TRPC6 Knockout Mice Research Articles

Abstract P2163: The Genetic And Pharmacological Inhibition Of Classic Transient Receptor Potential Channels 3 And 6 (trpc3/c6) Improve Pulmonary Arterial Hypertension.

Background: Pulmonary artery hypertension (PAH) is a progressive and fatal disease. Currently, there are only three types of PAH drugs, targeting the endothelin pathway, NO pathway, and prostacyclin pathway, and all these drugs are pulmonary vasodilators. However, the pathogenesis of PAH develops through various mechanisms, including vascular endothelial dysfunction, pulmonary artery smooth muscle cell (PASMC) proliferation, fibrosis and inflammation as well as vasoconstriction. Recently, calcium signaling has been reported to be involved in these multiple mechanisms in PAH. Classic transient receptor potential channel 3 and 6 (TRPC3/C6), known as receptor-operated calcium channels, have been reported to be involved in the pathogenesis of PAH. Methods and Results: Initially, we evaluated TRPC3 knockout mice (TRPC3KO), TRPC6 knockout mice (TRPC6KO), and TRPC3/C6 double-knockout mice (dKO) in the PAH models, to evaluate the effects of genetic inhibition of TRPC3/C6. All of TRPC3KO, TRPC6KO, and dKO have weakened PAH phenotypes such as the systolic pulmonary artery pressure, the right ventricular hypertrophy, and the tunica media thickening of the pulmonary artery in monocrotaline pyrrole-induced PAH models. On the other hand, in the hypoxia-induced PAH model, TRPC6KO and dKO ameliorated these PAH phenotypes, but TRPC3KO didn’t. Next, we evaluated the effect of pharmacological inhibition of TRPC3/C6 in PAH model rats. We have developed a novel selective TRPC3/C6 inhibitor, L862. This compound showed excellent pharmacokinetic and safety properties. L862 ameliorated monocrotaline-induced and hypoxia-induced PAH in rat models on prevention protocol. Moreover, L862 significantly suppressed PDGF-BB-induced proliferation of human PASMC derived from idiopathic PAH patients in a dose-dependent manner. L862 also improved PAH in Sugen/hypoxia rat model on the therapeutic protocol. Conclusions: Both genetic and pharmacological inhibition of TRPC3/C6 ameliorated PAH in several PAH models. These results suggested that TRPC3/C6 had essential roles in the pathogenesis of PAH and L862 had potential clinical application.

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.

Trpc6 Promotes Doxorubicin-Induced Cardiomyopathy in Male Mice With Pleiotropic Differences Between Males and Females.

Background: Doxorubicin is a widely used and effective chemotherapy, but the major limiting side effect is cardiomyopathy which in some patients leads to congestive heart failure. Genetic variants in TRPC6 have been associated with the development of doxorubicin-induced cardiotoxicity, suggesting that TRPC6 may be a therapeutic target for cardioprotection in cancer patients.Methods: Assessment of Trpc6 deficiency to prevent doxorubicin-induced cardiac damage and function was conducted in male and female B6.129 and Trpc6 knock-out mice. Mice were treated with doxorubicin intraperitoneally every other day for a total of 6 injections (4 mg/kg/dose, cumulative dose 24 mg/kg). Cardiac damage was measured in heart sections by quantification of vacuolation and fibrosis, and in heart tissue by gene expression of Tnni3 and Myh7. Cardiac function was determined by echocardiography.Results: When treated with doxorubicin, male Trpc6-deficient mice showed improvement in markers of cardiac damage with significantly reduced vacuolation, fibrosis and Myh7 expression and increased Tnni3 expression in the heart compared to wild-type controls. Similarly, male Trpc6-deficient mice treated with doxorubicin had improved LVEF, fractional shortening, cardiac output and stroke volume. Female mice were less susceptible to doxorubicin-induced cardiac damage and functional changes than males, but Trpc6-deficient females had improved vacuolation with doxorubicin treatment. Sex differences were observed in wild-type and Trpc6-deficient mice in body-weight and expression of Trpc1, Trpc3 and Rcan1 in response to doxorubicin.Conclusions: Trpc6 promotes cardiac damage following treatment with doxorubicin resulting in cardiomyopathy in male mice. Female mice are less susceptible to cardiotoxicity with more robust ability to modulate other Trpc channels and Rcan1 expression.

