Role of Formyl Peptide Receptors and ß-Arrestin-1 in suPAR Signal Transduction in Mouse Podocytes: Interactions with αVß3-Integrin.

The soluble urokinase plasminogen activator receptor (suPAR) has been implicated in a wide range of pathological conditions including primary nephrotic syndromes and acute kidney injuries. suPAR can trigger transduction cascades in podocytes by outside-in activation of αVß3-integrin, but there is evidence that the functional cell surface response element is actually a complex of different types of receptors, which may also include the receptor for advanced glycation end-products (RAGE) and formyl peptide receptors (FPRs). Here we observed that ROS accumulation and Src activation could be evoked by continuous 24 h exposure to either suPAR or the FPR agonist fMLF. Responses to suPAR and fMLF were completely blocked by either the FPR antagonist WRW4 or by the αV-integrin inhibitor cilengitide. Moreover, endogenous podocyte mouse Fpr1 co-immunoprecipitates with ß3-integrin, suggesting that these receptors occur as a complex on the cell surface. suPAR- and fMLF-evoked activation of Src and ROS differed in time course. Thus, robust pertussis toxin (PTX)-sensitive responses were evoked by 60 min exposures to fMLF but not to suPAR. By contrast, responses to 24 h exposures to either suPAR or fMLF were PTX-resistant and were instead abolished by knockdown of ß-arrestin-1 (BAR1). FPRs, integrins, and RAGE (along with various Toll-like receptors) can all function as pattern-recognition receptors that respond to "danger signals" associated with infections and tissue injury. The fact that podocytes express such a wide array of pattern-recognition receptors suggests that the glomerular filter is designed to change its function under certain conditions, possibly to facilitate clearance of toxic macromolecules.

The small GTPase regulatory protein Rac1 drives podocyte injury independent of cationic channel protein TRPC5.

Transient receptor potential canonical channels (TRPCs) are non-selective cationic channels that play a role in signal transduction, especially in G -protein-mediated signaling cascades. TRPC5 is expressed predominantly in the brain but also in the kidney. However, its role in kidney physiology and pathophysiology is controversial. Some studies have suggested that TRPC5 drives podocyte injury and proteinuria, particularly after small GTPase Rac1 activation to induce the trafficking of TRPC5 to the plasma membrane. Other studies using TRPC5 gain-of-function transgenic mice have questioned the pathogenic role of TRPC5 in podocytes. Here, we show that TRPC5 over-expression or inhibition does not ameliorate proteinuria induced by the expression of constitutively active Rac1 in podocytes. Additionally, single-cell patch-clamp studies did not detect functional TRPC5 channels in primary cultures of podocytes. Thus, we conclude that TRPC5 plays a role redundant to that of TRPC6 in podocytes and is unlikely to be a useful therapeutic target for podocytopathies.

Capsazepine (CPZ) Inhibits TRPC6 Conductance and Is Protective in Adriamycin-Induced Nephropathy and Diabetic Glomerulopathy.

Reactive oxygen species (ROS), which excessively arise in diabetes and systemic inflammatory diseases, modify cellular lipids and cellular lipid composition leading to altered biophysical properties of cellular membranes. The impact of lipid peroxidation on transmembrane signaling routes is not yet well studied. The canonical transient receptor potential channel 6 (TRPC6) is implicated in the pathogenesis of several forms of glomerular diseases. TRPC6 is sensitive to membrane stretch and relies on a distinct lipid environment. This study investigates the effect of oxidative alterations to plasma membrane lipids on TRPC6 activity and the function of the glomerular filter. Knockout of the anti-oxidative, lipid modifying enzyme paraoxonase 2 (PON2) leads to altered biophysical properties of glomerular epithelial cells, which are called podocytes. Cortical stiffness, quantified by atomic force microscopy, was largely increased in PON2-deficient cultured podocytes. PON2 deficiency markedly enhanced TRPC6 channel currents and channel recovery. Treatment with the amphiphilic substance capsazepine in micromolar doses reduced cortical stiffness and abrogated TRPC6 conductance. In in vivo studies, capsazepine reduced the glomerular phenotype in the model of adriamycin-induced nephropathy in PON2 knockout mice and wildtype littermates. In diabetic AKITA mice, the progression of albuminuria and diabetic kidney disease was delayed. In summary, we provide evidence that the modification of membrane characteristics affects TRPC6 signaling. These results could spur future research to investigate modification of the direct lipid environment of TRPC6 as a future therapeutic strategy in glomerular disease.

