The present study was performed to investigate whether transient receptor potential (TRPC)6 participated in Ca 2ϩ signaling of glomerular mesangial cells (MCs) and expression of this protein was altered in diabetes. Western blots and real-time PCR were used to evaluate the expression level of TRPC6 protein and mRNA, respectively. Cell-attached patch-clamp and fura-2 fluorescence measurements were utilized to assess angiotensin II (ANG II)-stimulated membrane currents and Ca 2ϩ responses in MCs. In cultured human MCs, high glucose significantly reduced expression of TRPC6 protein, but there was no effect on either TRPC1 or TRPC3. The high glucose-induced effect on TRPC6 was time and dose dependent with the maximum effect observed on day 7 and at 30 mM glucose, respectively. In glomeruli isolated from streptozotocin-induced diabetic rats, TRPC6, but not TRPC1, was markedly reduced compared with the glomeruli of control rats. Furthermore, TRPC6 mRNA in MCs was also significantly decreased by high glucose as early as 1 day after treatment with maximal reduction on day 4. Patch-clamp experiments showed that ANG II-stimulated membrane currents in MCs were significantly attenuated or enhanced by knockdown or overexpression of TRPC6, respectively. Fura-2 fluorescence measurements revealed that the ANG II-induced Ca 2ϩ influxes were markedly inhibited in MCs with TRPC6 knockdown, reminiscent of the impaired Ca 2ϩ entry in response to ANG II in high glucose-treated MCs. These results suggest that the TRPC6 protein expression in MCs was downregulated by high glucose and the deficiency of TRPC6 protein might contribute to the impaired Ca 2ϩ signaling of MCs seen in diabetes.

This study examined the effect of H2O2 on the TRPC6 channel and its underlying mechanisms using a TRPC6 heterologous expression system. In TRPC6-expressing HEK293T cells, H2O2 significantly stimulated Ca2+ entry in a dose-dependent manner. Electrophysiological experiments showed that H2O2 significantly increased TRPC6 channel open probability and whole-cell currents. H2O2 also evoked a robust inward current in A7r5 vascular smooth muscle cells, which was nearly abolished by knockdown of TRPC6 using a small interfering RNA. Catalase substantially attenuated arginine vasopressin (AVP)-induced Ca2+ entry in cells co-transfected with TRPC6 and AVP V1 receptor. N-Ethylmaleimide and thimerosal were able to simulate the H2O2 response. Dithiothreitol or glutathione-reduced ethyl ester significantly antagonized the response. Furthermore, both N-ethylmaleimide- and H2O2-induced TRPC6 activations were only observed in the cell-attached patches but not in the inside-out patches. Moreover, 1-oleoyl-2-acetyl-sn-glycerol effect on TRPC6 was significantly greater in the presence of H2O2. Biotinylation assays revealed a significant increase in cell surface TRPC6 in response to H2O2. Similarly, in cells transfected with TRPC6-EGFP, confocal microscopy showed a significant increase in fluorescence intensity in the region of the cell membrane and adjacent to the membrane. AVP also increased the fluorescence intensity on the surface of the cells co-transfected with TRPC6-EGFP and V1 receptor, and this response was inhibited by catalase. These data indicate that H2O2 activates TRPC6 channels via modification of thiol groups of intracellular proteins. This cysteine oxidation-dependent pathway not only stimulates the TRPC6 channel by itself but also sensitizes the channels to diacylglycerol and promotes TRPC6 trafficking to the cell surface.

Contractility of mesangial cells (MC) is tightly controlled by [G lomerular mesangial cells (MC) are located within glomerular capillary loops and contribute to the physiologic regulation of glomerular hemodynamics (1). Altered responsiveness of MC to hormones is one of the major causes that lead to various renal diseases. Ca 2ϩ influx across the plasma membrane is a major component of MC responses to vasoconstrictors (1). Several types of Ca 2ϩ -conductive channels in the plasma membrane are involved in the physiologic processes. These channels include voltage-operated Ca 2ϩ channel (VOCC), receptor-operated channel (ROC), and recently found store-operated channel (SOC) (1,2). In contrast to the widely known VOCC, the molecular identity, physiologic significance, and regulatory mechanism of ROC and SOC in the glomerular contractile cells remain unknown.Recently, the channel proteins from a new family, canonical transient receptor potential (TRPC), were found in a variety of cells (3). TRPC family includes seven related members, designated as TRPC1 through 7 (3). Pharmacologic and electrophysiologic studies in conjunction with molecular biologic tools and Ca 2ϩ imagings have demonstrated that TRPC channel activity is tightly linked to the signaling cascade of G protein-coupled receptor or receptor tyrosine kinase (4,5), supporting the current hypothesis that TRPC proteins are potential candidates for ROC and SOC. TRPC proteins have been identified in glomeruli and glomerular MC (6 -8). Our previous work also demonstrated that human MC selectively express TRPC1,3,4, and 6 (9). However, the function, regulation, and physiologic relevance of these glomerular TRPC are unexplored at large extent. In this study, we focused on TRPC1 and investigated its contribution to mesangial contraction in vitro and in vivo. Our results indicate that TRPC1 is an important component mediating contractile responses of MC. The TRPC1-involved mesangial contraction is attributed to TRPC1-associated Ca 2ϩ influx. Materials and Methods AnimalsTwo-to 3-mo-old male Sprague-Dawley rats were used in this study. All rats were purchased from Harlan (Indianapolis, IN). Care and use of all animals in this study were in strict agreement with the guidelines set forth by the University of North Texas Health Science Center. Measurement of GFR and Renal Blood FlowGFR and renal blood flow (RBF) were estimated by measurement of inulin and para-aminohippurate (PAH) plasma clearances as described

Although Orai1 protein was recently identified as the component of CRAC channels in hematopoietic cells, store-operated channels (SOC) in other cell types may have a different molecular entity. Also, the activation mechanism of SOC remains unclear, in general. In the present study, we tested the hypothesis that TRPC1 and TRPC4 proteins were functional subunits of SOC in glomerular mesangial cells (MCs) and that STIM1 was required for the channel activation through interaction with the TRPC proteins. In cultured human MCs, cell-attached patch clamp and fura-2 fluorescence measurements showed that single knockdown of either TRPC1 or TRPC4 significantly attenuated thapsigargin-induced membrane currents and Ca2+ entry as well as Ang II-induced channel activity. Double knockdown of both TRPCs resulted in a comparable inhibition on store-operated Ca2+ entry with single knockdown of either TRPC. Consistent with our previous report, co-immunoprecipitation showed a physical interaction between TRPC1 and TRPC4. Furthermore, we found that knockdown of STIM1 using RNAi significantly reduced the thapsigargin-stimulated membrane currents. Co-immunoprecipitation showed that STIM1 interacted with TRPC4, but not TRPC1. In addition, simultaneous inhibition of STIM1 and TRPC1 resulted in a comparable effect on SOC with single inhibition of either one of them. Taken together, we conclude that in glomerular mesangial cells, the TRPC1/TRPC4 complexes constitute the functional subunits of SOC and that the interaction between STIM1 and TRPC4 may be the mechanism for the activation of the channels.

Du J, Ding M, Sours-Brothers S, Graham S, Ma R. Mediation of angiotensin II-induced Ca 2ϩ signaling by polycystin 2 in glomerular mesangial cells.