Sex differences in cardio-oncology: utilizing a genetic variant as a therapeutic target doxorubicin-induced cardiomyopathy

Abstract Introduction Doxorubicin is an anthracycline used as a chemotherapeutic drug for the treatment of a wide range of adult and pediatric cancers. Doxorubicin is associated with an increased risk of cardiomyopathy and heart failure with up to 10% of patients developing cardiac complications. A GWAS conducted of 1,191 patients from the N9831 clinical trial identified that cardiac gene expression and genetic variants of TRPC6 were associated with a decline in left ventricular ejection fraction (LVEF) (p=0.005 and p=1.6x10–6, respectively). TRPC6 is a non-selective cation channel expressed in heart and vascular tissue. TRPC6 participates in the pathogenesis of cardiac hypertrophy as a pathological response to chronic mechanical stress. Chronic activation has been found to promote cardiac fibrosis leading to heart failure. Purpose TRPC6 variants could be associated with increased risk of doxorubicin-induced cardiotoxicity. Data/tests to determine which patients may progress to cardiomyopathy and heart failure are currently lacking and there are no targeted treatments to prevent cardiomyopathy in these patients. Methods In preliminary in vivo data, B6.129 wild-type mice or TRPC6 knockout mice were treated with either 6x intraperitoneal saline or 4mg/kg doxorubicin injections (cumulative dose of 24mg/kg). Results We found doxorubicin increased cardiac vacuolation (male, p≤0.001 and female, p≤0.05) in WT mice compared to controls. Higher HW/BW ratio was observed in male TRPC6 knock out mice compared to wild-type mice when both were treated with doxorubicin (males, p=0.005 and females, p=0.19). Additionally, we found that doxorubicin-induced cardiac injury was significantly reduced in TRPC6 knock-out mice compared to wild-type mice based on reduced vacuolation (p=0.0004 males, p=0.03 females), with the effect being greater in male mice than female mice. Furthermore, a significant decrease in stroke volume (p=0.007), diastolic volume (p=0.01) and cardiac output (p=0.004) in wild-type male mice treated with doxorubicin compared to control and TRPC6 knock-out mice. Our in vitro preliminary data show that inhibition of TRPC6 using the TRPC6 inhibitor GsMTx-4 in human iPSC-derived cardiomyocytes significantly reduced doxorubicin-induced apoptosis (p<0.0001). Further we treated male mice with the TRPC6 inhibitor, GsMTx4, and found that they had less global longitudinal cardiac strain (p=0.04) and higher ejection fraction (p=0.01) compared to mice who were only treated with doxorubicin. Histologically we found that mice given the TRPC6 inhibitor had less fibrosis as measured by Trichrome stained heart sections (p=0.004) which could account for improvements in cardiac function. Conclusion TRPC6 could be a novel therapeutic target in the prevention of chemotherapy-induced cardiomyopathy and heart failure. Additionally genetic mapping of TRCP6 functional variants may provide a new screening tool to determine increased risk of developing heart failure. Funding Acknowledgement Type of funding sources: Private hospital(s). Main funding source(s): Mayo Clinic

Huangkui Capsule Ameliorates Renal Fibrosis in a Unilateral Ureteral Obstruction Mouse Model Through TRPC6 Dependent Signaling Pathways.

Renal fibrosis is the final common pathological manifestation of almost all progressive chronic kidney diseases (CKD). Transient receptor potential canonical (TRPC) channels, especially TRPC3/6, were proposed to be essential therapeutic targets for kidney injury. Huangkui capsule (HKC), an important adjuvant therapy for CKD, showed superior efficacy for CKD at stages 1–2 in clinical practice. However, its anti-fibrotic effect and the underlying mechanisms remain to be investigated. In the present study, we evaluated the efficacy of HKC on renal fibrosis in a mouse model of unilateral ureteral obstruction (UUO) and explored the potential underlying mechanism. Administration of HKC by intragastric gavage dose-dependently suppressed UUO-induced kidney injury and tubulointerstitial fibrosis. Similarly, HKC suppressed the expression level of α-smooth muscle actin (α-SMA), increased the expression of E-cadherin, and suppressed the mRNA expression of a plethora of proinflammatory mediators that are necessary for the progression of renal fibrosis. Mechanistically, HKC suppressed both canonical and non-canonical TGF-β signaling pathways in UUO mice as well as the TRPC6/calcineurin A (CnA)/nuclear factor of activated T cells (NFAT) signaling axis. In addition, TRPC6 knockout mice and HKC treated wild type mice displayed comparable protection on UUO-triggered kidney tubulointerstitial injury, interstitial fibrosis, and α-SMA expression. More importantly, HKC had no additional protective effect on UUO-triggered kidney tubulointerstitial injury and interstitial fibrosis in TRPC6 knockout mouse. Further investigation demonstrated that HKC could directly suppress TRPC3/6 channel activities. Considered together, these data demonstrated that the protective effect of HKC on renal injury and interstitial fibrosis is dependent on TRPC6, possibly through direct inhibition of TRPC6 channel activity and indirect suppression of TRPC6 expression.

Pressure-induced constriction of the middle cerebral artery is abolished in TrpC6 knockout mice.

