Unexpected Role of TRPC6 Channel in Familial Nephrotic Syndrome (2006)

Focal and segmental glomerulosclerosis (FSGS) is a leading cause of glomerulonephritis and chronic kidney disease (CKD) in children and young adults (1). As much as one fifth of the ESRD population carries this diagnosis, and the proportion of ESRD attributed to FSGS has increased more than 10-fold over the past two decades (2). The glomerular landmarks of FSGS can develop secondary to a variety of systemic conditions, including disorders that cause chronic hypoxemia, processes that augment glomerular blood flow, and diseases that reduce renal mass. The structural features of FSGS also develop in patients who lack recognized systemic diseases, so-called primary (or idiopathic) FSGS. Over the past three decades, the recognition of familial forms of FSGS has grown, and this information has been highlighted in recent years with characterization of several human genes that cause FSGS (3). Most recently, a mutation in a member of the canonical transient receptor potential (TRP) family of proteins, TRPC6, was identified to cause FSGS in a large kindred of autosomal dominant FSGS (4). The mutation in TRPC6, in contrast to genes that have been implicated in the pathogenesis of FSGS, seems to perturb cytosolic calcium ([Ca2+]i) signaling. The purpose of this review is to examine what is known about TRPC6 mutations in familial nephrotic syndrome and to explore how this new knowledge may be applied to the care of patients with glomerular diseases. Familial Forms of FSGS Exciting discoveries in the past 10 years have identified numerous genes that are important for human glomerular diseases. The study of Mendelian forms of FSGS has been the catalyst behind these discoveries, lending insight into the pathophysiologic processes that participate in this disease. Reports of familial forms of FSGS date back as far as 1956, with the observation of an autosomal recessive disease primarily within the Finnish population. The disease process is characterized by massive proteinuria in utero, with up to 20 to 30 g/d protein loss (5). Advances in gene mapping and molecular biology within the past decade helped localize the causative gene to chromosome 19q31.1 (6), with the subsequent identification of NPHS1 (7). NPHS1 encodes a gene product termed “nephrin,” within which numerous mutations, including deletions, insertions, nonsense, missense, and splicing errors, have been described (8). Structurally, nephrin is composed of eight Ig C2 motifs, a fibronectin III–like domain, and a single transmembrane segment (Figure 1). Nephrin localizes to lipid “rafts” within the slit diaphragm of the podocyte (9–11). Lipid rafts are specialized microdomains of the plasma membrane with a unique lipid content and a concentrated assembly of signal transduction molecules (12). Localization to lipid rafts suggests that mutations of the nephrin gene likely affect its role in regulating podocyte signaling pathways (13,14). Confirmation of the role that nephrin plays in the development of glomerular disease is demonstrated by mouse models for congenital nephrotic syndrome of the Finnish type (Finnish nephropathy). Targeted disruption of the nephrin gene in embryonic stem cells has resulted in mice homozygous for the mutant NPHS1 allele, and the phenotype of these mice mirrors the human disease with the early onset of massive proteinuria and neonatal death (15). Additional mouse models that mimic recurrence of congenital nephrotic syndrome in humans after renal transplant have been developed (16,17). These models have been created by injection of mice with mAb directed toward the extracellular domain of nephrin (18). The biologic effects of the anti-mouse nephrin antibodies highlight the importance of nephrin and the slit diaphragm in the regulation of glomerular permselectivity (18). Steroid-resistant nephrotic syndrome is another human disorder that is characterized by autosomal recessive nephrotic syndrome. This disorder manifests between 3 mo and 5 yr of age, rapid progression to ESRD, and with few cases of recurrence after renal transplantation. The gene product is podocin (NPHS2), located on 1q25–31 (19). The structure of podocin is characterized by a single membrane domain forming a hairpin-like structure, with both N- and C-terminal domains in the cytosol. Podocin is expressed exclusively in podocytes (20), and specifically localizes to the base of the foot processes on either side of the slit diaphragm as seen on electron microscopy (21). Mice that lack the podocin gene product have a prominent renal phenotype