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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 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 β3 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.

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.

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 (Trpc6del/del rats) and wild-type littermates (Trpc6wt/wt rats). Administration of NTS evoked albuminuria and proteinuria observed 4 and 28 days later that was equally severe in Trpc6wt/wt and Trpc6del/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 Trpc6wt/wt and Trpc6del/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 (β2-macroglobulin) were actually more severe in Trpc6del/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. Keywords: Chronic kidney disease, TRPC6, Renal fibrosis, Glomerulonephritis

BackgroundTransient receptor potential channel 5 (TRPC5) is a non-selective cationic ion channel expressed in brain, kidney and other organs where its activation underlies podocyte injury in chronic kidney diseases. Specifically, it has been suggested that a podocyte TRPC5 plasma membrane relocation and channel activation following injury results from activation of Rac-1, propagating podocyte dysfunction and proteinuria. However, previous TRPC5 transgenic mouse studies had questioned a pathogenic role for TRPC5 in podocytes. This investigation was designed to specifically evaluate podocyte Rac-1 activation in the context of functional TRPC5 or a TRPC5 pore mutant to assess effects on proteinuria.Materials and MethodsWe employed single cell patch-clamp studies of cultured podocytes and studied proteinuria in transgenic mouse models to characterize the effects of TRPC5 following podocyte Rac-1 activation.ResultsInhibition of TRPC5 by small molecules reportedly ameliorated proteinuria in murine models of proteinuric kidney diseases. In order to directly examine TRPC5 function following Rac-1-induced podocyte injury, we analyzed TRPC5 inhibition in podocyte specific Rac-1 (active) transgenic mice. In addition, we generated a double-transgenic mouse constitutively overexpressing either TRPC5 (TRPC5WT) or a TRPC5 dominant-negative pore mutant (TRPC5DN) in concert with podocyte specific and inducible activation of active Rac-1 (Rac-1Dtg). In electrophysiological experiments, active TRPC5 was detected in primary podocytes overexpressing TRPC5 but not in podocytes with endogenous TRPC5 expression, nor with Rac-1 overexpressing podocytes. TRPC5 inhibition did not change proteinuria in mice with active podocyte Rac-1, nor did an increase or loss of TRPC5 activity affected podocyte injury in Rac-1Dtg animals. Administration of TRPC5 inhibitors, ML204 and AC1903, did not alleviate podocyte Rac-1 induced proteinuria.ConclusionTRPC5 inhibition did not modify podocyte Rac-1 induced proteinuria in mice.Significance StatementTRPC5 is a calcium conducting ion channel involved in a plethora of biological functions in the brain, kidney and other organs. In proteinuric kidney diseases, others proposed a model that links activation of small GTPase Rac-1 in podocytes to activation of TRPC5 channels propagating cellular injury and eventually leading to progressive kidney disease. To test this hypothesis, we have developed a novel transgenic mouse model that employs podocyte Rac-1 activation in the presence or absence of a functional TRPC5 channel. Our data shows that transgenic mice with activated Rac-1 in podocytes did not enhance endogenous TRPC5 expression or its activity. Furthermore, TRPC5 blockade or activation did not modify Rac-1 induced proteinuria in mice.

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. Electronic supplementary material The online version of this article (10.1007/s00109-018-1648-3) contains supplementary material, which is available to authorized users.

Gain-of-function mutations of classic transient receptor potential channel 6 (TRPC6) were identified in familial FSGS, and increased expression of wild-type TRPC6 in glomeruli is observed in several human acquired proteinuric diseases. Synaptopodin, an actin binding protein that is important in maintaining podocyte function, is downregulated in various glomerular diseases. Here, we investigated whether synaptopodin maintains podocyte function by regulating podocyte surface expression and activity of TRPC6. We show indirect interaction and nonrandom association of synaptopodin and TRPC6 in podocytes. Knockdown of synaptopodin in cultured mouse podocytes increased the expression of TRPC6 at the plasma membrane, whereas overexpression of synaptopodin decreased it. Mechanistically, synaptopodin–dependent TRPC6 surface expression required functional actin and microtubule cytoskeletons. Overexpression of wild–type or FSGS–inducing mutant TRPC6 in synaptopodin-depleted podocytes enhanced TRPC6–mediated calcium influx and induced apoptosis. In vivo, knockdown of synaptopodin also caused increased podocyte surface expression of TRPC6. Administration of cyclosporin A, which stabilizes synaptopodin, reduced LPS-induced proteinuria significantly in wild-type mice but to a lesser extent in TRPC6 knockout mice. Furthermore, administration of cyclosporin A reversed the LPS-induced increase in podocyte surface expression of TRPC6 in wild-type mice. Our findings suggest that alteration in synaptopodin levels under disease conditions may modify intracellular TRPC6 channel localization and activity, which further contribute to podocyte dysfunction. Reducing TRPC6 surface levels may be a new approach to restoring podocyte function.

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 Ca 2+ -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 H 2 O 2 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 H 2 O 2 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 Ca 2+ signaling may contribute to loss of podocytes and progression of various forms of CKD.

PodocyteTRPC6 channels have been implicated in glomerular diseases. Syndecan-4 (Sdc4) is a membrane proteoglycan that can be cleaved to release a soluble ectodomain capable of paracrine and autocrine signaling. We have confirmed that overexpression of Sdc4 core protein increases surface abundance of TRPC6 channels in cultured podocytes, whereas Sdc4 knockdown has the opposite effect. Exposure to soluble Sdc4 ectodomain also increased the surface abundance of TRPC6, and increased cationic currents evoked by a diacylglycerol analog in podocytes. Sdc4 ectodomain increased generation of reactive oxygen species (ROS), reduced activation of RhoA, increased activation of Rac1, increased nuclear abundance of NFATc1, and increased total β3-integrin. The effects of Sdc4 ectodomain on cell-surface TRPC6 were blocked by the ROS quencher TEMPOL, and by the Rac1 inhibitor NSC-23766, but were not blocked by inhibition of calcineurin–NFATc1 signaling. The Sdc4 core protein co-immunoprecipitates with β3-integrin in cultured podocytes. Moreover, effects of Sdc4 ectodomain on TRPC6, ROS generation, Rac1 and RhoA modulation, and NFATc1 activation were blocked by cilengitide, a selective inhibitor of outside-in signaling through αv-containing integrins. Exposure to TNF, or serum from three patients with recurrent FSGS in relapse, increased shedding of podocyte Sdc4 ectodomains into the surrounding medium. This was also observed after treating podocytes with the metalloproteinase ADAM17 or after overexpression of the Sdc4 core protein. Increased concentrations of Sdc4 ectodomain were detected in urine of rats during acute puromycin aminonucleoside nephrosis. Locally generated Sdc4 may play a role in regulating TRPC6 channels, and may contribute to glomerular pathology.