Pressure-induced constriction (PIC) is an inherent response of small arteries and arterioles in which increases in intraluminal pressure evoke vasoconstriction. It is a critical mechanism of blood flow autoregulation in the kidney and brain. Degenerin (Deg) and transient receptor potential (Trp) protein families have been implicated in transduction of PIC because of evolutionary links to mechanosensing in the nematode and fly. While TrpC6 has been suggested to contribute to PIC signaling, direct supporting evidence is contradictory. Therefore, the aim of this study was to determine the importance of TrpC6 in PIC signaling using a mouse model lacking TrpC6. To address this aim, we evaluated graded pressure (20-90 mmHg), depolarization (4-80 mM KCl)-, and adrenergic receptor (phenylephrine; PE 10-7-10-4 M)-mediated constriction of isolated middle cerebral artery (MCA) segments from 9-wk-old male wild-type (TrpC6+/+, n = 7) and homozygous null (TrpC6-/-, n = 9) TrpC6 mice (Jackson Laboratories). Isolated MCA segments were cannulated and pressurized with physiological salt solution using pressure myography (Living Systems). Vasoconstrictor responses to KCl and PE were identical in TrpC6-/- and TrpC6+/+ mice. In contrast, PIC responses were totally abolished in TrpC6-/- mice. At 90 mmHg, the calculated myogenic tone was -0.8 ± 0.5 vs. 10.7 ± 1.7%, P = 0.0002 in TrpC6-/- and TrpC6+/+ mice, respectively. Additionally, there were no changes in mechanical properties of circumferential wall strain and stress or morphological properties of wall thickness and wall-to-lumen ratio at 50 mmHg between TrpPC6-/- and TrpC6+/+ mice. Although these results demonstrate that TrpC6 is critical for the integrated PIC response, they do not identify whether TrpC6 acts as a mechanosensor or a downstream signaling component.NEW & NOTEWORTHY Pressure-induced, but not agonist-induced, vasoconstriction is abolished in the middle cerebral artery (MCA) of TrpC6 null mice. TrpC6 localization in dissociated cerebral vascular smooth muscle cells is primarily cytoplasmic and not associated with the surface membrane where a mechanoelectrical coupler might be expected. These findings suggest that TrpC6 is required for transduction of pressure-induced constriction in the MCA; however, its role as a mechanoelectrical coupler or downstream signal amplifier remains unresolved.

Pressure‐Induced Constriction of the Middle Cerebral Artery is Abolished in TrpC6 Knockout Mice

Pressure‐induced constriction (PIC) is an inherent response of small arteries and arterioles in which increases in intraluminal pressure evoke vasoconstriction. It is a critical mechanism of blood flow autoregulation in the kidney and brain. Degenerin (Deg) and transient receptor potential (Trp) protein families have been implicated in transduction of PIC because of evolutionary links to mechanosensing in the nematode and fly. We hypothesize that Deg and Trp proteins associate to form a multi‐meric mechanosensing complex in vascular smooth muscle cells (VSMCs), in which Deg and Trp proteins play different, yet equally critical, roles as sensors and amplifiers, in PIC molecular signaling. Our laboratory has provided direct evidence that the Deg protein bENaC acts as a sensor by mediating mechanoreceptor currents in isolated VSMCs. While TrpC6 has been suggested to mediate PIC, direct supporting evidence is lacking. Therefore, the aim of this study was to determine the importance of TrpC6 in PIC signaling using a mouse model lacking TrpC6. To address this aim, we evaluated graded pressure (20–90 mm Hg), depolarization‐ (20–80 mM KCl) and adrenergic receptor‐(phenylephrine, PE 10−7–10−4 M) mediated constriction of isolated middle cerebral artery (MCA) segments from 9 weeks old male wild‐type (+/+, n=7) and homozygous null (−/−, n=9) TrpC6 mice (Jackson Laboratories). Isolated MCA segments were cannulated and pressurized with physiological salt solution using pressure myography (Living Systems). The data were analyzed preliminarily using independent 2‐tailed t‐test. Normalized MCA vasoconstrictor responses to KCl (80 mM, 45.5±1.1% vs 40.9±1.2%, p=0.2) and PE (10−4 M, 37.4±1.7% vs 38.7.0±1.2%, p=0.8) were similar in TrpC6−/− and TrpC6+/+ mice, respectively. In contrast, PIC responses were totally abolished in TrpC6 −/− mice; at 90 mmHg calculated myogenic tone was 0.8±0.5% vs 10.7±1.7%, p=0.0002, in TrpC6−/− and TrpC6+/+ mice, respectively. Additionally, there were no changes in mechanical properties of circumferential wall strain (0.2±0.02 vs. 0.2±0.02, p=0.8) and stress (2.7±0.1 vs. 3.1±0.4, p=0.4), at 90 mmHg; or morphological properties wall thickness (13.4±0.8μm vs. 14.3±1.5μm, p=0.6) and wall‐to‐lumen ratio (0.3±0.02 vs. 0.3±0.04, p=0.7), at 50 mmHg between TrpC6−/− and TrpC6+/+ mice. Although these results suggest that TrpC6 is required for the PIC response, they do not identify the role of TrpC6. Studies in the nematode suggest that Deg and Trp channels play different, yet equally critical roles in mechano‐signaling; Deg channels mediate mechanoreceptor currents, while Trp channels mediate post‐transduction signal amplification. Future studies will determine the role of TrpC6 in isolated VSMCs and cerebral blood flow autoregulation. This research was supported by P20GM104357, P20121334, P01HL051971, R01HL1136684.Support or Funding InformationP20GM104357, P20121334, P01HL051971, R01HL1136684.

