TRPC6 Mutations in Children with Steroid-Resistant Nephrotic Syndrome and Atypical Phenotype Article

Article

TRPC6 Mutations in Children with Steroid-Resistant Nephrotic Syndrome and Atypical Phenotype

Maddalena Gigante,*† Gianluca Caridi,‡ Eustacchio Montemurno,*† Mario Soccio,† Maria d’Apolito,§ Giuseppina Cerullo,* Filippo Aucella, Annalisa Schirinzi,*† Francesco Emma,¶ Laura Massella,¶ Giovanni Messina,** Tommaso De Palo,** Elena Ranieri,*† Gian Marco Ghiggeri,‡ and Loreto Gesualdo*

Summary *Department of Background and objectives Mutations in the TRPC6 gene have been recently identified as the cause of late- Biomedical Sciences, onset autosomal-dominant focal segmental glomerulosclerosis (FSGS). To extend the screening, we analyzed †Interdepartmental TRPC6 in 33 Italian children with sporadic early-onset SRNS and three Italian families with adult-onset Research Center FSGS. BioAgromed, and §Institute of Pediatrics, University of Foggia, Design, setting, participants, & measurements TRPC6 mutation analysis was performed through PCR and Foggia, Italy; ‡Division sequencing. The effects of the detected amino acid substitutions were analyzed by bioinformatics tools and of Nephrology, functional in vitro studies. The expression levels of TRPC6 and nephrin proteins were evaluated by confocal Laboratory on microscopy. Pathophysiology of Uremia, G. Gaslini Institute, Genoa, Italy; Results Three heterozygous missense mutations (c.374AϾG_p.N125S, c.653AϾT_p.H218L, c.2684GϾT_p.R895L) ʈDivision of were identified. The first new mutation, p.H218L, was found in a 18-year-old boy who presented a severe Nephrology, Casa form of FSGS at the age of 8 years. The second, p.R895L, a new de novo mutation, was identified in a girl Sollievo della Sofferenza Hospital, with collapsing glomerulosclerosis at the age of 2 years. The former mutation, p.N125S, was found in two IRCCS, San Giovanni siblings with early-onset steroid-resistant nephrotic syndrome (SRNS) at the ages of 4 and 14 years. Renal Rotondo, Italy; immunofluorescence revealed upregulated expression of TRPC6 and loss of nephrin in glomeruli. The intra- ¶Department of cellular calcium concentrations were significantly higher in the cells expressing all mutant TRPC6 channels Nephrology and Urology, Bambino compared with cells expressing wild-type TRPC6. Gesu`Children’s Hospital and Research Conclusions Our findings suggest that TRPC6 variants can also be detected in children with early-onset Institute, Rome Italy; and sporadic SRNS (4 of 33 patients). Moreover, in one patient a new de novo TRPC6 mutation was as- and **Division of Nephrology, Giovanni sociated with a rare severe form of childhood collapsing glomerulosclerosis with rapid progression to XXIII Hospital and uremia. Department of Clin J Am Soc Nephrol 6: 1626–1634, 2011. doi: 10.2215/CJN.07830910 Biomedicine of Developing Age, University of Bari, Bari, Italy Introduction of the intracellular calcium concentration in re- The discovery of genes involved in podocyte devel- sponse to phospholipase C (PLC) activation and are Correspondence: Dr. opment and function was crucial for understanding involved in many different signaling cascades of Loreto Gesualdo, cell growth and mechanosensation (13,14). TRPC6, Department of mechanisms of glomerular kidney diseases that Biomedical Sciences, cause various degrees of proteinuria and, poten- TRPC3, and TRPC7 are directly activated by diac- University of Foggia, tially, the progression to ESRD (1). The list of genes ylglycerol (DAG) responding to PLC-mediated sig- Viale Pinto, 1, 71100 involved in nephrotic syndrome (NS) is rapidly nals (15). In the kidney, TRPC6 is expressed in renal Foggia, Italy. Phone: 39- 0881-587101; Fax: 39- increasing (NPHS1, NPHS2, WT1, PLCE1, CD2AP, tubules and glomeruli with predominance in podo- 0881-736001; E-mail: ACTN4, and TRPC6) and includes at least seven cytes. So far, 11 different mutations in the TRPC6 [email protected] molecules associated with variable inheritance (2– gene have been identified in eight families from Dr. Maddalena Gigante Department of 9). Heterozygous mutations of TRPC6 have been different ethnic backgrounds and in three sporadic Biomedical Sciences, recently identified as the cause of late-onset auto- patients (8–12). As many as five of these mutations University of Foggia, somal-dominant focal segmental glomerulosclero- (R895C, E897K, P112Q, Q889K, M132T) were gain- Viale Pinto, 1, 71100 sis (FSGS) (8–12). The TRPC6 gene is located at the of-function mutations that resulted in increased cal- Foggia, Italy. Phone: 39- 0881-587101; Fax: 39- long arm of chromosome 11 (11q22.1) and codes for cium current amplitudes; three of them did not 0881-736001; E-mail: the transient receptor potential cation channel, sub- modify calcium influx, even if genetic data sup- mgigante@medicina. family C, member 6. The TRPC subfamily (TRPC1 ported their role as pathogenic variants; and the unifg.it through TRPC7) is a group of calcium-permeable other three were evaluated by an in silico approach. cation channels that are important for the increase In general, considering the length and the high cost

