Mechanisms of decreased tubular flow-induced nitric oxide in Dahl salt-sensitive rat thick ascending limbs

Abstract

Dahl salt-sensitive (SS) rat kidneys produce less nitric oxide (NO) than those of salt-resistant (SR) rats. Thick ascending limb (TAL) NO synthase 3 (NOS3) is a major source of renal NO, and luminal flow enhances its activity. We hypothesized that flow-induced NO is reduced in TALs from SS rats primarily due to NOS uncoupling and diminished NOS3 expression rather than scavenging. Rats were fed normal-salt (NS) or high-salt (HS) diets. We measured flow-induced NO and superoxide in perfused TALs and performed Western blots of renal outer medullas. For rats on NS, flow-induced NO was 35 ± 6 arbitrary units (AU)/min in TALs from SR rats but only 11 ± 2 AU/min in TALs from SS (P < 0.008). The superoxide scavenger tempol decreased the difference in flow-induced NO between strains by about 36% (P < 0.020). The NOS inhibitor N-nitro-l-arginine methyl ester (l-NAME) decreased flow-induced superoxide by 36 ± 8% in TALs from SS rats (P < 0.02) but had no effect in TALs from SR rats. NOS3 expression was not different between strains on NS. For rats on HS, the difference in flow-induced NO between strains was enhanced (SR rats: 44 ± 10 vs. SS: 9 ± 2 AU/min, P < 0.005). Tempol decreased the difference in flow-induced NO between strains by about 37% (P < 0.012). l-NAME did not significantly reduce flow-induced superoxide in either strain. HS increased NOS3 expression in TALs from SR rats but not in TALs from SS rats (P < 0.003). We conclude that 1) on NS, flow-induced NO is diminished in TALs from SS rats mainly due to NOS3 uncoupling such that it produces superoxide and 2) on HS, the difference is enhanced due to failure of TALs from SS rats to increase NOS3 expression.NEW & NOTEWORTHY The Dahl rat has been used extensively to study the causes and effects of salt-sensitive hypertension. Our study suggests that more complex processes other than simple scavenging of nitric oxide (NO) by superoxide lead to less NO production in thick ascending limbs of the Dahl salt-sensitive rat. The predominant mechanism involved depends on dietary salt. Impaired flow-induced NO production in thick ascending limbs most likely contributes to the Na⁺ retention associated with salt-sensitive hypertension.

Keywords: hypertension; kidney; reactive oxygen species; salt.

Graphical abstract

Figure 1.

Flow-induced nitric oxide (NO) production by thick ascending limbs from Dahl salt-resistant (SR) and salt-sensitive (SS) rats fed normal salt. Data were analyzed by an unpaired t test (n = 8 SR rats and 6 SS rats, P < 0.008). AU, arbitrary units.

Figure 2.

Effect of the ${\text{O}}_2^{-}$ scavenger tempol on flow-induced nitric oxide (NO) production by thick ascending limbs from Dahl salt-sensitive (SS) rats fed normal salt. Data on SS rats from Fig. 1 are repeated here for comparison with tempol-treated tubules. Data were analyzed by an unpaired t test (n = 6 for untreated tubules and n = 9 for tempol-treated tubules, each tubule came from a different rat, P < 0.020). AU, arbitrary units.

Figure 3.

Effect of the nitric oxide synthase inhibitor N-nitro-l-arginine methyl ester (l-NAME) on flow-induced ${\text{O}}_2^{-}$ production by thick ascending limbs from Dahl salt-resistant (SR) and salt-sensitive (SS) rats fed normal salt. Data analyzed by a post hoc paired t test after two-way ANOVA are shown (n = 5 SR rats, no significance; n = 6 SS rats, P < 0.02). AU, arbitrary units; Con, control period.

Figure 4.

Nitric oxide synthase isoform 3 (NOS3) expression by thick ascending limbs from Dahl salt-resistant (SR) and salt-sensitive (SS) rats fed normal salt. A: representative blot. B: mean data of NOS3 expression normalized to the loading control GAPDH. Data were analyzed by an unpaired t test (n = 5 rats for each strain).

Figure 5.

Effect of a 6–8 days of high-salt diet on systolic blood pressure of Dahl salt-resistant (SR) and salt-sensitive (SS) rats. Data were analyzed by an unpaired t test (n = 7 SR rats and 6 SS rats, P < 0.0001).

Figure 6.

Flow-induced nitric oxide (NO) production by thick ascending limbs from Dahl salt-resistant (SR) and salt-sensitive (SS) rats fed high salt. Data were analyzed by an unpaired t test (n = 6 rats for each strain, P < 0.005). AU, arbitrary units.

Figure 7.

Effect of the ${\text{O}}_2^{-}$ scavenger tempol on flow-induced nitric oxide (NO) production by thick ascending limbs from Dahl salt-sensitive (SS) rats fed high salt. Data on SS rats from Fig. 6 are repeated here for comparison with tempol-treated tubules from SS rats. Data were analyzed by an unpaired t test (n = 6 rats for each group, P < 0.012). AU, arbitrary units.

Figure 8.

Effect of the nitric oxide synthase inhibitor N-nitro-l-arginine methyl ester (l-NAME) on flow-induced ${\text{O}}_2^{-}$ production by thick ascending limbs from Dahl salt-resistant (SR) and salt-sensitive (SS) rats fed high salt. Data analyzed by two-way ANOVA showed no significance (n = 7 rats for each strain). AU, arbitrary units; Con, control period.

Figure 9.

Nitric oxide synthase isoform 3 (NOS3) expression by thick ascending limbs from Dahl salt-resistant (SR) and salt-sensitive (SS) rats fed high salt. A: representative blot. B: mean data of NOS3 expression normalized to the loading control GAPDH. Data were analyzed by an unpaired t test (n = 5 rats for each strain, P < 0.003).

Figure 10.

Nitric oxide synthase isoform 3 (NOS3) expression by thick ascending limbs from Dahl salt-resistant (SR) and salt-sensitive (SS) rats fed normal salt (NS) or high salt (HS). A: representative blots. B: mean data of NOS3 expression normalized to the loading control vinculin. Data analyzed by a post hoc unpaired t test after ANOVA are shown (n = 5 rats for each group, P < 0.04 for NS vs. HS in SR rats).