TNF has been implicated in glomerular diseases, but its actions on podocytes are not well understood. Endogenous TNF expression is markedly increased in mouse podocytes exposed to sera from patients with recurrent focal segmental glomerulosclerosis, and TNF is able to increase its own expression in these cells. Exposure of podocytes to TNF increased phosphorylation of NF-κB p65-RelA followed by increased tyrosine phosphorylation of STAT3. STAT3 activation was blocked by the NF-κB inhibitor JSH-23 and by the STAT3 inhibitor stattic, whereas TNF-evoked NF-κB activation was not affected by stattic. TNF treatment increased nuclear accumulation of nuclear factor of activated T cells (NFAT)c1 in podocytes, a process that occurred downstream of STAT3 activation. TNF also increased expression of cyclin D1 but had no effect on cyclin-dependent kinase 4, p27kip, or podocin. Despite its effects on cyclin D1, TNF treatment for up to 72 h did not cause podocytes to reenter the cell cycle. TNF increased total expression of transient receptor potential (TRP)C6 channels through a pathway dependent on NFATc1 and increased the steady-state expression of TRPC6 subunits on the podocyte cell surface. TNF effects on TRPC6 trafficking required ROS. Consistent with this, La3+-sensitive cationic currents activated by a diacylglycerol analog were increased in TNF-treated cells. The effects of TNF on NFATc1 and TRPC6 expression were blocked by cyclosporine A but were not blocked by the pan-TRP inhibitor SKF-96365. TNF therefore influences multiple pathways previously implicated in podocyte pathophysiology and is likely to sensitize these cells to other insults.

Primary forms of focal and segmental glomeruloslerosis (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 24 hr 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 αvβ3-integrin signaling. These data suggest that primary FSGS is a heterogeneous condition mediated by multiple circulating factors, and support TRPC6 and αvβ3-integrin as potential therapeutic targets.

The transcription factor signal transducer and activator of transcription-3 (STAT3) is activated by proinflammatory cytokines and circulating factors in many cell types. Synaptopodin (Synpo) is a cytoskeleton regulatory protein expressed in podocyte foot processes that regulates the dynamics of actin filaments and the stability of small GTPases. Here we show that inhibition of STAT3 signaling using the small-molecule inhibitor benzo[b]thiophene,6-nitro-,1,1-dioxide (Stattic), or by STAT3 knockdown by small interfering RNA, caused a decrease in Synpo mRNA and protein in an immortalized mouse podocyte cell line. This loss of Synpo, which occurred in 30-80 minutes, was also seen after treatment with the translational inhibitor cycloheximide. The loss of Synpo protein after Stattic or cycloheximide treatment did not occur when podocytes were simultaneously exposed to 1-[N-[(l-3-trans-carboxyoxirane-2-carbonyl)-l-leucyl]amino]-4-guanidinobutane (E-64), an inhibitor of thiol proteases such as cathepsin L. Treatment with interleukin-6 (IL-6) increased tyrosine phosphorylation of STAT3 and evoked a parallel increase in Synpo levels in podocytes. The stimulatory effect of IL-6 on Synpo was completely inhibited by pretreatment with Stattic. By contrast, 30-60-minute exposure to angiotensin II (Ang II) inhibited STAT3 signaling and concurrently reduced Synpo protein levels. The Ang II-evoked loss of Synpo was prevented by E-64 but not by inhibition of calcineurin or blockade of transient receptor potential cation channels. Inhibition of STAT3 by Stattic caused marked changes in the distribution of podocyte actin filaments, and caused a nearly complete suppression of the migration of these cells in wound assays, consistent with the loss of Synpo. Stattic treatment also caused loss of RhoA protein.

Angiotensin II (AII) plays a major role in the progression of chronic kidney diseases. Podocytes are essential components of the ultrafiltration apparatus, and are targets for AII signaling. AII has been shown to increase generation of reactive oxygen species (ROS) in podocytes. Canonical transient receptor potential-6 (TRPC6) channels stimulate Ca(2+) influx in podocytes, and have been implicated in glomerular disease. We observed that AII increased cationic currents in rat podocytes in an isolated glomerulus preparation in which podocytes are still attached to the underlying capillary. This effect was completely blocked by SKF-96365, by micromolar La(3+) , and by siRNA knockdown of TRPC6, indicating that TRPC6 is the primary source of Ca(2+) influx mobilized by endogenously expressed angiotensin II receptors in these cells. These responses were also blocked by the AT1R antagonist losartan, the phospholipase C inhibitor D-609, and by inhibition of G protein signaling. The pan-protein kinase C inhibitor chelerythrine had no effect. Importantly, pretreating podocytes with the ROS quencher manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) eliminated AII activation of TRPC6. Significant reductions of AII effects on podocyte TRPC6 were also observed after pretreatment with NADPH oxidase inhibitors apocynin or diphenylene iodonium (DPI). These data suggest that ROS production permits activation of TRPC6 channels by G protein and PLC-dependent cascades initiated by AII acting on AT1Rs in podocytes. This pathway also provides a basis whereby two forms of cellular stress-oxidative stress and Ca(2+) overload-converge on common pathways relevant to disease.

