Differential effects of NO inhibition in renal epithelial and endothelial cells in mono-culture vs. co-culture conditions.
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(G)monomethyl-arginine (L-NMMA) and a cofactor-based-inhibitor, H4-amino-biopterin (4-ABH(4)) 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-ABH(4). Furthermore we detected significant basal iNOS protein expression in unstimulated HK-2 cells. NOS inhibition by 4-ABH(4) 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-ABH(4). 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.
Department of Internal Medicine I, Medical University of Innsbruck, Innsbruck, Austria.
Cristina
C
Bertocchi
Marianne
M
Schmid
Julia
J
Hasslacher
Stefan
S
Dunzendorfer
Josef R
JR
Patsch
Michael
M
Joannidis
D017209:Apoptosis; D001708:Biopterins / Q000494:pharmacology; D018920:Coculture Techniques; D016207:Cytokines / Q000378:metabolism; D042783:Endothelial Cells / Q000201:enzymology / Q000378:metabolism*; D004847:Epithelial Cells / Q000201:enzymology / Q000378:metabolism*; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019323:omega-N-Methylarginine / Q000494:pharmacology
D016207:Cytokines; D015850:Interleukin-6; D016209:Interleukin-8; D001708:Biopterins; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II