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WO2000061630A1 - Methode pour stimuler la formation de tubules renaux - Google Patents

Methode pour stimuler la formation de tubules renaux Download PDF

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Publication number
WO2000061630A1
WO2000061630A1 PCT/US1999/007745 US9907745W WO0061630A1 WO 2000061630 A1 WO2000061630 A1 WO 2000061630A1 US 9907745 W US9907745 W US 9907745W WO 0061630 A1 WO0061630 A1 WO 0061630A1
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wnt
polypeptide
kidney
mesenchyme
mammal
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Andrew P. Mcmahon
Andreas Kispert
Seppo Vainio
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Harvard University
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Harvard University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • the Wnt polypeptide is Wnt-4
  • 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.
  • sulfated glycosaminoglycans are co-administered with the Wnt compositions.
  • the mammal to be treated is characterized as suffering from a kidney disorder.
  • 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.
  • the method is used to treat or prevent renal tubule epithelial cell degeneration associated with HIV-1 infection.
  • Wnt compositions are local or systemic.
  • 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.
  • 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.
  • a Wnt composition e.g, a substantially pure polypeptide or an isolated nucleic acid
  • 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.
  • 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.
  • 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.
  • the fragment contains at least residues 1- 180 of naturally-occurring Wnt-1 (SEQ ID NO:l).
  • 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • operably linked is meant able to promote transcription of an mRNA corresponding to a polypeptide-encoding sequence located downstream on the same DNA strand.
  • a Wnt polypeptide 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.
  • 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
  • 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.
  • 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
  • mice Wnt-3a (GENBANK ® P27467)
  • mouse Wnt-4 (GENBANK ® P22724 and M89787)
  • mouse Wnt-7a (GENBANK ® M89802)
  • 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.
  • 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.
  • Wnt-4 is a key auto-regulator of the mesenchymal to epithelial transformation that leads to tubulogenesis and nephrogenesis .
  • 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.
  • 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 is an amount which is capable of producing a medically desirable result, e.g., tubulogenesis, in a treated animal.
  • 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.
  • compositions of the invention can be administered locally i.e., at the site of organ damage or systemically.
  • 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) .
  • PEG polyethylene glycol
  • 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 .
  • sustained-release matrices examples 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.
  • 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.
  • 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.
  • 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.
  • enzymes which affect glycosylation e.g., mammalian glycosylating or deglycosylating enzymes.
  • 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 ) .
  • the peptide bonds of a peptide may be replaced with an alternative type of covalent bond (a "peptide mimetic").
  • a peptide mimetic an alternative type of covalent bond
  • 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.
  • mimetics, and methods of incorporating them into polypeptides are well known in the art.
  • 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.
  • 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.
  • kidney tissue or dissociated cells derived from kidney tissue or embryonic tissue
  • kidney tissue or dissociated cells 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.
  • 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 .
  • kidney tissue 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.
  • kidney cells e.g., renal mesenchymal cells or endothelial cells
  • 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
  • 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.
  • 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.
  • 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.
  • a target cell e.g., a mesenchymal or epithelial kidney cell
  • 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.
  • 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 10 6 to 10 22 copies of the DNA molecule.
  • the compositions of the invention may be administered locally or systemically.
  • 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.
  • tissue-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.
  • nucleic acids are expressed under the control of the cytomegalovirus (CMV) ⁇ -actin promoter for general constitutive expression.
  • CMV cytomegalovirus
  • a screening assay to identify compounds which are capable of inducing or increasing Wnt polypeptide expression in kidney tissue of a post-natal mammal 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • Wnt-11, Wnt-4 and other Wnt family members were 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.
  • 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
  • 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.
  • 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 ⁇ l of medium on polycarbonate filter and grown for 18-24 h at 37°C in 5% C0 2 . Organ culture techniques
  • Metanephric kidneys from SW or Wnt-4 intercrosses were dissected in phosphate buffered saline (PBS) .
  • PBS phosphate buffered saline
  • the tissue is dissected and enzymatically digested.
  • 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.
  • 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.
  • 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
  • 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.
  • Wnt-4 mutant mesenchyme 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
  • 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.
  • 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.
  • 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
  • NIH3T3 cells expressing Wnt-4 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.
  • 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.
  • 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 ⁇ m, 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.
  • 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
  • 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
  • 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) .
  • 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.
  • Table 5 Induction of tubulogenesis in Wnt-4/Wnt-4 mutant metanephric mesenchyme by NIH3T3 cells expressing Wnt-4 or Wnt-1
  • 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.
  • 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.
  • the metanephric mesenchyme undergoes tubulogenesis upon a permissive stimulus from the ureter. Signals required for induction of tubulogenesis
  • Wnt-4 In addition to Wnt-4, other Wnts may replace Wnt-4 activity in the mesenchyme.
  • 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.
  • 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.
  • 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.
  • a secondary signal from the ureter tip might be necessary to induce Wnt-4 expression in aggregating mesenchyme.
  • Wnt-11 does not play a role as a ureteric signal for mesenchymal aggregation.
  • tubulogenesis was not detected with cells expressing Wnt-11.
  • Wnt-4 is a mesenchymal signal for tubulogenesis
  • Wnt-4 mutants have 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.
  • 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.
  • FGF basic fibroblast growth factor
  • EGF epidermal growth factor
  • 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.
  • ECM extracellular matrix
  • 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

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Abstract

L'invention concerne une méthode destinée à stimuler la formation de tubules rénaux chez un mammifère après sa naissance, cette méthode consistant notamment à administrer à ce mammifère un polypeptide Wnt sensiblement pur ou un agoniste Wnt.
PCT/US1999/007745 1999-04-08 1999-04-08 Methode pour stimuler la formation de tubules renaux Ceased WO2000061630A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010018185A1 (fr) * 2008-08-14 2010-02-18 Hospital Clinic I Provincial De Barcelona Wnt1 en tant que biomarqueur de lésion rénale
US20140309157A1 (en) * 2011-08-04 2014-10-16 Caregen Co., Ltd. Wnt family-derived peptides and use thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5686289A (en) * 1993-10-08 1997-11-11 The University Of Michigan Method and compositions of a bioartificial kidney suitable for use in vivo or ex vivo
WO1998006747A2 (fr) * 1996-08-16 1998-02-19 Genentech Inc Utilisation de polypeptides wnt

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5686289A (en) * 1993-10-08 1997-11-11 The University Of Michigan Method and compositions of a bioartificial kidney suitable for use in vivo or ex vivo
WO1998006747A2 (fr) * 1996-08-16 1998-02-19 Genentech Inc Utilisation de polypeptides wnt

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010018185A1 (fr) * 2008-08-14 2010-02-18 Hospital Clinic I Provincial De Barcelona Wnt1 en tant que biomarqueur de lésion rénale
ES2341419A1 (es) * 2008-08-14 2010-06-18 Hospital Clinic I Provincial De Barcelona Wnt1 como biomarcador de daño renal.
ES2341419B1 (es) * 2008-08-14 2011-05-03 Hospital Clinic I Provincial De Barcelona Wnt1 como biomarcador de daño renal.
US20140309157A1 (en) * 2011-08-04 2014-10-16 Caregen Co., Ltd. Wnt family-derived peptides and use thereof
US9295629B2 (en) * 2011-08-04 2016-03-29 Caregen Co., Ltd. WNT family-derived peptides and use thereof

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