(en)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.ApplicationNumber: US-201615170916-A
1.PublishNumber: US-2016376655-A1
2.Date Publish: 20161229
3.Inventor: WINN MICHELLE
PERICAK-VANCE MARGARET A.
VANCE JEFFERY M.

4.Inventor Harmonized: WINN MICHELLE(US)
PERICAK-VANCE MARGARET A(US)
VANCE JEFFERY M(US)

5.Country: US
6.Claims:
(en)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.

7.Description:
(en)RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 12/782,075, filed May 18, 2010, which is a continuation of U.S. patent application Ser. No. 11/716,050, filed Mar. 9, 2007, now U.S. Pat. No. 7,745,597, which is a divisional of U.S. patent application Ser. No. 11/417,113, filed May 4, 2006, now abandoned, which claims priority from U.S. Provisional Patent Application No. 60/677,825, filed May 5, 2005. The entire contents of these applications are incorporated herein by reference in their entirety.


GOVERNMENT SUPPORT
The invention of the present application was made using funds from the U.S. government. The U.S. government may therefore retain certain rights under the terms of grant 5K08-DK02815-04.


TECHNICAL FIELD OF THE INVENTION
This invention is related to the area of kidney disease. In particular, it relates to diagnosis and treatment and drug discovery for kidney disease.
BACKGROUND OF THE INVENTION
FSGS is a significant cause of end-stage renal disease world-wide and up to one-fifth of dialysis patients have this diagnosis (1, 2). The prevalence of FSGS is increasing yearly and the incidence is particularly high in the black population (1, 3). FSGS is a pathological entity in which the glomerulus is primarily targeted. Typical manifestations of FSGS include proteinuria, hypertension, renal insufficiency and eventual kidney failure. Our understanding of the pathogenesis of FSGS is incomplete and there are no consistently effective treatments.
Analysis of disease-causing mutations in hereditary FSGS and congenital nephrotic syndromes has provided striking new insights into the pathogenesis of nephrotic syndrome. The previous identification of at least three genes causing familial FSGS and hereditary nephrotic syndromes underscores the significant genetic heterogeneity in this disorder (4-6). These studies have highlighted the importance of abnormalities in the podocyte and the slit diaphragm of the glomerulus to the development of the severe proteinuria that characterizes the nephrotic syndrome.
Previously, we ascertained and characterized a large New Zealand family of British origin with autosomal dominant hereditary FSGS ( FIGS. 4 ) (7). The character of the disease in this family is particularly aggressive. Affected individuals typically present with high-grade proteinuria in their 3 rd or 4 th decade and approximately 60% progress to end-stage renal disease (ESRD). The average time between initial presentation and the development of ESRD is 10 years. A genomic screen performed on this kindred localized the disease-causing mutation to chromosome 11q (8). However, this genomic region is very large and contains hundreds of genes.
There is a continuing need in the art to identify genes and proteins which are associated with or causative of kidney disease, so that they can be used to more accurately and effectively diagnose and treat kidney disease.
SUMMARY OF THE INVENTION
The present invention has many aspects. A first aspect of the invention is a cell-free preparation of a mutant TRPC6 protein comprising a glutamine at residue 112 of TRPC6.
Another aspect of the invention is 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.
An additional aspect of the invention is 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.
In one embodiment of the invention a cell culture comprising a human cell is provided. The human cell comprises a polynucleotide which encodes a human TRPC6 protein comprising a P112Q substitution.
According to another aspect of the invention a cell-free preparation of an antisense polynucleotide is provided. The polynucleotide comprises 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.
An additional embodiment of the invention provides a method of inhibiting TRPC6 channels in a kidney of a glomerulonephritis patient. An inhibitor of TRPC6 channels is administered to the patient. Calcium ion influx is thereby reduced.
Yet another embodiment of the invention provides a method of inhibiting expression of TRPC6 channels in the kidney of a glomerulonephritis patient. An antisense polynucleotide is administered to a kidney of the patient. Expression of TRPC6 channels is thereby inhibited. The antisense polynucleotide comprises 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.
Still another aspect of the invention is a method of identifying a subject at increased risk of developing Focal and Segmental Glomerulosclerosis (FSGS). A sequence feature of a TRPC6 gene in a subject is determined. The determined sequence feature of the gene of the subject is compared to the sequence feature in a reference wild-type TPRC6 gene. The subject is identified as being at increased risk of developing FSGS if the sequence feature of the gene of the subject differs from the reference or if it matches a mutation associated with FSGS.
Another embodiment of the invention provides another method of identifying a person at increased risk of developing Focal and Segmental Glomerulosclerosis (FSGS). A sequence feature of a TRPC6 protein in a subject is determined. The determined sequence feature of the protein of the subject is compared to the sequence feature in a reference wild-type TPRC6 protein. The subject is identified as being at increased risk of developing FSGS if the sequence feature of the protein of the subject differs from the reference or if it matches a mutation associated with FSGS.
Another embodiment of the invention is a container comprising a set of primer pairs for amplifying all or part of TRPC6 sequences. The set comprises a pair which amplifies all or part of exon 2 sequences of TRPC6.
Still another aspect of the invention is a probe for detecting a C335A mutation. The probe comprises a single stranded or double stranded polynucleotide of at least 15 nucleotides which are complementary to a contiguous portion of a human TRPC6 gene which comprises nucleotide 335 of the coding sequence.
According to another embodiment of the invention a cell-free preparation of an antibody is provided. The antibody preferentially binds to a TRPC6 protein with a P112Q substitution relative to a TRPC6 protein with a proline at residue 112.
Another aspect of the invention is embodied by a method of screening for candidate agents useful for treating FSGS. A wild-type or mutant form of TRPC6 protein is contacted with a test substance. Activity of the form of TRPC6 protein is measured. A test substance is identified as a candidate agent for treating FSGS if it inhibits activity of the TRPC6 protein.