Ion channels and channelopathies in glomeruli.

An essential step in renal function entails the formation of an ultrafiltrate that is delivered to the renal tubules for subsequent processing. This process, known as glomerular filtration, is controlled by intrinsic regulatory systems and by paracrine, neuronal, and endocrine signals that converge onto glomerular cells. In addition, the characteristics of glomerular fluid flow, such as the glomerular filtration rate and the glomerular filtration fraction, play an important role in determining blood flow to the rest of the kidney. Consequently, disease processes that initially affect glomeruli are the most likely to lead to end-stage kidney failure. The cells that comprise the glomerular filter, especially podocytes and mesangial cells, express many different types of ion channels that regulate intrinsic aspects of cell function and cellular responses to the local environment, such as changes in glomerular capillary pressure. Dysregulation of glomerular ion channels, such as changes in TRPC6, can lead to devastating glomerular diseases, and a number of channels, including TRPC6, TRPC5, and various ionotropic receptors, are promising targets for drug development. This review discusses glomerular structure and glomerular disease processes. It also describes the types of plasma membrane ion channels that have been identified in glomerular cells, the physiological and pathophysiological contexts in which they operate, and the pathways by which they are regulated and dysregulated. The contributions of these channels to glomerular disease processes, such as focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy, as well as the development of drugs that target these channels are also discussed.

The Effects of TRPC6 Knockout in Animal Models of Kidney Disease.

Diseases that induce a loss of renal function affect a substantial portion of the world's population and can range from a slight decline in the glomerular filtration rate or microalbuminuria to complete kidney failure. Kidney disorders can be acute or chronic, but any significant reduction in renal function is associated with increased all-cause morbidity and mortality, especially when the conditions become chronic. There is an urgent need for new therapeutic approaches to slow or halt the progression of kidney disease. One potential target of considerable interest is the canonical transient receptor potential-6 (TRPC6) channel. TRCP6 is a cationic channel with a significant permeability to Ca2+. It is expressed in several tissues, including in multiple cell types of the kidney in glomeruli, microvasculature, and tubules. Here, we will describe TRPC6 channels and their roles in signal transduction, with an emphasis on renal cells, and the studies implicating TRPC6 channels in the progression of inherited and acquired kidney diseases. We then describe studies using TRPC6 knockout mice and rats subjected to treatments that model human diseases, including nephrotic syndromes, diabetic nephropathy, autoimmune glomerulonephritis, and acute kidney injuries induced by renal ischemia and by obstruction of the urinary tract. TRPC6 knockout has been shown to reduce glomerular manifestations of disease in several of these models and reduces renal fibrosis caused by urinary tract obstruction. TRPC6 knockout has proven to be less effective at reducing diabetic nephropathy in mouse and rat models. We also summarize the implications of these studies for drug development.

Paraoxonase 2 (PON2) Deficiency Reproduces Lipid Alterations of Diabetic and Inflammatory Glomerular Disease and Affects TRPC6 Signaling.

