Differential Effects of NO Inhibition in Renal Epithelial and Endothelial Cells in Mono-Culture vs. Co-Culture Conditions

Abstract

Nitric oxide (NO) plays a critical role in the regulation of renal hemodynamics and tubular function after post-ischemic damage or sepsis. Diminished NO bioavailability contributes to endothelial dysfunction and may be caused by reduced NO synthesis due to substrate or co-factor deficiency. The aim of this study was to investigate the effects of NOS inhibition and NO depletion in a renal endo-epithelial bilayer model compared to monolayers of proximal tubular epithelial (HK-2) cells and endothelial cells of venous origin (EA.hy 926) with respect to cellular integrity, apoptosis and cytokine release. Two different NOS inhibitors have been used: an arginine-based-inhibitor, L-N^Gmonomethyl-arginine (L-NMMA) and a cofactor-based-inhibitor, H4-amino-biopterin (4-ABH₄) showing iNOS selectivity. We found significantly higher basal NO production by epithelial than by endothelial monolayers, which was significantly reduced by both NOS-inhibitors with a stronger effect demonstrated by 4-ABH₄. Furthermore we detected significant basal iNOS protein expression in unstimulated HK-2 cells. NOS inhibition by 4-ABH₄ was associated with increased LDH release, apoptosis and reduced IL-6 production in epithelial but not in endothelial monolayers. These effects on epithelial cells were abolished under co-culture conditions. In contrast, endothelial cells showed higher IL-6 and IL-8 release under co-culture conditions than in monolayers, with IL-8 production being largely suppressed by L-NMMA but not by 4-ABH₄. In conclusion, inhibition of basal NO production in epithelial monolayers shows detrimental effects on cell integrity and viability. Under co-culture conditions interrelation between epithelial and endothelial cells appears to counteract these potentially harmful effects of epithelial NOS inhibition.

Comparative Characterization of Shiga Toxin Type 2 and Subtilase Cytotoxin Effects on Human Renal Epithelial and Endothelial Cells Grown in Monolayer and Bilayer Conditions

“…In line with our results, Bertocchi et al have shown that under coculture conditions interrelation between epithelial and endothelial cells appears to counteract the potentially harmful effects of epithelial NOS inhibition [ 47 ]. Nevertheless, Gomez et al have demonstrated that Stx2 caused an oxidative imbalance in an in vivo model.…”

Protective Effect of Nebivolol against Oxidative Stress Induced by Aristolochic Acids in Endothelial Cells

“…Our studies have previously demonstrated that AAs induce oxidative stress in endothelial cells [10]. Excessive production of reactive oxygen species (ROS) may contribute to decreased nitric oxide (NO) bioavailability, leading to endothelial dysfunction and apoptosis or necrosis [11,12]. Moreover, Yang et al demonstrated that the protein expression of endothelial nitric oxide synthase (eNOS) was reduced by AAs in a mouse model of AAN [13].…”

Einfluss des eNOS T-786C - Polymorphismus auf Morbidität und Mortalität kardiochirurgischer Patienten.

“…Unabhängig der durchgeführten Operationen konnte auch innerhalb des renalen Systems ein bestehender Einfluss des NO auf dessen Funktion nachgewiesen werden (Bertocchi et al 2010, Kon et al 1990…”