Another aspect of the invention is embodied by a method of screening for candidate agents useful for treating glomerulonephritis. A wild-type or mutant form of TRPC6 protein is contacted with a test substance. Activity of the form of TRPC6 protein is measured. A test substance is identified as a candidate agent for treating glomerulonephritis if it inhibits activity of the TRPC6 protein.
An additional embodiment of the invention is a method of classifying a patient with Focal and Segmental Glomerulosclerosis (FSGS). A sequence feature of a TRPC6 gene in a subject with FSGS is determined. The determined sequence feature of the gene of the subject is compared to the sequence feature in a reference wild-type TPRC6 gene. The subject is identified as having a TRPC6 mutation if the sequence feature of the gene of the subject differs from the sequence feature in the reference or if it matches a mutation associated with FSGS.
Still another embodiment of the invention is a method of classifying a patient with Focal and Segmental Glomerulosclerosis (FSGS). A sequence feature of a TRPC6 protein in a subject with FSGS is determined. The determined sequence feature of the gene of the subject is compared to the sequence feature in a reference wild-type TPRC6 protein. The subject is identified as having a TRPC6 mutation if the sequence feature of the protein of the subject differs from the sequence feature in the reference or if it matches a mutation associated with FSGS.
These and other embodiments which will be apparent to those of skill in the art upon reading the specification provide the art with reagents and methods for detection, diagnosis, therapy, prognosis, and drug screening pertaining to glomerulonephritis and FSGS.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1B . ( FIG. 1 . A) Minimal candidate region (MCR) of FSGS on human chromosome 11q. The area of interest is flanked by markers D11S1876 and D11S1339. The genetic distance from the centromere (cent) was obtained from the website at UCSC Genome Browser: ucsc.edu. Black squares indicate common alleles among affected individuals. Light squares indicate recombination. Individual 165 is the parent of individual 9044. Individual 9014 provides an example of the ancestral affected haplotype. All individuals represented are affected. The MCR is defined by individual 9044. Microsatellite markers in bold indicate original flanking markers. Gray box indicates candidate gene region. tel=telomere. ( FIG. 1 . B) Sequence chromatogram of exon 2 of TRPC6. The arrows highlight the C/A mutation.
FIGS. 2A-2F . Immunofluorescence staining and fluorescent in-situ hybridization of normal human renal cortical tissue for TRPC6. ( FIG. 2A ) Immunofluorescent staining of normal human renal cortical tissue with rabbit antibody against human TRPC6. In this representative photomicrograph, specific staining within a glomerulus (G) and the epithelium of surrounding tubules (T) is easily seen. ( FIG. 2B ) Negative control of an adjacent section also stained with the primary anti-TRPC6 antibody in the presence of the immunizing peptide. There is minimal non-specific staining. Bars in panels A and B=25 Fluorescent in situ hybridization (FISH) of TRPC6 mRNA in normal human renal cortex. ( FIG. 2C ) TRPC6 antisense probe generated from nucleotides 2301-3621 from TRPC6 mRNA [Accession number AJ006276]. ( FIG. 2D ) Hybridization with the corresponding TRPC6 sense probe. Scale bar=90 microns. ( FIG. 2E ) and ( FIG. 2F ) High power photomicrographs of the same sense and antisense probes. Scale bar=40 microns. Widespread expression of TRPC6 mRNA was detected throughout the kidney in both glomeruli and tubular epithelia. Background staining in panels D and F reflects autofluorescence from red blood cells trapped at the time of kidney harvest. Arrows highlight glomeruli. Asterisks are centered in renal tubules.
FIGS. 3A-3D . The TRPC6 P112Q mutant enhances the influx of calcium into cells via DAG-mediated and receptor-operated pathways. ( FIG. 3A ) Intracellular calcium concentrations were measured after OAG perfusion. TRPC6 P112Q transfected cells had significantly higher calcium concentrations than cells transfected with WT TRPC6. The peak influx [Ca 2+ ]i is depicted in the bar graph below the tracing. ( FIG. 3B ) Angiotensin-II induced intracellular calcium concentrations were measured. The peak influx [Ca 2+ ]i is depicted in the bar graph below the tracing. Again, TRPC6 P112Q transfected cells had significantly higher calcium concentrations than cells transfected with WT TRPC6. Each trace represents the mean value derived from 15-20 cells in a single experiment, each experiment was replicated three times, with similar results. The error bars represent standard deviation. ( FIG. 3C ) Whole cell current recordings of HEK 293 cells expressing either WT TRPC6 or TRPC6 P112Q protein. Considerable inward currents in normal Na + extracellular solution were observed in WT TRPC6 cells. However, inward currents were significantly larger in TRPC6 P112Q cells. When cells were perfused with 100 μM UTP, even larger inward currents were obtained. Cells expressing TRPC6 P112Q mutation conducted 2-3 times more current than the WT TRPC6 expressing cells as depicted in the bar graph next to the current recordings. ( FIG. 3D ) Surface expression experiments in HEK 293 cells transfected with TRPC6 protein. Biotinylation was used to quantitate cell surface expression of TRPC6 proteins. Cells expressing TRPC6-V5 or TRPC6 P112Q -V5 were incubated with biotin-SS reagent followed by pull-down with streptaviden agarose beads. Immunoblotting with an anti-V5 antibody of surface and whole cell lysates demonstrate increased surface expression of the TRPC6 P112Q compared to wild-type TRPC6 protein. Immunoblotting with an anti-transferrin receptor antibody (TfR) is located in the middle row and shows no difference in the surface expression of the constitutively active plasma membrane receptor (95 kD band). Each experiment was replicated four times, with similar results. Densitometry measurement in relative units are depicted in the bar graph next to the immunoblot (the results from all four replicants are quantitated). The error bars represent standard error.
FIG. 4 . Simplified version of pedigree for family 6530. Apparent autosomal dominant inheritance. Family structures have been changed for anonymity (1).