that includes proteinuria and early death from renal failure (22). Podocin most likely functions in the structural organization of the slit diaphragm and regulation of its filtration function (20). It has been shown to interact in vivo with both nephrin and CD2-associated protein (CD2AP), a cytoplasmic binding partner of nephrin (23). Podocin recruits nephrin to lipid rafts in the slit diaphragm (13,23,24). Recruitment of nephrin to lipid rafts, through the direct interaction of podocin with its cytoplasmic tail, is required for the proper initiation of nephrin signaling. Targeted disruptions of podocin inhibits both nephrin trafficking and nephrin-initiated signal transduction (24). The nephrin-podocin complex seems crucial to nephrin signal transduction and maintenance of normal podocyte biology, but mutations that compromise the structural integrity of the podocyte also seem to result in foot process effacement and lesions that are characteristic of FSGS. Mutations in the α-actinin 4 gene (ACTN4), which localizes to chromosome 19q13, have been associated with autosomal dominant FSGS, characterized by adult-onset disease of variable severity and rate of progression to ESRD (25,26). ACTN4 is one of four actinin genes, which encodes a 100-kD actin cross-linking protein. Although ACTN4 is expressed in a wide range of tissues, it is very highly expressed in podocytes, functioning in the regulation of the podocyte cytoskeleton. Transgenic mice that express mutant ACTN4 as well as ACTN4-deficient mice develop renal disease that resembles FSGS (27,28). Fractions of the mutant protein have been shown to form large aggregates within podocytes, ultimately compromising the function of the normal actin cytoskeleton, through both its abnormal function and its toxic accumulation (29). The most recently reported disease-causing mutation for hereditary FSGS has been localized to chromosome 11q21-22, with the subsequent identification by Winn et al. (30) of transient receptor potential cation channel, subfamily C, member 6 (TRPC6) as the disease-causing gene. The missense mutation changes a highly conserved proline in the first ankyrin repeat of TRPC6 to a glutamine at position 112 (P112Q). Additional work was reported by Reiser et al. (31), who corroborated findings implicating TRPC6 in the pathogenesis of familial FSGS. Whereas previously reported mutations such as NPHS1, NPHS2, and ACTN4 have emphasized the importance of cytoskeletal and structural proteins in glomerular diseases, TRPC6-related FSGS suggests an additional mechanism for renal disease pathogenesis. Knowledge of TRPC6-mediated calcium entry into cells may offer unique insights into therapeutic options for glomerular diseases. Biology of TRP Channels The TRP channels have an extensive family of mammalian homologues (32). These channels have been implicated in diverse biologic functions such as cell growth, ion homeostasis, mechanosensation, and phospholipase C (PLC)-dependent calcium entry into cells. TRP channels were originally identified in the Drosophila visual system (33). It was observed that in Drosophila with the mutated TRP channel, blindness after intense and prolonged light exposure as a result of sustained Ca2+ entry occurred (34). There are common structural motifs in TRP channels. In addition to sequence homology, they are permeable to cations, especially calcium. The common feature among these channels is six transmembrane-spanning domains. The fifth and sixth transmembrane domains form tetramers that line the pore of the ion channel, and both the N- and C-termini are intracellular. Another frequent feature in some TRP channels is ankyrin binding repeats in the N-terminus. The regulation and biologic functions of TRP channels have not yet been defined clearly. Hereafter, we concentrate on the TRPC family of proteins and, more specifically, TRPC6. The TRPC family of proteins is made of seven different proteins with four subfamilies (TRPC1, TRPC3,6,7, TRPC4,5, and TRPC2) based on sequence homology and operative similarities. The TRPC3,6,7 subfamily are nearly 75% identical via sequence homology. The TRPC family of proteins is widely expressed in human tissues. It is known that most TRP channels are nonselective cation channels and permit Na+ as well as Ca2+ entry into cells; the TRPC3,6,7 subfamily has selectivity on the order of PCa/PNa 1.5 to 6:1, with TRPC6 being the most selective (32). TRPC6 can be activated either via a G protein–coupled receptor (GPCR) pathway or by application of exogenous analogues such as diacylglycerol (DAG) and act independently of [Ca2+]i store