pore sizes.

Supernatants from Wnt-4 expressing cells alone did not induce tubulogenesis, suggesting that cell contact is required. Wnt-4 may act as an insoluble cell bound factor or it may associate with the extracellular matrix (ECM) . It is unlikely that Wnt-4 mediated induction occurs through a secondary, soluble factor. Wnt-4 signaling requires sulphated glvcosaminoglycans

Experiments were carried out to determine whether Wnt signaling for tubule induction depends on sulfated glvcosaminoglycans (GAG) s which might act as cofactors for binding the Wnt protein on the responsive cell . Accordingly, studies were undertaken to see evaluate whether the presence of 30 mM NaCl0₃ (a competitive inhibitor of sulfation of GAGs) affects tubule induction. NIH3T3 cells expressing Wnt-4 were recombined with isolated metanephric mesenchyme in a transfilter set-up with addition of 30 mM NaC10₃ in the medium. NaC10₃ was added to cultures at the start of transfilter culture, or 24 and 48 h later. As a control, chlorate was omitted completely. Induction was scored after 4 d by Pax-8 expression using whole mount in si tu hybridization analysis. Addition of 30 mM NaC10₃ after 24 h or 48 h of culture did not affect tubule induction compared to untreated controls, whereas administration of 30 mM NaCl0₃ at the beginning of the culture abrogated tubule induction completely (Table 4) .

Table 4 : Induction of tubulogenesis in isolated metanephric mesenchyme by NIH3T3 cells expressing Wnt-4 in presence of 30 mM NaClO^

30 mM NaC10₃ added after h in culture #induced/#total

0 h 0/19

24 h 12/19

48 h 14/17

12/15

When chlorate was added at 0 h, mesenchyme degenerated and Pax-8 expression was consequently negative. However, addition of chlorate after 24 h did not influence Pax-8 expression. Hence, GAGs are not involved in tubule maturation and differentiation. Tubule induction does, however, depend on sulfated GAGs in the first 24 h, the period essential for complete induction by the spinal cord.

The chlorate inhibition experiments define a critical period of 24 h for induction. Further differentiation, i.e. aggregation and epithelialization of mesenchymal cells is only initiated when a certain number of cells (a small community) has received the Wnt- 4 signal. At this time, mesenchymal development is independent of ureteric signaling.

Chlorate acts as a competitive inhibitor of sulphotransferases and inhibits the sulphation of glvcosaminoglycans . The inhibition studies point to a critical role of these ECM compounds in tubulogenesis. Numerous studies have shown that branching morphogenesis of the ureter as well as branching of other epithelia requires an intact ECM. Since presence of chlorate after 24 h does not influence tubulogenesis, GAGs do not seem to be involved in tubule maturation and differentiation. Tubule induction does, however, depend on sulfated GAGs in the first 24 h, the period essential for complete induction by the spinal cord. GAGs may act as co- receptors, facilitating presentation or increasing the local concentration of the ligand. Wnt-4 signaling as a trigger for tubulogenesis

In order to test whether Wnt-4 expressing cells can rescue a Wnt-4 mutant mesenchyme, direct recombination experiments were carried out in culture. Induction of tubulogenesis in wild-type and Wnt-4 mutant metanephric mesenchyme by NIH3T3 cells stably expressing Wnt-4 was evaluated as follows. Isolated metanephric mesenchyme was placed on top of NIH3T3 cells expressing Wnt-4 which were supported by a nucleopore filter. After 48 h and 96 h, cultures were monitored as whole mounts using bright field microscopy; after 144 h, the cultures were monitored as histological sections. Induction of tubulogenesis in wild-type and Wnt-4/Wnt-4 mutant metanephric mesenchyme by Wnt-4 expressing cells were indistinguishable. Wnt-4-expressing cells were equally efficient at inducing tubule formation in wild type or Wnt-4 mutant metanephric mesenchyme (Table 5) .

Brightfield microscopy and histological analysis of specimen After 6 d in culture revealed the full spectrum of tubular differentiation including glomerulus formation.

As with spinal cord mediated induction, Wnt-4 expression in the mesenchyme itself is not required for tubule formation, but supplying Wnt-4 in adjacent cells is sufficient to trigger the inductive process. These results suggest that whereas Wnt-4 plays an essential role in initial tubulogenesis, it may not be required for later morphogenesis of the tubule. As shown in Table 5, Wnt-1 expressing cells were also sufficient to trigger tubulogenesis in mesenchyme mutant for Wnt-4.

