Hydrogen peroxide induces dimerization of protein kinase G type Iα subunits and increases albumin permeability in cultured rat podocytes

Abstract

Podocytes help regulate filtration barrier permeability in the kidneys. They express contractile proteins that are characteristic of smooth muscle cells as well as receptors for vasoactive factors such as angiotensin II and atrial natriuretic peptide (ANP). The later one generates intracellular cGMP, with subsequent activation of cGMP-dependent protein kinase; PKG (isoform PKGIα and PKGIβ). In this study, we asked whether hydrogen peroxide (H(2)O(2)), a physiological vasorelaxing factor, affected podocyte permeability and the podoctye PKGIα signaling pathway. Expression of PKGIα was confirmed in cultured rat podocytes using RT-PCR, immunofluorescence, and Western blotting. Exposure of podocytes to exogenous H(2)O(2) (100 µM) in non-reducing conditions increased the formation of PKGIα interprotein disulfide bonds, affected the phosphorylation of PKG target proteins, namely MYPT1 (maximal increase of about 57% at 30 min) and MLC (maximal decrease of about 62% at 10 min). Furthermore, H(2)O(2) increased the permeability of a layer of podocytes to albumin: Transmembrane flux for albumin increased five-fold (106.6 ± 5.2 µg/ml vs. 20.2 ± 2.5 µg/ml, P < 0.05, n = 5), and the PKG inhibitor Rp-8-Br-cGMPS (100 µM) prevented the flux increase. These data suggest that oxidative modulation of PKGIα in podocytes plays an important

Protein kinase G oxidation is a major cause of injury during sepsis

“…3B). This concurs with recent work showing PKG Iα disulfide formation increased kidney podocyte permeability (15), further supporting the idea that oxidative activation is a crucial, causative mediator of this classical feature of sepsis. CLP resulted in PKG Iα disulfide dimerization in cardiac tissue at 6 and 24 h (Fig.…”

“…Because oxidants can activate cGMP-dependent protein kinase 1 alpha (PKG Iα) to lower blood pressure (12)(13)(14), we hypothesized this process underlies sepsis-induced hypotension and consequential organ injury. Because PKG couples to enhance endothelial permeability (6,15), oxidative activation would also account for the enhanced microvascular leak that would further exacerbate the hypotension. PKG is also negatively inotropic (13,16); therefore, oxidative activation might also explain the attenuated cardiac output characteristic of sepsis, further exacerbating the hypotension.…”

Multiple myeloma cells’ capacity to decompose H2O2 determines lenalidomide sensitivity

“…Increased intermolecular disulfide bond formation (protein disulfide dimers) is a consequence and hallmark of elevated intracellular H 2 O 2 due to cysteine oxidation. 21,22 Using MM.1S, we observed increased formation of IgL-l dimers after 3 hours of treatment with lenalidomide or H 2 O 2 (supplemental Figure 2C). Because the degree of dimerization was somewhat low after 3 hours of lenalidomide, we subsequently prolonged lenalidomide exposure to enhance dimerization.…”

Combined effect of insulin and high glucose concentration on albumin permeability in cultured rat podocytes

“…Experiments were performed with podocytes that had been cultivated for 12e20 days. The phenotype of the podocytes and cell viability were determined as described previously, based on immunocytochemical methods [15]. For the different experiments, cells were cultured for five days without or with insulin (300 nM) in normal D-glucose (NG, 5.6 mM) or high D-glucose (HG, 30 mM) concentrations.…”