Journal
HYPERTENSION
Volume 41, Issue 5, Pages 1136-1142Publisher
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1161/01.HYP.0000068201.48340.3B
Keywords
cyclosporin; bradykinin; nitric oxide; cyclic GMP; antioxidants
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Funding
- NHLBI NIH HHS [P01 HL-41618, R01 HL-56791] Funding Source: Medline
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Cyclosporin A (CsA) is used to reduce transplant rejection rates. Chronic use, however, has a destructive toxic effect on the kidney, resulting in hypertension. In this study, we investigated the effects of CsA treatment on the bradykinin/soluble guanylate cyclase signaling cascade and the involvement of superoxide in LLC-PK1 porcine kidney proximal tubule cells. Treatment with 1 mumol/L CsA for 24 hours increased basal cGMP levels by 41%, whereas CsA inhibited bradykinin-stimulated cGMP production by 26%. Western blotting showed increased expression of eNOS, but no other protein in the bradykinin/soluble guanylate cyclase (sGC) pathway was affected. Using lucigenin-dependent chemiluminescence, we found that CsA treatment significantly increased superoxide production. Production of O-2(-) was not significantly reduced by 10 mumol/L oxypurinol or 30 mumol/L ketoconazole. However, it was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium chloride (10 mumol/L) as well as the O-2(-) scavenger superoxide dismutase (SOD) (100 U). On treatment with 50 mumol/L quercetin, 10 mmol/L N-acetyl-cysteine, both antioxidants, as well as the O-2(-) scavenger Tiron (10 mmol/L), concomitant with 1 mumol/L CsA for 24 hours the activation of cGMP production, was restored in combination with a reduction in O-2(-). Incubation with 100 mumol/L menadione, a reactive oxygen generator, and 10 nmol/L bradykinin showed similar effects on the level of cGMP as with CsA. CsA treatment was found to increase nitrotyrosine levels. These findings suggest that CsA activates a NADPH oxidase that releases O-2(-) and disrupts the bradykinin/soluble guanylate cyclase pathway, probably by binding with NO to form peroxynitrite (ONOO-).
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