of TRPC6 analysis, these mutations have been mainly detected and analyzed in adults, in particular in those presenting familial forms with an autosomal-dominant trait. In this study, we show that TRPC6 mutations can also be detected in children with early-onset and sporadic steroid- resistant nephrotic syndrome (SRNS) and describe for the first time a de novo TRPC6 mutation in a severe form of pediatric collapsing glomerulosclerosis.

Materials and Methods Patients Thirty-three Italian patients with early-onset SRNS and three Italian families with adult-onset autosomal-dominant FSGS were enrolled (16 patients affected). Overall, all pe- Ͼ T p. R895L CYTOP Ͼ T p.H218L ANK4 Ͼ G p.N125S ANK1 diatric patients presented steroid resistance and early- (1 to Ͼ G p.N125S ANK1 5 years) or late- (6 to 12 years) childhood onset of disease.

Resistance to therapy was considered as the persistence of Mutation Protein Change Protein Domain proteinuria in a nephrotic range after 2 months of prednisone treatment (2 mg/kg per day) and after 6 months of calcineurin inhibitors (cyclosporine 5 mg/kg per day). The kidney biopsy report was available and showed FSGS or Years ESRD minimal change disease in all index patients, except for (Yes/No)/ a patient with a collapsing variant of glomerulosclerosis and another patient with IgA nephropathy with a membranoproliferative glomerulonephritis-like pattern. FSGS diagnosis was based on the histologic diagnosis of primary FSGS as defined by the Columbia FSGS classification system. As many as 2 of 33 pediatric patients were affected siblings of the same family. All patients were also screened for mutations of relevant genes involved in inherited SRNS (Steroids/CNI) Response to Drugs and FSGS, such as NPHS1, NPHS2, CD2AP, ACTN4, and exons 8 to 9 of WT1. A normal control group of 170 healthy donors was also recruited and studied. All participants or their parents or guardians provided informed consent to 1 Negative/Negative Yes/2 c0.2684G 8 Negative/Negative No c0.653A molecular analysis. Our Institutional Review Board ap- 14 Negative/Positive No c0.374A proved the study. Clinical details for all patients with (Years) TRCP6 variants are given in Table 1. Age at Diagnosis

Mutation Analysis Genomic DNA was extracted from peripheral blood sam- SRNS SRNS ples using a Wizard Genomic DNA Purification Kit (Pro- SRNS mega, Madison, WI). Molecular analyses of the 13 exons of the TRPC6 gene (NM_004621.4; GI 51511727) were performed by PCR, denaturing HPLC (DHPLC), and direct sequencing. TRPC6 flanking intronic primers were selected on the basis of published sequences or designed using the primer3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). PCR reactions were run in a total volume of 25 ␮l with a Biopsy Phenotype final magnesium chloride concentration of 1.5 mM and containing 100 ng of template DNA, 1ϫ reaction buffer, 0.2 mM of each nucleotide, 30 pmol of primers, and 0.1 U of pattern DNA polymerase (TaqGold, Applied Biosystems). The PCR reactions were carried out for 30 to 35 cycles with denaturation at 94°C for 15 seconds, annealing at approx- Age imately 55°C to 60°C for 15 seconds, and extension at 72°C (Years) for 30 seconds. All PCR programs included an initial denaturation of 10 minutes at 94°C, and the extension lasted 7 minutes in the last cycle. DHPLC analysis was carried out on a WAVE DNA Patient ID 13-MS 2 CG 5-RB 18 FSGS 19-PR 15 IgAN with MPGN-like Table 1. Clinical features of patients and TRPC6 mutations MCD, minimal change disease;glomerulosclerosis; IgAN CG, with collapsing MPGN-like glomerulopathy; pattern,CYTOP, SRNS, IgA cytoplasmic steroid-resistant nephropathy domain. nephrotic with syndrome; a CNI, membranoproliferative calcineurin glomerulonephritis inhibitor; (MPGN)-like ANK1, pattern; first FSGS, ankyrin focal repeat; segmental ANK4, fourth ankyrin repeat; fragment analysis system (Transgenomic, Crewe, United 18-PG 10 MCD SRNS 4 Negative/Positive No c0.374A 1628 Clinical Journal of the American Society of Nephrology