Definition of Disease Status:
Affected—Family members who had a renal biopsy demonstrating FSGS without evidence of other systemic diseases that have been known to cause FSGS, were on dialysis or had undergone renal transplantation.

Probably affected—Greater than or equal to 3+-4+proteinuria by qualitative urinalysis, in the absence of other systemic diseases likely to lead to proteinuria.

Unknown—Less than 1-2+proteinuria or 500 mg of proteinuria on 24-hour urine collection.

Unaffected—If they had no detectable proteinuria on qualitative urinalysis or were unrelated married-in spouses.

FIG. 5 . Proline 112 is highly conserved in evolution and is present in TRPC protein homologs from Mus musculus, Rattus norvegicus, Drosophila melanogaster, Caenorhabditis elegans and Cavia porcellus (SEQ ID NOS: 1-6). The arrow indicates the mutated proline. Comparative alignments with the mouse, human and rat sequences indicate that a homolog of the TRPC protein family is also present in zebrafish ( Danio rerio ) and pufferfish ( Fugu rubripes ) and that this proline is also conserved in these organisms (see the world wide web site at ensembl.org). Within the TRPC family, TRPC3, −6 and −7 all contain the conserved proline. TRPC4 and −5 have an alanine at this position, which, like proline, is a non-polar amino acid, while TRPC1 has a tyrosine at this position. TRPC2 is a pseudogene in humans. Genebank Accession Numbers: TRPC6 Homo sapiens , N_004612; Mus musculus , Q61143; Rattus norvegicus , N_446011; Caenorhabditis elegans , N_498881; Drosophila Melanogaster , N_476895; Cavia porcellus , CAC06051.
FIG. 6 . Western blot of protein extracts from HEK 293 cells transfected with empty vector (lane 1), TRPC6 P112Q (lane 2) or TRPC6 (lane3). Blots were incubated with specific antibodies against TRPC6 and actin. The immunoblot reveals three different bands and while there are different isoforms of TRPC6, there is evidence that this is glycosylation as TRPC6 is known to be heavily glycosylated (8).
FIG. 7 . OAG-stimulated barium transients were measured using the Fura-2 method in HEK293 cells transfected with WT TRPC6 (blue) or TRPC6P112Q (red). Fura fluorescence shifts in a similar pattern with barium and calcium, barium was used as a surrogate for calcium in these assays (9). As expected, OAG perfusion increased late barium transients in cells transfected with WT TRPC6. In contrast, barium influx was significantly enhanced in TRPC6P112Q-expressing cells exposed to OAG, reflecting dramatic exaggeration of intracellular cation concentration in cells expressing the mutant protein (Δ340/380 ratio TRPC6P112Q=1.30±0.7 vs. WT TRPC6=0.49±0.18; p=0.01). There are fluctuations of barium entry in the HEK 293 cells with the mutant construct. We postulate that this mutation causes disordered and dysregulated calcium entry. Y-axis is wavelength. The mean change in fluorescence is depicted by the bar graph. Error bars represent standard deviation.
FIG. 8 . Angiotensin II-induced barium transients were measured in HEK 293 cells transfected with AT1 receptor alone (green) or co-transfected with the AT1 receptor along with wild-type TRPC6 (blue) or TRPC6 P112Q (red). Upon exposure to angiotensin II, large barium transients were triggered in both WT- and TRPC6 P112Q -transfected cells and the amplitude of the initial barium signal tended to be higher in the TRPC6 P112Q cells. The Y-axis is wavelength. Moreover, the duration of the signal was prolonged in the TRPC6 P112Q cells (1054±282 seconds) compared with cells transfected with WT TRPC6 (271±121 seconds; p <0.01). The duration and amplitude are depicted in the bar graph below the tracing. Error bars represent standard deviation.
FIG. 9 . Table 1 showing primer sequences for TRPC6 (SEQ ID NO: 7-62).
FIG. 10 . Table 2 showing SNPs in mRNA.
FIG. 11 . Table 3 showing SNPs in gene.



DETAILED DESCRIPTION OF THE INVENTION
We have identified TRPC6 as a disease-gene causing hereditary FSGS. Because ion channels such as TRPC6 tend to be amenable to pharmacological manipulation, TRPC6 is identified as a useful therapeutic target in chronic kidney disease, including glomerulonephritis. Glomerulonephritis may be associated with any of the following conditions, without limitation: Focal Segmental Glomerulosclerosis (FSG), Goodpasture's syndrome, an IgA nephropathy, IgM mesangial proliferative glomerulonephritis, Lupus nephritis, Membranoproliferative glomerulonephritis I, Membranoproliferative glomerulonephritis II, Membranoproliferative glomerulonephritis III, post-streptococcal glomerulonephritis, rapidly progressive (crescentic) glomerulonephritis, membranous nephropathy, and diabetic nephropathy.
A cell-free preparation according to the present invention may comprise either polynucleotide or protein. Typically, the polynucleotide or protein will be extracted from the cells by breaking the cell membrane and optionally removing non-desired components. For example, proteins or nucleic acids can be removed if not desired using enzymatic degradation. Alternatively, desired proteins or nucleic acids can be purified using sequence-specific reagents, including but not limited to oligonucleotide probes, primers, and antibodies. Lysozyme and/or detergents and/or pressure can be used to break cells, for example. Techniques for isolating cell-free preparations are well known in the art, and any that are convenient can be used.
The P112Q mutant TRPC6 protein of the invention has a glutamine at residue 112 in place of the proline which is found in normal, non-affected humans. See SEQ ID NO: 63 to determine which residue is residue 112. See also SEQ ID NO: 65 which contains a PISS single nucleotide polymorphism at residue 15. Both of these are considered to be wild-type with respect to FSGS. Additional polymorphisms which do not affect calcium ion channel function, as described herein, may be present in addition to the P112Q substitution mutation. With respect to the TRPC6 gene, over 600 SNPs are known which do not affect the encoded protein. These include two synonymous SNPs at codons 904 and 561, as well as numerous SNPs in untranslated regions and in introns. See Tables 2 and 3. These, too, are considered wild-type with respect to FSGS.