depletion (35). All members of the TRPC family have inositol 1,4,5 triphosphate receptor (IP3R) binding sites in the C-terminus, and calmodulin binds to this peptide sequence in a Ca2+-dependent manner (36). As such, a mechanism for channel function that is coupled to intracellular calcium concentration can be inferred from these findings. TRPC3,6,7 seem to co-assemble when heterologously expressed (37). It is now also believed that TRP channels form a signaling complex, or “signalplex,” with other molecules such as the scaffolding protein INAD; in turn, INAD interacts with immunophilins (38). TRPC6-deficient mice display a higher contractility in tracheal and aortic rings after stimulation with agonists such as methacholine and phenylephrine (39). They also have a modest elevation in blood pressure; we speculate that this may be due to a vascular phenotype and may have an impact on the human disease. In addition, constitutively active TRPC3 ion channels are upregulated in TRPC6-deficient smooth muscle cells and do not replace TRPC6 functionally, therefore indicating a nonredundant role (39). Given the previous emphasis on structural mutations as a cause for the glomerular pathology seen in FSGS, a new paradigm that features podocyte GPCR and downstream cellular signaling now can be added to the emerging mechanisms for this disorder. PLC-Coupled Receptors on Podocytes Vasoactive hormones are important regulators of glomerular ultrafiltration and may promote glomerular injury in disease states (40). As a result, the receptor systems and effector pathways that are activated by these hormones have been an intense area of investigation. The effects of vasoactive agents are typically mediated by binding to cell surface receptors that belong to the large superfamily of seven transmembrane-spanning receptor proteins (40,41). As shown in Table 1, podocytes express numerous heptahelical receptor systems (43–61). The majority of these receptors activate intracellular effector pathways (Table 1) by coupling to G proteins (41,62). An important exception may be the type 2 angiotensin II receptor (AT2). Cloning of the AT2 receptor (63,64) suggested that it belonged to a unique group of heptahelical receptors that do not couple to G proteins (64). The precise role of GPCR in regulating podocyte function is incompletely understood. A large body of evidence, however, suggests that vasoactive hormones modulate glomerular filtration by modulating efferent and afferent arterial tone as well as altering the glomerular ultrafiltration coefficient Kf (40). A role for the podocyte in altering Kf has been proposed by several investigators (40,65,66). In this regard, the podocyte foot process contains a contractile apparatus (67) that may respond to vasoactive hormones (40,65,66). Although direct evidence for such a contractile response is lacking, it has been postulated that Kf may be regulated, in part, by modulating the size of the filtration slits through both calcium- and cAMP-dependent signaling pathways (40,65,66). As shown in Table 1, a large number of GPCR in podocytes are coupled to either cAMP or calcium signaling cascades; therefore, these GPCR systems have the potential to modulate podocyte contractility and may play a role in disease pathogenesis. Because many of these GPCR systems are Gq coupled they have the capacity to activate TRPC6 (68). The Km values suggests that GPCR on podocytes have a high affinity for ligand; therefore, the receptors are physiologically relevant binding sites. The Bmax provides an index of receptor density that, in most receptor systems, correlates with the magnitude of physiologic response to agonist. Vasoactive hormones, such as angiotensin II (Ang II), also have the capacity to modulate the surface charge of the podocyte and, in turn, alter glomerular permselectivity (69). This change in glomerular permselectivity may play a role in disease pathogenesis by enhancing protein excretion and progression of kidney injury in disease states (69,70). Indeed, GPCR systems expressed by podocytes are likely to play an important role in glomerular disease processes as discussed below. Glomerular podocytes express numerous GPCR that are implicated in the pathogenesis of glomerular diseases, including receptors for Ang II (AT1 receptor), receptor), and Although the signaling pathways that are activated by these GPCR are common to of these receptor systems is of through G proteins that belong to the Gq family Although the precise mechanisms are incompletely receptors are of TRPC6 This seems to be mediated by stimulation of and of the and inositol