Table 5: Induction of tubulogenesis in Wnt-4/Wnt-4 mutant metanephric mesenchyme by NIH3T3 cells expressing Wnt-4 or Wnt-1

#Exp # Recombinants #induced/#total

+/+ Wnt-4/+ Wnt-4/Wnt-4

with NIH3T3 cells expressing Wnt-4:

4 42 7/7 18/18 17/17

with NIH3T3 cells expressing Wnt-1:

2 20 5/5 11/12 3/3

Mammalian kidney development

Metanephric development is a highly coordinated process characterized by a continuous interaction of the epithelial ureter and the surrounding metanephric mesenchyme. Classical organ culture experiments have pointed to the fact that these two compartments achieve coordinated development by use of reciprocal signaling systems. First, the metanephric blastema induces a bud from the adjacent nephric duct which invades and branches into the mesenchyme. This process appears to be mediated by GDNF which is secreted by the metanephric mesenchyme and sensed by the c-ret/GDNFRa receptor complex on the ureter. Next, the metanephric mesenchyme undergoes tubulogenesis upon a permissive stimulus from the ureter. Signals required for induction of tubulogenesis

In addition to Wnt-4, other Wnts may replace Wnt-4 activity in the mesenchyme. Using cell lines expressing various Wnt genes, Wnt-1, Wnt-3a, Wnt-7a, Wnt-7b, were shown to evoke tubulogenesis in isolated metanephric mesenchyme. The results described herein suggest a different interpretation of the use of kidney cultures to elucidate the nature of the ureteric signal involved in inducing the mesenchyme. Experiments which have used heterologous sources of tubule inducers, e.g., the spinal cord, may not have been investigating the nature of ureteric signaling, but rather the mesenchymal action of signals such as Wnt-4. At present, the exact nature of ureteric signaling remains obscure. A primary signal might be required for a sufficient length of time to allow auto-induction of the mesenchyme by Wnt-4. Alternatively, a secondary signal from the ureter tip might be necessary to induce Wnt-4 expression in aggregating mesenchyme. In contrast to earlier studies, the data presented herein indicate that Wnt-11 does not play a role as a ureteric signal for mesenchymal aggregation. In the present studies, tubulogenesis was not detected with cells expressing Wnt-11. Wnt-4 is a mesenchymal signal for tubulogenesis

Analysis of Wnt-4 mutants has demonstrated a critical role for Wnt-4 in kidney development. Homozygous pups die 24 h after birth due to small agenic kidneys consisting of undifferentiated mesenchyme intermingled with collecting duct tissue. Histological and marker analysis revealed that primary condensation of mesenchymal cells around the ureter tips as well as ureteric branching occurs normally. However, mutant kidneys quickly become growth retarded and the mesenchyme remains undif erentiated lacking pretubular cell aggregates and epithelial tubules. Since kidney size as well as cell death initially remain unaffected, proliferation is unlikely to be controlled by Wnt-4. Rather, the lack of Wnt-4 expression itself and of epithelial structures in the mutant mesenchyme indicates that Wnt-4 may autoinduce the epithelialization of condensed mesenchyme. Mesenchymally-derived Wnt-4 is not only required but also sufficient for induction of tubulogenesis in the mammalian kidney. Judging by histological and molecular markers, Wnt-4 can elicit the complete program of tubular differentiation in isolated metanephric mesenchyme. The activity of Wnt-4 contrasts with other factors thought to regulate mesenchymal development. For example, basic fibroblast growth factor (FGF) and epidermal growth factor (EGF) can both support mesenchymal survival but are not sufficient for tubulogenesis. Like Wnt-4, BMP-7 has been suggested to induce tubules, but loss-of-function studies indicate it is not essential for tubule formation in vivo as some glomeruli form in BMP7 mutants. In contrast, loss of Wnt-4 led to a complete absence of glomeruli.

Wnt-4 activity shows all the characteristics which have previously been ascribed to induction by dorsal spinal cord tissue. Signaling is cell-contact dependent. Below a certain pore size in the transfilter assay the formation of cellular processes which penetrate the filter pores is inhibited and isolated mesenchyme degenerates. Cell contact is required for induction of tubulogenesis, and Wnt proteins may interact with extracellular matrix (ECM) components.

Wnt-4 expression in the metanephric mesenchyme is initiated in the aggregating mesenchyme and maintained in the comma shaped bodies before it is downregulated in S- shaped bodies. Therefore, Wnt-4 likely has a later function in tubulogenesis. Table 15 : Human Wnt - 4 -encoding nucleic acid

TGCAAGTGTC ACGGGGTGTC AGGCTCCTGT GAGGTAAAGA CGTGCTGGCG AGCCGTGCCG CCCT CCGCC AGGTGGGTCA CGCACTGAAG GAGAAGTTTG ATGGTGCCAC TGAGGTGGAG CCACGCCGCG TGGGCTCCTC CAGGGCACT GTGCCACGCA ACGCACAGTT CAAGCCGCAC ACAGATGAGG ACCTGGTGTA CTTGGAGCCT AGCCCCGACT TCTGTGAGCA GGACATGCGC AGCGGCGTGC: TGGGCACGAG GGGCCGCACA TGCAACAAGA CGTCCAAGGC CATCGACGGC: TGTGAGCTGC TGTGCTGTGG CCGCGGCTTC CACACGGCGC AGGTGGAGCT GGCTGAACGC TOCAGCTGCA AATTCCACTG GTGCTTGTTC TTGAGTCGAC