Kingdom) equipped with a DNASep Column (Trans- ified sequence (forward: 5Ј-GAAGAATGCCACTCACT- genomic, Crewe, United Kingdom). CAGCGTTAACTGTGTGGATTAC-3Ј and reverse: 5Ј-GTA- PCR products showing an abnormal elution profile were ATCCACACAGTTAACGCTGAGTGAGTGGCATTCTTC-3Ј reamplified, purified, and sequenced using the Big Dye for the N125S mutation; forward: 5Ј-GGGACACGGTT- Terminator version 3.1 cycle sequencing kit on a 3130 CTCCCTTGATGTGACTCCAATC-3Ј and reverse: 5Ј-GA- Genetic Analyzer (Applied Biosystems). Sequences were TTGGAGTCACATCAAGGGAGAACCGTGTCCC-3Ј for the analyzed by the SeqScape program (Applied Biosystems). H218L mutation; forward: 5Ј-GGACATCTCAAGTCTCCTC- TRPC6 novel missense mutations were checked on the TATGAACTCCTTGAAG-3Ј and reverse: 5Ј-CTTCAAGG- DNA of 170 healthy controls by DHPLC analysis. As a AGTTCATAGAGGAGACTTGAGATGTCC-3Ј for the R895L second indirect demonstration, a comparative analysis in mutation). The presence of desired mutations in selected five different species (human, mouse, rat, guinea pig, and colonies was verified by sequencing. Human embryonic zebrafish) of TRPC6 amino acid sequences was done by kidney 293 (HEK293) cells were successively transfected ClustalW software. Moreover, the effect of the detected with mutant or wild-type (WT) TRPC6 cDNAs. Briefly, amino acid substitution was analyzed by two different in HEK293 cells were seeded onto 35-mm plates containing silico approaches: the SIFT (Sorting Intolerant From Toler- DMEM (Life Technologies) with 10% FBS (Life Technolo- ant) program (http://blocks.fhcrc.org/sift/SIFT.html) and gies). When a density of 70% to 80% was achieved, cells the PolyPhen program (www.bork.embl-heidelberg.de/ were incubated with 3 ␮g of TRPC6 plasmid (mutant or PolyPhen). All patients were previously analyzed for WT) or 1 ␮g of pCMV6 empty vector (C-terminal Myc- NPHS1, NPHS2, CD2AP, ACTN4, and exons 8 to 9 of WT1 DDK-tagged) for the transient transfection using Turbo- mutations by PCR and direct sequencing. COQ2 and Fectin Transfection Reagent (Origene). The transfection ef- PDSS2 genes were also analyzed in the patient with a ficiency was evaluated with confocal laser scanner collapsing variant of glomerulosclerosis. When avail- microscopy using an anti-DDK tag monoclonal antibody able, DNA samples of other family members were also (Origene, 1:200). obtained and analyzed. Ten highly polymorphic fluores- ceinated markers belonging to the human genome were Intracellular Calcium Concentration Measurement analyzed to ensure paternity and maternity in the case of Transfected HEK293 cells were plated in 96-well culture de novo mutation. plates, and 24 hours after plating cells were washed once with HEPES-buffered saline (HBS) containing 145 mM so- Tissue Immunofluorescence dium chloride, 2.5 mM potassium chloride, 10 mM HEPES, Expression levels of TRPC6 and nephrin proteins were 3.3 potassium dihydrogen phosphate, 10 mM d-glucose, evaluated by indirect immunofluorescence and confocal 1.0 mM magnesium chloride, and 1.0 mM calcium chlo- microscopy analysis on renal biopsies from patients with ride. Cells were then loaded with 5 ␮M Fluo-4 (Molecular TRPC6 variants and from control subjects. Briefly, the Probes) in HBS for 30 minutes at 37°C. After washing with slides were incubated overnight with the primary antibod- HBS, the fluorescence was detected with a Wallac 1420 ies at room temperature (rabbit anti-TRPC6 polyclonal an- multilabel counter (PerkinElmer). Fluo-4 fluorescence, ex- tibody, Sigma-Aldrich, 1:100; guinea pig anti-nephrin an- cited at 485 nm, was detected at 535 nm. After baseline tibody, Progen Biotechnik, 1:100), washed in PBS, and then readouts for 30 seconds, cells were then stimulated with incubated with goat anti-rabbit IgG-Alexa Fluor 488 con- 100 ␮M 1-oleoyl-2-acetyl-sn-glycerol (Sigma-Aldrich). Flu- jugate antibody and chicken anti-guinea pig IgG-Alexa orescence was collected at different time points (0, 30, 60, ϩ Fluor 488 conjugate antibody (Molecular Probes, Eugene, and 120 seconds). Calcium concentration ([Ca2 ]) was ob- 2ϩ ϭ ϫ Ϫ OR), respectively. The slides, mounted with an antifading tained using the following equation: [Ca ] Kd ([F Ϫ aqueous medium (Gel/Mount, Biomeda), were examined Fmin]/[Fmax F]), where Kd is the ion dissociation constant 2ϩ under a fluorescence microscope equipped with appropri- (Kd [Ca ] of Fluo-4 is 345 nM); Fmin, recorded after addi- ate filters (Leica TCS SP5; Leica, Wetzlar, Germany). Con- tion of 5 mM EGTA (Sigma-Aldrich), is the fluorescence focal images were taken at 500-nm intervals through the intensity of the indicator in the absence of calcium; Fmax, z-axis of the section, encompassing a total of 4 ␮m in depth. measured after addition of 0.1% Triton X-100 (Sigma- Images from individual optical planes and multiple serial Aldrich), is the fluorescence intensity of the calcium- optical sections were analyzed, and the images were se- saturated indicator; and F is the fluorescence intensity of quentially scanned. Image analysis was performed on all measured samples (10). acquired fields. Negative controls were performed by re- placing the primary antibody with nonimmune serum of Results the equivalent concentration. Each experiment was carried Mutation Analysis out 3 times. We analyzed 33 Italian patients with early-onset NS and three Italian families with adult-onset autosomal-dominant FSGS. Site-Directed Mutagenesis and Transfection Three heterozygous missense mutations (c.374AϾG_p.N125S, Site-directed mutagenesis experiments were carried out c.653AϾT_p.H218L, c.2684GϾT_p.R895L) were identified in to insert the identified mutations into a full-length human two sporadic patients and in two siblings (5-RB, 13-MS, 18-PG, TRPC6 cDNA plasmid (pCMV6-Vector, Myc-DDK-tagged; 19-PR) with NS in early (n ϭ 2) or late (n ϭ 2) childhood (Table Origene) using the Quickchange Kit (Stratagene), accord- 1). A few additional innocuous single-nucleotide polymor- ing to the manufacturer’s instructions, and three primer phisms (SNPs; i.e., P15S [SNP: rs3802829] and A404V [SNP: pairs containing the desired mutations and flanked unmod- rs36111323]) with an amino acid change were found in 11 pa- Clin J Am Soc Nephrol 6: 1626–1634, July, 2011 TRPC6 Mutations and SRNS, Gigante et al. 1629