Gain-of-function mutations in the calcium channel TRPC6 lead to autosomal dominant focal segmental glomerulosclerosis and podocyte expression of TRPC6 is increased in some acquired human glomerular diseases, particularly in membranous nephropathy. These observations led to the hypothesis that TRPC6 overactivation is deleterious to podocytes through pathological calcium signaling, both in genetic and acquired diseases. Here, we show that the effects of TRPC6 on podocyte function are context-dependent. Overexpression of TRPC6 alone did not directly affect podocyte morphology and cytoskeletal structure. Unexpectedly, however, overexpression of TRPC6 protected podocytes from complement-mediated injury, whereas genetic or pharmacological TRPC6 inactivation increased podocyte susceptibility to complement. Mechanistically, this effect was mediated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activation. Podocyte-specific TRPC6 transgenic mice showed stronger CaMKII activation, reduced podocyte foot process effacement and reduced levels of proteinuria during nephrotoxic serum nephritis, whereas TRPC6 null mice exhibited reduced CaMKII activation and higher levels of proteinuria compared with wild type littermates. Human membranous nephropathy biopsy samples showed podocyte staining for active CaMKII, which correlated with the degree of TRPC6 expression. Together, these data suggest a dual and context dependent role of TRPC6 in podocytes where acute activation protects from complement-mediated damage, but chronic overactivation leads to focal segmental glomerulosclerosis.

Gain-of-function mutations in the transient receptor potential (TRP) cation channel subfamily C member 6 ( TRPC6) gene and mutations in the NPHS2 gene encoding podocin result in nephrotic syndromes. The purpose of this study was to determine the functional significance of biochemical interactions between these proteins. We observed that gating of TRPC6 channels in podocytes is markedly mechanosensitive and can be activated by hyposmotic stretch or indentation of the plasma membrane. Stretch activation of cationic currents was blocked by small interfering RNA knockdown of TRPC6, as well as by SKF-96365 or micromolar La3+. Stretch activation of podocyte TRPC6 persisted in the presence of inhibitors of phospholipase C (U-73122) and phospholipase A2(ONO-RS-082). Robust stretch responses also persisted when recording electrodes contained guanosine 5′- O-(2-thiodiphosphate) at concentrations that completely suppressed responses to ANG II. Stretch responses were enhanced by cytochalasin D but were abolished by the peptide GsMTx4, suggesting that forces are transmitted to the channels through the plasma membrane. Podocin and TRPC6 interact at their respective COOH termini. Knockdown of podocin markedly increased stretch-evoked activation of TRPC6 but nearly abolished TRPC6 activation evoked by a diacylglycerol analog. These data suggest that podocin acts as a switch to determine the preferred mode of TRPC6 activation. They also suggest that podocin deficiencies will result in Ca2+overload in foot processes, as with gain-of-function mutations in the TRPC6 gene. Finally, they suggest that mechanical activation of TRP family channels and the preferred mode of TRP channel activation may depend on whether members of the stomatin/prohibitin family of hairpin loop proteins are present.

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 La3+, 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-βS, 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.

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 Ca2+ 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.

International audience; 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.

Insulin receptors in podocytes are essential for normal kidney function. Here, we show that insulin evokes a rapid increase in the surface expression of canonical transient receptor potential-6 channel (TRPC6) channels in cultured podocytes, but caused a decrease in surface expression of TRPC5. These effects are accompanied by a marked increase in outwardly rectifying cationic currents that can be blocked by 10 μM SKF96365 or 100 μM La3+. Application of oleoyl-2-acetyl- sn-glycerol (OAG) also increased SKF96365- and La3+-sensitive cationic currents in podocytes. Importantly, current responses to a combination of OAG and insulin were the same amplitude as those evoked by either agent applied alone. This occlusion effect suggests that OAG and insulin are targeting the same population of channels. In addition, shRNA knockdown of TRPC6 markedly reduced cationic currents stimulated by insulin. The effects of insulin on TRPC6 were mimicked by treating podocytes with H2O2. Insulin treatment rapidly increased the generation of H2O2 in podocytes, and it increased the surface expression of the NADPH oxidase NOX4 in cultured podocytes. Basal and insulin-stimulated surface expression of TRPC6 were reduced by pretreatment with diphenylene iodonium, an inhibitor of NADPH oxidases and other flavin-dependent enzymes, by siRNA knockdown of NOX4, and by manganese (III) tetrakis (4-benzoic acid) porphyrin chloride, a membrane-permeable mimetic of superoxide dismutase and catalase. These observations suggest that insulin increases generation of ROS in part through activation of NADPH oxidases, and that this step contributes to modulation of podocyte TRPC6 channels.

The growth of many human breast tumors requires the proliferative effect of estrogen acting via the estrogen receptor α (ERα). ERα signaling is therefore a clinically important target for breast cancer prevention and therapeutics. Although extensively studied, the mechanism by which ERα promotes proliferation remains to be fully established. We observed an up-regulation of transcript encoding the pH-sensitive two-pore domain potassium channel KCNK5 in a screen for genes stimulated by 17β-estradiol (E2) in the ERα(+) breast cancer cell lines MCF-7 and T47D. KCNK5 mRNA increased starting 1 h after the onset of E2 treatment, and protein levels followed after 12 h. Estrogen-responsive elements are found in the enhancer region of KCNK5, and chromatin immunoprecipitation assays revealed binding of ERα to the KCNK5 enhancer in E2-treated MCF-7 cells. Cells treated with E2 also showed increases in the amplitude of pH-sensitive potassium currents, as assessed by whole-cell recordings. These currents are blocked by clofilium. Although confocal microscopy suggested that most of the channels are located in intracellular compartments, the increase in macroscopic currents suggests that E2 treatment increases the number of active channels at the cell surface. Application of small interfering RNA specific for KCNK5 decreased pH-sensitive potassium currents and also reduced the estrogen-induced proliferation of T47D cells. We conclude that E2 induces the expression of KCNK5 via ERα(+) in breast cancer cells, and this channel plays a role in regulating proliferation in these cell lines. KCNK5 may therefore represent a useful target for treatment, for example, of tamoxifen-resistant breast cancer.