Diabetes and inflammatory diseases are associated with an altered cellular lipid composition due to lipid peroxidation. The pathogenic potential of these lipid alterations in glomerular kidney diseases remains largely obscure as suitable cell culture and animal models are lacking. In glomerular disease, a loss of terminally differentiated glomerular epithelial cells called podocytes refers to irreversible damage. Podocytes are characterized by a complex ramified cellular architecture and highly active transmembrane signaling. Alterations in lipid composition in states of disease have been described in podocytes but the pathophysiologic mechanisms mediating podocyte damage are unclear. In this study, we employ a genetic deletion of the anti-oxidative, lipid-modifying paraoxonase 2 enzyme (PON2) as a model to study altered cellular lipid composition and its effects on cellular signaling in glomerular disease. PON2 deficiency reproduces features of an altered lipid composition of glomerular disease, characterized by an increase in ceramides and cholesterol. PON2 knockout mice are more susceptible to glomerular damage in models of aggravated oxidative stress such as adriamycin-induced nephropathy. Voltage clamp experiments in cultured podocytes reveal a largely increased TRPC6 conductance after a membrane stretch in PON2 deficiency. Correspondingly, a concomitant knockout of TRPC6 and PON2 partially rescues the aggravated glomerular phenotype of a PON2 knockout in the adriamycin model. This study establishes PON2 deficiency as a model to investigate the pathophysiologic mechanisms of podocyte dysfunction related to alterations in the lipid composition, as seen in diabetic and inflammatory glomerular disease. Expanding the knowledge on these routes and options of intervention could lead to novel treatment strategies for glomerular disease.

miR-155 Is a Positive Regulator of FcεRI-Induced Cyclooxygenase-2 Expression and Cytokine Production in Mast Cells.

MicroRNA-155 (miR-155) has been implicated in IgE-dependent allergic disease including asthma and atopic dermatitis. A few roles for miR-155 have been described in mast cells and some specifically related to IgE receptor signaling, but it is not completely understood. Here, we demonstrate by miRNA seq profiling and quantitative RT-PCR that miR-155 expression is significantly increased in human skin-derived mast cells (SMCs) and mouse bone marrow-derived mast cells (BMMCs) following FcεRI crosslinking with antigen. We demonstrate that FcεRI-induced expression of cyclooxygenase-2 (COX-2) was significantly inhibited in miR-155 knockout (KO) BMMCs whereas arachidonate-5-lipoxygenase (ALOX-5) expression and leukotriene C4 (LTC4) biosynthesis, and degranulation were unaffected. FcεRI-induced cytokine production (TNF, IL-6, and IL-13) from miR-155 KO BMMCs was also significantly diminished. Correspondingly, Akt phosphorylation, but not protein expression, was inhibited in the absence of miR-155 whereas p38 and p42/44 were unaffected. Interesting, lipopolysaccharide (LPS)-induced cytokine production was increased in miR-155 KO BMMCs. Together, these data demonstrate that miR-155 specifically targets the FcεRI-induced prostaglandin and cytokine pathways, but not the leukotriene or degranulation pathways, in mast cells. The data further suggest that miR-155 acts indirectly by targeting a repressor of COX-2 expression and a phosphatase that normally blocks Akt phosphorylation. Overall, this study reveals the role of miR-155 as a positive regulator of mast cell function.

TRPC6 Inactivation Reduces Albuminuria Induced by Protein Overload in Sprague Dawley Rats.

Canonical transient receptor potential-6 (TRPC6) channels have been implicated in familial and acquired forms of focal and segmental glomerulosclerosis (FSGS), and in renal fibrosis following ureteral obstruction in mice. TRPC6 channels also appear to play a role in driving glomerular disease in aging and in autoimmune glomerulonephritis. In the present study, we examine the role of TRPC6 in the proteinuric state caused by prolonged albumin overload (AO) in Sprague Dawley rats induced by daily injections of exogenous albumin. This was assessed in rats with a global and constitutive inactivation of TRPC6 channels (Trpc6del/del rats) and in wild-type littermates (Trpc6wt/wt rats). AO for 14 and 28 days caused increased urine albumin excretion that was significantly attenuated in Trpc6del/del rats compared to Trpc6wt/wt controls. AO overload did not induce significant glomerulosclerosis or azotemia in either genotype. AO induced mild tubulointerstitial disease characterized by fibrosis, hypercellularity and increased expression of markers of fibrosis and inflammation. Those changes were equally severe in Trpc6wt/wt and Trpc6del/del rats. Immunoblot analysis of renal cortex indicated that AO increased the abundances of TRPC3 and TRPC6, and caused a nearly complete loss of TRPC5 in Trpc6wt/wt rats. The increase in TRPC3 and the loss of TRPC5 occurred to the same extent in Trpc6del/del rats. These data also suggest that TRPC6 plays a role in the normal function of the glomerular filtration barrier. However, whether TRPC6 inactivation protects the tubulointerstitial compartments in Sprague Dawley rats depends on the disease model examined.

