Angiotensin II-mediated hypertension impairs nitric oxide-induced NKCC2 inhibition in thick ascending limbs

Abstract

In thick ascending limbs (THALs), nitric oxide (NO) decreases NaCl reabsorption via cGMP-mediated inhibition of Na-K-2Cl cotransporter (NKCC2). In angiotensin (ANG II)-induced hypertension, endothelin-1 (ET-1)-induced NO production by THALs is impaired. However, whether this alters NO's natriuretic effects and the mechanisms involved are unknown. In other cell types, ANG II augments phosphodiesterase 5 (PDE5)-mediated cGMP degradation. We hypothesized that NO-mediated inhibition of NKCC2 activity and stimulation of cGMP synthesis are blunted via PDE5 in ANG II-induced hypertension. Sprague-Dawley rats were infused with vehicle or ANG II (200 ng·kg^-1·min^-1) for 5 days. ET-1 reduced NKCC2 activity by 38 ± 13% (P < 0.05) in THALs from vehicle-treated rats but not from ANG II-hypertensive rats (Δ: -9 ± 13%). A NO donor yielded similar results as ET-1. In contrast, dibutyryl-cGMP significantly decreased NKCC2 activity in both vehicle-treated and ANG II-hypertensive rats (control: Δ-44 ± 15% vs.

Ang ii: Δ-41 ± 10%). NO increased cGMP by 2.08 ± 0.36 fmol/μg protein in THALs from vehicle-treated rats but only 1.06 ± 0.25 fmol/μg protein in ANG II-hypertensive rats (P < 0.04). Vardenafil (25 nM), a PDE5 inhibitor, restored NO's ability to inhibit NKCC2 activity in THALs from ANG II-hypertensive rats (Δ: -60 ± 9%, P < 0.003). Similarly, NO's stimulation of cGMP was also restored by vardenafil (vehicle-treated: 1.89 ± 0.71 vs. ANG II-hypertensive: 2.02 ± 0.32 fmol/μg protein). PDE5 expression did not differ between vehicle-treated and ANG II-hypertensive rats. We conclude that NO-induced inhibition of NKCC2 and increases in cGMP are blunted in ANG II-hypertensive rats due to PDE5 activation. Defects in the response of THALs to NO may enhance NaCl retention in ANG II-induced hypertension.

Keywords: cGMP; endothelin-1; kidney; phosphodiesterase 5; sodium transport.

Fig. 1.

Effect of 1 nM endothelin (ET)-1 on Na-K-2Cl cotransporter (NKCC2) activity. Nai, intracellular Na; AFU/s, arbitrary fluorescence U/s. A: Nai measured as AFU vs. time in isolated and perfused thick ascending limbs (THALs) from vehicle-treated rats (n = 5). B: same data as A but NKCC2 activity is displayed as the rate of increase in Nai in vehicle-treated rats (n = 5). C: Nai measured as AFU vs. time in isolated and perfused THALs from ANG II-induced hypertensive rats (n = 6). D: same data as C but NKCC2 activity is displayed as the rate of increase in Nai in ANG II-induced hypertensive rats (n = 6).

Fig. 2.

Effect of ET-1 on NKCC2 activity in the presence of tempol in THALs from ANG II-induced hypertensive rats. Effect of 1 nM ET-1 on NKCC2 activity in the presence of 100 μM tempol measured in isolated and perfused THALs (n = 5).

Fig. 3.

Effect of NONOate (NO) on NKCC2 activity. NKCC2 activity was measured in isolated and perfused THALs before and after treatment with 100 μM spermine NONOate. A: vehicle-treated rats (n = 6). B: ANG II-induced hypertensive rats (n = 6).

Fig. 4.

Effect of db-cGMP on NKCC2 activity. NKCC2 activity was measured in isolated and perfused THALs before and after treatment with 500 μM db-cGMP. A: vehicle-treated rats (n = 5). B: ANG II-induced hypertensive rats (n = 4).

Fig. 5.

Effect of NONOate (NO) on cGMP levels in THALs from vehicle- and ANG II-induced hypertensive rats. THAL suspensions were incubated in the presence and absence of 100 μM spermine NONOate for 10 min (vehicle n = 6; ANG II n = 8). cGMP was measured by enzyme immunoassay.

Fig. 6.

Effect of NO on NKCC2 activity in the presence of the phosphodiesterase 5 (PDE5) inhibitor vardenafil. Effect of 100 μM spermine NONOate on NKCC2 activity in the presence of 25 nM vardenafil in isolated and perfused THALs. A: vehicle-treated rats (n = 4). B: ANG II-induced hypertensive rats (n = 5).

Fig. 7.

Effect of NO on cGMP levels in the presence of the PDE5 inhibitor vardenafil in THALs from vehicle- and ANG II-induced hypertensive rats. THAL suspensions were preincubated with 25 nM vardenafil for 10 min and then treated with 100 μM spermine NONOate for 10 min (vehicle n = 6; ANG II n = 8). cGMP was measured by enzyme immunoassay.

Fig. 8.

PDE5 protein levels in THALs from vehicle- and ANG II-induced hypertensive rats. Top: representative Western blot for PDE5 and β-tubulin. Bottom: cumulative data showing PDE5/β-tubulin ratio (n = 5).