Loss or dysfunction of podocytes is a major cause of glomerular kidney disease. Several genetic forms of glomerular disease are caused by mutations in genes that encode structural elements of the slit diaphragm or the underlying cytoskeleton of podocyte foot processes. The recent discovery that gain-of-function mutations in Ca2+-permeable canonical transient receptor potential-6 channels (TRPC6) underlie a subset of familial forms of focal segmental glomerulosclerosis (FSGS) has focused attention on the basic cellular physiology of podocytes. Several recent studies have examined the role of Ca2+dynamics in normal podocyte function and their possible contributions to glomerular disease. This review summarizes the properties of TRPC6 and related channels, focusing on their permeation and gating properties, the nature of mutations associated with familial FSGS, and the role of TRPC channels in podocyte cell biology as well as in glomerular pathophysiology. TRPC6 interacts with several proteins in podocytes, including essential slit diaphragm proteins and mechanosensitive large-conductance Ca2+-activated K+channels. The signaling dynamics controlling ion channel function and localization in podocytes appear to be quite complex.

Large conductance Ca2+-activated K+(BKCa) channels encoded by the Slo1 gene (also known as KCNMA1) are physiologically important in a wide range of cell types and form complexes with a number of other proteins that affect their function. We performed a yeast two-hybrid screen to identify proteins that interact with BKCachannels using a bait construct derived from domains in the extreme COOH-terminus of Slo1. A protein known as membrane-associated guanylate kinase with inverted orientation protein-1 (MAGI-1) was identified in this screen. MAGI-1 is a scaffolding protein that allows formation of complexes between certain transmembrane proteins, actin-binding proteins, and other regulatory proteins. MAGI-1 is expressed in a number of tissues, including podocytes and the brain. The interaction between MAGI-1 and BKCachannels was confirmed by coimmunoprecipitation and glutathione S-transferase pull-down assays in differentiated cells of a podocyte cell line and in human embryonic kidneys (HEK)293T cells transiently coexpressing MAGI-1a and three different COOH-terminal Slo1 variants. Coexpression of MAGI-1 with Slo1 channels in HEK-293T cells results in a significant reduction in the surface expression of Slo1, as assessed by cell-surface biotinylation assays, confocal microscopy, and whole cell recordings. Partial knockdown of endogenous MAGI-1 expression by small interfering RNA (siRNA) in differentiated podocytes increased the surface expression of endogenous Slo1 as assessed by electrophysiology and cell-surface biotinylation assays, whereas overexpression of MAGI-1a reduced steady-state voltage-evoked outward current through podocyte BKCachannels. These data suggest that MAGI-1 plays a role in regulation of surface expression of BKCachannels in the kidney and possibly in other tissues.

Hyperpolarization activated cyclic nucleotide modulated (HCN) ion channel currents are activated by hyperpolarization and modulated in response to changes in cytosolic adenosine 3',5'-cyclic monophosphate (cAMP) concentrations. A cDNA chimera combining the rat HCN2 cyclic nucleotide binding domain and the DNA binding domain of the cAMP receptor protein (CRP) from E. coli and the histidine tag (HCN2/CRP) was expressed and purified. The construct is capable of forming only non-ligand dependent dimers because the C-linker region of the channel is not present in this construct. The construct binds 8-[[2-[(fluoresceinylthioureido) amino] ethyl] thio] adenosine-3',5'-cyclic monophosphate (8-fluo cAMP) with a Kd of 0.299 microM as determined with a monomer binding model. The Ki values of 20 ligands related to cAMP were measured in order to determine the properties necessary for a ligand to bind to the HCN2 binding domain. This is the first report of cAMP and gunaosine 3',5'-cyclic monophosphate (cGMP) affinities to the HCN2 binding domain being equivalent, even though they modulate the channel with a 10-fold difference in K0.5. Furthermore, the array of ligands measured allows the preference rank order for each purine ring position to be determined: position 1, H > NH2 > O; position 2, NH2 > Cl > H > O; position 6, NH2 > Cl > H > O; and position 8, NH2 > Cl > H > O. Finally, the ability of HCN2/CRP to bind cyclic nucleotide pyrimidine rings at concentrations approximately 1.33 times greater than cAMP suggests that ribofuranose is key for binding.

Photoreceptors are non-spiking neurons, and their synapses mediate the continuous release of neurotransmitters under the control of L-type voltage-gated calcium channels (VGCCs). Photoreceptors express endogenous circadian oscillators that play important roles in regulating photoreceptor physiology and function. Here, we report that the L-type VGCCs in chick cone photoreceptors are under circadian control. The L-type VGCC currents are greater when measured during the subjective night than during the subjective day. Using antibodies against the VGCCα1C and VGCCα1D subunits, we found that the immunofluorescence intensities of both VGCCα1C and VGCCα1D in photoreceptors are higher during the subjective night. However, the mRNA levels of VGCCα1D, but not VGCCα1C, are rhythmic. Nocturnal increases in L-type VGCCs are blocked by manumycin A, PD98059, and KN93, which suggest that the circadian output pathway includes Ras, Erk, and calcium-calmodulin dependent kinase II. In summary, four independent lines of evidence show that the L-VGCCs in cone photoreceptors are under circadian control.

