Role of angiotensin in renal dihydrofolate reductase, GTP-cyclohydrolase 1 and nitric oxide synthase expression in renal ischemic reperfusion

Abstract

Renal ischemic reperfusion (IR) enhances angiotensin II (ANG II) level. In cell culture study, ANG II suppresses dihydrofolate reductase (DHFR) but not modulate GTP-cyclohydrolase 1 (GTP-CH 1) expression. Both enzymes are required for synthesis of BH4 which plays a crucial role in dimerization of nitric oxide synthase (NOS). There is no simultaneous study the role of ANG II on renal DHFR, GTP-CH 1, NOS gene and protein expression during IR. Male Wistar rats were divided into two main groups: sham (S) or IR (30-minute renal pedical occlusion and reperfusion for 1 day). Both groups were further treated with 1) water, or 2) angiotensin converting enzyme inhibitor (ACEI; Enalapril ; 5 mg/kg/day), or 3)angiotensin II receptor type 1 blocker (ARB; Losartan ; 10 mg/kg/day) for one day before S or IR and continuously for 1 day after the operation. On each experimental due date, 24-hr urine and blood samples were collected. The serum was determined for electrolytes, blood urea nitrogen, creatinine (Cr), and Cr clearance (CCr). The kidneys were removed for RNA isolation, detection of mRNA expression (RT-PCR), and protein abundance (Western blot) for DHFR, GTP-CH 1, endothelial NOS (eNOS), and inducible NOS (iNOS). The data show that IR decreased DHFR mRNA and protein levels (p<0.01) which were restored by ACEI (p<0.05) or ARB (p<0.01); whereas GTP-CH 1 expression was remained. IR suppressed the eNOS dimer (p<0.01) while enhanced the monomer (p<0.01) which were corrected by ACEI or ARB (p<0.01). The renal eNOS mRNA and total eNOS protein levels did not change in all experimental groups. IR increased iNOS mRNA, total iNOS protein, and iNOS monomer (p<0.01) which reduced by ACEI or ARB (p<0.01). The present study demonstrates the first evidence indicating that IR, via stimulation of ANG II, suppresses renal DHFR but activates iNOS both mRNA and protein expression. IR enhances the monomer while diminishes the dimer form of eNOS. Inhibition of ANG II by using ACEI or ARB could restore DHFR and eNOS dimer whereas attenuate the heightened levels of iNOS expression and NOS monomer. IR has no effect on GTP-CH 1 expression. The present result also indicates that angiotensin II receptor type 1 play a crucial role in modulation of DHFR and NOS expression.