and calcium by TRPC6 evidence suggests that the effects of on TRPC6 may be mediated by binding to a complex that is composed of TRPC6 and the receptor by of calcium from the intracellular This process of TRPC6 from the intracellular to the plasma membrane and, in turn, a sustained in [Ca2+]i the FSGS and structural proteins have previously been recognized as important in hereditary kidney diseases. The of a calcium channel in the pathogenesis of FSGS suggests an different cause of glomerular disease pathogenesis. of TRPC6 has been to be in the including podocytes and and cells of TRPC6 in is as abnormal podocyte function seems to be a common pathway in a variety of kidney diseases In addition, the mutation increased and prolonged calcium into cells; an of the mutant TRPC6 protein in cells that are with either the or mutant protein (4). The of protein in the plasma membrane is than protein. calcium as a result of mutations in TRPC6 are in with by The importance of Ang II as a of kidney injury was The mutation affect Ang calcium signaling. This mutation higher intracellular Ca2+ changes in cells in response to stimulation by Ang It is that when the mutation in TRPC3 is in the in calcium entry is observed after Ang II Additional work by has the findings. Reiser et al. also demonstrated evidence that TRPC6 is an important of the slit diaphragm These also evidence of mutations in TRPC6 in with familial FSGS. They evidence that TRPC6 is expressed the kidney and specifically in podocytes in the TRPC6 in the and foot processes of the TRPC6 with and podocin in mouse podocytes and with nephrin and TRPC6 to be upregulated in calcium in two of mutations in either the N- or the of the TRPC6 protein cause the changes in calcium suggests that mechanisms TRPC6 such as of the ion channel, or interaction with other proteins, with the that this in glomerular cell function or It is how mutations in TRPC6 cause FSGS. many different as a is a effector of cellular functions such as cell and It is known that intracellular calcium are calcium signaling by the mutation may glomerular cell function or may cause that are by Ang II and are known to promote kidney injury and proteinuria may be by the mutant protein. Podocytes are known to be highly as they to glomerular filtration TRPC6 is to protein such as nephrin or podocin in the filtration in response to Another mechanism is that the and sustained in intracellular calcium by the TRPC6 mutation activate the is to of through of the protein A observation in these is that the TRPC6 mutations cause sustained in [Ca2+]i after stimulation altering [Ca2+]i (4). The on stimulation for increased [Ca2+]i that that the relevant GPCR systems may be for the development of renal disease in that common signaling such as TRPC6 be an for podocyte injury by of GPCR These with previously highlight the importance of [Ca2+]i in podocyte The of the podocyte to [Ca2+]i seems to play a role in glomerular disease Because ion channels are to these the that [Ca2+]i by TRPC6 may be a for not familial disease but also patients with other primary and secondary forms of FSGS. and TRPC6 in FSGS and Glomerular The between TRPC6 mutations and FSGS of the pathogenesis of familial forms of but this also two that may to patients with other forms of glomerular of TRPC6 channels for many glomerular diseases and that the that including of TRPC6 is to the of the podocyte in Because some TRPC6 mutations result in FSGS and in intracellular it is to that the TRPC6 as the therapeutic TRPC6 channels as nonselective for with selectivity for Ca2+ over Na+ As previously TRPC6 is by analogues of intracellular calcium of a complex of the receptor and TRPC6 It is that analogues calcium by TRPC6 of protein C a common of Additional knowledge of the TRPC6 or its of by binding of the inositol receptor may new to the channel The to develop TRPC6 be however, by a few Although the calcium may be in some TRPC6 the TRPC channels of calcium channel In addition, members of the and the gene may some that is for TRPC6 may also to TRPC3 and which are very in function to TRPC6. development of for TRPC6 be function of the podocyte TRPC6 channel may be for at of the effects of that the of and receptor are that they reduce proteinuria and progression of renal disease not in FSGS but also in chronic glomerular diseases such as and The also reduce protein excretion and progression to ESRD in renal diseases that with when the podocyte is including and autosomal dominant disease As TRPC6 channels and participate in both the and the of [Ca2+]i in the podocyte after of the of this response