KCa 3.1 channels maintain endothelium-dependent vasodilatation in isolated perfused kidneys of spontaneously hypertensive rats after chronic inhibition of NOS

Abstract

Background and purpose: The purpose of the study was to investigate renal endothelium-dependent vasodilatation in a model of severe hypertension associated with kidney injury.

Experimental approach: Changes in perfusion pressure were measured in isolated, perfused kidneys taken from 18-week-old Wistar-Kyoto rat (WKY), spontaneously hypertensive rats (SHR) and SHR treated for 2 weeks with N(ω) -nitro-L-arginine methyl ester in the drinking water (L-NAME-treated SHR, 6 mg·kg(-1) ·day(-1) ).

Key results: Acetylcholine caused similar dose-dependent renal dilatation in the three groups. In vitro administration of indomethacin did not alter the vasodilatation, while the addition of N(w) -nitro-L-arginine (L-NA) produced a differential inhibition of the vasodilatation, (inhibition in WKY > SHR > L-NAME-treated SHR). Further addition of ODQ, an inhibitor of soluble guanylyl cyclase, abolished the responses to sodium nitroprusside but did not affect the vasodilatation to acetylcholine. However, the addition of TRAM-34 (or charybdotoxin) inhibitors of Ca(2+) -activated K(+) channels of intermediate conductance (K(Ca) 3.1), blocked the vasodilatation to acetylcholine, while apamin, an inhibitor of Ca(2+) -activated K(+) channels of small conductance (K(Ca) 2.3), was ineffective. Dilatation induced by an opener of K(Ca) 3.1/K(Ca) 2.3 channels, NS-309, was also blocked by TRAM-34, but not by apamin. The magnitude and duration of NS-309-induced vasodilatation and the renal expression of mRNA for K(Ca) 3.1, but not K(Ca) 2.3, channels followed the same ranking order (WKY < SHR < L-NAME-treated SHR).

Conclusions and implications: In SHR kidneys, an EDHF-mediated response, involving activation of K(Ca) 3.1 channels, contributed to the mechanism of endothelium-dependent vasodilatation. In kidneys from L-NAME-treated SHR, up-regulation of this pathway fully compensated for the decrease in NO availability.

Figure 1

Bolus injections of increasing doses of (A) acetylcholine and (B) sodium nitroprusside during vasoconstrictions caused by methoxamine in isolated perfused kidneys from WKY, SHR and SHR treated with L-NAME (SHR L-NAME). The results are shown as % dilatation of the increase in perfusion pressure induced by methoxamine, and values are expressed as means ± SEM. (n= 9–10). *P < 0.05, significantly different from the WKY group; $P < 0.05, significant difference between the SHR and L-NAME-treated SHR groups; two-way anova, followed by a Bonferroni post hoc test, 15 and 12 multiple comparisons for acetylcholine and sodium nitroprusside experiments, respectively.

Figure 2

Effects of indomethacin (INDO) and L-NA on the dilatation due to acetylcholine (A) and sodium nitroprusside (B) of isolated perfused kidney from WKY, SHR and SHR treated with L-NAME (SHR L-NAME). The results are shown as % dilatation of the increase in perfusion pressure induced by methoxamine, and values are expressed as means ± SEM. (n= 5–10). *P < 0.05, significant difference between the indomethacin and the indomethacin + L-NA groups; two-way anova followed by a Bonferroni post hoc test, 15 and 12 multiple comparisons for acetylcholine and sodium nitroprusside experiments respectively.

Figure 3

Effects of ODQ on the dilatation evoked by acetylcholine and sodium nitroprusside in isolated perfused kidneys from L-NAME-treated SHR (in the presence of L-NA plus indomethacin). The results are shown as % dilatation of the increase in perfusion pressure induced by methoxamine and values are expressed as mean ± SEM (n= 5–8). *P < 0.05, significantly different from control; two-way anova, followed by a Bonferroni post hoc test, five and four multiple comparisons for acetylcholine and sodium nitroprusside experiments respectively.

Figure 4

Effects of TRAM-34 (0.5, 1 and 5 µmol·L⁻¹, top) and apamin (0.5 µmol·L⁻¹, bottom) on the dilatation due to acetylcholine in isolated perfused kidneys from WKY, SHR and L-NAME-treated SHR (n= 3–7). The experiments were performed in the presence of indomethacin (3 µM) and L-NA (100 µM). The results are shown as % dilatation of the increase in perfusion pressure induced by methoxamine and values are expressed as means ± SEM (n≥ 3). *P < 0.05, significantly different from control; two-way anova, followed by a Bonferroni post hoc test, 10–15 and 5 multiple comparisons for TRAM-34 and apamin experiments respectively.

Figure 5

Bolus injection of NS 309 (10 nmol) in isolated perfused kidneys from normotensive WKY, SHR and L-NAME-treated SHR (n= 5–7). The experiments were performed in the presence of indomethacin plus L-NA. The results are shown as % dilatation of the increase in perfusion pressure induced by methoxamine and values are expressed as mean ± SEM. *P < 0.05, AUC significantly different from control; $P < 0.05, significant difference between SHR and L-NAME-treated SHR; one-way anova, followed by a Bonferroni post hoc test, three multiple comparisons.

Figure 6

Effects of TRAM-34 (0.5, 1 and 5 µM) on the dilatation due to NS 309 (10 nmol) in isolated perfused kidneys from WKY, SHR and L-NAME-treated SHR (n= 3–7). The experiments were performed in the presence of indomethacin plus L-NA. The results are shown as % dilatation of the increase in perfusion pressure induced by methoxamine, and values are expressed as mean ± SEM. *P < 0.05, AUC significantly different from control; one-way anova followed by a Bonferroni post hoc test, 2–3 and 1 multiple comparisons for TRAM-34 and apamin experiments respectively.

Figure 7

Quantitative RT-PCR for mRNA for K_Ca2.3 and K_Ca3.1 protein in kidneys of WKY, SHR and L-NAME-treated SHR (n= 8–13). The values are normalized to the geometric mean of the values obtained with three internal controls: HPRT, β-actin and GAPDH. Values are expressed as mean ± SEM. *P < 0.05, significant difference between L-NAME-treated SHR and WKY kidneys; one-way anova-1 followed by a Bonferroni post hoc test, three multiple comparisons.