Proteinuria is a common feature of kidney dysfunction of glomerular origin and is itself a risk factor for both renal and extra-renal disease1
. A variety of recent evidence supports the notion of the glomerular podocyte as a central component of the renal filtration barrier 2
. Podocytes (glomerular visceral epithelial cells) are located within the glomerulus. Their complex cytoarchitecture includes cellular extensions (foot processes) that connect to the glomerular basement membrane on the outer aspect of the glomerular capillaries. Together with the interposed slit diaphragm, a specialized multi-protein junction, they form a critical component of the ultrafiltration barrier 3
. This explains why structural damage of the podocyte may lead to the development of proteinuria. Mutations in ACTN4
(nephrin) and NPHS2
(podocin) have all been shown to lead to the development of proteinuric kidney disease 4
. Winn and colleagues identified a family with autosomal dominant FSGS in which disease co-segregated with a point mutation in the TRPC6
gene on chromosome 11q 5,6
. This prompted us to examine the renal expression and interactions of TRPC6 as well as the spectrum and function of TRPC6
genetic variants in FSGS families.
TRPC6 is a member of the transient receptor potential (TRP) superfamily of cation-selective ion channels. The TRPC subfamily (TRPC1-7) is a group of calcium-permeable cation channels that are important for the increase in intracellular [Ca2+] following engagement of G-protein-coupled receptors and receptor tyrosine kinases 7
. TRPCs form homo-and heterotetramers that can interact with a variety of other proteins 8
. Because all previously described genes mutated in FSGS and nephrotic syndrome are highly expressed in the glomerular podocyte, we examined the expression of TRPC6 in the kidney to define its localization. Confocal microscopy of adult rat kidney sections revealed wide expression of TRPC6 throughout the kidney in tubules and glomeruli (). This observation is consistent with recent reports detecting TRPC6 mRNA in glomeruli 6,9
. Most of TRPC6 expression within the glomerulus was confined to podocytes (, left panel) as shown by immunofluorescent double labeling with the podocyte marker synaptopodin (, middle panel) resulting in a yellow staining pattern (, right panel) 10
. In addition, we found a signal in glomerular endothelial cells. Whereas TRPC6 labeling with the anti-TRPC6 antibody produced a strong glomerular staining and staining within tubules ( left panel), the pre-incubation of TRPC6 antibody with a TRPC6 control peptide resulted in a negative signal ( right panel). Next, we studied the expression of TRPC1-6 mRNAs in isolated glomeruli and cultured murine podocytes by RT-PCR (). Whereas TRPC1-6 are all expressed in the glomerulus, only TRPC1, 2, 5 and 6 were found to be expressed in cultured podocytes. We analyzed TRPC6 expression in a cultured mouse podocyte cell line 11
. Labelling was detected at the cell membrane (). To determine the precise subcellular localization of TRPC6, we carried out immunogold labeling of ultrathin frozen sections from adult kidney cortex (). Gold particles were found in the cell body of podocytes and in primary processes (, white arrows). In podocyte foot processes gold particles labeled areas in close vicinity to the slit diaphragm region (, black arrows). We also detected TRPC6 expression in glomerular endothelial cells () and on a few mesangial cells (data not shown). In order for TRPC6 to reach the slit diaphragm in podocyte foot processes, TRPC6 protein has to be transported from the cell body through the major processes into the foot processes. Membrane proteins with high protein turnover can be found in various subcellular localizations. Similarly, podocalyxin, a major sialoprotein in podocytes is detected intracellularly throughout the entire exocytotic pathway consistent with a high rate synthesis 12
. High power view of a section through the slit diaphragm area shows the close association of TRPC6 to the slit diaphragm (, black arrows).
FIGURE 1 TRPC6 expression in the kidney glomerulus. (a) Confocal microscopy shows TRPC6 (red) expression in the glomerulus. TRPC6 co-localizes with the podocyte marker Synaptopodin (green) resulting in a yellow overlap. (b) Compared to the TRPC6 antibody labeling, (more ...)
Next, we tested whether TRPC6 co-localizes with the human disease-associated slit diaphragm proteins nephrin, podocin and CD2AP. Since available antibodies against TRPC6 and slit diaphragm proteins are all rabbit-polyclonal, we transfected cultured podocytes with GFP-tagged TRPC6. Confocal microscopy of GFP-TRPC6 transfected podocytes stained with antibodies against nephrin, podocin and CD2AP () revealed expression of GFP-TRPC6 at the podocyte cell membrane partially co-localizing with nephrin, podocin and CD2AP 13–15
. These findings suggest that within podocytes, TRPC6 is at least in part associated with the slit diaphragm.