Myasthenia gravis can be induced in mice by injecting the extracellular domain of rat muscle-specific kinase (MuSK), a transmembrane receptor tyrosine kinase involved in agrin signaling at the neuromuscular junction. About 5-10% of human myasthenia gravis patients have autoantibodies against MuSK. Here we have examined mouse neuromuscular junctions following MuSK immunization in two groups of muscles that can be distinguished on the basis of the timing of neuromuscular synaptogenesis and their response to perturbation of agrin signaling. We used confocal microscopy to characterize the distribution and expression of nicotinic acetylcoline receptors and of two presynaptic makers, neurofilament protein and synaptophysin. We observed disruption of neuromuscular junctions in all muscles examined in this model of myasthenia gravis. However delayed-synapsing muscles, including the diaphragm, sternomastoid and tibialis posterior, were significantly more severely affected than fast-synapsing muscles, including the intercostal, adductor longus and tibialis anterior. These results suggest a basis for the differential susceptibility of muscles in different classes of myasthenia gravis patients, including patients with autoantibodies against MuSK.

In developing ciliary ganglion (CG) neurons, movement of functional large-conductance (BK type) Ca2+-activated K+( KCa) channels to the cell surface is stimulated by the endogenous growth factors TGFβ1 and β-neuregulin-1 (NRG1). Here we show that a brief NRG1 treatment (0.5–1.5 h) mobilizes KCachannels in a post-Golgi compartment, but longer treatments (>3.5 h) mobilize KCachannels located in the endoplasmic reticulum or Golgi apparatus. Specifically, the effects of 3.5 h NRG1 treatment were completely blocked by treatments that disrupt Golgi apparatus function. These include inhibition of microtubules, or inhibition of the ADP-ribosylation factor-1 (ARF1) system by brefeldin A, by over-expression of dominant-negative ARF1, or over-expression of an ARF1 GTPase-activating protein that blocks ARF1 cycling between GTP- and GDP-bound states. These treatments had no effect on stimulation of KCaevoked by 1.5 h treatment with NRG1, indicating that short-term responses to NRG1 do not require an intact Golgi apparatus. By contrast, both the acute and sustained effects of NRG1 were inhibited by treatments that block trafficking processes that occur close to the plasma membrane. Thus mobilization of KCawas blocked by treatments than inhibit ADP-ribosylation factor-6 (ARF6) signaling, including overexpression of dominant-negative ARF6, dominant-negative ARNO, or dominant-negative phospholipase D1. TGFβ1, the effects of which on KCaare much slower in onset, is unable to selectively mobilize channels in the post-Golgi pool, and its effects on KCaare completely blocked by inhibition of microtubules, Golgi function and also by plasma membrane ARF6 and phospholipase D1 signaling.

The trafficking of large-conductance Ca2+-activated K+ channels (K(Ca)) in chick ciliary ganglion neurons is regulated by growth factors. Here we show that a canonical p38 cascade inhibits K(Ca) trafficking in ciliary ganglion neurons. Two different p38 inhibitors (SB202190 or SB203580) or over-expression of dominant-negative forms of several components of the p38 cascade increased K(Ca) in ciliary neurons. Inhibition of protein synthesis or Golgi processing had no effect on this phenomenon, suggesting that p38 is acting at a distal step of the trafficking pathway. Depolymerization of filamentous actin (F-actin) increased functional expression of K(Ca), whereas stabilization of F-actin inhibited the effect of SB202190 on K(Ca) trafficking. SB202190 also caused an immunochemically detectable increase in K(Ca) on the plasma membrane. Inhibition of p38 decreased the extent of cortical F-actin in ciliary neurons. Macroscopic K(Ca) is suppressed by transforming growth factor (TGF) beta3. Application of TGFbeta3 increased the phosphorylation of p38 in ciliary neurons and increased cortical F-actin. Thus, the p38 signaling cascade endogenously suppresses development of functional K(Ca), in part by stabilizing an F-actin barrier that prevents plasma membrane insertion of functional channel complexes. This cascade also appears to mediate inhibitory effects of TGFbeta3 on the expression of K(Ca).

The protein kinase Akt is a crucial regulator of neuronal survival and apoptosis. Here we show that Akt activation is necessary for mobilization of large-conductance KCachannels in ciliary ganglion (CG) neurons evoked by β-neuregulin-1 (NRG1) and transforming growth factor-β1 (TGFβ1). Application of NRG1 to embryonic day 9 (E9) CG neurons increased Akt phosphorylation, as observed previously for TGFβ1. NRG1- and TGFβ1-evoked stimulation of KCais blocked by inhibitors of PI3K by overexpression of a dominant-negative form of Akt, by overexpression of CTMP, an endogenous negative regulator of Akt, and by application of the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-( R)-2- O-methyl-3- O-octadecylcarbonate (HIMO). Conversely, overexpression of a constitutively-active form of Akt was sufficient by itself to increase mobilization of functional KCachannels. NRG1 and TGFβ1 evoked an Akt-dependent increase in cell-surface SLO α-subunits. These procedures have no effect on voltage-activated Ca2+currents. Thus Akt plays an essential role in the developmental regulation of excitability in CG neurons.

Endogenous beta-neuregulin-1 is required for the plasma membrane expression of large-conductance (BK-type) Ca2+-activated K+ channels in developing chick ciliary neurons of the chick ciliary ganglion. During normal development, beta-neuregulin-1 acts in concert with transforming growth factor-beta1 to stimulate movement of large-conductance Ca2+-activated K+ channels from intracellular stores into the plasma membrane, although these two growth factors preferentially act on different intracellular pools. We have previously shown that actions of transforming growth factor-beta1 on ciliary neurons require activation of phosphoinositol 3-kinase and Akt, as well as a parallel cascade composed of the small GTPase Ras and a mitogen-activated protein kinase (extracellular signal-regulated kinase). In addition, we have shown that the actions of beta-neuregulin-1 require activation of phosphoinositol 3-kinase and the protein kinase Akt. Here we examine whether beta-neuregulin-1-evoked mobilization of large-conductance Ca2+-activated K+ channels also requires activation of a Ras-extracellular signal-regulated kinase signaling cascade. We observed that application of beta-neuregulin-1 caused a robust and MEK1/2-dependent increase in extracellular signal-regulated kinase diphosphorylation that indicates activation of this signaling cascade in ciliary ganglion neurons, similar to what we have previously observed for transforming growth factor-beta1. However, activation of this cascade is not necessary for beta-neuregulin-1-evoked mobilization because stimulation of macroscopic large-conductance Ca2+-activated K+ channels persisted in cells treated with the MEK1/2 inhibitors PD98059 or U0126, in cells over-expressing dominant-negative forms of extracellular signal-regulated kinase, and in cells treated with the Ras inhibitor FTI-277. These results indicate that the mechanisms that underlie beta-neuregulin-1 and transforming growth factor-beta1 mobilization of large-conductance Ca2+-activated K+ channels are only partly overlapping, possibly because they cause recruitment of spatially distinct signaling complexes.