Role of TRPC6 on Single Cell Mechanics in Mouse Cardiomyocytes

An increase in preload induces a biphasic response in the heart. The short-term increase in preload rapidly augments contractile force owing to the Frank-Starling mechanism (FSM) that accelerates Ca2+ sensitivity of the contraction protein. A further long-term increase in preload for several minutes causes the increase in [Ca2+]i, leading to slow force response to stretch (SFR), a further increase in the contractile force. The authors’ previous study identified the involvement of TRPC3 and TRPC6, mechanosensitive non-selective cation channels, in SFR. However, the participation of TRPC3 and TRPC6 in FSM remains unclear. To clarify the role of TRPC3 and TRPC6 in FSM, cardiomyocytes were enzymatically isolated from either wild-type (WT), TRPC3 knockout (KO), or TRPC6 KO mice. Both cell ends were held by two pairs of carbon fibers attached to both upper and bottom cell surfaces to apply stretch to the cells. The cells were paced at 4 Hz and superfused in normal Tyrode solution at 37 °C. Cell length and active/passive force calculated from carbon fiber bending were recorded in six different preload conditions. The effect of each genetic deletion on cellular contractility and elastance was assessed by the slope of end-systolic force-length relation curve (ESFLR) and end-diastolic force-length relation curve (EDFLR). The slope of ESFLR was significantly steeper in TRPC6 KO mice than in WT mice, though it remained unchanged in TRPC3 KO mice (0.47 ± 0.1; 0.35 ± 0.14; 0.36 ± 0.14 mN∙mm−2∙μm−1). Meanwhile, the slope of EDFLR was not significantly different between WT, TRPC3 KO, and TRPC6 KO mice (0.16 ± 0.046; 0.19 ± 0.11; 0.19 ± 0.13 mN∙mm−2∙μm−1). These results suggest that TRPC6 regulates the contractile property via modulating the increase in the contractile force controlled by FSM, while TRPC3 is not involved in FSM.

The TRPC6 inhibitor, larixyl acetate, is effective in protecting against traumatic brain injury-induced systemic endothelial dysfunction

BackgroundThe incidence of traumatic brain injuries (TBIs) is on the rise in the USA. Concussions, or mild TBIs without skull fracture, account for about 75% of all TBIs. Mild TBIs (mTBIs) lead to memory and cognitive deficits, headaches, intraocular pressure rises, axonal degeneration, neuroinflammation, and an array of cerebrovascular dysfunctions, including increased vascular permeability and decreased cerebral blood flow. It has been recently reported that besides vascular dysfunction in the cerebral circulation, mTBI may also cause a significant impairment of endothelial function in the systemic circulation, at least within mesenteric microvessels. In this study, we investigated whether mTBI affects endothelial function in aortas and determined the contribution of transient receptor potential canonical (TRPC) channels to modulating mTBI-associated endothelial dysfunction.MethodsWe used a model of closed-head mTBI in C57BL/6, 129S, 129S-C57BL/6-F2 mice, and 129S-TRPC1 and 129S-C57BL/6-TRPC6 knockout mice to determine the effect of mTBI on endothelial function in mouse aortas employing ex vivo isometric tension measurements. Aortic tissue was also analyzed using immunofluorescence and qRT-PCR for TRPC6 expression following mTBI.ResultsWe show that in various strains of mice, mTBI induces a pronounced and long-lasting endothelial dysfunction in the aorta. Ablation of TRPC6 protects mice from mTBI-associated aortic endothelial dysfunction, while TRPC1 ablation does not impact brain injury-induced endothelial impairment in the aorta. Consistent with a role of TRPC6 activation following mTBI, we observed improved endothelial function in wild type control mice subjected to mTBI following 7-day in vivo treatment with larixyl acetate, an inhibitor of TRPC6 channels. Conversely, in vitro treatment with the pro-inflammatory endotoxin lipopolysaccharide, which activates endothelial TRPC6 in a Toll-like receptor type 4 (TLR4)-dependent manner, worsened aortic endothelial dysfunction in wild type mice. Lipopolysaccharide treatment in vitro failed to elicit endothelial dysfunction in TRPC6 knockout mice. No change in endothelial TRPC6 expression was observed 7 days following TBI.ConclusionsThese data suggest that TRPC6 activation may be critical for inducing endothelial dysfunction following closed-head mTBI and that pharmacological inhibition of the channel may be a feasible therapeutic strategy for preventing mTBI-associated systemic endothelial dysfunction.