SEQ ID NO : 10

Table 16 : Human Wnt - 7a-encoding nucleic acid

TGTAAGTGTC ACGGCGTGTC AGGCTCGTGC ACCACCAAGA CGTGCTGGAC CACACTGCCA CAGTTTCGGG AGCTGGGCTA CGTGCTCAAG GACAAGTACA ACGAGGCCGT TCACGTGGAG CCTGTGCGTG CCAGCCGCAA CAAGCGGCCC ACCTTCCTGA AGATCAAGAA GCCACTGTCG TACCGCAAGC CCATGGACAC GGACCTGGTG TACATCGAGA AGTCGCCCAA CTACTGCGAG GGGGACCCGG TGACCGGCAG TGTGGGCACC CAGGGCCGCG CCTGCAACAA GACGGCTCCC. CAGGCCAGCG GCTGTGACCT CATGTGCTGT GGGCGTGGCT ACAACACCCA CCAGTACGCC CGCGTGTGGC AGTGCAATTG TAAGTTCCAT TGGTGC

SEQ ID NO : 11 Table 17 : Human Wnt - 7b- encoding nucleic acid

GTAAAATGTC ACGGCGTGTC TGGCTCCTGC ACCACCAAAA CCTGCTGGAC CACGCTGCCC AAGTTCCGAG AGGTGGGCCA CCTGCTGAAG GAGAAGTACA ACGCGGCCGT GCAGGTGGAG GTGGTGCGGG CCAGCCGTCT GCGGCAGCCC ACCTTCCTGC GCATCAAACA GCTGCGCAGC TATCAGAAGC CCATGGAGAC AGACCTGGTG TACATTGAGA AGTCGCCCAA CTACTGCGAG GAGGACGCG3 CCACGGGCAG CGTGGGCACG CAGGGCCGTC TCTGCAACCG CACGTCGCCC GGCGCGGACG GCTGTGACAC CATGTGCTGC GGCCGAGGCT ACAACACCCΛ CCAGTACACC AAGGTGTGGC AGTGCAACTG CAAATTCCAC TGGTGCTGC^r." CTAG

SEQ ID NO : 12

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. What is claimed is:

Claims

1. A method of stimulating kidney tubule formation in a post-natal mammal, comprising administering to said mammal a substantially pure Wnt polypeptide or a Wnt agonist, wherein said Wnt polypeptide is not Wnt-11.

2. The method of claim 1, wherein said mammal is characterized as suffering from a kidney disorder.

3. The method of claim 1, wherein said mammal is an adult mammal.

4. The method of claim 2, wherein said disorder is chronic renal failure.

5. The method of claim 2, wherein said disorder is renal cell carcinoma.

5. The method of claim 2, wherein said disorder is polycystic kidney disease.

6. The method of claim 2, wherein said disorder is chronic obstructive uropathy.

7. The method of claim 2 , wherein said disorder is virus-induced nephropathy.

8. The method of claim 7, wherein said virus is HIV-1.

9. The method of claim 1, wherein said Wnt polypeptide is a Wnt-1 class polypeptide.

10. The method of claim 1, wherein said Wnt polypeptide is selected from the group consisting of Wnt- 3a, Wnt-4, Wnt-7a, and Wnt-7b.

11. The method of claim 1, wherein said Wnt polypeptide is Wnt-4.

12. The method of claim 1, wherein said Wnt agonist is HLDAT86.

13. The method of claim 1, further comprising administering a sulfated glycosaminoglycan.

14. The method of claim 1, wherein said Wnt polypeptide or Wnt agonist is administered locally to a renal tissue.

15. The method of claim 14, wherein said Wnt polypeptide or Wnt agonist administered by retrograde perfusion of said renal tissue.

16. The method of claim 1, wherein said Wnt polypeptide or Wnt agonist is administered ex vivo to an explanted renal tissue.

17. The method of claim 1, wherein said Wnt agonist is a peptide mimetic.

18. The method of claim 1, wherein said Wnt polypeptide has an amino acid sequence at least 85% identical to SEQ ID N0:1, 2, 3, 4, or 5, and wherein said Wnt polypeptide induces tubulogenesis.

19. A method of stimulating kidney tubule formation in a post-natal mammal, comprising administering to said mammal a substantially pure nucleic acid encoding a Wnt polypeptide or a Wnt agonist.

20. An ex vivo mammalian kidney comprising an substantially pure exogenous Wnt polypeptide.