Podocyte insulin sensitivity is critical for glomerular function, and the loss of appropriate insulin signaling leads to alterations and disorders featuring diabetic nephropathy. Energy-sensing pathways, such as AMP-dependent protein kinase (AMPK) and protein deacetylase SIRT1, have been shown to play an important role in insulin resistance. The absence of a stimulating effect of insulin on glucose uptake into podocytes after exposure to hyperglycemic conditions has been demonstrated to be related to a decreased level and activity of SIRT1 protein, leading to reduced AMPK phosphorylation. The present work was undertaken to investigate metformin's ability to restore the insulin responsiveness of podocytes by regulating SIRT1 and AMPK activities. Primary rat podocytes cultured with standard or high glucose concentrations for 5days were transfected with siRNAs targeting SIRT1, AMPKα1, or AMPKα2. SIRT1 activity was measured by a fluorometric method. Insulin-stimulated changes in glucose uptake were used to detect insulin resistance. Podocyte permeability was measured by a transmembrane albumin flux assay to examine podocytes functioning. Our results demonstrated that metformin activated SIRT1 and AMPK, prevented hyperglycemia-induced reduction of SIRT1 protein levels, ameliorated glucose uptake into podocytes, and decreased glomerular filtration barrier permeability. Furthermore, metformin activated AMPK in a SIRT1-independent manner, as the increase in AMPK phosphorylation after metformin treatment was not affected by SIRT1 downregulation. Therefore, the potentiating effect of metformin on insulin-resistant podocytes seemed to be dependent on AMPK, as well as SIRT1 activity, establishing multilateral effects of metformin action.

Podocytes are dynamic polarized cells on the surface of glomerular capillaries and an essential component of the glomerular filtration barrier. Insulin increases the activation of protein kinase G type Iα (PKGIα) subunits, leading to podocyte dysfunction. In addition, accumulating evidence suggests that TRPC6 channels are crucial mediators of podocyte calcium handling and involved in the regulation of glomerular filtration. Therefore, we investigated whether TRPC6 is involved in the regulation of filtration barrier permeability by insulin via the PKGIα-dependent manner. TRPC channel inhibitor SKF96365 abolished insulin-dependent glomerular albumin permeability and transepithelial albumin flux in cultured rat podocytes. Insulin-evoked albumin permeability across podocyte monolayers was also blocked using TRPC6 siRNA. The effect of insulin on albumin permeability was mimicked by treating podocytes with TRPC channel activator (oleolyl-2-acetyl-sn-glycerol, OAG). Insulin or OAG treatment rapidly increased the superoxide generation through activation of NADH oxidase. TRPC inhibitor SKF96365 or siRNA knockdown of TRPC6 attenuated insulin-dependent increase of ROS production. Furthermore, TRPC inhibitor or downregulation of TRPC6 blocked insulin-induced rearrangement of the actin cytoskeleton and attenuated oxidative activation of PKGIα and changes in the phosphorylation of PKG target proteins MYPT1 and MLC. Moreover insulin regulated the PKGIα interaction with TRPC6 in cultured rat podocytes. Taken together, our data suggest a key role of TRPC6 channels in the mediation of insulin-dependent activation of PKGIα signaling pathways. Overall, we have identified a potentially important mechanism that may explain disturbances in filtration barrier permeability in many diseases with increased expression of TRPC6 and chronic Ca overload.

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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.

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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.

Soft tissue calcification is a pathological phenomenon that often occurs in end-stage chronic kidney disease (CKD), which is caused by diabetic nephropathy, among other factors. Hyperphosphatemia present during course of CKD contributes to impairments in kidney function, particularly damages in the glomerular filtration barrier (GFB). Essential elements of the GFB include glomerular epithelial cells, called podocytes. In the present study, we found that human immortalized podocytes express messenger RNA and protein of phosphate transporters, including NaPi 2c (SLC34A3), Pit 1 (SLC20A1), and Pit 2 (SLC20A2), which are sodium-dependent and mediate intracellular phosphate (Pi) transport, and XPR1, which is responsible for extracellular Pi transport. We found that cells that were grown in a medium with a high glucose (HG) concentration (30 mM) expressed less Pit 1 and Pit 2 protein than podocytes that were cultured in a standard glucose medium (11 mM). We found that exposure of the analyzed transporters in the cell membrane of the podocyte is altered by HG conditions. We also found that the activity of tissue nonspecific alkaline phosphatase increased in HG, causing a rise in Pi generation. Additionally, HG led to a reduction of the amount of ectonucleotide pyrophosphatase/phosphodiesterase 1 in the cell membrane of podocytes. The extracellular concentration of pyrophosphate also decreased under HG conditions. These data suggest that a hyperglycemic environment enhances the production of Pi in podocytes and its retention in the extracellular space, which may induce glomerular calcification.

Cellobiose dehydrogenase (CDH), a secreted flavocytochrome produced by a number of wood-degrading fungi, was detected in the culture supernatant of a biotechnologically important strain of Cerrena unicolor grown in a modified cellulose-based liquid medium. The enzyme was purified as two active fractions: CuCDH-FAD (flavin domain) (1.51-fold) with recovery of 8.35 % and CuCDH (flavo-heme enzyme) (21.21-fold) with recovery of 73.41 %. As CDH from other wood-rotting fungi, the intact form of cellobiose dehydrogenase of C. unicolor is a monomeric protein containing one flavin and one heme b with molecular mass 97 kDa and pI = 4.55. The enzyme is glycosylated (8.2 %) mainly with mannose and glucosamine residues. Moreover, the cellobiose dehydrogenase gene cdh1 and its corresponding cDNA from the fungus C. unicolor were isolated, cloned, and characterized. The 2316-bp full-length cDNA of cdh1 encoded a mature CDH protein containing 771 amino acids preceded by a signal peptide consisting of 18 amino acids. Moreover, both active fractions were characterized in terms of kinetics, temperature and pH optima, and antioxidant properties.