FIGURE 2 TRPC6 co-localizes and directly interacts with slit diaphragm proteins. (a) GFP-TRPC6 co-localizes with CD2AP, nephrin and podocin at the cell membrane of cultured podocytes as shown by confocal microscopy (arrows). (b) GFPTRPC6 associates with FLAG-tagged (more ...)
To test whether TRPC6 interacts with nephrin, podocin or CD2AP, we performed co-immunoprecipitation studies (). Therefore, we co-expressed recombinant mouse GFP-TRPC6 with FLAG-tagged mouse nephrin, podocin, and CD2AP, respectively, in human embryonic kidney (HEK293) cells. FLAG-fusion proteins from cell lysates were immunoprecipitated using anti-FLAG-M2 beads and eluates were analyzed by immunoblotting using anti-FLAG and anti-GFP antibodies. To detect the interaction of GFP-TRPC6 with FLAG-tagged nephrin, podocin or CD2AP, we analyzed eluates with anti-GFP antibody. Immunoblotting showed that GFP-TRPC6 was absent from the eluates derived from cells co-transfected with FLAG-CD2AP (). In contrast, GFP-TRPC6 was present in eluates derived from cells co-transfected with FLAG-nephrin and FLAG-podocin, indicating a direct biochemical interaction of TRPC6 with nephrin and podocin but not with CD2AP. We also performed the reverse co-immunoprecipitation where we immunoprecipitated the GFP-fusion proteins first (data not shown). This set of experiment yielded identical results. We also performed endogenous co-immunoprecipitation using whole cellular extracts from cultured differentiated podocytes known to express the vital slit diaphragm components nephrin, podocin and CD2AP 16
. The immunoprecipitation with anti-TRPC6 antibody revealed an interaction of TRPC6 with nephrin and podocin but not with CD2AP ().
Nephrin is a central component of the slit diaphragm and mice deficient in the nephrin gene suffer from proteinuria and partial foot process effacement 17,18
. We therefore examined the effect of nephrin deficiency on the localization of TRPC6 in the glomerulus (). Neonatal wild type mice displayed a low expression of glomerular TRPC6 (, left upper panel). Some of the expression is found in podocytes as revealed by immunofluorescence double labeling with the podocyte marker synaptopodin (, middle upper panel), resulting in a partial yellow overlap (, right upper panel). The targeted deletion of nephrin leads to an induction of podocyte TRPC6 expression and to focal accumulation of the TRPC6 protein (). This data suggest that the lack of nephrin induces podocyte TRPC6 expression and leads to altered cellular localization of TRPC6.
FIGURE 3 TRPC6 is upregulated in 2 days old nephrin-KO mouse glomeruli as shown by fluorescence microscopy and immunogold labeling. Weak TRPC6 expression was detected in glomeruli of 2 days old wt-mice (upper panel), TRPC6 was found to be upregulated in glomeruli (more ...)
To explore the role of TRPC6 gene variation in kidney disease, we screened probands of 71 pedigrees with familial FSGS for alterations in the TRPC6 gene by DNA sequence analysis. Of these 71 families screened, 43 showed evidence of disease in members of multiple generations, and 28 showed evidence of disease in 2 or more members of a single generation. Probands of 49% of the families were of western European ancestry; 5% of African ancestry, and 27% described themselves as Hispanic. The phenotype in affected members of largest of these families (FS-Z) showed cosegregation only with chromosome 11q markers (with 2 point lod score examining linkage of affected individuals with the disease-associated haplotype equal to 1.8 at θ=0). We identified different heterozygous sequence variants in five unrelated families with adult onset disease, all of which predicted changes in the encoded gene product: N143S, S270T, K874Stop, R895C and E897K (). We genotyped other available family members for the relevant variants. In all families, patterns of TRPC6 variant and disease inheritance followed a pattern of cosegregation (with less than complete penetrance). In each family, inheritance was consistent with an autosomal dominant pattern (; see Supplementary Note for clinical details). Each of the observed amino acid substitutions occurred in evolutionarily conserved residues (). Two mutations predict amino-acid substitutions in the N-terminal intracellular domain of TRPC6, two predict amino-acid substitutions in the C-terminal intracellular domain, and one encodes a premature stop codon near the C terminus (). We genotyped 180 control individuals for the disease-associated variants. None of these disease associated variants were identified in the controls. In addition, none of these substitutions were found in the public SNP databases.
