Effects of asymmetric dimethylarginine on renal arteries in portal hypertension and cirrhosis

Abstract

Aim: To evaluate the effects of asymmetric dimethylarginine (ADMA) in renal arteries from portal hypertensive and cirrhotic rats.

Methods: Rat renal arteries from Sham (n = 15), pre-hepatic portal hypertension (PPVL; n = 15) and bile duct ligation and excision-induced cirrhosis (BDL; n = 15) were precontracted with norepinephrine, and additional contractions were induced with ADMA (10^-6-10^-3 mol/L), an endogenous inhibitor of nitric oxide (NO) synthase. Concentration-response curves to acetylcholine (1 × 10^-9-3 × 10^-6 mol/L) were determined in precontracted renal artery segments with norepinephrine in the absence and in the presence of ADMA. Kidneys were collected to determine the protein expression and activity of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that catabolizes ADMA.

Results: In renal arteries precontracted with norepinephrine, ADMA caused endothelium-dependent contractions. The pD₂ values to ADMA were similar in the Sham and PPVL groups (4.20 ± 0.08 and 4.11 ± 0.09, P > 0.05, respectively), but were lower than those of the BDL group (4.79 ± 0.16, P < 0.05). Acetylcholine-induced endothelium-dependent relaxation that did not differ, in terms of pD₂ and maximal relaxation, among the 3 groups studied. Treatment with ADMA (3 × 10^-4 mol/L) inhibited acetylcholine-induced relaxation in the 3 groups, but the inhibition was higher (P < 0.05) in the BDL group compared with that for the Sham and PPVL groups. The mRNA and protein expression of DDAH-1 were similar in kidneys from the three groups. Conversely, DDAH-2 expression was increased (P < 0.05) in PPVL and further enhanced (P < 0.05) in the BDL group. However, renal DDAH activity was significantly decreased in the BDL group.

Conclusion: Cirrhosis increased the inhibitory effect of ADMA on basal- and induced-release of NO in renal arteries, and decreased DDAH activity in the kidney.

Keywords: Asymmetric dimethylarginine; Cirrhosis; Dimethylarginine dimethylaminohydrolase; Nitric oxide; Nitric oxide inhibitors; Portal hypertension.

Figure 1

Effects of portal hypertension and cirrhosis on contractile effects induced by high extracellular concentrations of KCl in rat renal arteries. PPVL: Pre-hepatic portal hypertension; BDL: Bile duct ligation.

Figure 2

Effects of nitric oxide synthase inhibitors on basal nitric oxide release in renal artery. Contractions induced by L-NAME (n = 8) and ADMA (n = 8) on rings of rat renal artery with endothelium from Sham, PPVL, and BDL groups in the absence and in the presence of L-arginine (L-arg, 10^-3 mol/L, n = 6). Contractions were determined after evoking submaximal tone with 10^-7-3 × 10^-7 mol/L norepinephrine. PPVL: Pre-hepatic portal hypertension; BDL: Bile duct ligation; ADMA: Asymmetric dimethylarginine; L-NAME: N^G-nitro-L-arginine methyl ester.

Figure 3

Effects of nitric oxide synthase inhibitors on acetylcholine-induced relaxation in renal artery. A: Concentration-response curves to acetylcholine on rings of rat renal artery from Sham, PPVL, and BDL groups in artery rings with endothelium (n = 8) and without endothelium (n = 6) and in artery rings with endothelium in the presence of L-NAME (3 × 10^-4 mol/L; n = 8) or ADMA (3 × 10^-4 mol/L; n = 8). Relaxation is expressed as a percentage of the contraction in response to norepinephrine; B: Difference between the areas under curves (AUCs) from artery rings with endothelium (Control) and treated with L-NAME or ADMA. ^aP < 0.05 vs L-NAME treated group and ^cP < 0.05 vs Sham and PPVL groups treated with ADMA. PPVL: Pre-hepatic portal hypertension; BDL: Bile duct ligation.

Figure 4

DDAH1 and DDAH2 expression in kidneys from portal hypertensive and cirrhotic rats. A and B: DDAH1 and DDAH2 mRNA expression in kidney from Sham, PPVL, and BDL groups normalized to the expression of GAPDH, which was used as an endogenous reference gene; C and D: Immunoblot analysis in single kidney probed with antibodies against DDAH1, DDAH2 or β-actin, as indicated; E and F: Graphs show the results of densitometric analyses from pooled data, plotted as optical densitometry relative to the signal obtained by β-actin. Each data set represents the mean ± SEM derived from 6 independent experiments. ^aP < 0.05 vs Sham group and ^cP < 0.05 vs PPVL group. DDAH: Dimethylarginine dimethylaminohydrolase.

Figure 5

Effects of portal hypertension and cirrhosis on renal dimethylarginine dimethylaminohydrolase activity. Bar graphs represent DDAH activity in kidney from Sham, PPVL, and BDL groups. Each data set represents the mean ± SEM derived from 6 independent experiments. ^aP < 0.05 vs Sham group and ^cP < 0.05 vs PPVL group. DDAH: Dimethylarginine dimethylaminohydrolase.