Sequence features, according to the present invention, can be determined using any techniques which detect directly or indirectly a change in a protein or nucleic acid sequence. Thus, for example, if a mutation causes premature truncation, such a sequence feature can be detected by determining the size of the encoded mRNA or protein. Directly determining amino acid or nucleotide sequences can be used, and these techniques are well known in the art. Antibodies that are specific for a sequence feature can be used for probing mutant proteins. Probes and or primers that hybridize to wild-type or a particular mutation can be used. Any technique which detects such hybridization or the lack thereof can be used without limitation.
Polypeptides according to the present invention which contain residue 112 of TRPC6 protein can be used inter alfa for raising antibodies. Such polypeptides are typically less than full-length, 931 residue proteins. Preferably such residues are at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 19, 21, 23, or 25 residues in length. As an example, if the polypeptide is 6 residues in length, than it can comprises residues 112-117, 111-116, 110-115, 109-114, 108-113, or 107-112. Sufficient residues are desired to form a good immunogen or blocking antigen for use in assays. It may be desirable to conjugate or genetically fuse additional sequences to the polypeptide, for example, to boost immunogenicity, to enhance purification, to facilitate production or expression, or to facilitate detection. Any sequences as are convenient may be used for these or other purposes. The size of these additional sequences may vary greatly, but typically will be at least 2, 4, 6, or 8 amino acid residues in length. The polypeptide may contain either proline (wild-type) or glutamine (mutant) residue at position 112.
While particular nucleotide sequences which are found in humans are disclosed herein (see, e.g., SEQ ID NO: 64 and the SNPs shown in Tables 2 and 3) any nucleotide sequences may be used which encode a desired form of TRPC6. Thus non-naturally occurring sequences may be used. These may be desirable, for example, to enhance expression in heterologous expression systems of polypeptides or proteins of TRPC6. Computer programs for generating degenerate coding sequences are available and can be used for this purpose. Pencil, paper, the genetic code, and a human hand can also be used to generate degenerate coding sequences. For production purposes, it may be desirable to genetically engineer a coding sequence of a TRPC6 protein or polypeptide into an expression vector. Such vectors will typically contain an origin of replication, either of viral or plasmid origin. Such polynucleotides and/or vectors can be replicated and/or expressed in cell culture. Preferably the cultures will be of mammalian cells, and more preferably of human cells. However, other cell types may be advantageous for production, including but not limited to yeast cells, insect cells, and avian cells.
Antisense constructs, antisense oligonucleotides, RNA interference constructs or siRNA duplex RNA molecules can be used to interfere with expression of TRPC6. Typically at least 15, 17, 18, 19, or 21 nucleotides of the complement of TRPC6 mRNA sequence are sufficient for an antisense molecule. Typically at least 18, 19, 21, 22, or 23 nucleotides of TRPC6 are sufficient for an RNA interference molecule. Preferably an RNA interference molecule will have a 2 nucleotide 3′ overhang. If the RNA interference molecule is expressed in a cell from a construct, for example from a hairpin molecule or from an inverted repeat of the desired TRPC6 sequence, then the endogenous cellular machinery will create the overhangs. siRNA molecules can be prepared by chemical synthesis, in vitro transcription, or digestion of long dsRNA by Rnase III or Dicer. These can be introduced into cells by transfection, electroporation, or other methods known in the art. See Hannon, G J, 2002, RNA Interference, Nature 418: 244-251; Bernstein E et al., 2002, The rest is silence. RNA 7: 1509-1521; Hutvagner G et al., RNAi: Nature abhors a double-strand. Curr. Opin. Genetics & Development 12: 225-232; Brummelkamp, 2002, A system for stable expression of short interfering RNAs in mammalian cells. Science 296: 550-553; Lee N S, Dohjima T, Bauer G, Li H, Li M-J, Ehsani A, Salvaterra P, and Rossi J. (2002). Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nature Biotechnol. 20:500-505; Miyagishi M, and Taira K. (2002). U6-promoter-driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells. Nature Biotechnol. 20:497-500; Paddison P J, Caudy A A, Bernstein E, Hannon G J, and Conklin D S. (2002). Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes & Dev. 16:948-958; Paul C P, Good P D, Winer I, and Engelke D R. (2002). Effective expression of small interfering RNA in human cells. Nature Biotechnol. 20:505-508; Sui G, Soohoo C, Affar E-B, Gay F, Shi Y, Forrester WC, and Shi Y. (2002). A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA 99(6):5515-5520; Yu J-Y, DeRuiter S L, and Turner D L. (2002). RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA 99(9):6047-6052.
Antisense or RNA interference molecules can be delivered in vitro to cells or in vivo, e.g., to tumors of a mammal. Typical delivery means known in the art can be used. For example, delivery to a diseased kidney can be accomplished by direct intrarenal injections. Other modes of delivery can be used without limitation, including: intravenous, intramuscular, intraperitoneal, intraarterial, local delivery during surgery, endoscopic, subcutaneous, and per os. Vectors can be selected for desirable properties for any particular application. Vectors can be viral or plasmid. Adenoviral vectors are useful in this regard. Tissue-specific, cell-type specific, or otherwise regulatable promoters can be used to control the transcription of the inhibitory polynucleotide molecules. Non-viral carriers such as liposomes or nanospheres can also be used.
