Role of Transient Receptor Potential Cation Channel 6 in Podocyte Injury
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Abstract:
Recent studies have shown that transient receptor potential cation channel 6(TRPC6) expressed in podocytes,and located in slit diaphragm,it colocalized with nephrin,podocin and CD2AP,participating in signal transduction and maintaining podocytes normal construction and function.Mutations in TRPC6 cause autosomal dominant familial focal segmental glomerulosclerosis which is characterized by grade proteinuria and podocyte injury.In vitro studies,it also indicates that overexpression of TRPC6 leads to podocyte dysfunction,maybe through elevating calcium influx,suggesting that TRPC6 associates with the onset and development of proteinuria.This article will describe the role of TRPC6 in podocyte injury,maybe provide us the new targets for diagnosing and theraping renal disorders in the future.Keywords:
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Physiologic permeability of the glomerular capillary depends on the normal structure of podocyte foot processes forming a functioning slit diaphragm in between. Mutations in several podocyte genes as well as specific molecular pathways have been identified as the cause for progressive kidney failure with urinary protein loss. Podocyte injury is a hallmark of glomerular disease, which is generally displayed by the rearrangement of the podocyte slit diaphragm and the actin cytoskeleton. Recent studies demonstrate a unique role for the Ca2+-permeable ion channel protein TRPC6 as a regulator of glomerular ultrafiltration. In both genetic and acquired forms of proteinuric kidney disease, dysregulation of podocyte TRPC6 plays a pathogenic role. This article illustrates how recent findings add to emerging concepts in podocyte biology, particularly mechanosensation and signaling at the slit diaphragm.
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Podocytes (terminally differentiated epithelial cells of the glomeruli) play a key role in the maintenance of glomerular structure and permeability and in the incipiency of various renal abnormalities. Injury to podocytes is considered a major contributor to the development of kidney disease as their loss causes proteinuria and progressive glomerulosclerosis. The physiological function of podocytes is critically dependent on proper intracellular calcium handling; excessive calcium influx in these cells may result in the effacement of foot processes, apoptosis, and subsequent glomeruli damage. One of the key proteins responsible for calcium flux in the podocytes is transient receptor potential cation channel, subfamily C, member 6 (TRPC6); a gain-of-function mutation in TRPC6 has been associated with the onset of the familial forms of focal segmental glomerulosclerosis (FSGS). Recent data also revealed a critical role of this channel in the onset of diabetic nephropathy. Therefore, major efforts of the research community have been recently dedicated to unraveling the TRPC6-dependent effects in the initiation of podocyte injury. This mini-review focuses on the TRPC6 channel in podocytes and colligates recent data in an attempt to shed some light on the mechanisms underlying the pathogenesis of TRPC6-mediated glomeruli damage and its potential role as a therapeutic target for the treatment of chronic kidney diseases.
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Podocyte injury and the appearance of proteinuria are key features of several progressive kidney diseases. Genetic deletion or selective inhibition of TRPC5 channels with small-molecule inhibitors protects podocytes in rodent models of kidney disease, but less is known about the human relevance and translatability of TRPC5 inhibition. Here, we investigate the effect of TRPC5 inhibition in puromycin aminonucleoside (PAN)-treated rats, human iPSC-derived podocytes, and kidney organoids. We first established that systemic administration of the TRPC5 inhibitor AC1903 was sufficient to protect podocyte cytoskeletal proteins and suppress proteinuria in PAN-induced nephrosis rats, an established model of podocyte injury. TRPC5 current was recorded in the human iPSC-derived podocytes and was blocked by AC1903. PAN treatment caused podocyte injury in human iPSC-derived podocytes and kidney organoids. Inhibition of TRPC5 channels reversed the effects of PAN-induced injury in human podocytes in both 2D and 3D culture systems. Taken together, these results revealed the relevance of TRPC5 channel inhibition in puromycin-aminonucleoside induced nephrosis models, highlighting the potential of this therapeutic strategy for patients.
