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.

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

“…Recently, we showed that activation of the NOX4 subunit of NAD(P)H oxidase induced changes in MYPT1 and MLC phosphorylation by activating PKGIa [13]. When we used changes in MLC phosphorylation to indicate PKG activity, we confirmed that INS activated PKGIa.…”

“…In that model, both PKGIa and NOX4 expression were elevated. Together, those studies suggested that insulin may regulate filtration-barrier permeability, and this mechanism may be dysregulated in diabetes [13].…”

“…Recently, we demonstrated that insulin increased albumin permeability in both isolated rat glomeruli and podocytes. That study demonstrated that insulin stimulation of reactive oxygen species (ROS) generation in podocytes could result in cell membrane localization and dimerization of the cGMP-dependent protein kinase G, type Ia (PKGIa) [13]. Moreover, high glucose concentrations also induced an increase in podocyte permeability to albumin through a similar mechanism, which depended on the expression of two proteins: the NAD(P)H oxidase-4 (NOX4), which regulates ROS generation; and protein kinase G, isoform Ia (PKGIa), which regulates smooth muscle relaxation [14].…”

Insulin increases glomerular filtration barrier permeability through PKGIα-dependent mobilization of BKCa channels in cultured rat podocytes

“…We previously demonstrated that insulin increased the activation of PKGIα subunits, which led to podocyte dysfunction [21]. We found relationships between PKGIα activation, oxidative stress, actin reorganization, and changes in the permeability of the filtration layer to albumin [22].…”

The TRPC6-AMPK Pathway is Involved in Insulin-Dependent Cytoskeleton Reorganization and Glucose Uptake in Cultured Rat Podocytes

“…Actin reorganization leads to changes in podocyte structure, and insulin receptor stimulation causes the retraction of podocyte processes [5]. Some groups have suggested that insulin plays a role in the control of podocyte contractility, which may contribute to glomerular permeability [6][7][8][9]. We recently demonstrated that insulin remodels the actin cytoskeleton and increases the albumin permeability of both isolated rat glomeruli and podocytes; we further showed that the underlying mechanism is calcium-dependent [10,11].…”