Because the channel activity of the disease-associated variant appears to cause increased calcium channel activity, inhibitors of such channel activity can be used therapeutically. Any inhibitor known in the art can be used, including but not limited to 2-aminoethoxy diphenyl borate, gadolinium, and SKF96365. Such inhibitors may have a therapeutic benefit in glomerulonephritis, whether or not the patient carries the P112Q variant. Thus a patient with any glomerulonephritis may be treated with inhibitor. Such patients include those with Focal Segmental Glomerulosclerosis (FSG), Goodpasture's syndrome, an IgA nephropathy, IgM mesangial proliferative glomerulonephritis, Lupus nephritis, Membranoproliferative glomerulonephritis I, Membranoproliferative glomerulonephritis II, Membranoproliferative glomerulonephritis III, post-streptococcal glomerulonephritis, rapidly progressive (crescentic) glomerulonephritis, membranous nephropathy, and diabetic nephropathy.
Additional calcium channel activity inhibitors can be screened using assays described in the examples below. Cells transfected with a coding sequence for the P112Q variant or cells transfected with a coding sequence for the wild-type TRPC6 can be used as test cells. Ion currents or intracellular calcium accumulation can be measured. The effects of various test substances on the ion currents or intracellular calcium accumulation can be determined and inhibitors thereby identified. Inhibitors are candidate therapueutic agents for treating glomerulonephritis.
Because FSGS associated with the TRPC6 mutation first manifests itself in adults, the identification of a mutation in TRPC6 can be used to predict which children or young adults are likely to manifest the disease. This is especially true among family members of affected individuals. Similarly, identifying family members who do not carry a mutation can also be useful so that anxiety and monitoring and preventive steps can be diminished. Any altered sequence in the TRPC6 gene or protein relative to normal unaffected controls will suggest an increased risk. Further functional tests of the mutant protein may be used to confirm a phenotype of any new mutations identified. Disease causing mutations will cause channel activity similar to that of the P112Q mutation, i.e., increased calcium ion influx into cells. A mutation can be detected either at the nucleic acid or at the protein level. As indicated above, synonymous mutations are unlikely to cause any changed phenotype associated with disease.
Primer pairs are provided in a single divided or undivided container. Each primer may be packaged separately or in pairs. Similarly, each pair may be packaged separately or in sets. Primers are useful for amplifying regions of TRPC6, especially exon 2, so that mutations can be identified in test samples. Preferably the primers include a pair which amplifies a segment containing codon 112 of TRPC6. Primers can be allele specific, e.g., they will amplify only in the presence of a specific allele, or they may amplify more than one allelic form. An allele- specific primer might hybridize, e.g., to a nucleotide at position 335, either a Cytosine or an Adenine. Probes similarly can be allele specific or not. Probes typically contain a readily detectable moiety, such as a fluorescent, bioluminescent, enzymatic, or radioactive moiety. Probes and primers are typically at least 12, 14, 16, 18, 20, 22, or 25 nucleotides in length.
As indicated above, antibodies can be used to detect TRPC6 proteins in general, or particular variants. Antibodies can be selected to preferentially bind to particular variants relative to wild-type. The amount of the binding preference may be at least two-fold, at least five-fold, at least ten-fold, or at least twenty-fold. One antibody which is particularly useful is one which preferentially binds to the P112Q variant relative to the wild-type. An antibody with the converse specificity may also be diagnostically useful. Antibodies according to the invention can be polyclonal or monoclonal. Methods of making both types of antibodies are well known in the art. Typically generating antibodies of either type begins with immunization of a sheep, rabbit, mouse, goat, etc., with a specific immunogen which is enriched for the antigen or epitope to which antibodies are desired. Adjuvants may also be used to enhance the immune response to the immunogen. Antibodies can be labeled with a moiety which facilitates detection. Such moieties include enzymatic, radioactive, fluorescent, and luminescent moieties.
Determining the presence of a mutant form of TRPC6 may be useful, as described above, to identify affected individuals prior to the manifestation of symptoms. In addition, identification of mutation carriers may be useful in assigning patients to treatment regimes. For example, a patient with an P112Q variant is an excellent candidate for receiving TRPC6 inhibitor therapy. In addition, identification of a TRPC6 mutation can be used during clinical trials to stratify patients for drug testing.
Mutations in several other proteins have been identified in familial nephrotic syndrome and hereditary FSGS. Nephrin (NPHS1), the cause of Finnish nephropathy, is a protein of unknown function that localizes to the glomerular slit diaphragm and appears to form a “zipper-like structure” (20). Podocin (NPHS2) appears to anchor elements of the slit diaphragm to the cytoskeleton (21). Lastly, mutations in alpha-actinin 4 (ACTN4) may alter functions of the actin cytoskeleton in the podocyte (6, 22). CD2-associated protein (CD2AP) has been implicated in glomerular function on the basis of mouse studies (23). CD2AP also appears to have important interactions with nephrin and podocin at the slit diaphragm.
TRP channels have become the object of intense interest as their role in diverse biological functions emerge. They have been associated with cell growth, ion homeostasis, mechanosensation and PLC-dependent calcium entry into cells. Interestingly, calcium as a second messenger affects many of these same cellular functions. Although the applicants do not wish to be bound by any particular theory or mechanism of operation, the exaggerated calcium signaling conferred by the TRPC6 P112Q mutation may disrupt glomerular cell function or may cause apoptosis (24). Moreover, the mutant TRPC6 P112Q protein may amplify injurious signals triggered by ligands such as angiotensin II that promote kidney injury and proteinuria.
Clinical manifestations of renal disease do not appear until the 3 rd decade in individuals with the TRPC6 P112Q mutation. This contrasts with Finnish nephropathy and steroid-resistant nephrotic syndrome, whose sufferers typically develop proteinuria in utero or at birth (5). The delay of onset in those carrying the TRPC6 P112Q mutation may reflect the difference between these recessive disorders and the autosomal dominant mechanism of inheritance in our New Zealand family; the presence of one normal TRPC6 allele may postpone the onset of kidney injury. Patients with autosomal dominant FSGS due to mutations in the ACTN4 gene also have a delayed onset of kidney disease.
The above disclosure generally describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.
