TRPC6 involved in glomerulonephritis

Abstract

Focal and segmental glomerulosclerosis (FSGS) is a kidney disorder of unknown etiology and up to 20% of patients on dialysis have this diagnosis. A large family with hereditary FSGS carries a missense mutation in the TRPC6 gene on chromosome 11q, encoding the ion channel protein Transient Receptor Potential Cation Channel 6. The missense mutation is a P112Q substitution, which occurs in a highly conserved region of the protein, enhances TRPC6-mediated calcium signals in response to agonists such as angiotensin II, and alters the intracellular distribution of TRPC6 protein. Previous work has emphasized the importance of cytoskeletal and structural proteins in proteinuric kidney diseases. Our findings suggest a novel mechanism for glomerular disease pathogenesis.

1 . A cell-free preparation of a mutant TRPC6 protein comprising a glutamine at residue 112 of TRPC6.  
     
     
         2 . The preparation of  claim 1  which is free of wild-type TRPC6 having a proline at residue 112 of TRPC6.  
     
     
         3 . A cell-free preparation of a polypeptide comprising at least six contiguous amino acid residues of a P112Q mutant of TRPC6, wherein the polypeptide comprises residue 112.  
     
     
         4 . The cell-free preparation of  claim 3  wherein the polypeptide comprises at least ten contiguous amino acid residues of a P112Q mutant of TRPC6.  
     
     
         5 . The cell-free preparation of  claim 3  wherein the polypeptide comprises at least twelve contiguous amino acid residues of a P112Q mutant of TRPC6.  
     
     
         6 . The cell-free preparation of  claim 3  wherein the polypeptide comprises at least thirteen contiguous amino acid residues of a P112Q mutant of TRPC6.  
     
     
         7 . The cell-free preparation of  claim 3  wherein the polypeptide comprises at least fifteen contiguous amino acid residues of a P112Q mutant of TRPC6.  
     
     
         8 . A cell-free preparation of a polynucleotide which encodes a human TRPC6 polypeptide of at least six contiguous amino acid residues, said polypeptide comprising a P112Q substitution.  
     
     
         9 . The cell-free preparation of  claim 8  wherein the polynucleotide comprises an origin of replication.  
     
     
         10 . The cell-free preparation of  claim 8  wherein the polynucleotide comprises a plasmid origin of replication.  
     
     
         11 . The cell-free preparation of  claim 8  wherein the polynucleotide comprises a viral origin of replication.  
     
     
         12 . The cell-free preparation of  claim 8  wherein the polypeptide comprises at least eight amino acid residues.  
     
     
         13 . The cell-free preparation of  claim 8  wherein the polypeptide comprises at least ten contiguous amino acid residues.  
     
     
         14 . The cell-free preparation of  claim 8  wherein the polypeptide comprises at least twelve contiguous amino acid residues.  
     
     
         15 . The cell-free preparation of  claim 8  wherein the polypeptide comprises at least fourteen contiguous amino acid residues.  
     
     
         16 . The cell-free preparation of  claim 8  wherein the polypeptide comprises at least sixteen contiguous amino acid residues.  
     
     
         17 . The cell-free preparation of  claim 8  wherein the polypeptide is a fusion of a first and a second portion, wherein the first portion is said at least six contiguous amino acids residues of human TRPC6, and said second portion comprises at least six contiguous amino acid residues of a protein which is not a human TRPC6.  
     
     
         18 . The cell-free preparation of  claim 8  wherein the polypeptide comprises a full-length human TRPC6.  
     
     
         19 . The cell-free preparation of  claim 8  which comprises the sequence shown in SEQ ID NO: 64, except at codon 112 where the codon comprises a C335A substitution.  
     
     
         20 . A cell culture comprising a human cell which comprises a polynucleotide which encodes a human TRPC6 protein comprising a P112Q substitution  
     
     
         21 . A cell-free preparation of an antisense oligonucleotide comprising at least 18 contiguous nucleotides which are complementary to a human TRPC6 coding sequence selected from the group consisting of wild-type and a P112Q mutant.  
     
     
         22 . The cell-free preparation of  claim 21  which is complementary to a region of the coding sequence comprising the initiating ATG codon.  
     
     
         23 . A method of inhibiting TRPC6 channels in the kidney of a glomerulonephritis patient, comprising: 
 administering an inhibitor of TRPC6 channels to the patient, whereby calcium influx is reduced.    
     
     
         24 . The method of  claim 23  wherein the inhibitor is 2-aminoethoxy diphenyl borate.  
     
     
         25 . The method of  claim 23  wherein the inhibitor is gadolinium.  
     
     
         26 . The method of  claim 23  wherein the inhibitor is SKF96365.  
     
     
         27 . The method of  claim 23  wherein the patient has Focal Segmental Glomerulosclerosis (FSG).  
     
     
         28 . The method of  claim 23  wherein the patient has Goodpasture's syndrome.  
     
     
         29 . The method of  claim 23  wherein the patient has an IgA nephropathy.  
     