tients, whereas a few others (p.N561N, p.F843F, p.T714T, This substitution was previously described in a 42-year-old and p.Q904Q) that did not result in an amino acid change woman with adult-onset FSGS by Santín et al. (11). p.N125S were finally detected in a significant portion of our patient occurs in a moderately conserved site in the multisequence cohort (n ϭ 15). alignment (Figure 1D) and in a conserved functional do- The missense mutation c.374AϾG was found in two main (ANK1); it is a nontolerated change affecting protein siblings (18-PG and 19-PR; Table 1) with early-onset NS at function with a score of 0.01 by the SIFT program (http:// the ages of 4 and 14 years, respectively. It results in an blocks.fhcrc.org/sift/SIFT.html) and is predicted to be asparagine-to-serine substitution (p.N125S) within the first “probably damaging” by the PolyPhen program (www. ankyrin repeat (ANK1) of the TRPC6 protein (Figure 1A). bork.embl-heidelberg.de/PolyPhen) (11). Moreover, it was

Figure 1. | (A through C) Electropherograms of TRPC6 mutations and (D through F) multisequence alignments by ClustalW software. 1630 Clinical Journal of the American Society of Nephrology

not found in any of the public SNP databases or in 340 control chromosomes. Thus, according to our data and published data, this missense substitution could be classi- fied as a high-probability pathogenic variant. Interestingly, our patients also presented a heterozygous variant (c.2491CϾT_p.R831C) in the NPHS1 gene. Blood samples from parents were not available for these patients. The second mutation, c.653AϾT, results in an histidine- to-leucine substitution (p.H218L) within the fourth ankyrin repeat (ANK4) of the TRPC6 protein (Figure 1B). This substitution, which is described in this report for the first time, occurs in a highly conserved site in the multisequence alignment (Figure 1E) and is predicted to be a tolerated change with a score of 0.16 by the SIFT program but as “probably damaging” by the PolyPhen program. p.H218L was found in an 18-year-old boy who developed SRNS at the age of 8 years. Treatment with prednisone had no effects and the renal biopsy showed FSGS with negative immunofluorescence and podocyte foot process effacement. The healthy mother was WT for the detected variant, but the father’s blood sample was not available. The third mutation changes a guanine to a thymine in exon 13 (c.2684GϾT), which substitutes an arginine for a leucine (p.R895L; Figure 1C). This amino acid is highly conserved through evolution (Figure 1F) and is located in the cytoplasmic domain of the TRPC6 protein. p.R895L is a nontolerated change that affects protein function with a score of 0.00 by the SIFT program and is predicted to be Figure 2. | Immunofluorescence showing expression levels of “probably damaging” by the PolyPhen program. It is the TRPC6 (b, e, h, and k) and nephrin (c, f, i, and l) on renal biopsy first de novo mutation described in the TRPC6 gene. It has from (b and c) a wild-type (WT) subject; (e and f) a patient with been detected in a 1-year-old child with the unusual pa- focal segmental glomerulosclerosis (FSGS) without TRPC6 muta- thology feature of collapsing glomerulosclerosis with rapid tion; and two patients ([h and i] 5-RB and [k and l] 13-MS) with novel progression to ESRD. Peritoneal dialysis was started at the TRPC6 mutations (p.H218L and p.R895L, respectively). Glomerular age of 2 years. Both healthy parents resulted WT (Figure and tubular expression of TRPC6 protein is markedly upregulated in the carriers of TRPC6 mutations (h and k) with respect to the patient 1C) and microsatellite markers were analyzed to ensure with FSGS without TRPC6 mutation and the WT subject (b and e), paternity and maternity. whereas an irregular distribution along the capillary loops was