Example 1
Identification of Altered Gene in Affected Kindred
Haplotype analyses reduced the minimal candidate region to an approximate 2.1 centiMorgan (cM) area defined by critical recombination events at D11S1390 and D11S1762 ( FIG. 1A ). This region contains several known genes as well as multiple novel and predicted genes which were systematically screened for mutations by direct sequencing. After examination of 42 other candidate genes, TRPC6 (GenBank Accession No. NP_004612) emerged as a candidate based on reports of detection of TRPC6 mRNA in the kidney (9, 10). We therefore sequenced each of the 13 exons of the TRPC6 gene along with their intron/exon boundaries. Primer sequences are provided ( FIG. 9 ). As shown in FIG. 1B , we discovered a missense mutation (C335A) in exon 2, from affected individuals, causing a proline to glutamine substitution at position 112 within the first ankyrin repeat of the TRPC6 protein. This variant was present in all of the affected individuals (20 affected; 1 probably affected) in our kindred and there were no non-penetrant carriers. The change was not found in any of the public single nucleotide polymorphism (SNP) databases. Furthermore, we found no evidence of the substitution in 614 chromosomes screened from a group of Caucasian controls without known renal disease, 33 of whom were from New Zealand. The allele frequencies from all markers used for linkage in this kindred and the New Zealand controls are similar to those in the other Caucasian controls. Proline 112 is highly conserved in evolution and is present in TRPC protein homologues from multiple species ( FIG. 5 ).
Example 2
Immunohistological Determination of Protein Expression and FISH Determination of mRNA Expression
Our previous finding that familial FSGS does not recur in affected patients after renal transplantation indicates a critical role for the kidney in disease pathogenesis (11). While expression of TRPC6 mRNA has been reported in multiple tissues including the kidney, its distribution in kidney is not clear (9, 10). Therefore, to define the spatial distribution of TRPC6 protein expression in human kidney, we performed immunohistochemistry of normal human renal cortex with rabbit antibody raised against a specific human TRPC6 peptide ( FIGS. 2A and 2B ). Immunofluorescence staining revealed TRPC6 expression throughout the kidney in glomeruli and tubules. This is consistent with a recent study detecting TRPC6 mRNA in isolated glomeruli (12). Expression of TRPC6 in glomeruli is particularly noteworthy as abnormal podocyte function appears to be a final common pathway in a variety of proteinuric kidney diseases (13). To verify these immunofluorescence findings, we carried out fluorescent in situ hybridization (FISH) in human kidney sections ( FIGS. 2C-2F ). These studies confirmed diffuse expression of TRPC6 mRNA in glomeruli and tubules in a pattern that is virtually identical to that seen with anti-TRPC6 antibody staining.
Example 3
Mutation Activates Channel Activity
To determine the effect of the P112Q mutation on TRPC6 function, we studied HEK 293 cells (human kidney cells) transfected with mutant (TRPC6 P112Q ) or wild-type (WT) TRPC6 (14). The WT TRPC6 was cloned from a human kidney cDNA library. On Western blots, the abundance and mobility of the P112Q mutant was comparable to that of WT TRPC6 ( FIG. 6 ). Diacylglycerol (DAG) is a potent activator of TRPC6 (15). We therefore measured the intracellular calcium concentration ([Ca +2 ]i) using Fura fluorescence in HEK 293 cells expressing either the WT or TRPC6 P112Q after exposure to the DAG analogue OAG (1-oleoyl-2-acetyl-sn-glycerol). OAG perfusion increased late Ca +2 transients in cells transfected with WT TRPC6 as expected ( FIGS. 3A and FIG. 7 ). Peak intracellular concentrations were significantly higher in cells expressing the TRPC6 P112Q compared with WT controls ([Ca 2+ ]i TRPC6 P112Q =181±25 nM vs. [Ca 2 ]i WT TRPC6=106±15 nM; p<0.05).
Example 4
Mutation Enhances Receptor-Operated Calcium Signaling
Angiotensin II acting through its AT1 receptor plays a critical role in the generation of proteinuria and progression of kidney injury in a number of kidney diseases including FSGS (16). AT1 receptors, coupled to heterotrimeric G nucleotide-binding proteins, activate phospholipase C-beta isoforms that hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP 2 ). This triggers production of inositol 1,4,5-trisphosphate (InsP 3 ) and diacylglycerol (DAG) releases internal calcium stores and activates Ca +2 entry, respectively (17). We examined whether the P112Q mutation would affect angiotensin II-dependent (i.e., receptor-operated) calcium signaling. HEK 293 cells were co-transfected with the AT1 angiotensin receptor (AT-YFP) and either WT TRPC6 or TRPC6 P112Q . [Ca +2 ], changes were measured after exposure to angiotensin II ( FIGS. 3B and FIG. 8 ); similar to the OAG experiments, the peak angiotensin II-stimulated [Ca +2 ] i was higher in cells expressing the mutant protein compared with WT controls ([Ca 2+ ]i TRPC6 P112Q =640±66 nM vs. [Ca 2+ ]i WT TRPC6=357±46 nM; p<0.05). To establish that the P112Q mutation is not isolated to TRPC6 channels, we introduced the analogous mutation into the TRPC3 channel. Our results demonstrate the same augmented Ca +2 entry observed with the TRPC6 P112Q mutation.
Example 5
Mutation Increases Surface Expression of TRPC6
We also evaluated the subcellular localization of the mutant TRPC6 protein by performing surface biotinylation experiments ( FIG. 3D ). These confirmed that the relative distribution of TRPC6 P112 Q protein in the plasma membrane was significantly greater than the wild-type protein (densitometry—WT TRPC6=1210.33 vs. TRPC6 P112Q =23126.67 units; p=0.05). Because the transferrin receptor (TfR) is constitutively expressed on the cell surface, we probed the cell surface fraction with an antibody to TfR as a control. No difference was found in the surface expression of TfR in cells transfected with either WT TRPC6 or TRPC6 P112Q . Likewise, we probed the cell surface fraction with antibody to the cytosolic protein actin and found no non-specific labeling of intracellular proteins. Our findings are in accordance with reports by others (18, 19). This enhanced cell surface expression of TRPC6 P112Q protein suggests a mechanism of exaggerated calcium signaling and flux.