The functional expression of A-type K+ channels ( IA) was examined in chick lumbar motoneurons (LMNs) at embryonic days 6 and 11 (E6 and E11). We observed a threefold increase in IA density between E6 and E11 in spinal cord slices and acutely dissociated LMNs. There was no change in current density, kinetics, or voltage dependence of IA in E11 homozygous limbless mutants or in E11 embryos in which hindlimbs were surgically removed at E6. Moreover, chronic in ovo administration of d-tubocurarine, which causes an increase in motoneuron branching on the surface of target muscles, had no effect on IA. Electrical activity played an important role in IA regulation in LMNs in vitro and in ovo. Blocking spontaneous electrical activity of LMNs by chronic in ovo application of mecamylamine or muscimol reduced IA by 80%. LMNs cultured in the presence of TTX also failed to express normal densities of IA, even when the cultures also contained target tissues. The portion of IA that remained after in ovo or in vitro blockade of activity inactivated more quickly than the IA of LMNs that were allowed to discharge spikes. The developmental expression of LMN IA increases significantly during development, and this increase is activity dependent but does not require interactions with target tissues. Ongoing activity also seems to regulate the kinetics of IA inactivation.

Background: The Drosophila circadian clock is controlled by interlocked transcriptional feedback loops that operate in many neuronal and nonneuronal tissues. These clocks are roughly divided into a central clock, which resides in the brain and is known to control rhythms in locomotor activity, and peripheral clocks, which comprise all other clock tissues and are thought to control other rhythmic outputs. We previously showed that peripheral oscillators are required to mediate rhythmic olfactory responses in the antenna, but the identity and relative autonomy of these peripheral oscillators has not been defined. Results: Targeted ablation of lateral neurons by using apoptosis-promoting factors and targeted clock disruption in antennal neurons with newly developed dominant-negative versions of CLOCK and CYCLE show that antennal neurons, but not central clock cells, are necessary for olfactory rhythms. Targeted rescue of antennal neuron oscillators in cyc 01 flies through wild-type CYCLE shows that these neurons are also sufficient for olfaction rhythms. Conclusions: Antennal neurons are both necessary and sufficient for olfaction rhythms, which demonstrates for the first time that a peripheral tissue can function as an autonomous pacemaker in Drosophila . These results reveal fundamental differences in the function and organization of circadian oscillators in Drosophila and mammals and suggest that components of the olfactory signal transduction cascade could be targets of circadian regulation.

Large-conductance Ca(2+)-activated K(+) channels (K(Ca)) in chick ciliary ganglion neurons are regulated by target-derived TGFbeta1. Here we show that TGFbeta1 stimulation of K(Ca) expression was blocked by the structurally dissimilar Ras protein farnesyl transferase inhibitors manumycin-A and FTI-277. A similar effect was produced in ciliary neurons overexpressing RasN17, a widely used dominant-negative form of Ras. Moreover, TGFbeta1-evoked increases in phosphorylation of SMAD2 were reduced by manumycin-A, suggesting that Ras-dependent transduction cascades activated by TGFbeta1 feed back onto SMAD signaling. Thus, Ras is a mediator of pleiotropic TGFbeta1 signaling in developing neurons.

The affinity of cGMP-gated ion channels (CNGCs) for cGMP in chick retinal cone photoreceptors is under circadian control. Here we report that dopamine (DA) and D2 receptor agonists evoke phase-dependent shifts in the affinity of CNGCs for activating ligand. Inside-out patch recordings from cultured chick cones were performed at circadian time (CT) 4–7 and CT 16–19 on the second day of constant darkness. Exposing intact cells to DA or the D2 agonist quinpirole for 2 hr before patch excision caused a significant increase in the K D for cGMP during the night (CT 16–19) but had no effect during the day (CT 4–7). DA or quinpirole treatment had no effect on the Hill slope or the average number of channels per patch. The effect of DA was blocked by the D2 antagonist eticlopride and was not mimicked by D1 agonists or blocked by D1 antagonists. By contrast, a brief (15 min) exposure to DA or quinpirole caused a decrease in K D during the subjective day and had no effect during the subjective night. Thus, the effect of D2 agonists depends on both the duration of agonist exposure and the time of day. Application of DA or quinpirole evoked a transient activation of the MAP kinase Erk (extracellular signal-related kinase) during the day but caused a sustained inhibition during the night. Conversely, D2 agonists caused activation of Ca2+/calmodulin-dependent protein kinase II during the night and inhibited this enzyme during the day. A circadian oscillator in cones appears to regulate the nature of the transduction cascade used by D2 receptors.

The functional expression of large-conductance (BK-type) Ca2+-activated K+(KCa) channels was examined in developing chick lumbar motoneurons (LMNs) between embryonic day 6 (E6) and E13 using patch-clamp recording techniques. The macroscopic KCacurrent of E13 LMNs is inhibited by iberiotoxin and resistant to apamin. The average macroscopic KCadensity was low before E8 and increased 3.3-fold by E11, with an additional 1.8-fold increase occurring by E13. BK-type KCachannels could not be detected in inside-out patches from E8 LMNs but were readily detected at E11. The density of voltage-activated Ca2+currents did not change between E8 and E11. Surgical ablation of target tissues at E5 caused a significant reduction in average KCadensity in LMNs measured at E11. Conversely, chronicin ovoadministration ofd-tubocurarine, which causes an increase in motoneuron branching on the surface of the muscle target tissue, evoked a 1.8-fold increase in average LMN KCadensity measured at E11. Electrical activity also contributed to developmental regulation of LMN KCadensity. A significant reduction in E11 KCadensity was found after chronicin ovotreatment with the neuronal nicotinic antagonist mecamylamine or the GABA receptor agonist muscimol, agents that reduce activation of LMNsin ovo. Moreover, 3 d exposure to depolarizing concentrations of external K+to LMNs cultured at E8 caused an increase in KCaexpression. Conversely, tetrodotoxin caused a decrease in KCaexpression in cultured E8 LMNs developing for 3 d in the presence of neurotrophic factors that promote neuronal survival in the absence of target tissues.