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Over a decade ago, mutations in the gene encoding TRPC6 (transient receptor potential cation channel, subfamily C, member 6) were linked to development of familial forms of nephrosis. Since this discovery, TRPC6 has been implicated in the pathophysiology of non-genetic forms of kidney disease including focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, immune-mediated kidney diseases, and renal fibrosis. On the basis of these findings, TRPC6 has become an important target for the development of therapeutic agents to treat diverse kidney diseases. Although TRPC6 has been a major focus for drug discovery, more recent studies suggest that other TRPC family members play a role in the pathogenesis of glomerular disease processes and chronic kidney disease (CKD). This review highlights the data implicating TRPC6 and other TRPC family members in both genetic and non-genetic forms of kidney disease, focusing on TRPC3, TRPC5, and TRPC6 in a cell type (glomerular podocytes) that plays a key role in proteinuric kidney diseases.
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Abstract Objectives Diabetic nephropathy is a major complication of diabetes and a frequent cause of end‐stage renal disease and recent studies suggest that podocyte damage may play a role in the pathogenesis of this. At early onset of diabetic nephropathy there is podocyte drop‐out, which is thought to provoke glomerular albuminuria and subsequent glomerular injury; however, the underlying molecular mechanisms of this remain poorly understood. Here we report that we tested the hypothesis that early diabetic podocyte injury is caused, at least in part, by up‐regulation of transient receptor potential cation channel 6 ( TRPC 6), which is regulated by the canonical Wnt signalling pathway, in mouse podocytes. Materials and methods Mechanism of injury initiation in mouse podocytes, by high concentration of D‐glucose (HG, 30 mM ), was investigated by MTT, flow cytometry, real‐time quantitative PCR, and western blot analysis. Results HG induced apoptosis and reduced viability of differentiated podocytes. It caused time‐dependent up‐regulation of TRPC 6 and activation of the canonical Wnt signalling pathway, in mouse podocytes. In these cells, blockade of the Wnt signalling pathway by dickkopf related protein 1 (Dkk1) resulted in effective reduction of TRPC 6 up‐regulation and amelioration of podocyte apoptosis. Furthermore, reduction of cell viability induced by HG was attenuated by treatment with Dkk1. Conclusion These findings indicate that the Wnt/β‐catenin signalling pathway may potentially be active in pathogenesis of TRPC 6‐mediated diabetic podocyte injury.
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Transient receptor potential cation channel 6(TRPC6) is a nonselective cation channel,located in the podocyte cell membrane and contains six membrane-spanning domains.TRPC6 is associated with slit diaphragm proteins-nephrin and podocin,the three proteins composing the slit diaphragm complex.Mutant TRPC6 may affect the function of this complex,leading to abnormalities in podocyte foot processes.Research suggested that mutant TRPC6 closely correlated with the mechanism of hereditary and non-hereditary nephropathies,possibly by altering podocyte dynamics and decreasing podocyte number.Blocking TRPC6 channels might be of therapeutic effect in treating kidney disease with proteinuria,and it may translate into long-lasting clinical benefits in patients with nephropathy.
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Transient receptor potential channel 6 (TRPC6) is a Ca2+ ion transport channel associated with the slit diaphragm of podocytes, and is indispensable for regulation of structural components of podocytes and renal function.1 Since the discovery of a TRPC6 gain-of-function mutation that causes familial FSGS, TRPC6 activation has been associated with several progressive glomerular diseases.2,3 In fact, TRPC6 in the context of kidney disease has become quite ubiquitous, and its activity is being considered by many as an indicator of podocyte vulnerability and progressive kidney disease.4 It is of no surprise that researchers began investigating blocking TRPC6 activity, thereby reducing deleterious Ca2+ influx in an effort to adjust the podocyte cytoskeleton. In the current issue of the Journal of the American Society of Nephrology, Sonneveld et al.5 found sildenafil (i.e., Viagra) has antiproteinuric effects via a mechanism involving peroxisome proliferator-activated receptor γ (PPAR-γ) in mouse podocytes, and may be an effective regulator of TRPC6 signaling for use in treating glomerular disease. As a successful erectile dysfunction drug, sildenafil occupies the active site of phosphodiesterase type 5 (PDE5), giving rise to