Abstract P192: Trpc6 Deficiency Causes Obesity, Impaired Glucose Tolerance and Leptin Resistance

Transient receptor potential cation channel subfamily C member 6 (TPRC6) is a receptor-operated cation channel that contributes to changes in cytosolic free Ca 2+ concentration. TRPCs have been suggested to play a role in the regulation of neurite outgrowth, synapse formation, and neuronal survival. However, the role of TRPC6 in controlling metabolic and cardiovascular functions is still unclear. We therefore investigated the impact of TRPC6 on regulation of energy balance, metabolic and cardiovascular functions and anorexic responses to leptin in male and female TRPC6 knockout mice and control B6/129 mice, including assessment of body weight (BW), food intake (FI), body fat/lean composition, responses to acute leptin injection, glucose tolerance tests (GTTs), and blood pressure (BP) and heart rate (HR). TRPC6 null mice of both sexes were heavier than control mice from 6 to 16 weeks of age when fed a standard diet (39.5 ± 1.5 vs 31.1 ± 1.2 g in males and 31.1 ± 1.2 vs 22.9 ± 1.0 in females at 16 weeks of age, n=7, p <0.05). EchoMRI scans showed that the higher body weight in TRPC6 null mice was mainly due to increased body fat compared to controls (30.0 % vs 22.6 % of fat mass/BW in males and 32.7 % vs 21.0 % in females at 16 weeks of age), and was associated with increased daily FI (3.4 ± 0.1 vs 2.7 ± 0.2 g in males and 2.7 ± 0.1 vs 2.0 ± 0.1 in females at 16 weeks of age, n=7, p <0.05). Acute leptin injections (5 mg/kg, i.p. at 18 weeks of age, n=7) significantly reduced 24-hr FI by 41 % and 32 % in male and female control mice, while only an 8 % and 18 % reduction in FI were observed in male and female TRPC6 null mice. At 20 weeks of age, male and female TRPC6 null mice also exhibited impaired glucose tolerance during GTT compared to controls (AUC: 16,138 ± 1,877 vs 8,016 ± 1,476 mg/dL x 120 min in males, and 6,152 ± 1,173 vs 1,975 ± 512 mg/dL x 120 min in females, n=7, p <0.05). BP and HR measured by telemetry for 5 consecutive days at 22 weeks of age were similar between groups. Our results indicate that TRPC6 plays an important role in normal control of food intake, body weight and glucose homeostasis as well as for leptin’s anorexic action. Although TRPC6 deficiency caused obesity and metabolic abnormalities, BP and HR did not increase.

A NOX4/TRPC6 Pathway in Podocyte Calcium Regulation and Renal Damage in Diabetic Kidney Disease.

Background Loss of glomerular podocytes is an indicator of diabetic kidney disease (DKD). The damage to these cells has been attributed in part to elevated intrarenal oxidative stress. The primary source of the renal reactive oxygen species, particularly H2O2, is NADPH oxidase 4 (NOX4). We hypothesized that NOX4-derived H2O2 contributes to podocyte damage in DKD via elevation of podocyte calcium.Methods We used Dahl salt-sensitive (SS) rats with a null mutation for the Nox4 gene (SSNox4-/-) and mice with knockout of the nonselective calcium channel TRPC6 or double knockout of TRPC5 and TRPC6. We performed whole animal studies and used biosensor measurements, electron microscopy, electrophysiology, and live calcium imaging experiments to evaluate the contribution of this pathway to the physiology of the podocytes in freshly isolated glomeruli.Results Upon induction of type 1 diabetes with streptozotocin, SSNox4-/- rats exhibited significantly lower basal intracellular Ca2+ levels in podocytes and less DKD-associated damage than SS rats did. Furthermore, the angiotensin II-elicited calcium flux was blunted in glomeruli isolated from diabetic SSNox4-/- rats compared with that in glomeruli from diabetic SS rats. H2O2 stimulated TRPC-dependent calcium influx in podocytes from wild-type mice, but this influx was blunted in podocytes from Trpc6-knockout mice and, in a similar manner, in podocytes from Trpc5/6 double-knockout mice. Finally, electron microscopy revealed that podocytes of glomeruli isolated from Trpc6-knockout or Trpc5/6 double-knockout mice were protected from damage induced by H2O2 to the same extent.Conclusions These data reveal a novel signaling mechanism involving NOX4 and TRPC6 in podocytes that could be pharmacologically targeted to abate the development of DKD.