RAGE and αVß3-integrin are essential for suPAR signaling in podocytes.

The soluble urokinase plasminogen activator receptor (suPAR) has been implicated in the pathogenesis of kidney diseases including primary and recurrent focal and segmental glomerulosclerosis (FSGS), diabetic nephropathy, and acute kidney injuries (AKI). Elevated serum suPAR concentration is a negative prognostic indicator in multiple critical clinical conditions. This study has examined the initial transduction steps used by suPAR in cultured mouse podocytes. We now report that the receptor for advanced glycation end-products (RAGE) co-immunoprecipitates with αV and ß3 integrin subunits, which have been previously shown to initiate suPAR signal transduction at the podocyte cell surface. siRNA knock-down of RAGE attenuated Src phosphorylation evoked by either suPAR or by glycated albumin (AGE-BSA), a prototypical RAGE agonist. suPAR effects on Src phosphorylation were also blocked by the structurally dissimilar RAGE antagonists FPS-ZM1 and azeliragon, as well as by cilengitide, an inhibitor of outside-in signaling through αV-integrins. FPS-ZM1 also blocked Src phosphorylation evoked by AGE-BSA. FPS-ZM1 blocked increases in cell surface TRPC6 abundance, cytosolic reactive oxygen species (ROS) and activation of the small GTPase Rac1 evoked by either suPAR or AGE-BSA. In addition, FPS-ZM1 inhibited Src phosphorylation evoked by serum collected from a patient with recurrent FSGS during a relapse. The magnitude of this inhibition was indistinguishable from the effect produced by a neutralizing antibody against suPAR. These data suggest that orally bioavailable small molecule RAGE antagonists could represent a useful therapeutic strategy for a wide range of clinical conditions associated with elevated serum suPAR, including primary FSGS and AKI.

Effects of TRPC6 Inactivation on Glomerulosclerosis and Renal Fibrosis in Aging Rats.

Canonical transient receptor potential 6 (TRPC6) channels have been implicated in familial and acquired forms of focal and segmental glomerulosclerosis (FSGS) in patients and animal models, as well as in renal fibrosis following ureteral obstruction in mice. Aging also evokes declines in renal function owing to effects on almost every renal compartment in humans and rodents. Here, we have examined the role of TRPC6 in driving inflammation and fibrosis during aging in Sprague-Dawley rats. This was assessed in rats with non-functional TRPC6 channels owing to CRISPR-Cas9 deletion of a portion of the ankyrin repeat domain required for the assembly of functional TRPC6 channels (Trpc6del/del rats). Wild-type littermates (Trpc6wt/wt rats) were used as controls. Animals were evaluated at 2 months and 12 months of age. There was no sign of kidney disease at 2 months of age, regardless of genotype. However, by 12 months of age, all rats examined showed declines in renal function associated with albuminuria, azotemia and increased urine excretion of ß2-microglobulin, a marker for proximal tubule pathology. These changes were equally severe in Trpc6wt/wt and Trpc6del/del rats. We also observed age-related increases in renal cortical expression of markers of fibrosis (α-smooth muscle actin and vimentin) and inflammation (NLRP3 and pro-IL-1ß), and there was no detectable protective effect of TRPC6 inactivation. Tubulointerstitial fibrosis assessed from histology also appeared equally severe in Trpc6wt/wt and Trpc6del/del rats. By contrast, glomerular pathology, blindly scored from histological sections, suggested a significant protective effect of TRPC6 inactivation, but only within the glomerular compartment.

TRPC channels: Regulation, dysregulation and contributions to chronic kidney disease.