observed for nephrin protein in all patients with respect to the WT subject. Panels a, d, g, and j represent negative controls. Each Tissue Immunofluorescence experiment was carried out 3 times. TRPC6 is an essential component of the podocyte slit diaphragm, where it is integrated into a signaling complex These findings strongly suggest that p.H218L and that interacts with nephrin and podocin. TRPC6 is ex- p.R895L could be gain-of-function variations, resulting pressed in renal tubules and glomeruli, with predomi- in overly active channels, increased levels of podocyte nance in podocytes. The expression of the above muta- calcium influx, and dysregulated actin cytoskeleton as- tions was analyzed by immunofluorescence on renal sembly. tissue to determine glomerular distribution of TRPC6 and nephrin, the key component of slit diaphragm struc- Transfection and Intracellular Calcium Measurement ture. In Figure 2, expression levels of TRPC6 and neph- HEK293 cells were transfected with mutant or WT rin in the renal biopsies of two patients carrying the two TRPC6 cDNAs. The transfection efficiency and the correct new mutations here reported (Figure 2, h and i, for membrane expression of WT and mutant TRPC6 was eval- p.H218L and Figure 2, k and l, for p.R895L) are com- uated by confocal microscopy and anti-DDK tag monoclo- pared with a WT subject (Figure 2, b and c) and a patient nal antibody (Figure 3, C through F). with FSGS without TRPC6 mutation (Figure 2, e and f). To determine the effect of mutations on TRPC6 function, ϩ Glomerular and tubular expression of TRPC6 protein we measured the intracellular [Ca2 ] of HEK293 cells was markedly upregulated in the carriers of TRPC6 transfected with cDNA encoding WT TRPC6 or N125S or changes, whereas a significant downregulation and an H218L or R895L mutant TRPC6. TRPC6 is directly acti- irregular distribution along the capillary loops was ob- vated by DAG responding to PLC-mediated signals; there- ϩ served for nephrin protein in all patients (Figure 2, f, i, fore, we measured the intracellular [Ca2 ] by collecting and l) with respect to the WT subject (Figure 2 c). The Fluo-4 fluorescence in transfected cells stimulated with strong upregulation of TRPC6 in crescentic epithelial 1-oleoyl-2-acetyl-sn-glycerol, an analog of DAG. As shown ϩ cells in the unique patient with collapsing and extracel- in Figure 4, the intracellular [Ca2 ] in cells expressing lular proliferation (Figure 2k) is noteworthy. mutant and WT cells was increased upon stimulation com- Clin J Am Soc Nephrol 6: 1626–1634, July, 2011 TRPC6 Mutations and SRNS, Gigante et al. 1631

Figure 3. | Confocal laser scanner microscopy using an anti-DDK tag monoclonal antibody (Origene, 1:200) to evaluate the correct membrane expression of WT and mutant TRPC6: (A) nontransfected human embryonic kidney 293 (HEK293) cells, (B) HEK293 cells transfected with pCMV6 empty vector, (C) HEK293 cells transfected with WT TRPC6, (D) HEK293 cells transfected with N125S TRPC6, (E) HEK293 cells transfected with H218L TRPC6, and (F) HEK293 cells transfected with R895L TRPC6. 1632 Clinical Journal of the American Society of Nephrology

been described so far, emphasizing the key roles of these domains for the protein function. The former mutation, p.N125S, was found in two siblings who had early-onset NS (at the ages of 4 and 14 years, respectively) and presented different glomerular lesions: minimal change disease in one patient and proliferation and thickening of the glomerular basement membrane with an IgA deposition that may be defined as an atypical IgA glomerulonephritis (Table 1) in the other patient. A plausible explanation of this finding could be the genetic contribution of other susceptibility genes. In fact, even if mutations affecting other genes involved in inherited NS