Increased glomerular filtration rate and impaired contractile function of mesangial cells in TRPC6 knockout mice

The present study was conducted to determine if TRPC6 regulates glomerular filtration rate (GFR) and the contractile function of glomerular mesangial cells (MCs). GFR was assessed in conscious TRPC6 wild type and knockout mice, and in anesthetized rats with and without in vivo knockdown of TRPC6 in kidneys. We found that GFR was significantly greater, and serum creatinine level was significantly lower in TRPC6 deficient mice. Consistently, local knockdown of TRPC6 in kidney using TRPC6 specific shRNA construct significantly attenuated Ang II-induced GFR decline in rats. Furthermore, Ang II-stimulated contraction and Ca2+ entry were significantly suppressed in primary MCs isolated from TRPC6 deficient mice, and the Ca2+ response could be rescued by re-introducing TRPC6. Moreover, inhibition of reverse mode of Na+-Ca2+ exchange by KB-R7943 significantly reduced Ca2+ entry response in TRPC6-expressing, but not in TRPC6-knocked down MCs. Ca2+ entry response was also significantly attenuated in Na+ free solution. Single knockdown of TRPC6 and TRPC1 resulted in a comparable suppression on Ca2+ entry with double knockdown of both. These results suggest that TRPC6 may regulate GFR by modulating MC contractile function through multiple Ca2+ signaling pathways.

Inhibition of TRPC6 channels ameliorates renal fibrosis and contributes to renal protection by soluble klotho

Fibrosis is an exaggerated form of tissue repair that occurs with serious damage or repetitive injury and ultimately leads to organ failure due to the excessive scarring. Increased calcium ion entry through the TRPC6 channel has been associated with the pathogenesis of heart and glomerular diseases, but its role in renal interstitial fibrosis is unknown. We studied this by deletion of Trpc6 in mice and found it decreased unilateral ureteral obstruction-induced interstitial fibrosis and blunted increased mRNA expression of fibrosis-related genes in the ureteral obstructed kidney relative to that in the kidney of wild-type mice. Administration of BTP2, a pyrazol derivative known to inhibit function of several TRPC channels, also ameliorated obstruction-induced renal fibrosis and gene expression in wild-type mice. BTP2 inhibited carbachol-activated TRPC3 and TRPC6 channel activities in HEK293 cells. Ureteral obstruction caused over a 10-fold increase in mRNA expression for TRPC3 as well as TRPC6 in the kidneys of obstructed relative to the sham-operated mice. The magnitude of protection against obstruction-induced fibrosis in Trpc3 and Trpc6 double knockout mice was not different from that in Trpc6 knockout mice. Klotho, a membrane and soluble protein predominantly produced in the kidney, is known to confer protection against renal fibrosis. Administration of soluble klotho significantly reduced obstruction-induced renal fibrosis in wild-type mice, but not in Trpc6 knockout mice, indicating that klotho and TRPC6 inhibition act in the same pathway to protect against obstruction-induced renal fibrosis. Thus klotho and TRPC6 may be pharmacologic targets for treating renal fibrosis.

Acute effects of angiotensin II on calcium influx in the podocytes of the freshly isolated glomeruli of the wild type and TRPC6‐deficient mice (892.1)

Podocytes play a key role in regulating glomerular permeability and maintaining glomerular structure. It was previously reported that angiotensin (Ang II) enhances albuminuria and increases calcium flux in podocytes by activating TRPC6 channels. The goal of this study was to examine acute effects of Ang II on intracellular calcium levels and endogenous ion channels activity in their native setting, podocytes of freshly isolated mouse glomeruli. Single channel patch‐clamp experiments demonstrated that an ion channel with distinct TRPC6 properties (22 pS) was present in the wild type animals, however it was not identified in TRPC6 knockout mice. Ang II acutely activated these native channels in the podocytes of wild type, but not TRPC6 deficient mice; the effect was mediated by changes in the channel open probability. Data obtained in CHO cells transiently expressed with TRPC6 and AT1R confirmed these findings. We further measured alterations in intracellular calcium stimulated by Ang II in the presence and absence of the TRPC inhibitors gadolinium chloride and SKF 96365, and TRPC6 activator flufenamic acid; these experiments were performed in Fluo4/FuraRed loaded podocytes of the intact glomeruli of the wild type and TRPC6‐deficient mice. Our data have provided the first direct evidence of Ang II‐dependent activation of TRPC6 channels and calcium influx in podocytes, which might play a significant role in the pathogenesis of glomerular diseases.Grant Funding Source: Supported by NIH HL108880, DK079968, Z01‐ES‐101684, ADA 1‐10‐BS‐168, ASN Ben J Lipps Fellowship