     
         30 . The method of  claim 23  wherein the patient has IgM mesangial proliferative glomerulonephritis.  
     
     
         31 . The method of  claim 23  wherein the patient has Lupus nephritis.  
     
     
         32 . The method of  claim 23  wherein the patient has Membranoproliferative glomerulonephritis I.  
     
     
         33 . The method of  claim 23  wherein the patient has Membranoproliferative glomerulonephritis II.  
     
     
         34 . The method of  claim 23  wherein the patient has Membranoproliferative glomerulonephritis III.  
     
     
         35 . The method of  claim 23  wherein the patient has post-streptococcal glomerulonephritis.  
     
     
         36 . The method of  claim 23  wherein the patient has rapidly progressive (crescentic) glomerulonephritis.  
     
     
         37 . The method of  claim 23  wherein the patient has membranous nephropathy.  
     
     
         38 . The method of  claim 23  wherein the patient has diabetic nephropathy.  
     
     
         39 . A method of inhibiting expression of TRPC6 channels in the kidney of a glomerulonephritis patient, comprising: 
 administering to a kidney of the patient an antisense polynucleotide according to  claim 21  whereby said expression is inhibited.    
     
     
         40 . The method of  claim 39  wherein the antisense polynucleotide is administered to the glomeruli of the kidney.  
     
     
         41 . The method of  claim 39  wherein the antisense polynucleotide is administered to the tubules of the kidney.  
     
     
         42 . A method of identifying a subject at increased risk of developing Focal and Segmental Glomerulosclerosis (FSGS), comprising: 
 determining a sequence feature of a TRPC6 gene in a subject;    comparing the determined sequence feature of the gene of the subject to a reference wild-type TPRC6 gene;    identifying the subject as being at increased risk of developing FSGS if the sequence feature of the gene of the subject differs from the reference or identifying the subject as being at no demonstrable increased risk if the sequence feature of the gene of the subject does not differ from the reference.    
     
     
         43 . A method of identifying a person at increased risk of developing Focal and Segmental Glomerulosclerosis (FSGS), comprising: 
 determining a sequence feature of a TRPC6 protein in a subject;    comparing the determined sequence feature of the subject to a reference wild-type TPRC6 protein;    identifying the subject as being at increased risk of developing FSGS if the sequence feature of the protein of the subject differs from the reference or identifying the subject as being at no demonstrable increased risk if the sequence feature of the protein of the subject does not differ from the reference.    
     
     
         44 . The method of  claim 42  wherein the sequence feature is a non-synonymous difference with respect to the reference wild-type TPRC6 gene.  
     
     
         45 . The method of  claim 42  wherein the sequence feature is an adenine at nucleotide 335.  
     
     
         46 . The method of  claim 42  wherein the sequence feature is a codon which encodes glutamine at residue 112.  
     
     
         47 . The method of  claim 43  wherein the sequence feature is a glutamine at residue 112.  
     
     
         48 . The method of  claim 43  wherein the sequence feature is detected using an antibody which preferentially binds to a TPRC6 protein with a glutamine at residue 112 relative to a proline at residue 112.  
     
     
         49 . The method of  claim 42  wherein oligonucleotide probes are used to detect the sequence feature.  
     
     
         50 . The method of  claim 42  wherein an amplification reaction is used to determine the sequence feature.  
     
     
         51 . A container comprising a set of primer pairs for amplifying all or part of TRPC6 sequences, said set comprising a pair which amplifies exon 2 sequences.  
     
     
         52 . The container of  claim 51  wherein said set amplifies nucleotide 335 of the coding sequence of TRPC6.  
     
     
         53 . The container of  claim 51  wherein said set amplifies all thirteen exons of TRPC6.  
     
     
         54 . The container of  claim 51  wherein said set comprises primers TRPC6 — 2bF, TRPC6 — 2bR, or both TRPC6 — 2bF and TRPC6 — 2bR.  
     
     
         55 . The container of  claim 51  wherein said set comprises at least one primer as shown in FIG.  
     
     
         56 . The container of  claim 51  wherein said set comprises all primers as shown in  FIG. 9 .  
     
     
         57 . A probe for detecting a C335A mutation comprising: a single stranded or double stranded polynucleotide of at least 15 nucleotides which are complementary to a contiguous portion of human TRPC6 which comprises nucleotide 335 of the coding sequence.  
     
     
         58 . The probe of  claim 57  which further comprises a detectably labeled moiety  
     
     
         59 . The probe of  claim 57  which comprises C335A or its complement.  
     
     
         60 . A cell-free preparation of an antibody which preferentially binds to a TRPC6 protein with a P112Q substitution relative to a TRPC6 protein with a proline at residue 112.  
     
     
         61 . The cell-free preparation of  claim 60  wherein the antibody is a polyclonal antibody.  
     
     
         62 . The cell-free preparation of  claim 60  wherein the antibody is a monoclonal antibody.  
     