Mutations in the gene encoding canonical transient receptor potential-6 (TRPC6) channels result in severe nephrotic syndromes that typically lead to end-stage renal disease. Many but not all of these mutations result in a gain in the function of the resulting channel protein. Since those observations were first made, substantial work has supported the hypothesis that TRPC6 channels can also contribute to progression of acquired (non-genetic) glomerular diseases, including primary and secondary FSGS, glomerulosclerosis during autoimmune glomerulonephritis, and possibly in type-1 diabetes. Their regulation has been extensively studied, especially in podocytes, but also in mesangial cells and other cell types present in the kidney. More recent evidence has implicated TRPC6 in renal fibrosis and tubulointerstitial disease caused by urinary obstruction. Consequently TRPC6 is being extensively investigated as a target for drug discovery. Other TRPC family members are present in kidney. TRPC6 can form a functional heteromultimer with TRPC3, and it has been suggested that TRPC5 may also play a role in glomerular disease progression, although the evidence on this is contradictory. Here we review literature on the expression and regulation of TRPC6, TRPC3 and TRPC5 in various cell types of the vertebrate kidney, the evidence that these channels are dysregulated in disease models, and research showing that knock-out or pharmacological inhibition of these channels can reduce the severity of kidney disease. We also summarize several areas that remain controversial, and some of the large gaps of knowledge concerning the fundamental role of these proteins in regulation of renal function.

TRPC6 inactivation does not affect loss of renal function in nephrotoxic serum glomerulonephritis in rats, but reduces severity of glomerular lesions.

Canonical transient receptor potential-6 (TRPC6) channels have been implicated in a variety of chronic kidney diseases including familial and acquired forms of focal and segmental glomerulosclerosis (FSGS) and renal fibrosis following ureteral obstruction. Here we have examined the role of TRPC6 in progression of inflammation and fibrosis in the nephrotoxic serum (NTS) model of crescentic glomerulonephritis. This was assessed in rats with non-functional TRPC6 channels due to genomic disruption of an essential domain in TRPC6 channels (Trpc6 del/del rats) and wild-type littermates (Trpc6 wt/wt rats). Administration of NTS evoked albuminuria and proteinuria observed 4 and 28 days later that was equally severe in Trpc6 wt/wt and Trpc6 del/del rats. By 28 days, there were dense deposits of complement and IgG within glomeruli in both genotypes, accompanied by severe inflammation and fibrosis readily observed by standard histological methods, and also by increases in renal cortical expression of multiple markers (α-smooth muscle actin, vimentin, NLRP3, and CD68). Tubulointerstitial fibrosis appeared equally severe in Trpc6 wt/wt and Trpc6 del/del rats. TRPC6 inactivation did not protect against the substantial declines in renal function (increases in blood urea nitrogen, serum creatinine and kidney:body weight ratio) in NTS-treated animals, and increases in a urine maker of proximal tubule pathology (ß2-macroglobulin) were actually more severe in Trpc6 del/del animals. By contrast, glomerular pathology, blindly scored from histology, and from renal cortical expression of podocin suggested a partial but significant protective effect of TRPC6 inactivation within the glomerular compartment, at least during the autologous phase of the NTS model.

TRPC6 inactivation does not protect against diabetic kidney disease in streptozotocin (STZ)-treated Sprague-Dawley rats.

Canonical transient receptor potential-6 (TRPC6) channels have been implicated in the progression of several forms of kidney disease (1). While there is strong evidence that glomerular TRPC6 channels are dysregulated in diabetic nephropathy (DN), there is no consensus as to whether deletion or inactivation of TRPC6 is protective in animal models of DN. A previous study in Dahl salt-sensitive rats suggests that TRPC6 knockout has a modest protective effect in streptozotocin (STZ)-induced DN (2). In the present study, we examined whether inactivation of TRPC6 channels by CRISPR/Cas9 editing (Trpc6 del/del rats) affects progression of STZ-induced DN in Sprague-Dawley rats. Wild-type littermates (Trpc6 wt/wt rats) were used as controls. We observed that a single injection of STZ resulted in severe hyperglycemia that was sustained over a 10-week period, accompanied by a marked reduction in circulating C-peptide, dyslipidemia, and failure to gain weight compared to vehicle-treated animals. Those effects were equally severe in Trpc6 wt/wt and Trpc6 del/del rats. STZ treatment resulted in increased urine albumin excretion at 4, 8, and 10 weeks after injection, and this effect was equally severe in Trpc6 wt/wt and Trpc6 del/del rats. TRPC6 inactivation had no effect on blood urea nitrogen (BUN), plasma creatinine concentration, urine nephrin excretion, or kidney weight:body weight ratio measured 10 weeks after STZ injection. STZ treatment evoked modest and equivalent mesangial expansion in Trpc6 wt/wt and Trpc6 del/del rats. In summary, we observed no protective effect of TRPC6 inactivation on STZ-induced DN in rats on the Sprague-Dawley background.