Angiotensin II has acute effects on TRPC6 channels in podocytes of freshly isolated glomeruli

A key role for podocytes in the pathogenesis of proteinuric renal diseases has been established. Angiotensin II causes depolarization and increased intracellular calcium concentration in podocytes; members of the cation TRPC channels family, particularly TRPC6, are proposed as proteins responsible for calcium flux. Angiotensin II evokes calcium transient through TRPC channels and mutations in the gene encoding the TRPC6 channel result in the development of focal segmental glomerulosclerosis. Here we examined the effects of angiotensin II on intracellular calcium ion levels and endogenous channels in intact podocytes of freshly isolated decapsulated mouse glomeruli. An ion channel with distinct TRPC6 properties was identified in wild type, but was absent in TRPC6 knockout mice. Single channel electrophysiological analysis found that angiotensin II acutely activated native TRPC-like channels in both podocytes of freshly isolated glomeruli and TRPC6 channels transiently overexpressed in CHO cells; the effect was mediated by changes in the channel open probability. Angiotensin II evoked intracellular calcium transients in the wild type podocytes, which was blunted in TRPC6 knockout glomeruli. Pan-TRPC inhibitors gadolinium and SKF 96365 reduced the response in wild type glomerular epithelial cells, whereas the transient in TRPC6 knockout animals was not affected. Thus, angiotensin II-dependent activation of TRPC6 channels in podocytes may have a significant role in the development of kidney diseases.

Mechanosensitive ATP release from hemichannels and Ca²⁺ influx through TRPC6 accelerate wound closure in keratinocytes.

Cutaneous wound healing is accelerated by exogenous mechanical forces and is impaired in TRPC6-knockout mice. Therefore, we designed experiments to determine how mechanical force and TRPC6 channels contribute to wound healing using HaCaT keratinocytes. HaCaT cells were pretreated with hyperforin, a major component of a traditional herbal medicine for wound healing and also a TRPC6 activator, and cultured in an elastic chamber. At 3 h after scratching the confluent cell layer, the ATP release and intracellular Ca(2+) increases in response to stretching (20%) were live-imaged. ATP release was observed only in cells at the frontier facing the scar. The diffusion of released ATP caused intercellular Ca(2+) waves that propagated towards the rear cells in a P2Y-receptor-dependent manner. The Ca(2+) response and wound healing were inhibited by ATP diphosphohydrolase apyrase, the P2Y antagonist suramin, the hemichannel blocker CBX and the TRPC6 inhibitor diC8-PIP2. Finally, the hemichannel-permeable dye calcein was taken up only by ATP-releasing cells. These results suggest that stretch-accelerated wound closure is due to the ATP release through mechanosensitive hemichannels from the foremost cells and the subsequent Ca(2+) waves mediated by P2Y and TRPC6 activation.

DAG-sensitive and Ca(2+) permeable TRPC6 channels are expressed in dentate granule cells and interneurons in the hippocampal formation.

Members of the transient receptor potential (TRP) cation channel family play important roles in several neuronal functions. To understand the precise role of these channels in information processing, their presence on neuronal elements must be revealed. In this study, we investigated the localization of TRPC6 channels in the adult hippocampal formation. Immunostainings with a specific antibody, which was validated in Trpc6 knockout mice, showed that in the dentate gyrus, TRPC6 channels are strongly expressed in granule cells. Immunogold staining revealing the subcellular localization of TRPC6 channels clarified that these proteins were predominantly present on the membrane surface of the dendritic shafts of dentate granule cells, and also in their axons, often associated with intracellular membrane cisternae. In addition, TRPC6 channels could be observed in the dendrites of some interneurons. Double immunofluorescent staining showed that TRPC6 channels were present in the dendrites of hilar interneurons and hippocampal interneurons with horizontal dendrites in the stratum oriens expressing mGlu1a receptors, whereas parvalbumin immunoreactivity was revealed in TRPC6-expressing dendrites with radial appearance in the stratum radiatum. Electron microscopy showed that the immunogold particles depicting TRPC6 channels were located on the surface membranes of the interneuron dendrites. Our results suggest that TRPC6 channels are in a key position to alter the information entry into the trisynaptic loop of the hippocampal formation from the entorhinal cortex, and to control the function of both feed-forward and feed-back inhibitory circuits in this brain region. © 2012 Wiley Periodicals, Inc.