Characteristics of TRPC6 mutations
Familial FSGS pedigrees. TRPC6 variants segregating within each family are indicated. Genotyped individuals are indicated as carrying (+) or not carrying (–) the variant identified in each family.
Sequence alterations. TRPC channel sequence alignments (using T-COFFEE). Mutated residues all occur in highly conserved amino acid residues.
To study whether mutations in TRPC6
affect calcium channel function we expressed either wild type or each of the five mutant TRPC6 channels in HEK293-M1 cells stably transfected with the Gαq-coupled M1 muscarinic receptor. TRPC6 currents were recorded before and after activation of M1 receptors by carbachol. Although we were able to measure currents from the N143S, S270T and K874Stop TRPC6 channels, the currents did not differ noticeably from wild type TRPC6 currents (data not shown). In contrast, currents from R895C and E897K mutant channels were significantly larger than wild type TRPC6 currents (). Similarly, Winn and coworkers identified increased current amplitude with the TRPC6 P112Q channel 5,6
. We also noted subtle differences in the rectification of the current-voltage relationship of these two mutants. However, we believe these changes in rectification result from the increased current density rather than directly from structurally related changes in channel gating or permeation. If the currents through R895C and E897K TRPC6 channels are similarly increased in vivo, these mutations could lead to a gain-of-function alteration in activity and thus increased calcium influx.
FIGURE 6 Representative whole-cell currents measured from HEK293-M1 cells transiently transfected with WT TRPC6 (a), R895C (b) or E897K (c) cDNA. Current traces were recorded as cells were perfused with control bath solution (CTRL, gray traces) or 100 μM (more ...)
Three of the TRPC6 mutations identified did not produce apparent changes in current amplitude. Nevertheless, we believe these to be disease causing because of the nature of the changes (substitutions in very highly conserved residues or premature stop codon), cosegregation with the disease phenotype, and the absence of these changes from control individuals. This suggests that an abnormality other than increased current amplitude is the cause of disease in the individuals with these mutations. The several possibilities include altered channel regulation (despite normal amplitude), altered interaction with other slit-diaphragm proteins, and altered protein turnover.
Mutations in several podocyte genes have been identified as culprits for progressive renal failure or increased glomerular disease susceptibility 4
. The presence of TRPC6
mutations co-segregating with kidney disease, the evolutionary conservation of the altered amino acids, slit-diaphragm localization and interactions, and the gain-of-function changes observed in two mutants suggest that TRPC6 channel function is essential for normal renal ultrafiltration. The question remains why the onset of kidney disease in patients with TRPC6
mutations occurs at a relatively advanced age. Similarly to the adult onset and dominantly inherited form of FSGS due to mutations in the widely expressed protein α-actinin-4, gain-of-function mutations in TRPC6
may produce subtle changes in intracellular function that lead to irreversibly altered cell behavior only after time and in the presence of other renal insults19
. In addition, podocytes express several other TRPC channels including TRPC1,2,5 and 6 (). Partial functional redundancy may also help account for the late onset of glomerular disease. The ability of TRPC6 to form heteromers with other TRPC channels suggests a complex cellular regulation of calcium homeostasis.
Podocyte electrophysiology has been under investigation for many years20
. However, published work has been limited to measurements of the membrane potential and whole-cell conductance in rodent podocytes and their response to vasoactive agonists 20
. The location of podocytes within the glomerulus, surrounding the glomerular capillaries, exposes these cells to the transmural hydrostatic pressure driving ultrafiltration. Podocyte foot processes contain a contractile apparatus that may be regulated by slit-diaphragm-derived calcium signaling 21
. Mature podocytes express several types of receptors and second messenger systems 3
. These include receptors for muscarine, angiotensin, prostaglandin E2, and atrial natriuretic peptide, which all activate intracellular Ca2+
, phospholipase C, inositol 1,4,5-triphosphate, cAMP and cGMP signaling cascades. Thus, podocytes may employ foot process and slit diaphragm-derived intracellular signals to respond to their cellular environment. Nephrin by itself has been shown to be a signalling molecule generating podocyte survival signals 22
. Fyn kinase is associated with nephrin 23
and known to regulate TRPC6 channel opening by tyrosine phosphorylation 24
. The disruption of nephrin from the slit diaphragm as has been reported in secondary forms of FSGS25
may mediate its effects on podocytes by modulation of TRPC6 originated calcium flux. Changes in TRPC6 calcium currents in podocyte foot processes appear to be central to the ability of the podocyte to regulate its intracellular and cytoskeletal behavior.