cGMP, which initiates smooth muscle relaxation and increased blood flow. Although sildenafil is a well known PDE5 blocker in penile and cardiac tissue, its role in podocytes is poorly defined. Sonneveld et al. contribute to ongoing research efforts that have demonstrated the renoprotective effects of sildenafil by more clearly defining a pathway in which sildenafil blocks PDE5 leading to cGMP accumulation, protein kinase G-1 (PKG-1) activation, and in turn, PPAR-γ activation to downregulate TRPC6 expression.5 In podocytes, cGMP accumulation is known to suppress renal disease,6 and podocyte responsiveness to PDE5 blockers has opened the door to cGMP regulation as a means of treating podocyte injury. Several groups have extensively studied components of pathways activated by cGMP accumulation in podocytes, and a great deal of evidence supports TRPC6 inactivation as a mechanism to treat kidney disease. For example, cGMP accumulation has been linked to podocyte contractility, mobility, and cytoskeletal structure7; PKG-1 is associated with poor clinical outcome in renal cell carcinoma8; PPAR-γ agonists have been shown to protect podocytes from nephropathies9; and overexpression of TRPC6 alone is sufficient to cause podocyte damage and subsequent glomerulopathies.10,11 Collectively, these findings highlight the importance of crosstalk among cGMP, PKG-1, PPAR-γ, and TRPC6, and demonstrate the importance of TRPC6 signaling in kidney disease. Sonneveld et al. found PDE5 is expressed in podocytes, and sildenafil can have antiproteinuric effects through PPAR-γ to decrease TRPC6 expression levels, and ultimately minimize deleterious Ca2+ influx.5 Transcriptional downregulation of TRPC6 via PPAR-γ in response to sildenafil ameliorated podocyte injury, and was deemed more important than affecting TRPC6 channel functionality directly, as previously reported.12 cGMP accumulation had no effect on Ca2+ influx in the presence of PPAR-γ antagonists, suggesting PKG-1–mediated binding of PPAR-γ to the TRPC6 promoter is the pivotal interaction regulating Ca2+ homeostasis. In the context of clinically approved drugs, this study is a reminder of the potential benefits of repurposing approved drugs. After all, sildenafil was not intended as an erectile dysfunction drug and is, in essence, a side effect drug itself. Complex pathways with influential secondary messengers, like cGMP, need to be broken down to be fully understood. Although feedback mechanisms and TRPC6 channel dynamics in response to cGMP accumulation in podocytes remain unclear, studying each component of the cGMP–PKG-1–PPAR-γ–TRPC6 pathway individually, and in combination, helped define functional roles in podocytes. Blocking PDE5 with sildenafil clearly initiates a signaling cascade that downregulates TRPC6 transcription via PPAR-γ, subsequently reducing Ca2+ influx to reduce podocyte injury and presumably glomerular disease. Despite this newly discovered mechanism by which sildenafil operates in podocytes, there are some limitations to the study. There does not seem to be substantial evidence to support the notion that PPAR-γ–mediated transcription of TRPC6 is the principal mechanism regulating TRPC6 activity in response to sildenafil treatment, given TRPC6 channel dynamics were not evaluated. Without considering the phosphorylation state of TRPC6 at Thr69, a recently reported response to sildenafil that inhibits TRPC6 channel function, the number of channels at the membrane is only problematic in the context of podocyte injury if the channels are open and allowing Ca2+ influx.12 Thus, activation of each component of the sildenafil-initiated signaling cascade should be compared directly before determining the most crucial step. Is reducing TRPC6 expression or inhibiting TRPC6 channel activity (or both) the mechanism of PDE5-mediated renoprotection? The interplay between mechanical deactivation of TRPC6 via phosphorylation and TRPC6 membrane expression (i.e., number of active channels) has yet to be explored to provide insight on channel conformation, activity, and openness probability in response to sildenafil treatment, and the contribution of each to Ca2+ homeostasis and podocyte injury. A compensatory or cooperative response of other TRPC channels also merits consideration. TRPC channel expression can influence activity of other TRP channels, which may have adverse effects in podocytes. In this study, TRPC1 was also downregulated in podocytes treated with sildenafil, and Kiso et al.13 showed sildenafil decreases TRPC1, TRPC3, and TRPC6 expression in cardiomyocytes; both of which draw concern because of the undefined role of TRPC1 in glomerular disease. Of note, TRPC6 protein levels were not assessed by Western blot, making it difficult to evaluate the outcomes of decreased transcriptional activity of the TRPC6 promoter. As