title: Insulin increases glomerular filtration barrier permeability through dimerization of protein kinase G type Iα subunits.
abstract: The increase in the permeability of the glomerular barrier filtration to albumin is a well-known feature of diabetic microvasculature and a negative prognostic factor for vascular complications. However, the underlying mechanisms are incompletely understood. We demonstrated recently that superoxide anion generation increases dimerization of protein kinase G type Iα (PKGIα) subunits, leading to podocyte dysfunction. Here we investigated whether high insulin concentration is involved in PKGI-dependent hyperpermeability of the diabetic glomerular filtration barrier. We assessed changes in insulin-induced glomerular permeability by measuring glomerular capillary permeability to albumin in isolated glomeruli from Wistar and obese and lean Zucker rats and transmembrane albumin flux in cultured rat podocytes. Expression of PKGIα and upstream proteins was confirmed in the podocytes using Western blotting and immunofluorescence. Insulin (300nM, 5min) increased NAD(P)H-dependent glomerular albumin permeability in Wistar rats and PKGI-dependent transmembrane albumin flux in cultured podocytes. Podocyte exposure to insulin in non-reducing conditions increased PKGIα interprotein disulfide bond formation, altered the phosphorylation of the PKG target proteins MYPT1 and MLC, and disrupted the actin cytoskeleton. The role of NADPH oxidase (NOX) in insulin-induced reactive oxygen species (ROS) generation and insulin-evoked increases in albumin permeability in podocytes was confirmed with NOX2 and NOX4 siRNA. Glomerular albumin permeability was increased in hyperinsulinemic Zucker obese rats with isolated glomeruli showing increased expression of PKGIα and NOX4. Taken together, these data demonstrate that insulin increases glomerular barrier albumin permeability via a PKGI-dependent mechanism involving NAD(P)H-dependent generation of superoxide anion. These findings reveal a role for insulin in the pathophysiology of diabetic glomerular nephropathy.
authors: Mossakowski Medical Research Centre Polish Academy of Sciences, Laboratory of Molecular and Cellular Nephrology, Gdańsk, Poland. apiwkowska@cmdik.pan.pl
authors: Agnieszka
authors: A
authors: Piwkowska
authors: Dorota
authors: D
authors: Rogacka
authors: Małgorzata
authors: M
authors: Kasztan
authors: Stefan
authors: S
authors: Angielski
authors: Maciej
authors: M
authors: Jankowski
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D062965:Cyclic GMP-Dependent Protein Kinase Type I / Q000378:metabolism*; D005260:Female; D005919:Glomerular Filtration Rate; D007328:Insulin / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000378:metabolism; D010539:Permeability; D050199:Podocytes / Q000166:cytology / Q000378:metabolism*; D055503:Protein Multimerization; D054645:Protein Phosphatase 1 / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011924:Rats, Zucker; D012709:Serum Albumin / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism
chemical_list: D007328:Insulin; D008562:Membrane Glycoproteins; D012709:Serum Albumin; D013481:Superoxides; C500593:Cybb protein, rat; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498911:Nox4 protein, rat; D062965:Cyclic GMP-Dependent Protein Kinase Type I; C569721:Prkg1 protein, rat; C499850:Ppp1r12a protein, rat; D054645:Protein Phosphatase 1