Mechanisms underlying modulation of podocyte TRPC6 channels by suPAR: Role of NADPH oxidases and Src family tyrosine kinases.

The soluble urokinase receptor (suPAR) has been implicated in the pathogenesis of chronic kidney diseases (CKD) and may function as a circulating "permeability factor" driving primary focal and segmental glomerulosclerosis (FSGS). Here we examined the mechanisms whereby suPAR causes mobilization and increased activation of Ca2+-permeable TRPC6 channels, which are also implicated in FSGS. Treatment of immortalized mouse podocytes with recombinant suPAR for 24 h caused a marked increase in cytosolic reactive oxygen species (ROS) that required signaling through integrins. This effect was associated with increased assembly of active cell surface NADPH oxidase 2 (Nox2) complexes and was blocked by the Nox2 inhibitor apoycynin. Treatment with suPAR also evoked a functionally measurable increase in TRPC6 channels that was blocked by concurrent treatment with the ROS-quencher TEMPOL as well as by inhibition of Rac1, an essential component of active Nox2 complexes. Elevated ROS evoked by exposing cells to suPAR or H2O2 caused a marked increase in the abundance of tyrosine-phosphorylated proteins including Src, and suPAR-evoked Src activation was blocked by TEMPOL. Moreover, mobilization and increased activation of TRPC6 by suPAR or H2O2 was blocked by concurrent exposure to PP2, an inhibitor of Src family tyrosine kinases. These data suggest that suPAR induces oxidative stress in podocytes that in turn drives signaling through Src family kinases to upregulate TRPC6 channels. The combination of oxidative stress and altered Ca2+ signaling may contribute to loss of podocytes and progression of various forms of CKD.

Permeation and Rectification in Canonical Transient Receptor Potential-6 (TRPC6) Channels.

Transient receptor potential-6 channels are widely expressed cation channels that play a role in regulating Ca2+ dynamics, especially during G protein-coupled receptor signaling. The permeation of cations through TRPC6 is complex and the relative permeability to Ca2+ relative to monovalent cations appears to be highly voltage-dependent and is reduced upon membrane depolarization. Many investigators have observed complex current-voltage (I-V) relationships in recordings of TRPC6 channels, which often manifest as flattening of I-V curves between 0 and +40 mV and negative to -60 mV. These features are especially common in recordings from TRPC6 channels expressed in heterologous expression systems. Indeed, it is sometimes argued that marked rectification at both negative and positive membrane potentials is a defining feature of TRPC6, and that recordings in which these features are reduced or absent cannot reflect activity of TRPC6. Here we present a review of the literature to show that complex rectification is not seen in every cell type expressing TRPC6, even when comparing recordings made from the same groups of investigators, or in recordings from what is nominally the same heterologous expression system. Therefore other criteria, such as gene knockout or knockdown, or the use of newly emerging selective blockers, must be used to ascertain that a given current reflects activity of endogenously expressed TRPC6 channels. We also discuss the possibility that complex rectification may not be an intrinsic property of TRPC6 in cells where it is observed, and may instead reflect presence of endogenous substances that cause voltage-dependent inhibition of the channels.

Trpc6 inactivation confers protection in a model of severe nephrosis in rats.

