Renal response to L-arginine in diabetic rats. A possible link between nitric oxide system and aquaporin-2

Abstract

The aim of this study was to evaluate whether L-Arginine (L-Arg) supplementation modifies nitric oxide (NO) system and consequently aquaporin-2 (AQP2) expression in the renal outer medulla of streptozotocin-diabetic rats at an early time point after induction of diabetes. Male Wistar rats were divided in four groups: Control, Diabetic, Diabetic treated with L-Arginine and Control treated with L-Arginine. Nitric oxide synthase (NOS) activity was estimated by [14C] L-citrulline production in homogenates of the renal outer medulla and by NADPH-diaphorase staining in renal outer medullary tubules. Western blot was used to detect the expression of AQP2 and NOS types I and III; real time PCR was used to quantify AQP2 mRNA. The expression of both NOS isoforms, NOS I and NOS III, was decreased in the renal outer medulla of diabetic rats and L-Arg failed to prevent these decreases. However, L-Arg improved NO production, NADPH-diaphorase activity in collecting ducts and other tubular structures, and NOS activity in renal homogenates from diabetic rats. AQP2 protein and mRNA were decreased in the renal outer medulla of diabetic rats and L-Arg administration prevented these decreases. These results suggest that the decreased NOS activity in collecting ducts of the renal outer medulla may cause, at least in part, the decreased expression of AQP2 in this model of diabetes and constitute additional evidence supporting a role for NO in contributing to renal water reabsorption through the modulation of AQP2 expression in this pathological condition. However, we cannot discard that another pathway different from NOS also exists that links L-Arg to AQP2 expression.

Figure 1. NOS activity measured as pmol [¹⁴C] L-citrulline/g tissue/min.

Two-way ANOVA showed no statistically significant interaction between the effects of Diabetes and L-Arg treatment on NOS activity. The effects of Diabetes and L-Arg were considered extremely significant (p<0.001). ***p<0.001 vs. control untreated rats; ^###p<0.001 vs. control rats treated with L-Arg; ^&&&p<0.001 vs. diabetic untreated rats. Data are mean ± SEM (n = 8).

Figure 2. NOS I expression in homogenates of the renal outer medulla.

A. Representative western blot analysis of NOS I (155 kDa band) and tubulin (50 kDa band) in the renal outer medulla; B. NOS I expression indicated as NOS I/tubulin ratio fold change from control untreated rats. Two-way ANOVA showed no statistically significant interaction between the effects of Diabetes and L-Arg treatment on NOS I expression. The effect of Diabetes was considered very significant (p<0.01), the effect of L-Arg was not significant. **p<0.01 vs. control untreated rats, ##p<0.01 vs. control rats treated with L-Arg. Data are mean ± SEM (n = 6).

Figure 3. NOS III expression in homogenates of the renal outer medulla.

A. Representative western blot analysis of NOS III (140 kDa band) and tubulin (50 kDa band) in the renal outer medulla; B. NOS III expression indicated as NOS III/tubulin ratio fold change from control untreated rats. Two-way ANOVA showed no statistically significant interaction between the effects of Diabetes and L-Arg treatment on NOS III expression. The effect of Diabetes was considered significant (p<0.05), the effect of L-Arg was not significant. *p<0.05 vs. control untreated rats, #p<0.05 vs. control rats treated with L-Arg. Data are mean ± SEM (n = 6).

Figure 4. AQP2 expression in homogenates of the renal outer medulla.

A. epresentative western blot analysis of AQP2 (28 and 35–40 kDa bands; unglycosylated and glycosylated forms respectively) and tubulin (50 kDa band) in the renal outer medulla. B. AQP2 expression indicated as the ratio AQP2/tubulin fold change from control untreated rats. Two-way ANOVA showed no statistically significant interaction between the effects of Diabetes and L-Arg treatment on the expression of AQP2. The effects of Diabetes and L-Arg on AQP2 expression were considered very significant (p<0.01). **p<0.01 vs. control untreated rats; ##p<0.01 vs. control rats treated with L-Arg, &&p<0.01 vs. diabetic untreated rats. Data are mean ± SEM (n = 6).

Figure 5. AQP2 mRNA levels in the renal outer medulla.

AQP2 mRNA levels are expressed as relative values from control untreated rats. The following formula was applied: ΔΔCT = (CTAQP2−CTGAPDH) experimental−(CT AQP2−CT GAPDH)control untreated rats. Two-way ANOVA showed a statistically significant (p<0.001) interaction between the effects of Diabetes and L-Arg treatment on AQP2 mRNA expression. Bonferroni’s post- tests: *p<0.05 vs. control untreated rats; ***p<0.001 vs. control untreated rats; &&&p<0.001 vs. diabetic untreated rats. Data are mean ± SEM (n = 6).