Impaired myogenic constriction of the renal afferent arteriole in a mouse model of reduced βENaC expression

Abstract

Previous studies demonstrate a role for β epithelial Na(+) channel (βENaC) protein as a mediator of myogenic constriction in renal interlobar arteries. However, the importance of βENaC as a mediator of myogenic constriction in renal afferent arterioles, the primary site of development of renal vascular resistance, has not been determined. We colocalized βENaC with smooth muscle α-actin in vascular smooth muscle cells in renal arterioles using immunofluorescence. To determine the importance of βENaC in myogenic constriction in renal afferent arterioles, we used a mouse model of reduced βENaC (βENaC m/m) and examined pressure-induced constrictor responses in the isolated afferent arteriole-attached glomerulus preparation. We found that, in response to a step increase in perfusion pressure from 60 to 120 mmHg, the myogenic tone increased from 4.5 ± 3.7 to 27.3 ± 5.2% in +/+ mice. In contrast, myogenic tone failed to increase with the pressure step in m/m mice (3.9 ± 0.8 to 6.9 ± 1.4%). To determine the importance of βENaC in myogenic renal blood flow (RBF) regulation, we examined the rate of change in renal vascular resistance following a step increase in perfusion pressure in volume-expanded animals. We found that, following a step increase in pressure, the rate of myogenic correction of RBF is inhibited by 75% in βENaC m/m mice. These findings demonstrate that myogenic constriction in afferent arterioles is dependent on normal expression of βENaC.

Fig. 1.

β−Epithelial Na⁺ channel (βENaC) labeling is decreased in afferent arterioles and distal nephron segments of a mouse model of reduced βENaC (βENaC m/m). All images were obtained in the cortex. A: localization of smooth muscle (SM) α-actin (green) and βENaC (red) in a cross-sectional image of a renal arteriole from a βENaC +/+ (top) and m/m (bottom) mouse. In the merged image, the arteriolar lumen is identified by an “L,” and the vascular smooth muscle cell (VSMC) bodies are identified by an asterisk (*). B: localization of SM α-actin (green) and βENaC (red) in a longitudinal section of a renal arteriole from a βENaC +/+ (top) and m/m (bottom) mouse. Dashed lines in merged image represent the glomerular border. Colocalization of βENaC and SM α-actin is identified as yellow labeling in the merged images in A and B. C: βENaC labeling in portions of the distal nephron in βENaC +/+ and m/m mice. Images are representative of 2 trials. Scale bar is equivalent to 8 μm.

Fig. 2.

Myogenic constriction in the afferent arteriole and interlobar artery is impaired in βENaC m/m mice. A–D: myogenic constriction in the afferent arteriole. A: image of the isolated afferent arteriole-glomerular preparation. B: inner diameter response to incubation at 60 and 120 mmHg under Ca²⁺-containing (solid lines) and Ca²⁺-free (broken lines) conditions in βENaC +/+ (■) and m/m (□) mice (n = 6). C: calculated myogenic tone is inhibited in βENaC m/m mice at 120 mmHg. D: constriction to the α-adrenergic agonist norepinephrine (NE) is similar between +/+ and m/m animals. E–G: myogenic constriction in the renal interlobar artery is impaired in βENaC mutant mice. For these experiments, m/m and m/+ mice were grouped together because responses were similar. E: calculated %myogenic tone is reduced in βENaC m/m or +/m (□, n = 5) compared with +/+ (■, n = 3) mice with stepwise increases in intraluminal pressure. F and G: vasoconstrictor responses to KCl (F) and phenylephrine (G) were identical. Data are presented as means ± SE. †Statistically different from 60 mmHg using paired, 2-tailed t-test, P < 0.05. NS, not significantly different. *Statistically different from +/+ using unpaired, 2-tailed t-test at P value indicated or 2-way repeated-measures ANOVA, with Student-Newman-Keuls post hoc test, P < 0.05.

Fig. 3.

Myogenic regulation of renal blood flow (RBF) and renal vascular resistance (RVR) is altered in βENaC m/m mice. Time course of the regulatory response of mean arterial pressure (MAP; A and D), RBF (B and E), and RVR (C and F) in wild-type (+/+, filled symbols, n = 6) and mutant (m/m, open symbols, n = 7) mice 10 s before and 30 s after the step increase in pressure. Data are presented as absolute values in A–C and normalized changes to minimize variance in D–F. The change in MAP, RBF, and RVR from 20 to 30 s to baseline is shown on right in A–C. Baseline values of MAP (mmHg), RBF (ml·min⁻¹·g kidney wt⁻¹), and RVR were not different between +/+ and m/m mice (see Table 1). Following a similar increase in MAP, the transient increase in RBF and decrease in RVR are similar between +/+ and m/m mice. Immediately following the transient drop, RVR begins to increase in the +/+, but remains low in the m/m. The inset in F shows the rate of increase in RVR during the first 5 s following the drop in RVR is significantly greater in +/+ vs. m/m mice (P = 0.014). By 20–30 s following the step increase in MAP, RBF in the +/+ is completely corrected while RBF remains elevated in m/m animals (P = 0.024, B, right). By 20–30 s following the step increase in MAP, the change in RVR from baseline is significantly greater in the +/+ vs. m/m (P = 0.027, C, right). Data are means ± SE. *Significantly different from βENaC +/+ group at the P value indicated.

Fig. 4.

Hypothetical model of a vascular mechanosensor containing βENaC. This model is based on the degenerin mechanosensor in the nematode Caenorhabditis elegans. In our model, we hypothesize that βENaC is a component of the ion-conducting pore. The pore is tethered to the extracellular matrix (ECM) and possibly the cytoskeleton, either directly or indirectly. Gating of the mechanosensor by mechanical forces is proposed to lead to Na⁺ influx, membrane depolarization, and activation of downstream signaling, leading to VSMC contraction.