ϩ were excluded in our patients, the implication of other Figure 4. | Intracellular calcium concentration ([Ca2 ]) measurement of HEK293 cells transfected with cDNA encoding wild type unknown molecules explaining the different kidney biopsy (WT) TRPC6 or N125S or H218L or R895L mutant TRPC6. Each bar findings in the two siblings cannot be ruled out a priori.In represents the mean calcium ion concentration plus the SEM of three any case, the clinical phenotype in both siblings was SRNS separate experiments (each with n ϭ 6) obtained by measuring that improved with calcineurin inhibitor treatment. A con- Fluo-4 fluorescence after stimulation with 1-oleoyl-2-acetyl-sn-glyc- stitutive activation of the calcineurin-NFAT (nuclear fac- 2ϩ ϭ ϫ Ϫ erol at 30 seconds and using the equation [Ca ] Kd ([F tor of activated T cells) pathway as a consequence of Ϫ 2ϩ Fmin]/[Fmax F]). The peak intracellular [Ca ] was significantly FSGS-associated mutations in TRPC6 was recently iden- increased in the cells expressing N125S, H218L, and R895L mutant tified by Schlo¨ndorff and colleagues (16). They showed TRPC6 compared with cells expressing WT TRPC6 (peak at 90 seconds after stimulation, P Ͻ 0.05). Ctr, control. that the activation of NFAT by TRPC6 mutants is blocked by inhibitors of calcineurin, calmodulin-dependent kinase II, and phosphatidylinositol-3-kinase (16). These data, together pared with the nontransfected control, but the peak intra- with our findings, suggest that calcineurin inhibitors could be ϩ cellular [Ca2 ] was significantly higher in the cells express- further investigated in the treatment of TRPC6-associated ing N125S, H218L, and R895L mutant TRPC6 compared FSGS and open up new avenues for research focusing on the with cells expressing WT TRPC6 (Figure 4; peak at 90 calcineurin-NFAT pathway in modulating glomerular func- 2ϩ N125S seconds after stimulation; [Ca ] of TRPC6 ϭ 56.46 Ϯ tion. This may be an important clue as to pathogenesis and 2ϩ H218L ϭ Ϯ 2ϩ 13.22 nM; [Ca ] of TRPC6 74.39 8.94 nM; [Ca ] future treatment of these patients. It is also of note that R895L ϭ Ϯ 2ϩ of TRPC6 119.83 2.18 nM; versus [Ca ]ofWT p.N125S occurred in compound heterozygosity with a muta- ϭ Ϯ Ͻ TRPC6 9.81 1.96 nM; P 0.05). tion (c.2491CϾT_p.R831C) in the NPHS1 gene that has been associated with congenital NS of the Finnish type (17). Discussion The second mutation, which affected ANK4 (i.e., This is the first report describing TRPC6 mutations in a p.H218L), was found in a boy who developed a severe pediatric population with NS that is resistant to drugs. The form of NS at the age of 8 years with complete resistance to unique description of TRPC6 mutation in a child was made prednisone and calcineurin inhibitors. Renal biopsy by Heeringa et al. (12), who described a family in which showed FSGS and podocyte foot process effacement. several individuals presented the same defect. Overall, the A further interesting aspect of the study presented here detection of TRPC6 variants in 4 children of a small cohort is related to the pathology picture associated with p.R895L of 33 children with SRNS suggests that this gene is in- mutation because we observed collapsing glomerulone- volved, more frequently than expected, in very serious phritis with many foci of extracapillary proliferation. This forms of NS and that it is a candidate for screening in these patient presented with a very early onset of NS (at 6 patients. months) with rapid progression toward renal failure re- As many as three heterozygous missense mutations quiring dialysis. Collapsing glomerulosclerosis is rare in (c.374AϾG_p.N125S, c.653AϾT_p.H218L, c.2684GϾT_p.R895L) were identified in two sporadic patients and in two siblings who children (two patients have been reported so far) and has presented with NS in their early or late childhood (Table 1). been uniquely associated with mitochondrial defects of the In the kidney, TRPC6 is expressed in renal tubules and genes encoding for the coenzyme Q10 (CoQ10) cascade glomeruli, with predominance in podocytes. It consists of (i.e., COQ2 (18) and PDSS2 (19)). Although mutations of three or four amino-terminal ankyrin repeats, six putative genes of the CoQ10 cascade are generally associated transmembrane domains, a short sequence termed the with severe neurologic symptoms and brain anomalies “TRP box” (of unknown function), and potential coiled- (nuclear magnetic resonance with bilateral symmetric coil structures in the amino and carboxy sequences. All areas of increased T2 and decreased T1 signal intensity in detected mutations occurred at highly conserved sites in the basal ganglia (19,20)), the child described here had a the multisequence alignment and are predicted by two pure renal phenotype. p.R895L mutation is located at the different in silico approaches to be nontolerated changes carboxy terminal in the same potential coiled-coil do- that affect protein function. Two mutations here described main where other anomalies (p.R895C, p.E897K, and are located in the ankyrin repeats (p.N125S, p.H218L) and p.Q889K) producing gain-of-function changes with an one in the cytoplasmic domain (p.R895L). The same do- increase of protein expression and calcium ion influx are mains are affected by 10 of 11 TRCP6 mutations that have reported (9,10). Clin J Am Soc Nephrol 6: 1626–1634, July, 2011 TRPC6 Mutations and SRNS, Gigante et al. 1633

