Podocytes constitute the outer layer of the renal glomerular filtration barrier. Their energy requirements strongly depend on efficient oxidative respiration, which is tightly connected with mitochondrial dynamics. We hypothesized that hyperglycemia modulates energy metabolism in glomeruli and podocytes and contributes to the development of diabetic kidney disease. We found that oxygen consumption rates were severely reduced in glomeruli from diabetic rats and in human podocytes that were cultured in high glucose concentration (30 mM; HG). In these models, all of the mitochondrial respiratory parameters, including basal and maximal respiration, ATP production, and spare respiratory capacity, were significantly decreased. Podocytes that were treated with HG showed a fragmented mitochondrial network, together with a decrease in expression of the mitochondrial fusion markers MFN1, MFN2, and OPA1, and an increase in the activity of the fission marker DRP1. We showed that markers of mitochondrial biogenesis, such as PGC-1α and TFAM, decreased in HG-treated podocytes. Moreover, PINK1/parkin-dependent mitophagy was inhibited in these cells. These results provide evidence that hyperglycemia impairs mitochondrial dynamics and turnover, which may underlie the remarkable deterioration of mitochondrial respiration parameters in glomeruli and podocytes.

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.

Podocytes, the principal component of the glomerular filtration barrier, regulate glomerular permeability to albumin via their contractile properties. Both insulin- and high glucose (HG)-dependent activation of protein kinase G type Iα (PKGIα) cause reorganization of the actin cytoskeleton and podocyte disruption. Vasodilator-stimulated phosphoprotein (VASP) is a substrate for PKGIα and involved in the regulation of actin cytoskeleton dynamics. We investigated the role of the PKGIα/VASP pathway in the regulation of podocyte permeability to albumin. We evaluated changes in high insulin- and/or HG-induced transepithelial albumin flux in cultured rat podocyte monolayers. Expression of PKGIα and downstream proteins was confirmed by western blot and immunofluorescence. We demonstrate that insulin and HG induce changes in the podocyte contractile apparatus via PKGIα-dependent regulation of the VASP phosphorylation state, increase VASP colocalization with PKGIα, and alter the subcellular localization of these proteins in podocytes. Moreover, VASP was implicated in the insulin- and HG-dependent dynamic remodelling of the actin cytoskeleton and, consequently, increased podocyte permeability to albumin under hyperinsulinaemic and hyperglycaemic conditions. These results indicate that insulin- and HG-dependent regulation of albumin permeability is mediated by the PKGIα/VASP pathway in cultured rat podocytes. This molecular mechanism may explain podocytopathy and albuminuria in diabetes.