Insulin increases glomerular filtration barrier permeability through PKGIα-dependent mobilization of BKCa channels in cultured rat podocytes

AbstractPodocytes are highly specialized cells that wrap around glomerular capillaries and comprise a key component of the glomerular filtration barrier. They are uniquely sensitive to insulin; like skeletal muscle and fat cells, they exhibit insulin-stimulated glucose uptake and express glucose transporters. Podocyte insulin signaling is mediated by protein kinase G type I (PKGI), and it leads to changes in glomerular permeability to albumin. Here, we investigated whether large-conductance Ca2+-activated K+ channels (BKCa) were involved in insulin-mediated, PKGIα-dependent filtration barrier permeability.Insulin-induced glomerular permeability was measured in glomeruli isolated from Wistar rats. Transepithelial albumin flux was measured in cultured rat podocyte monolayers. Expression of BKCa subunits was detected by RT-PCR. BKCa, PKGIα, and upstream protein expression were examined in podocytes with Western blotting and immunofluorescence. The BKCa–PKGIα interaction was assessed with co-immunoprecipitation.RT-PCR showed that primary cultured rat podocytes expressed mRNAs that encoded the pore-forming α subunit and four accessory β subunits of BKCa. The BKCa inhibitor, iberiotoxin (ibTX), abolished insulin-dependent glomerular albumin permeability and PKGI-dependent transepithelial albumin flux. Insulin-evoked albumin permeability across podocyte monolayers was also blocked with BKCa siRNA. Moreover, ibTX blocked insulin-induced disruption of the actin cytoskeleton and changes in the phosphorylation of PKG target proteins, MYPT1 and RhoA.These results indicated that insulin increased filtration barrier permeability through mobilization of BKCa channels via PKGI in cultured rat podocytes. This molecular mechanism may explain podocyte injury and proteinuria in diabetes

Metformin reduces NAD(P)H oxidase activity in mouse cultured podocytes through purinergic dependent mechanism by increasing extracellular ATP concentration

Hyperglycemia affects the functioning numbers of podocytes and leads to a gradual decline of renal function. The normalization of glucose level is a principle therapeutic goal in diabetic patients and metformin is a popular hypoglycemic drug used in type 2 diabetes mellitus. Metformin activates AMP-activated kinase (AMPK) and decreases NAD(P)H oxidase activity in podocytes leading to reduction of free radical generation. Similar effects are observed after activation of P2 receptors. Therefore, we investigated whether metformin increases extracellular ATP concentration and affects the activities of NAD(P) H oxidase and AMPK through P2 receptors. Experiments were performed on cultured mouse podocytes. NAD(P) H oxidase activity was measured by chemiluminescence and changes in AMPK activity were estimated by immunoblotting against AMPKα-Thr 172 -P. Metformin increased extracellular ATP concentration by reduction of ectoATPase activity, decreased NAD(P)H oxidase activity and increased AMPK phosphorylation. A P2 receptor antagonist, suramin (300 µM), prevented metformin action on NAD(P)H oxidase and AMPK phosphorylation. The data suggests a novel mechanism of metformin action, at least in podocytes. Metformin, which increases extracellular ATP concentration leads to activation of P2 receptors and consequent modulation of the podocytes' metabolism through AMPK and NAD(P)H oxidase which, in turn, may affect podocyte functioning

Role of Klotho in Hyperglycemia:Its Levels and Effects on Fibroblast Growth Factor Receptors, Glycolysis, and Glomerular Filtration

Hyperglycemic conditions (HG), at early stages of diabetic nephropathy (DN), cause a decrease in podocyte numbers and an aberration of their function as key cells for glomerular plasma filtration. Klotho protein was shown to overcome some negative effects of hyperglycemia. Klotho is also a coreceptor for fibroblast growth factor receptors (FGFRs), the signaling of which, together with a proper rate of glycolysis in podocytes, is needed for a proper function of the glomerular filtration barrier. Therefore, we measured levels of Klotho in renal tissue, serum, and urine shortly after DN induction. We investigated whether it influences levels of FGFRs, rates of glycolysis in podocytes, and albumin permeability. During hyperglycemia, the level of membrane-bound Klotho in renal tissue decreased, with an increase in the shedding of soluble Klotho, its higher presence in serum, and lower urinary excretion. The addition of Klotho increased FGFR levels, especially FGFR1/FGFR2, after their HG-induced decrease. Klotho also increased levels of glycolytic parameters of podocytes, and decreased podocytic and glomerular albumin permeability in HG. Thus, we found that the decrease in the urinary excretion of Klotho might be an early biomarker of DN and that Klotho administration may have several beneficial effects on renal function in DN

The TRPC6-AMPK Pathway is Involved in Insulin-Dependent Cytoskeleton Reorganization and Glucose Uptake in Cultured Rat Podocytes

Background/Aims: Podocytes are dynamic polarized cells on the surface of glomerular capillaries that are an essential part of the glomerular filtration barrier. AMP-activated protein kinase (AMPK), a key regulator of glucose and fatty acid metabolism, plays a major role in obesity and type 2 diabetes. Accumulating evidence suggests that TRPC6 channels are crucial mediators of calcium transport in podocytes and are involved in regulating glomerular filtration. Here we investigated whether the AMPK-TRPC6 pathway is involved in insulin-dependent cytoskeleton reorganization and glucose uptake in cultured rat podocytes. Methods: Western blot and immunofluorescence analysis confirmed AMPKα and TRPC6 expression, the phosphorylation of proteins associated with actin cytoskeleton reorganization (PAK, rac1, and cofilin), and the expression of insulin signaling proteins (Akt, Insulin receptor). Coimmunoprecipitation and immunofluorescence results demonstrated AMPKα/TRPC6 interaction. To ask whether TRPC6 is involved in the insulin regulation of glucose transport, we measured insulin-dependent (1, 2-3H)-deoxy-D-glucose uptake into podocytes after reducing TRPC6 activity pharmacologically and biochemically (TRPC6 siRNA). Results: The results suggested a key role for the TRPC6 channel in the mediation of insulin-dependent activation of AMPKα2 and glucose uptake. Moreover, AMPK and TRPC6 activation were required to stimulate the Rac1 signaling pathway. Conclusion: These results suggest a potentially important new mechanism that regulates glucose transport in podocytes and that could be injurious during diabetes