Several of the TRPC6 mutations that have been reported Disclosures so far are gain-of-function mutations leading to increased None. activity of ion channels by increasing calcium current amplitudes or by delaying channel inactivation. Moreover, it was also shown that the P112Q TRPC6 mutant was more References highly expressed at the cell surface, and it was speculated 1. Tryggvason K, Pikkarainen T, Patrakka J: Nck links nephrin to that the altered subcellular trafficking could explain the actin in kidney podocytes. Cell 125: 221–224, 2006 enhanced channel activity (8). 2. Kestila¨M, Lenkkeri U, Ma¨nnikko¨M, Lamerdin J, McCready P, Putaala H, Ruotsalainen V, Morita T, Nissinen M, Herva R, The expression of the novel TRPC6 mutations was ana- Kashtan CE, Peltonen L, Holmberg C, Olsen A, Tryggvason K: lyzed on renal tissue by immunofluorescence to determine Positionally cloned gene for a novel glomerular protein— glomerular distribution of TRPC6 and nephrin, the key nephrin—is mutated in congenital nephrotic syndrome. Mol component of slit diaphragm structure. Glomerular and Cell 1: 575–582, 1998 tubular expression of TRPC6 protein were markedly up- 3. Boute N, Gribouval O, Roselli S, Benessy F, Lee H, Fuchshuber A, Dahan K, Gubler MC, Niaudet P, Antignac C: regulated in the carriers of TRPC6 changes (Figure 2, h and NPHS2, encoding the glomerular protein podocin, is mutated k) with respect to the patient with FSGS without TRPC6 in autosomal recessive steroid-resistant nephrotic syndrome. defects and the WT subject (Figure 2, b and e). At variance, Nat Genet 24: 349–354, 2000 a significant downregulation and an irregular distribution 4. Hinkes B, Wiggins RC, Gbadegesin R, Vlangos CN, Seelow D, Nu¨rnberg G, Garg P, Verma R, Chaib H, Hoskins BE, along the capillary loops were observed for nephrin pro- Ashraf S, Becker C, Hennies HC, Goyal M, Wharram BL, tein in all patients (Figure 2, f, i, and l) with respect to the Schachter AD, Mudumana S, Drummond I, Kerjaschki D, WT subject (Figure 2c). Waldherr R, Dietrich A, Ozaltin F, Bakkaloglu A, Cleper R, Finally, to determine the effect of mutations on TRPC6 Basel-Vanagaite L, Pohl M, Griebel M, Tsygin AN, Soylu A, ϩ function, the intracellular [Ca2 ] was measured in HEK293 Mu¨ller D, Sorli CS, Bunney TD, Katan M, Liu J, Attanasio M, O’Toole JF, Hasselbacher K, Mucha B, Otto EA, Airik R, cells transfected with cDNA encoding WT TRPC6 or Kispert A, Kelley GG, Smrcka AV, Gudermann T, Holzman N125S, H218L, and R895L mutant TRPC6. The intracellular LB, Nu¨rnberg P, Hildebrandt F: Positional cloning uncovers ϩ [Ca2 ] was significantly higher in the cells expressing all mutations in PLCE1 responsible for a nephrotic syndrome mutant TRPC6 channels compared with cells expressing variant that may be reversible. Nat Genet 38: 1397–1405, 2006 WT TRPC6 (Figure 4). 5. Shih NY, Li J, Cotran R, Mundel P, Miner JH, Shaw AS: Therefore, most observations suggest that the high CD2AP localizes to the slit diaphragm and binds to nephrin abundance of WT TRPC6 channels in podocytes contrib- via a novel C-terminal domain. Am J Pathol 159: 2303–2308, utes to a similar pathophysiology as the presence of 2001 mutated, overly active channels. A possible explanation 6. Ruf RG, Schultheiss M, Lichtenberger A, Karle SM, Za- lewski I, Mucha B, Everding AS, Neuhaus T, Patzer L, could be that very high levels of calcium modify the Plank C, Haas JP, Ozaltin F, Imm A, Fuchshuber A, Bakka- cytoskeleton assembly. In fact, induction of TRPC6 ex- loglu A, Hildebrandt F; APN Study Group: Prevalence of pression in cultured podocytes causes a loss of actin WT1 mutations in a large cohort of patients with steroid- stress fibers and proteinuria in mice, thereby suggesting resistant and steroid-sensitive nephrotic syndrome. Kidney Int 66: 564–570, 2004 that TRPC6, in podocytes, is functionally connected to 7. Kaplan JM, Kim SH, North KN, Rennke H, Correia LA, Tong the actin cytoskeleton (21). The exact pathophysiology HQ, Mathis BJ, Rodríguez-Pe´rez JC, Allen PG, Beggs AH, through which dysregulated calcium influx in patients Pollak MR: Mutations in ACTN4, encoding alpha-actinin-4, with TRPC6 mutations leads to FSGS remains unclear. cause familial focal segmental glomerulosclerosis. Nat Genet The interaction of TRCP6, calcium, and actin in podo- 24: 251–256, 2000 8. Winn MP, Conlon PJ, Lynn KL, Farrington MK, Creazzo T, cytes should be defined. Hawkins AF, Daskalakis N, Kwan SY, Ebersviller S, Burchette In conclusion, this paper documents an important role of JL, Pericak-Vance MA, Howell DN, Vance JM, Rosenberg PB: the TRPC6 gene in sporadic SRNS with early onset and A mutation in the TRPC6 cation channel causes familial focal extends the associated pathology background to collapsing segmental glomerulosclerosis. Science 308: 1801–1804, 2005 glomerulosclerosis. Hyperinflux of calcium in podocytes, 9. Reiser J, Polu KR, Mo¨ller CC, Kenlan P, Altintas MM, Wei C, constitutive activation of the calcineurin-NFAT pathway, Faul C, Herbert S, Villegas I, Avila-Casado C, McGee M, Sug- and alterations of the actin assembly are potential mecha- imoto H, Brown D, Kalluri R, Mundel P, Smith PL, Clapham nisms for proteinuria associated with TRPC6 mutations. DE, Pollak MR: TRPC6 is a glomerular slit diaphragm-associated channel required for normal renal function. Nat Genet Acknowledgments 37: 739–744, 2005 10. Zhu B, Chen N, Wang ZH, Pan XX, Ren H, Zhang W, Wang This work was supported in part by grants “Progetto Giovani WM: Identification and functional analysis of a novel TRPC6 Ricercatori” (2007) from the University of Foggia (M.G.), “Pro- mutation associated with late onset familial focal segmental gramma per la Ricerca Sanitaria Finalizzata” (2005 to 2008) from glomerulosclerosis in Chinese patients. Mutat Res 664: 84– the Minister of Health (L.G.), and “PRIN 2009” (G.S.N.). G.C., 90, 2009 11. Santín S, Ars E, Rossetti S, Salido E, Silva I, García-Maset R, G.M.G., F.E., and L.M. acknowledge the financial support of the Gimeńez I, Ruíz P, Mendiza´bal S, Luciano Nieto J, Penã A, E-RARE Project “PodoNet: EU Consortium for Clinical, Genetic, Camacho JA, Fraga G, Cobo MA, Bernis C, Ortiz A, de Pab- and Experimental Research into Hereditary Diseases of the Podo- los AL, Sańchez-Moreno A, Pintos G, Mirapeix E, Fernańdez- cyte.” G.M.G. also acknowledges the contributions of “Fondazione Llama P, Ballarín J, Torra R; FSGS Study Group, Zamora I, la Nuova Speranza.” We thank Varraso Leonarda, Matera Rachele, Lo´pez-Hellin J, Madrid A, Ventura C, Vilalta R, Espinosa L, García C, Melgosa M, Navarro M, Gimeńez A, Cots JV, Alex- Pedicillo Maria Carmela, and Trunzo Roberta for technical sup- andra S, Caramelo C, Egido J, San Jose´MD, de la Cerda F, port. We also thank Chiara Di Giorgio (translator and medical Sala P, Raspall F, Vila A, Daza AM, Va´zquez M, Ecija JL, Es- writer, BioAgromed, University of Foggia) for linguistic revision. pinosa M, Justa ML, Poveda R, Aparicio C, Rosell J, Muley R, 1634 Clinical Journal of the American Society of Nephrology

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