Example 6
Materials and Methods
Ascertainment and Diagnostic Criteria
The ascertainment and clinical diagnosis are as previously described (1). Family 6530 was initially identified by the Department of Nephrology, Christchurch Hospital, Christchurch, New Zealand. All available renal biopsies and biopsy reports were independently reviewed by D.N.H. Evaluation of the family included a complete family history and an assay of serum creatinine and urinalysis where appropriate. Asymptomatic individuals were examined for proteinuria with qualitative urinalysis. The Duke University Medical Center (Durham, N.C.) Institutional Review Board and the Canterbury Ethics Committee approved this project and all participants gave signed informed consent prior to data and DNA collection.
Haplotype Analysis
Haplotype analysis was performed as previously described to identify critical recombination events (2). The haplotypes were constructed via visual inspection and by SIMWALK2 computerized haplotyping algorithm. Genetic and physical map distances as well as marker order were obtained from public databases (websites at genome.cse.ucsc.edu and research.marshfieldclinic.org/genetics/).
DNA Isolation and Genotyping
Genomic DNA was isolated from peripheral blood through the Center for Human Genetics, Duke University Medical Center using PureGene™ (Gentra). Genotyping was carried out as described by Vance et al., (3). Microsatellite markers within the area of interest were identified from a variety of sources, including, ensembl.org, and genome.cse.ucsc.edu. A Hitachi FMBIOII was used for detection, data was processed using Biolmage® and databased using Pedigene® (4).
TRPC6 Sequencing and Mutation Detection
TRPC6 exons were amplified using the polymerase chain reaction (PCR) with primers that were designed using the Primer 3 design software (website at genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) from known genomic sequence (website at genome.cse.ucsc.edu) using standard PCR protocols. Both strands of DNA were sequenced. Targeted sequence included all exons and 25-50 base pairs of the intronic sequence surrounding each exon. PCR products were purified using the QIAquick PCR purification kit (Qiagen). Sequencing reactions were performed using BigDye® Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems) and purified using Edge Biosystems Gel Filtration Columns (Edge Biosystems). Sequencing was carried out using the ABI 3730 DNA Analyzer (Applied Biosystems) and analyzed using Sequencher DNA sequencing analysis software (Gene Codes Corporation).
Immunofluorescent Staining
Normal human renal tissue was embedded in gelatin, snap frozen in liquid-nitrogen- cooled 2-methylbutane, and stored at −85° C. Frozen sections were cut at 4 μm, air dried, and fixed in acetone. For immunofluorescent staining, sections were incubated for 30-min at 25° C. with rabbit anti-TRPC6 (Chemicon) at a dilution of 1:50 and a mouse anti-synaptopodin monoclonal antibody (Progen Biotechnik GmBH) at 1:64 in phosphate-buffered saline, pH 7.4 (PBS) supplemented with 1% bovine serum albumin (PBS/BSA). The TRPC6 antibody was raised against a 16-amino-acid peptide that is identical to the human TRPC6 sequence in 15 of 16 residues (Mouse Peptide—RRNESQDYLLMDELG; SEQ ID NO: 68). The immunizing peptide does not align with any amino acid sequence other than TRPC6 according to a protein BLAST search (website at ncbi.nlm.nih.gov) of the human genome and according to printed material from the companies supplying the antibody. Additionally, we have found that this antibody does not recognize recombinant TRPC3, one of the members of the TRPC3/6/7 subfamily, on Western blot. The synaptopodin antibody has been well- characterized (5). Following two washes with PBS, biotinylated goat-anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) at a dilution of 1:400 and Cy3-conjugated donkey-anti-mouse IgG at 1:400 in PBS/BSA were added. After an additional 30-min incubation and two washes with PBS, Cy2-conjugated streptavidin (Jackson ImmunoResearch Laboratories) at a dilution of 1:400 was added. Following a final 30-min incubation and two additional washes with PBS, the sections were mounted with 80% glycerol in 0.25 M Tris buffer, pH 7.7, and examined using an epifluorescence microscope equipped with green excitation/red emission and blue excitation/green emission filter sets. As negative controls, additional sections were stained in parallel with either pre-immune immunoglobulin of the relevant species or anti-TRPC6 preincubated with a molar excess of the immunizing peptide substituted for the primary antibody. Sections were also incubated with rabbit anti-TRPC6 at a dilution of 1:50 and mouse anti-CD34 (BD Biosciences) at a dilution of 1:400 with staining carried out in the same manner as above.
Fluorescent In Situ Hybridization for TRPC6
Normal human renal tissue was fixed for 16 hours in paraformaldehyde, paraffin embedded, and sections cut at 4-5 microns in thickness. For in situ hybridization, sections were incubated with DIG-labeled probes corresponding to the sense and anti-sense strands of the of TRPC6 cloned from human kidney (Clontech). The probe was generated by digestion of the human kidney cDNA library with EcoRV and BamH1 (corresponding nucleotide 2301-5′ TATCACTTGG . . . (SEQ ID NO: 66); corresponding nucleotide 3621- . . . TTATTTCAGG 3′(SEQ ID NO: 67); accession number AJ006276). The resulting fragment was cloned into pBS. For DIG labeling, probes were generated by in vitro transcription according to the manufacturer's protocol (Roche). Serial washes in SSC were followed by a blocking step with 5% normal rabbit IgG (Dako) RT 30-40 min and then with (1:200) HRP-rabbit anti-DIG Ab RT 45-60 min (Roche). Sections were washed with TBST and then incubated with Cy3 conjugated tyramide for 5 minutes and then coverslipped. Sections were examined using confocal microscopy with a filter set optimized for Cy3. Background staining in the sense hybridization sections reflect autofluorescence from red blood cells trapped at the time of kdney harvest.