Mutations in canonical transient receptor potential-6 (TRPC6) channels give rise to rare familial forms of focal and segmental glomerulosclerosis (FSGS). Here we examined a possible role for TRPC6 in the progression of chronic puromycin aminonucleoside (PAN) nephrosis in Sprague-Dawley rats, a classic model of acquired nephrotic syndromes. We used CRISPR/Cas9 technology to delete a 239-bp region within exon 2 of the Trpc6 gene (Trpc6del allele). Trpc6del/del rats expressed detectable Trpc6 transcripts missing exon 2, and TRPC6 proteins could be detected by immunoblot of renal cortex. However, the abundance of Trpc6 transcripts and TRPC6 protein in renal cortex was much lower than in Trpc6wt/wt littermates, and functional TRPC6 channels could not be detected in whole-cell recordings from glomerular cells cultured from Trpc6del/del animals, possibly because of disruption of ankyrin repeats 1 and 2. During the chronic phase of PAN nephrosis, Trpc6del/del rats had reduced urine albumin excretion, reduced serum cholesterol and triglycerides, and improved azotemia compared to wild-type Trpc6wt/wt littermates. Glomerulosclerosis was severe during chronic PAN nephrosis in Trpc6wt/wt rats but was markedly reduced in Trpc6del/del littermates. Trpc6del/del animals also had less severe tubulointerstitial fibrosis as assessed by several biochemical and histological analyses, as well as reduced foot process effacement and glomerular basement thickening compared to Trpc6wtt/wt controls. None of the manipulations in this study affected the abundance of TRPC5 channels in renal cortex. TRPC3 was increased in PAN nephrosis and in Trpc6del/del rats. These data support a role for TRPC6 channels in driving an acquired form of secondary FSGS. KEY MESSAGES: We examined aminonucleoside nephrosis in rats with wild type and inactivated TRPC6. TRPC6 channels were inactivated by CRISPR/Cas9 editing of the Trpc6 gene. TRPC6 inactivation reduced albuminuria in the chronic but not the acute phase. TRPC6 inactivation reduced glomerulosclerosis and ultrastructural changes. TRPC6 inactivation also reduced interstitial changes and renal fibrosis.

Changes in podocyte TRPC channels evoked by plasma and sera from patients with recurrent FSGS and by putative glomerular permeability factors.

Primary forms of focal and segmental glomerulosclerosis (FSGS) are driven by circulating factors that cause dysfunction or loss podocytes. Rare genetic forms of FSGS can be caused by mutations in TRPC6, which encodes a Ca2+-permeable cationic channel expressed in mesangial cells and podocytes; and NPHS2, which encodes podocin, a TRPC6-binding protein expressed in podocyte slit diaphragm domains. Here we observed that exposing immortalized mouse podocytes to serum or plasma from recurrent FSGS patients for 24h increased the steady-state cell-surface abundance of TRPC6, accompanied by an increase in currents through endogenous TRPC6 channels evoked by a hypoosmotic stretch stimulus. These effects were mimicked by the soluble urokinase receptor (suPAR) and by tumor necrosis factor (TNF), circulating factors implicated in nephrotic syndromes. Most but not all of the recurrent FSGS plasma samples that we examined also caused a loss of podocin over a period of several hours. The loss of podocin was also seen following exposure to suPAR but not TNF. However, TNF increased the effects of suPAR on TRPC6 and podocin, and TNF and suPAR are required for the full effects of one of the recurrent FSGS plasma samples. The actions of FSGS plasma, suPAR and TNF on surface abundance of TRPC6 were blocked by cilengitide, an inhibitor of αvß3-integrin signaling. These data suggest that primary FSGS is a heterogeneous condition mediated by multiple circulating factors, and support TRPC6 and αvß3-integrin as potential therapeutic targets.

20-Hydroxyeicosatetraenoic Acid (20-HETE) Modulates Canonical Transient Receptor Potential-6 (TRPC6) Channels in Podocytes.

The arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) regulates renal function, including changes in glomerular function evoked during tubuloglomerular feedback (TGF). This study describes the cellular actions of 20-HETE on cultured podocytes, assessed by whole-cell recordings from cultured podocytes combined with pharmacological and cell-biological manipulations of cells. Bath superfusion of 20-HETE activates cationic currents that are blocked by the pan-TRP blocker SKF-96365 and by 50 µM La(3+), and which are attenuated after siRNA knockdown of TRPC6 subunits. Similar currents are evoked by a membrane-permeable analog of diacylgycerol (OAG), but OAG does not occlude responses to maximally-activating concentrations of 20-HETE (20 µM). Exposure to 20-HETE also increased steady-state surface abundance of TRPC6 subunits in podocytes as assessed by cell-surface biotinylation assays, and increased cytosolic concentrations of reactive oxygen species (ROS). TRPC6 activation by 20-HETE was eliminated in cells pretreated with TEMPOL, a membrane-permeable superoxide dismutase mimic. Activation of TRPC6 by 20-HETE was also blocked when whole-cell recording pipettes contained GDP-ßS, indicating a role for either small or heterotrimeric G proteins in the transduction cascade. Responses to 20-HETE were eliminated by siRNA knockdown of podocin, a protein that organizes NADPH oxidase complexes with TRPC6 subunits in this cell type. In summary, modulation of ionic channels in podocytes may contribute to glomerular actions of 20-HETE.

NMDA Receptors as Potential Therapeutic Targets in Diabetic Nephropathy: Increased Renal NMDA Receptor Subunit Expression in Akita Mice and Reduced Nephropathy Following Sustained Treatment With Memantine or MK-801.

N-methyl-d-aspartate (NMDA) receptors are expressed throughout the kidney, and the abundance of these receptors and some of their endogenous agonists are increased in diabetes. Moreover, sustained activation of podocyte NMDA receptors induces Ca(2+) influx, oxidative stress, loss of slit diaphragm proteins, and apoptosis. We observed that NMDA receptor subunits and their transcripts are increased in podocytes and mesangial cells cultured in elevated glucose compared with controls. A similar increase in NMDA subunits, especially NR1, NR2A, and NR2C, was observed in glomeruli and tubules of Akita mice. Sustained continuous treatment with the strong NMDA receptor antagonist dizocilpine (MK-801) for 28 days starting at 8 weeks of age reduced 24-h albumin excretion and mesangial matrix expansion and improved glomerular ultrastructure in Akita mice. MK-801 did not alleviate reduced Akita mouse body weight and had no effect on kidney histology or ultrastructure in DBA/2J controls. The structurally dissimilar NMDA antagonist memantine also reduced diabetic nephropathy, although it was less effective than MK-801. Inhibition of NMDA receptors may represent a valid therapeutic approach to reduce renal complications of diabetes, and it is possible to develop well-tolerated agents with minimal central nervous system effects. Two such agents, memantine and dextromethorphan, are already in widespread clinical use.

Second Messenger-Operated Calcium Entry Through TRPC6.

Canonical transient receptor potential 6 (TRPC6) proteins assemble into heteromultimeric structures forming non-selective cation channels. In addition, many TRPC6-interacting proteins have been identified like some enzymes, channels, pumps, cytoskeleton-associated proteins, immunophilins, or cholesterol-binding proteins, indicating that TRPC6 are engaged into macromolecular complexes. Depending on the cell type and the experimental conditions used, TRPC6 activity has been reported to be controlled by diverse modalities. For instance, the second messenger diacylglycerol, store-depletion, the plant extract hyperforin or H2O2 have all been shown to trigger the opening of TRPC6 channels. A well-characterized consequence of TRPC6 activation is the elevation of the cytosolic concentration of Ca(2+). This latter response can reflect the entry of Ca(2+) through open TRPC6 channels but it can also be due to the Na(+)/Ca(2+) exchanger (operating in its reverse mode) or voltage-gated Ca(2+) channels (recruited in response to a TRPC6-mediated depolarization). Although TRPC6 controls a diverse array of biological functions in many tissues and cell types, its pathophysiological functions are far from being fully understood. This chapter covers some key features of TRPC6, with a special emphasis on their biological significance in kidney and blood cells.