with and has been the most and to progression of many forms of of TRPC6 may also to the first line of in FSGS. either or have effects to cytoplasmic receptors that to the the of genes is including genes for and other pathways that participate in the response As a number of activate GPCR on podocytes and are of TRPC6 also at the of the by podocyte promote podocyte and in and may of the receptor was demonstrated to podocytes in when they were with an exposure that of glomerular cells and nephrotic syndrome in Although the importance of TRPC6 for the response has not been intracellular calcium is widely known to be an important in in the renal cell of patients who have primary FSGS and for as as 6 be to to and a to develop ESRD These patients other The FSGS in a population of patients with FSGS, suggested that a of with reduce the of proteinuria and kidney function with that were composed of also suggested that may have to to the of in patients who have FSGS and are to The of of and that the to the intracellular immunophilins and and that the complex interact and the of these to the system and to in FSGS is attributed to of and the in of in FSGS may on of TRP channels in the The for and are of binding with the of in patients with FSGS may be mediated by with the and TRPC6 The especially of patients with FSGS has investigators to explore other The of is these patients in a in that in that are with a to that in the FSGS or with and (68). As a of the of this for mutations associated with FSGS, including TRPC6, may an to the of known mutations that are associated with FSGS. of of with in the past few years have identified TRPC6 and other genes that renal these recognition of genes that are for the majority of cases of ESRD This likely from the that renal failure is a disease that manifests after of In addition, ESRD is the of a wide variety of and it is to that a single causative human gene can be The of a may also be by that are associated with of The proposed four of when in that to that kidney that promote progression of and that to of ESRD information from new genes such as TRPC6 of patients with and not be to with FSGS, it may be more to this new information to one of these Because the of FSGS in with the TRPC6 mutation is high in the to it may be that of TRPC6 act as or initiation for renal disease. additional to be in the human FSGS population. The of patients with primary FSGS suggests that knowledge of a of renal including TRPC6 have of patients who a of primary FSGS, including of and can be to who are at to develop ESRD to most or of also with renal Because of the for ESRD seems by these and characterization of TRPC6 other causative may early identification of patients with FSGS. knowledge of TRPC6 may also permit of in primary FSGS. of podocin mutations may be to to but it is not yet TRPC6 of ESRD, however, patients who have TRPC6 mutations and a renal are likely to develop FSGS. TRPC6 characterization may be in of potential kidney transplant and also in potential in that the disease. The in other chronic glomerular diseases are and such as TRPC6 may the may to be in this large population of the of gene has been proposed as a to a with early may be at to develop A information TRPC6 to such as or more such as early or Because many TRPC6 mutations described far are associated with cytosolic calcium to receptor these patients may also be for that with and These patients also be for for other podocyte GPCR when they are developed in the Over the past 10 familial nephrotic have insight into normal podocyte Mutations the podocyte proteins and α-actinin 4 have highlighted the importance of the maintenance of podocyte structure in the pathogenesis of glomerular disease. the of TRPC6 in familial nephrotic calcium signaling and the role of GPCR in regulating podocyte function now be at the of for FSGS, a disease that has been to regulation of podocyte [Ca2+]i suggests that TRPC6 agonists or direct of TRPC6 channels to be an for glomerular diseases. Although knowledge the function of podocyte proteins and role in normal in the the and that are in of disease progression and response to to be in a manner to permit of in the of familial nephrotic syndrome made decades has been made the importance of normal podocyte function on a molecular within the we a of this and therapeutic of a podocyte foot process and podocyte proteins with an important role in hereditary nephrotic receptor potential cation channel 6 (TRPC6) is to interact with CD2-associated protein (CD2AP), podocin and α-actinin G protein–coupled by receptors that may modulate TRPC6 function in