The transcription factor signal transducer and activator of transcription-3 (STAT3) is activated by proinflammatory cytokines and circulating factors in many cell types. Synaptopodin (Synpo) is a cytoskeleton regulatory protein expressed in podocyte foot processes that regulates the dynamics of actin filaments and the stability of small GTPases. Here we show that inhibition of STAT3 signaling using the small-molecule inhibitor benzo[b]thiophene,6-nitro-,1,1-dioxide (Stattic), or by STAT3 knockdown by small interfering RNA, caused a decrease in Synpo mRNA and protein in an immortalized mouse podocyte cell line. This loss of Synpo, which occurred in 30-80 minutes, was also seen after treatment with the translational inhibitor cycloheximide. The loss of Synpo protein after Stattic or cycloheximide treatment did not occur when podocytes were simultaneously exposed to 1-[N-[(l-3-trans-carboxyoxirane-2-carbonyl)-l-leucyl]amino]-4-guanidinobutane (E-64), an inhibitor of thiol proteases such as cathepsin L. Treatment with interleukin-6 (IL-6) increased tyrosine phosphorylation of STAT3 and evoked a parallel increase in Synpo levels in podocytes. The stimulatory effect of IL-6 on Synpo was completely inhibited by pretreatment with Stattic. By contrast, 30-60-minute exposure to angiotensin II (Ang II) inhibited STAT3 signaling and concurrently reduced Synpo protein levels. The Ang II-evoked loss of Synpo was prevented by E-64 but not by inhibition of calcineurin or blockade of transient receptor potential cation channels. Inhibition of STAT3 by Stattic caused marked changes in the distribution of podocyte actin filaments, and caused a nearly complete suppression of the migration of these cells in wound assays, consistent with the loss of Synpo. Stattic treatment also caused loss of RhoA protein.

In diabetic nephropathy (DN), podocyte cytoskeletal rearrangement occurs followed by podocyte effacement and the development of proteinuria. PTEN (phosphatase and tensin homologue) is a ubiquitously expressed phosphatase that plays a critical role in cell proliferation, cytoskeletal rearrangement, and motility. In mouse models of diabetes mellitus, PTEN expression is reportedly decreased in mesangial cells, contributing to expansion of the mesangial matrix, but how PTEN in the podocyte influences the development of DN is unknown. We observed that PTEN expression is down-regulated in the podocytes of diabetic db/db mice and patients with DN. In cultured podocytes, PTEN inhibition caused actin cytoskeletal rearrangement and this response was associated with unbalanced activation of the small GTPases Rac1/Cdc42 and RhoA. In mice treated with PTEN inhibitor, actin cytoskeletal rearrangement occurred in podocytes and was accompanied by increased albumin excretion. We also created mice with an inducible deletion of PTEN selectively in podocytes. These mice exhibited increased albumin excretion and moderate foot process effacement. When the mice were challenged with a high fat diet, podocyte-specific knockout of PTEN resulted in substantially increased proteinuria and glomeruloclerosis compared to control mice fed a high fat diet or mice with PTEN deletion fed a normal diet. These results indicate that PTEN is involved in the regulation of cytoskeletal rearrangement in podocytes and that loss of PTEN predisposes to the development of proteinuria and DN.

Diseases of the kidney filtration barrier are a leading cause of ESRD. Most disorders affect the podocytes, polarized cells with a limited capacity for self-renewal that require tightly controlled signaling to maintain their integrity, viability, and function. Here, we provide an atlas of in vivo phosphorylated, glomerulus-expressed proteins, including podocyte-specific gene products, identified in an unbiased tandem mass spectrometry-based approach. We discovered 2449 phosphorylated proteins corresponding to 4079 identified high-confidence phosphorylated residues and performed a systematic bioinformatics analysis of this dataset. We discovered 146 phosphorylation sites on proteins abundantly expressed in podocytes. The prohibitin homology domain of the slit diaphragm protein podocin contained one such site, threonine 234 (T234), located within a phosphorylation motif that is mutated in human genetic forms of proteinuria. The T234 site resides at the interface of podocin dimers. Free energy calculation through molecular dynamic simulations revealed a role for T234 in regulating podocin dimerization. We show that phosphorylation critically regulates formation of high molecular weight complexes and that this may represent a general principle for the assembly of proteins containing prohibitin homology domains.

Previous studies have shown that the transcription factor signal transducer and activator of transcription-3 (STAT3) in podocytes plays an important role in progression of HIV nephropathy and in collapsing forms of glomerulonephritis. Here, we have observed that application of 100 nM angiotensin II (Ang II) to cultured podocytes for 6-24 hours causes a marked increase in the phosphorylation of STAT3 on tyrosine Y705 but has no effect on phosphorylation at serine S727. By contrast, Ang II treatment of short periods (20-60 minutes) caused a small but consistent suppression of tyrosine phosphylation of STAT3. A similar biphasic effect was seen after treatment with the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG), an agent that causes activation of Ca(2+)-permeable canonical transient receptor potential-6 (TRPC6) channels in podocytes. The stimulatory effects of Ang II on STAT3 phosphorylation were abolished by small-interfering RNA knockdown of TRPC6 and also by inhibitors of the Ca(2+)-dependent downstream enzymes calcineurin and Ca(2+)-calmodulin-dependent protein kinase II. The stimulatory effects of Ang II appear to be mediated by secretion and accumulation of an unknown factor into the surrounding medium, as they are no longer detected when medium is replaced every 2 hours even if Ang II is continuously present. By contrast, the inhibitory effect of Ang II on STAT3 phosphorylation persists with frequent medium changes. Experiments with neutralizing and inhibitory antibodies suggest that the STAT3 stimulatory factor secreted from podocytes is not interleukin-6, but also suggest that this factor exerts its actions through a receptor system that requires glycoprotein 130.