stated, an increase in TRPC6 transcription may cause functional effects, but promoter activity does not translate to functional activity of a protein. Caution should be taken when assuming an increase in transcription leads to an increase in translational products that are effective within a given pathway. Post-translational modification, especially with a channel-forming, membrane-associated protein, has to be monitored. Additionally, sildenafil is associated with increased blood flow and GFR, although Sonneveld et al. suggest antiproteinuric effects of renal vasodilation are nonspecific because of the absence of TRPC6 in renal vasculature.5 Although transcriptional regulation of TRPC6 via PPAR-γ may be important in regulating Ca2+ influx, the TRPC6-mediated mechanisms and the effectiveness of sildenafil in treating glomerulopathies remain unclear. Evaluation of sildenafil treatment in the context of glomerular disease has been conducted with some success. In a clinical setting, administration of sildenafil improves kidney function, prevents disease progression, reduces proteinuria, and restores GFR in patients with conditions ranging from pulmonary hypertension to diabetic nephropathy (reviewed by Vasquez et al.14). In a laboratory setting, sildenafil treatment has been largely beneficial in reducing proteinuria, inflammation, oxidative stress, fibrosis, hypertension, and general renal damage in several kidney injury models (see Schinner et al.6 for a full review). Although the potential use of sildenafil to treat glomerulopathies is attractive, being the first and most well studied PDE5 blocker, mild but common side effects and adverse reactions to sildenafil treatment have been observed in some patients.14 In this regard, it is difficult to overlook the potential use of other approved PDE5 inhibitors or PPAR-γ agonists. Sonneveld et al. have identified an alternative mechanism for Viagra, and revealed a multistep pathway to downregulate TRPC6 and prevent harmful Ca2+ influx in podocytes.5 In so doing, they have uncovered other potential therapeutic entry points, such as the use of direct-acting PPAR-γ agonists like pioglitazone, with fewer side effects than PDE5 blockers. The data presented shows that pioglitazone is at least, if not more effective in all assays as a renoprotective agent than sildenafil. This result is likely because of the resource investment necessary for a sildenafil-initiated response requiring sequential responses from PDE5, cGMP, and PKG-1 to activate PPAR-γ. Using pioglitazone, TRPC6 is more efficiently and specifically blocked, having no effect on other TRPC channels, unlike sildenafil (example, TRPC1). Moving forward, the best option to reduce susceptibility of podocyte injury by maintaining Ca2+ homeostasis may still be by blocking TRPC6 directly. Although some groups have already identified potential TRPC6 inhibitors, in vivo efficacy has been inadequate.15 Thus, future research efforts will likely identify drugs that will be more effective than current treatment options, which downregulate TRPC6 activity by targeting distant upstream mediators. In fact, several regulators of TRPC6 in glomeruli have already been proposed.3 One would anticipate such drugs to come in the form of a small molecule or microRNA routed agent that targets structural components of the TRPC6 channel, thereby regulating mechanical processes that reduce Ca2+ influx and regulate the glomerular filter. Disclosures J.R. is cofounder of TRISAQ Inc., a biotech company dedicated to developing novel therapies for renal disease. He stands to gain royalties for commercialization of these products.
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Transient receptor potential channel 5 (TRPC5) is highly expressed in brain and kidney and mediates calcium influx and promotes cell migration. In the kidney, loss of TRPC5 function has been reported to benefit kidney filter dynamics by balancing podocyte cytoskeletal remodeling. However, in vivo gain-in-function studies of TRPC5 with respect to kidney function have not been reported. To address this gap, we developed two transgenic mouse models on the C57BL/6 background by overexpressing either wild-type TRPC5 or a TRPC5 ion-pore mutant. Compared with nontransgenic controls, neither transgenic model exhibited an increase in proteinuria at 8 months of age or a difference in LPS-induced albuminuria. Moreover, activation of TRPC5 by Englerin A did not stimulate proteinuria, and inhibition of TRPC5 by ML204 did not significantly lower the level of LPS-induced proteinuria in any group. Collectively, these data suggest that the overexpression or activation of the TRPC5 ion channel does not cause kidney barrier injury or aggravate such injury under pathologic conditions.
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