The Effects of Modulating Endothelial Nitric Oxide Synthase Activity and Coupling in Extracorporeal Shock Wave Lithotripsy

Extracorporeal shock wave lithotripsy (ESWL) treatment to ablate kidney stones has been clinically associated with the development of hypertension. This may be due to ESWL-induced vasoconstriction leading to endothelial dysfunction which is characterized by increased reactive oxygen species (ROS) and decreased endothelial-derived nitric oxide (NO) bioavailability. Normally, endothelial nitric oxide synthase (eNOS) forms NO in the presence of essential cofactor tetrahydrobiopterin (BH4). When the dihydrobiopterin (BH2):BH4 ratio is increased due to ESWL-induced ROS, eNOS becomes uncoupled producing superoxide (O2} and subsequently hydrogen peroxide (H2O2). Cell-permeable protein kinase C epsilon peptide activator (PKCϵ+, N-MyrHDAPIGYD) increases eNOS activity while PKCϵ peptide inhibitor (PKCϵ-, N-MyrEAVSLKPT) reduces eNOS activity. We studied the effects of combining PKCϵ+ (0.9 mg/kg) or PKCϵ- (0.8 mg/kg) with BH4 (0.8 mglkg) or BH2 (2 mg/kg) (n=5-6 per group). After a stable baseline was established, the drug cocktail or saline was given intravenously (i.v.) to anesthetized male Sprague-Dawley rats immediately after ESWL treatment (16kV shock waves, 500 at 60 shocks/min and 500 at 120 shocks/min) and compared to no-ESWL controls. Real-time blood NO and H202 levels were measured in left renal veins throughout the experiment (e.g., baseline, ESWL end, 30 min postESWL). ESWL with saline controls significantly decreased NO and increased H2O2 compared to no-ESWL controls (p4 infusion post-ESWL significantly increased NO and decreased H202 by 30 min compared to ESWL with saline controls (p2, post-ESWL, was similar to ESWL control in H202 and NO, suggesting that promotion of eNOS uncoupling and increased uncoupled eNOS activity cannot attenuate ESWL-induced oxidative stress and decreased NO bioavailability. BH2 may be nearing saturation to the eNOS binding site in this ESWL model. In contrast, post-ESWL infusion of PKCϵ- with either BH4 or BH2 resulted in decreased H202 and increased NO compared to ESWL with saline at 30 min (p\u3c0.05), suggesting that PKCϵ- attenuates ESWL-induced oxidative stress independent of the coupled/uncoupled eNOS status