Activation of ENaC in collecting duct cells by prorenin and its receptor PRR: involvement of Nox4-derived hydrogen peroxide

Abstract

The collecting duct (CD) has been recognized as an important source of prorenin/renin, and it also expresses (pro)renin receptor (PRR). The goal of this study was to examine the hypothesis that prorenin or renin via PRR regulates epithelial Na(+) channel (ENaC) activity in mpkCCD cells. Transepithelial Na(+) transport was measured by using a conventional epithelial volt-ohmmeter and was expressed as the calculated equivalent current (Ieq). Amiloride-inhibitable Ieq was used as a reflection of ENaC activity. Administration of prorenin in the nanomolar range induced a significant increase in Ieq that was detectable as early as 1 min, peaked at 5 min, and gradually returned to baseline within 15 min. These changes in Ieq were completely prevented by a newly developed PRR decoy inhibitor, PRO20. Prorenin-induced Ieq was inhibitable by amiloride. Compared with prorenin, renin was less effective in stimulating Ieq Prorenin-induced Ieq was attenuated by apocynin but enhanced by tempol, the latter effect being prevented by catalase. In response to prorenin treatment, the levels of total reactive oxygen species and H2O2 were both increased, as detected by spin-trap analysis and reactive oxygen species (ROS)-Glo H2O2 assay, respectively. Both siRNA-mediated Nox4 knockdown and the dual Nox1/4 inhibitor GKT137892 attenuated prorenin-induced Ieq Overall, our results demonstrate that activation of PRR by prorenin stimulates ENaC activity in CD cells via Nox4-derived H2O2.

Keywords: (pro)renin receptor; epithelial sodium channel; hydrogen peroxide; inner medullary collecting duct; renin activity.

Fig. 1.

Effect of prorenin/(pro)renin receptor (PRR) on epithelial Na⁺ channel (ENaC) activity in cultured collecting duct (CD) cells. Confluent mpkCCD cells grown on Transwells were pretreated for 30 min with PRO20 (1.5 μM) or losartan (1.0 μM) and then treated with prorenin (10 nM). The equivalent current (I_eq) was monitored for a 15-min period by using an epithelial volt-ohmmeter (EVOM). In a separate experiment, the time point of peak stimulation of Na⁺ transport at 1 min was chosen for measuring amiloride-sensitive I_eq, an index of ENaC activity. A: time course of I_eq changes in control (CTR), prorenin, and prorenin + PRO20 groups. B: corresponding amiloride-sensitive I_eq for experiments in A. C: time course of I_eq changes in control, prorenin, and prorenin + losartan (Los) groups. D: corresponding amiloride-sensitive I_eq for experiments in C. Data are means ± SE; n = 6–12 per group. *P < 0.05 vs. basal in the same group; #P < 0.05 vs. prorenin.

Fig. 2.

Effect of aliskiren on prorenin-induced Na⁺ transport in cultured CD cells. Confluent mpkCCD cells grown on Transwells were pretreated for 30 min with aliskiren (1.0 μM) and then treated with prorenin (10 nM). I_eq was and amiloride-sensitive I_eq were determined as described in Fig. 1. A: time course of I_eq changes in control, prorenin, and prorenin + aliskiren groups. Prorenin was added to the apical side. B: corresponding amiloride-sensitive I_eq for experiments in A. C: comparison of the effect of apical and basal administration of prorenin on I_eq. Data are means ± SE; n = 6–12 per group. *P < 0.05 vs. basal in the same group.

Fig. 3.

Effect of renin on ENaC activity in cultured CD cells. Confluent mpkCCD cells grown on a Transwell membrane were pretreated for 30 min with PRO20 (1.5 μM) and then treated with renin (10 nM). A: time course of I_eq changes in Control, renin, and renin + PRO20 groups. I_eq was monitored for 15 min. Renin was given at 10 nM. B: time course of I_eq changes in control and renin groups was monitored for 15 min. Renin was given at 100 nM. C: amiloride-sensitive I_eq in control, renin, and renin + PRO20 groups. Data are means ± SE; n = 6–12 per group. *P < 0.05 vs. basal in the same group.

Fig. 4.

Effect of antioxidants on prorenin-induced ENaC activity. A: mpkCCD cells were pretreated for 30 min with vehicle, apocynin (10 μM), tempol (2 mM), or tempol (2 mM) + catalase (1 μg/ml) and then treated with 10 nM prorenin. Amiloride-sensitive I_eq was taken as ENaC activity. B: mpkCCD cells were treated with vehicle, apocynin, tempol, or catalase alone at the same concentrations. C: time course of I_eq changes in control, prorenin, and prorenin + tempol groups. D: amiloride-sensitive I_eq in mpkCCD cells exposed to 1.5 μM H₂0₂ for 1 min. Data are means ± SE; n = 6–12 per group. *P < 0.05 vs. basal in the same group; #P < 0.05 vs. prorenin.

Fig. 5.

reactive oxygen species (ROS) generation in response to prorenin and renin. mpkCCD cells grown on a Transwell membrane were exposed to prorenin or renin each at 10 nM for 1 min in the presence or absence of 1.5 μM PRO20 or tempol. The samples were then subjected to spin trapping analysis of ROS (A and B) or the luciferase assay for H₂O₂ (C and D). A: effect of prorenin on ROS generation in the presence or absence of PRO20. B: effect of renin on ROS generation. EPR, electron paramagnetic resonance. C: time course of H₂O₂ generation after prorenin treatment. RLU, relative light units. D: effect of prorenin on H₂O₂ generation in the presence or absence of PRO20. E: effect of prorenin on H₂O₂ generation in the presence or absence of tempol. Data are means ± SE; n = 6–12 per group. *P < 0.05 vs. basal in the same group.

Fig. 6.

The role of Nox4 in prorenin-induced ENaC activity. mpkCCD cells were pretreated with a Nox1/4 inhibitor, GKT137892 (GKT), or transfected with Nox4 siRNA and then treated with 10 nM prorenin. H₂O₂ generation was determined by ROS-Glo H₂O₂ Assay. Transepithelial Na⁺ transport was measured by using an EVOM. Nox4 mRNA and protein were determined by quantitative RT-PCR and Western blotting, respectively. A: effect of GKT on prorenin-induced H₂O₂ level. B: effect of GKT on prorenin-induced I_eq. C: validation of Nox4 knockdown by quantitative RT-PCR. D: validation of Nox4 knockdown by Western blotting. Shown is a representative immunoblot of 3 independent experiments. The value underneath the blot shows the densitometry of Nox4 protein normalized by β-actin. E: effect of Nox4 siRNA knockdown on prorenin-induced H₂O₂ level. F: effect of Nox4 knockdown on prorenin-induced I_eq. Data are means ± SE: n = 3 per group for D and n = 6–8 per group for A–C, E, and F. *P < 0.05 vs. basal in the same group; #P < 0.05 vs. prorenin.