Cloning of TRPC6 and Mutagenesis of TRPC6 C335A
The open reading frame of human TRPC6 was cloned by PCR from a human kidney cDNA library (Clontech). PCR primers were designed to amplify several fragments and the overlapping clones were sub-cloned into the mammalian expression vector pcDNA3.1 (Invitrogen) with a CMV promoter. Site-directed mutagenesis was carried out using Quickchange kit (Stratagene) to construct the C335A mutation. The resulting mutant clone was sequenced to confirm the base change as well the entire full-length sequence. Where indicated the ORF for TRPC6 and TRPC6 P112Q were cloned into the pEF-V5 expression plasmid (Invitrogen) and pCMV-N1GFP expression plasmid (Clontech) as previously with TRPC3 (6).
Cell Transfection, Surface Expression and Imaging of TRPC6
HEK 293 cells were plated on glass bottom plates at near confluence. After 24 hours, the cells were transfected with plasmids in the presence of lipofectamine 2000 (Invitrogen). Here 3 μg of TRPC6 plasmid (WT or mutant) and 1 μg of ATR-YFP plasmid were incubated for 4 hours in Optimem 2000 media (Gibco) and then changed to 10% FBS/DMEM media. For biotinylation experiments, 24 hours after transfection with TRPC6-V5 and TRPC6 P112Q -V5 constructs, cells were washed with ice cold PBS and incubated with sulfo-NHS-SS-Biotin (2 mg/ml) (Pierce) for 30-min at 4° C. After 3 washes with PBS with 10 mM glycine, cells were incubated with RIPA lysis buffer for 30-min at 4° C. Lysates were passed through a 20G and then 25G needle for 25 passes. Samples were then collected by centrifuging at 20K G for 15-min at 4° C. Supernatants were then incubated overnight with streptavidin agaraose beads (Pierce), washed 3 times and then heated to 37° C. Samples were then subjected to SDS-PAGE and immunoblotting with an antibody raised against the V5 epitope tag (Invitrogen). Samples were then immunoblotted with a monoclonal anti-transferrin receptor antibody as a control (Sigma-Aldrich). For determining total cellular TRPC6 expression, protein extracts were collected prior to streptavidin bead incubation. For immunostaining experiments, cells were fixed with ice-cold methanol, permeabilized, and incubated with the TRPC6 antibody and followed by a FITC-conjugated secondary antibody. Standard epifluorescence microscopy was used to collect a series of Z images which were then stacked and processed using Metafluor image processing (Universal Imaging).
Single Cell Imaging of Divalent Cations
HEK 293 cells transfected with plasmids (TRPC6, TRPC6 P112Q and ATR-YFP) were attached to glass coverslips and loaded with 1-10 μM Fura-2 AM (Molecular Probes) for 45-min. Following a de-esterification period of 30-min, cells were imaged by an epifluorescent microscopy system designed to capture rapid calcium transients as previously described (6). Cells were then stimulated with angiotensin II (1 μM) or 100 μM 1-oleoyl-2-acetyl sn-glycerol (OAG) (Calbiochem) in a barium containing solution. The lambda DG4 (Sutter Instruments) provides for rapid excitation filter changes and 340/380 ratios are captured every 10-100 msec from a Cool SNAP HQ CCD camera (Roper Scientific). All components including the filter selection, stage temperature, shutters and motorized stage are controlled by Metafluor software (Universal Imaging).
Whole Cell Current Recordings and Patch Clamp
HEK 293 were plated on glass coverslips and transfected with TRPC6-GFP and TRPC6 P112 Q-GFP. Bath solution comprised of (in mM): 140 NaCl, 2 BaCl 2 , 2.8 KCl, 1.0 MgCl 2 , 10 HEPES, 10 dextrose, adjusted to pH 7.4 with NaOH. For Na + free solution, NaCl was replaced with 140 mM NMDG. The patch pipette solution was comprised of (in mM): 140 Cs aspartate, 5 NaCl, 10 HEPES, 10 BAPTA and 1 Mg 2 ATP (pH 7.3, KOH). Electrodes were pulled from type 7052 glass (Garner Glass Co.) and had resistances of 2-5 Mohms. The cells were voltage clamped using the whole-cell patch clamp technique (7). The electrodes were fire-polished and coated with Sylgard 184. Offset potentials between the solution and the bath were zeroed prior to seal formation. Currents were measured with a Dagon 3900 amplifier. Data acquisition and voltage pulses were controlled with pClamp 8.0 software and the Digidata 1300 system from Axon Instruments. All patch clamp recordings were at room temperature.
REFERENCES AND NOTES
The disclosure of each reference cited is expressly incorporated herein.
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Supporting Reference List for Example 6


1. M. P. Winn et al., Genomics 58, 113 (1999).
2. M. A. Pericak-Vance, in Current Protocols in Human Genetics , N. C. Dracopoli et al., Eds. (John Wiley And Sons, Inc., New York, 1997), p. 1.
3. J. M. Vance, K. Ben Othmane, in Approaches to Gene Mapping in Complex Human Diseases , J. L. Haines, M. A. Pericak-Vance, Eds. (Wiley-Liss, New York, 1998), pp. 213-228.
4. C. Haynes et al., paper presented at Am J Human Genet 1995.
5. L. Barisoni et al., Kidney Int. 58, 137 (2000).
6. P. Rosenberg et al., Proc. Natl. Acad. Sci. U. S. A 101, 9387 (2004).
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8. A. Dietrich et al., J. Biol. Chem. 278, 47842 (2003).
9. G. Vazquez, B. J. Wedel, M. Trebak, B. G. St John, J. W. Putney, Jr., J. Biol. Chem. 278, 21649 (2003).