     
         63 . The cell-free preparation of  claim 60  wherein the antibody is a detectably labeled antibody.  
     
     
         64 . The cell-free preparation of  claim 60  wherein the antibody binds to the TRPC6 protein with a P112Q substitution at least two times more than it binds to the TRPC6 protein with a proline at residue 112.  
     
     
         65 . The cell-free preparation of  claim 60  wherein the antibody binds to the TRPC6 protein with a P112Q substitution at least four times more than it binds to the TRPC6 protein with a proline at residue 112.  
     
     
         66 . The cell-free preparation of  claim 60  wherein the antibody binds to the TRPC6 protein with a P112Q substitution at least five times more than it binds to the TRPC6 protein with a proline at residue 112.  
     
     
         67 . The cell-free preparation of  claim 60  wherein the antibody binds to the TRPC6 protein with a P112Q substitution at least ten times more than it binds to the TRPC6 protein with a proline at residue 112.  
     
     
         68 . The cell-free preparation of  claim 60  wherein the antibody binds to the TRPC6 protein with a P112Q substitution at least twenty times more than it binds to the TRPC6 protein with a proline at residue 112.  
     
     
         69 . A method of screening for candidate agents useful for treating FSGS, comprising the steps of: 
 contacting a wild-type or mutant form of TRPC6 protein with a test substance;    measuring activity of the form of TRPC6 protein;    identifying a test substance as a candidate agent for treating FSGS if it inhibits activity of the TRPC6 protein.    
     
     
         70 . The method of  claim 69  wherein the form of TRPC6 protein is in a cell and inward currents of ions into the cell are measured.  
     
     
         71 . The method of  claim 69  wherein the form of TRPC6 protein is in a cell and intracellular calcium concentrations in the cell are measured.  
     
     
         72 . The method of  claim 69  wherein the form of TRPC6 protein is in a cell and intracellular barium concentrations in the cell are measured.  
     
     
         73 . The method of  claim 69  wherein the form is wild-type.  
     
     
         74 . The method of  claim 69  wherein the form is mutant.  
     
     
         75 . The method of  claim 69  wherein the form is a P112Q substitution.  
     
     
         76 . A method of screening for candidate agents useful for treating glomerulonephritis, comprising the steps of: 
 contacting a wild-type or mutant form of TRPC6 protein with a test substance;    measuring activity of the form of TRPC6 protein;    identifying a test substance as a candidate agent for treating glomerulonephritis if it inhibits activity of the TRPC6 protein.    
     
     
         77 . The method of  claim 76  wherein the form of TRPC6 protein is in a cell and inward currents of ions into the cell are measured.  
     
     
         78 . The method of  claim 76  wherein the form of TRPC6 protein is in a cell and intracellular calcium concentrations in the cell are measured.  
     
     
         79 . The method of  claim 76  wherein the form of TRPC6 protein is in a cell and intracellular barium concentrations in the cell are measured.  
     
     
         80 . The method of  claim 76  wherein the form is wild-type.  
     
     
         81 . The method of  claim 76  wherein the form is mutant.  
     
     
         82 . The method of  claim 76  wherein the form is a P112Q substitution.  
     
     
         83 . A method of classifying a patient with Focal and Segmental Glomerulosclerosis (FSGS), comprising: 
 determining a sequence feature of a TRPC6 gene in a subject;    comparing the determined sequence feature of the gene of the subject to a reference wild-type TPRC6 gene;    identifying the subject as having a TRPC6 mutation if the sequence feature of the gene of the subject differs from the reference or identifying the subject as not having a TRPC6 mutation if the sequence feature of the gene of the subject does not differ from the reference.    
     
     
         84 . A method of classifying a patient with Focal and Segmental Glomerulosclerosis (FSGS), comprising: 
 determining a sequence feature of a TRPC6 protein in a subject;    comparing the determined sequence feature of the subject to a reference wild-type TPRC6 protein;    identifying the subject as having a TRPC6 mutation if the sequence feature of the protein of the subject differs from the reference reference or identifying the subject as not having a TRPC6 mutation if the sequence feature of the protein of the subject does not differ from the reference.    
     
     
         85 . The method of  claim 83  wherein the sequence feature is a non-synonymous difference with respect to the reference wild-type TPRC6 gene.  
     
     
         86 . The method of  claim 83  wherein the sequence feature is an adenine at nucleotide 335.  
     
     
         87 . The method of  claim 83  wherein the sequence feature is a codon which encodes glutamine at residue 112.  
     
     
         88 . The method of  claim 83  wherein oligonucleotide probes are used to detect the sequence feature.  
     
     
         89 . The method of  claim 83  wherein an amplification reaction is used to determine the sequence feature.  
     
     
         90 . The method of  claim 84  wherein the sequence feature is a glutamine at residue 112.  
     
     
         91 . The method of  claim 84  wherein the sequence feature is detected using an antibody which preferentially binds to a TPRC6 protein with a glutamine at residue 112 relative to a proline at residue 112.