Canonical transient receptor potential-6 (TRPC6) channels have been implicated in the pathogenesis of kidney disease and in the regulation of vascular smooth muscle tone, podocyte function, and a variety of processes in other cell types. The question of whether their gating is intrinsically mechanosensitive has been controversial. In this study we have examined activation of two alleles of TRPC6 transiently expressed in CHO-K1 cells: the wild-type human TRPC6 channel, and TRPC6-N143S, an allele originally identified in a family with autosomal dominant familial focal and segmental glomerulosclerosis (FSGS). We observed that both channel variants carried robust cationic currents that could be evoked by application of membrane-permeable analogs of diacylglycerol (DAG) or by the P2Y receptor agonist ATP. The amplitudes and characteristics of currents evoked by the DAG analog or ATP were indistinguishable in cells expressing the two TRPC6 alleles. By contrast, hypoosmotic stretch evoked robust currents in wild-type TRPC6 channels but had no discernible effect on currents in cells expressing TRPC6-N143S, indicating that the mutant form lacks mechanosensitivity. Coexpression of TRPC6-N143S with wild-type TRPC6 or TRPC3 channels did not alter stretch-evoked responses compared with when TRPC3 channels were expressed by themselves, indicating that TRPC6-N143S does not function as a dominant-negative. These data indicate that mechanical activation and activation evoked by DAG or ATP occur through fundamentally distinct biophysical mechanisms, and they provide support for the hypothesis that protein complexes containing wild-type TRPC6 subunits can be intrinsically mechanosensitive.

Lipid peroxidation through electrophilic molecules of extracellular origin is involved in the pathogenesis of many inflammatory conditions. To counteract free radical actions at the plasma membrane, cells host a variety of antioxidative enzymes. Here we analyzed localization, membrane topology, and trafficking of PON2 a member of the paraoxonase family of 3 enzymatically active proteins (PON1-3) found to have antiatherogenic properties. Immunohistochemistry localized PON2 to the villous tip of human intestinal epithelial cells. Employing membrane preparations, surface biotinylation experiments, and mutational analyses in HEK 293T and HeLa cells, we demonstrate that PON2 is a type II transmembrane protein. A hydrophobic stretch in the N terminus was identified as single transmembrane domain of PON2. The enzymatically active domain faced the extracellular compartment, where it suppressed lipid peroxidation (P0.05) and regulated the glucosylceramide content, as demonstrated by mass spectrometry (P0.05). PON2 translocation to the plasma membrane was dependent on intracellular calcium responses and could be induced to10-fold as compared to baseline (P=0.0001) by oxidative stress. Taken together, these data identify the paraoxonase protein PON2 as a type II transmembrane protein, which is dynamically translocated to the plasma membrane in response to oxidative stress to counteract lipid peroxidation.

The development of functional Ca 2+ -activated K + channels (K Ca ) in chick ciliary ganglion (CG) neurons requires interactions with afferent preganglionic nerve terminals. Here we show that the essential preganglionic differentiation factor is an isoform of β-neuregulin-1. β-Neuregulin-1 transcripts are expressed in the midbrain preganglionic Edinger–Westphal nucleus at developmental stages that coincide with or precede the normal onset of macroscopic K Ca in CG neurons. Injection of β-neuregulin-1 peptide into the brains of developing embryos evoked a robust stimulation of functional K Ca channels at stages before the normal appearance of these channels in CG neurons developing in vivo . Conversely, injection of a neutralizing antiserum specific for β-neuregulin-1 inhibited the development of K Ca channels in CG neurons. Low concentrations of β-neuregulin-1 evoked a robust increase in whole-cell K Ca in CG neurons cocultured with iris target tissues. By contrast, culturing CG neurons with iris cells or low concentrations of β-neuregulin-1 by themselves was insufficient to stimulate K Ca . These data suggest that the preganglionic factor required for the development of K Ca in ciliary ganglion neurons is an isoform of β-neuregulin-1, and that this factor acts in concert with target-derived trophic molecules to regulate the differentiation of excitability.

The cyclic GMP-gated cationic channels of vertebrate photoreceptors are essential for visual phototransduction. We have examined the developmental regulation of cGMP-gated channels in morphologically identified cones in the chick retina. Expression of cone-type cGMP-gated channel mRNA can be detected at embryonic day 6 (E6), but expression of functional channels, as accessed by patch-clamp recordings, cannot be detected until E8. Plasma membrane channels in embryonic cones have a high turnover rate because inhibition of protein synthesis or disruption of the Golgi apparatus causes an almost complete loss of functional cGMP-gated channels within 12 hr. Different subpopulations of cones begin to express functional channels at different developmental stages, but all cones express channels by E10. Expression of cGMP-gated channels in at least one cone subpopulation appears to require one or more soluble differentiation factors, which are presumably present in the normal microenvironment of the developing retina. Application of chick embryo extract (CEE), a rich source of trophic factors, causes marked stimulation of cGMP-gated channel expression in chick cones at E8, but not at E6. Inhibition of MAP kinase (Erk) signaling using PD98059, or inhibition of PI3 kinase signaling by LY294002, blocked the stimulatory effects of CEE on E8 cones. Several recombinant trophic factors were also tested, but none could mimic the stimulatory effects of CEE on channel expression. In summary, the developmental expression of cGMP-gated cationic channels in embryonic cones appears to be regulated by epigenetic factors. The ability of cones to respond to these epigenetic factors is also developmentally regulated.