High glucose increases glomerular filtration barrier permeability by activating protein kinase G type Ice subunits in a Nox4-dependent manner

Hyperglycemia is a primary factor that disturbs podocyte function in the glomerular filtration process; this disturbance leads to the development of diabetic nephropathy, and ultimately, renal failure. Podocyte function may also be altered by biological agents that modify protein kinase activity, including the cGMP-activated protein kinase type In (PKGI alpha). We hypothesized that hyperglycemia-induced podocyte protein hyperpermeability was dependent on PKGI alpha activation, and that PKGI alpha was activated via dimerization induced by reactive oxygen species. This hypothesis was investigated in rat podocytes cultured in high glucose (HG, 30 mM). Protein expression was measured with Western blot and immunofluorescence. Podocyte permeability was measured with a transmembrane albumin flux assay. We found that HG increased podocyte permeability in long-term incubations (1, 3, and 5 days); permeability was increased by 66% on day 5. This effect was abolished with apocynin, a NAD (P)H inhibitor, and Rp-8-Br-cGMPS, a PKG inhibitor. It was also abolished by introducing small interfering RNAs (siRNAs) against Nox4 and PKGI alpha into cultured podocytes. Furthermore, HG increased PKGI alpha dimerization by 138% (0.23 +/- 0.04 vs. 0.54 +/- 0.09; P<0.05); this effect was abolished with a siRNA against Nox4. Our observations suggested that HG could increase albumin permeability across the podocyte filtration barrier via Nox4-dependent PKGI alpha dimerization. (C) 2013 Published by Elsevier Inc.