Mouse transient receptor potential channel 6: Role in hemostasis and thrombogenesis

Although changes in the intracellular levels of calcium (Ca2+) are a central step in platelet activation, the underlying mechanism of Ca2+ entry is still unclear. Previous studies have demonstrated that TRPC6, a member of the canonical transient receptor potential channel (TRPC) family is expressed in platelets in a significant amount, and is predominantly found on the plasma membrane. Based on these considerations, we hypothesized that TRPC6 plays a critical role in platelet function. To characterize the role of TRPC6 in platelet function in vivo, we employed a genetic approach, subjecting TRPC6 knockout mice to the tail bleeding time test and a carotid artery injury thrombosis model. We found that TRPC6-deficient animals displayed a prolonged bleeding time, and an increased time for occlusion of the injured carotid artery, compared to their wild-type littermates. Taken together, our data demonstrate for the first time, that TRPC6 deletion in mice results in defects in hemostasis and protection against thrombogenesis, suggesting a vital role in platelet function. Furthermore, TRPC6 may define a new therapeutic target for managing multiple thrombosis-based disorders.

Trpc2 depletion protects red blood cells from oxidative stress-induced hemolysis

Transient receptor potential (TRP) channels Trpc2 and Trpc3 are expressed on normal murine erythroid precursors, and erythropoietin stimulates an increase in intracellular calcium ([Ca(2+)](i)) through TRPC2 and TRPC3. Because modulation of [Ca(2+)](i) is an important signaling pathway in erythroid proliferation and differentiation, Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were utilized to explore the roles of these channels in erythropoiesis. Trpc2, Trpc3, and Trpc2/Trpc3 double knockout mice were not anemic, and had similar red blood cell counts, hemoglobins, and reticulocyte counts as wild-type littermate controls. Although the erythropoietin-induced increase in [Ca(2+)](i) was reduced, these knockout mice showed no defects in red cell production. The major phenotypic difference at steady state was that the mean corpuscular volume, mean corpuscular hemoglobin, and hematocrit of red cells were significantly greater in Trpc2 and Trpc2/Trpc3 double knockout mice, and mean corpuscular hemoglobin concentration was significantly reduced. All hematological parameters in Trpc3 knockout mice were similar to controls. When exposed to phenylhydrazine, unlike the Trpc3 knockouts, Trpc2 and Trpc2/Trpc3 double knockout mice showed significant resistance to hemolysis. This was associated with a significant reduction in hydrogen peroxide-induced calcium influx in erythroblasts. Although erythropoietin-induced calcium influx through TRPC2 or TRPC3 is not critical for erythroid production, these data demonstrate that TRPC2 plays an important role in oxidative stress-induced hemolysis, which may be related to reduced calcium entry in red cells in the presence of Trpc2 depletion.

Reactive Oxygen Species-mediated TRPC6 Protein Activation in Vascular Myocytes, a Mechanism for Vasoconstrictor-regulated Vascular Tone

Both TRPC6 and reactive oxygen species (ROS) play an important role in regulating vascular function. However, their interplay has not been explored. The present study examined whether activation of TRPC6 in vascular smooth muscle cells (VSMCs) by ROS was a physiological mechanism for regulating vascular tone by vasoconstrictors. In A7r5 cells, arginine vasopressin (AVP) evoked a striking Ca(2+) entry response that was significantly attenuated by either knocking down TRPC6 using siRNA or inhibition of NADPH oxidases with apocynin or diphenyleneiodonium. Inhibition of TRPC6 or ROS production also decreased AVP-stimulated membrane currents. In primary cultured aortic VSMCs, catalase and diphenyleneiodonium significantly suppressed AVP- and angiotensin II-induced whole cell currents and Ca(2+) entry, respectively. In freshly isolated and endothelium-denuded thoracic aortas, hyperforin (an activator of TRPC6), but not its vehicle, induced dose- and time-dependent constriction in TRPC6 wide type (WT) mice. This response was not observed in TRPC6 knock-out (KO) mice. Consistent with the ex vivo study, hyperforin stimulated a robust Ca(2+) entry in the aortic VSMCs from WT mice but not from KO mice. Phenylephrine induced a dose-dependent contraction of WT aortic segments, and this response was inhibited by catalase. Moreover, H(2)O(2) itself evoked Ca(2+) influx and inward currents in A7r5 cells, and these responses were significantly attenuated by either inhibition of TRPC6 or blocking vesicle trafficking. H(2)O(2) also induced inward currents in primary VSMCs from WT but not from TRPC6 KO mice. Additionally, H(2)O(2) stimulated a dose-dependent constriction of the aortas from WT mice but not from the vessels of KO mice. Furthermore, TIRFM showed that H(2)O(2) triggered membrane trafficking of TRPC6 in A7r5 cells. These results suggest a new signaling pathway of ROS-TRPC6 in controlling vessel contraction by vasoconstrictors.