Injury to podocytes is considered a major contributor to diabetic kidney disease: their loss causes proteinuria and progressive glomerulosclerosis. Podocyte depletion may result from improper calcium handling due to abnormal activation of the calcium permeant TRPC (Transient Receptor Potential Canonical) channels. Angiotensin II (Ang II) levels are found to be elevated in diabetes; furthermore, it was reported that Ang II causes activation of TRPC6 in podocytes. We hypothesized here that Ang II-mediated calcium influx is aggravated in the podocytes under the conditions of type 1 diabetic nephropathy (DN). Diabetes was induced in the Dahl Salt-Sensitive rats by an injection of streptozotocin (STZ-SS). Eleven weeks post treatment was sufficient for the animals to develop hyperglycemia, excessive urination, weight loss, microalbuminuria, nephrinuria and display renal histological lesions typical for patients with DN. Patch-clamp electrophysiology performed on podocytes of the freshly isolated glomeruli showed enhanced basal TRPC channel activity in the STZ-SS rats, and increased response to Ang II; total calcium influx triggered by Ang II application was also augmented in podocytes of these rats. Our studies have a strong potential for advancing the understanding of TRPC-mediated effects on podocytopenia in DN initiation.

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 HO, is NADPH oxidase 4 (NOX4). We hypothesized that NOX4-derived HO contributes to podocyte damage in DKD elevation of podocyte calcium. We used Dahl salt-sensitive (SS) rats with a null mutation for the gene (SS) 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. Upon induction of type 1 diabetes with streptozotocin, SS rats exhibited significantly lower basal intracellular Ca 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 SS rats compared with that in glomeruli from diabetic SS rats. HO stimulated TRPC-dependent calcium influx in podocytes from wild-type mice, but this influx was blunted in podocytes from 6-knockout mice and, in a similar manner, in podocytes from5/6 double-knockout mice. Finally, electron microscopy revealed that podocytes of glomeruli isolated from 6-knockout or5/6 double-knockout mice were protected from damage induced by HO to the same extent. These data reveal a novel signaling mechanism involving NOX4 and TRPC6 in podocytes that could be pharmacologically targeted to abate the development of DKD.

Dynamic remodeling of the actin cytoskeleton plays an essential role in cell migration and various signaling processes in the living cells. One of the critical factors that controls the nucleation of new actin filaments in eukaryotic cells is the actin related protein 2/3 (Arp2/3) complex. Recently, two novel classes of small molecules that bind to different sites on the Arp2/3 complex and inhibit its ability to nucleate F-actin have been discovered and described. The current study was aimed at investigating the effects of CK-0944666 (CK-666) and its analogs (CK-869 and inactive CK-689) on the reorganization of the actin microfilaments in the cortical collecting duct cell line, M-1. We have shown that treatment with CK-666 and CK869 results in the reorganization of F-actin and drastically affects cell motility rate. The concentrations of the compounds used in this study (100-200 μM) neither cause loss of cell viability nor influence cell shape or monolayer integrity; hence the effects of described compounds were not due to the structural side effects. Therefore, we conclude here that the Arp2/3 complex plays an important role in cell motility and F-actin reorganization in M-1 cells. Furthermore, CK-666 and its analogs are useful tools for the investigation of the Arp2/3 complex.

The actin cytoskeleton of podocytes plays a central role in the functioning of the filtration barrier in the kidney. Calcium entry into podocytes via TRPC6 (Transient Receptor Potential Canonical 6) channels leads to actin cytoskeleton rearrangement, thereby affecting the filtration barrier. We hypothesized that there is feedback from the cytoskeleton that modulates the activity of TRPC6 channels. Experiments using scanning ion-conductance microscopy demonstrated a change in migration properties in podocyte cell cultures treated with cytochalasin D, a pharmacological agent that disrupts the actin cytoskeleton. Cell-attached patch-clamp experiments revealed that cytochalasin D increases the activity of TRPC6 channels in CHO (Chinese Hamster Ovary) cells overexpressing the channel and in podocytes from freshly isolated glomeruli. Furthermore, it was previously reported that mutation in ACTN4, which encodes α-actinin-4, causes focal segmental glomerulosclerosis and solidifies the actin network in podocytes. Therefore, we tested whether α-actinin-4 regulates the activity of TRPC6 channels. We found that co-expression of mutant α-actinin-4 K255E with TRPC6 in CHO cells decreases TRPC6 channel activity. Therefore, our data demonstrate a direct interaction between the structure of the actin cytoskeleton and TRPC6 activity.