Angiotensin II regulation of renal vascular ENaC proteins

Abstract

Background

The importance of β and γ Epithelial Na⁺ Channel (ENaC) proteins in vascular smooth muscle cell mediated pressure-induced constriction in renal interlobar arteries has been demonstrated recently. In renal epithelial tissue, ENaC expression is regulated by angiotensin II (Ang II). However, whether or not Ang II regulates vascular ENaC expression has never been determined. Therefore, the goal of the current investigation was to determine if Ang II affects vascular ENaC expression and its contribution to pressure-induced constriction.

Methods

To address this goal, Sprague-Dawley rats were infused with Ang II (50 ng/kg/min) via osmotic mini-pump for 1 week. Mean arterial pressure was measured using radiotelemetry. Interlobar arteries were isolated from these animals to assess VSMC ENaC protein expression, pressure-induced constriction and agonist induced vascular reactivity.

Results

MAP was not different in control (113 ± 2) and Ang II (114 ± 2 mm Hg) infused mice. We found that Ang II infusion decreased renal VSMC β- and γENaC immunolabeling by 18%. Consistent with this finding, we also found that ENaC-dependant peak pressure-induced constriction was inhibited from 38 ± 3% to 25 ± 1% at 125 mm Hg. Vasoreactivity to KCl, phenylephrine and acetylcholine were unchanged.

Conclusions

Ang II suppression of pressure-induced constrictor responses in renal interlobar arteries may be mediated, at least in part, by inhibition of β and γENaC protein expression.

Introduction

Epithelial Na⁺ Channels (ENaC) are heteromultimeric channels formed by α, β and γENaC proteins. In the kidney, ENaC channels are found in the distal nephron where they regulate blood pressure by contributing to Na⁺ and water reabsorption. Expression of ENaC proteins is tightly regulated by several hormonal factors including steroid and peptide hormones that are critical to blood pressure regulation such as aldosterone and angiotensin II (1, 2).

β and γENaC proteins are also expressed in VSMC of renal vessels where they contribute to pressure-induced constriction in renal interlobar arteries. Pressure-induced constriction, also referred to as myogenic constriction, is initiated by pressure-induced vessel wall stretch. The molecular identity of molecule(s) transducing mechanical stretch into the cell-signaling event remains unknown, however mechanosensory complexes including β and γENaC proteins are one possibility (3–6). Myogenic constriction is a physiologically significant response because it contributes to blood flow autoregulation and may prevent transmission of systemic blood pressure to delicate microvessels (7, 8).

Although hormonal regulation of ENaC proteins has been addressed in epithelial cells, regulation of vascular ENaC remains unknown. We considered the possibility that Ang II may be an important regulator of vascular ENaC because Ang II is a potent regulator or vascular tone and regulates ENaC expression in epithelial tissue. In renal tubules, Ang II has contrasting effects on ENaC expression: stimulates αENaC, but inhibits β and γENaC expression (9). While renal epithelial ENaC expression is regulated by Ang II, whether or not Ang II regulates vascular ENaC expression has never been determined. Therefore, the goal of the current study was to determine if Ang II regulates vascular ENaC expression and function. To test this hypothesis Sprague Dawley rats received Ang II via osmotic mini-pump for 1 week. Renal interlobar arteries were isolated from these animals to assess VSMC ENaC protein expression and myogenic constrictor responses. Our findings suggest Ang II inhibits β and γENaC in renal VSMCs. Furthermore, Ang II inhibits myogenic constriction in renal interlobar arteries.

Methods

Experimental Groups

Male Sprague-Dawley rats (275–375 g body wt.; Harlan Industries) were divided into two groups for each experiment; control (saline, 0.5 μl/hr) and Ang II (50 ng/ml/kg). All animals were maintained on a high salt diet (8% NaCl) at least 1 wk prior to Ang II infusion to inhibit endogenous Ang II formation. Animals were housed 1/cage in a temperature-controlled room (23°C) with a 12:12 hour light/dark cycle. All protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center.

Telemetric Measure of Arterial Pressure in Conscious Rats

In a sub-group of rats, arterial pressure was chronically monitored in conscious rats by use of radio telemetry as previously described (10). Pressures were sampled at 500 Hz for 20 sec every 20 min and averaged in 60-minute blocks for analysis with arterial pressure averaged during 24-hour periods for daily values.

Ang II Infusion

Ang II was infused (50 ng/kg/min) intravenously for 1 wk via an osmotic mini-pump (model 2002, Alza Scientific, Palo Alto, CA) implanted abdominally under isofluorane anesthesia. Following this period, renal vessels were harvested for vessel function and expression studies.

Quantification of ENaC expression in freshly dispersed VSMCs

To determine the effect Ang II on ENaC expression, we used quantitative immunolabeling as previously described. For these studies, renal vessels are isolated, and VSMCs enzymatically dispersed and fixed as described previously (11). Samples were labeled with antibodies to α, β or γENaC and mouse anti-smooth muscle α-actin as previously described (11, 12). All samples were examined using quantitative fluorescence confocal microscopy (TCS-SP2, Leica Microsystems, Exton, PA). All samples were collected, labeled and imaged side-by-side under identical conditions. Background signal from samples where the primary antibody was omitted was subtracted from all samples. We have used this approach to quantify ENaC expression in VSMCs previously (11–13). Data were averaged from 3 animals in each group.

Cannulation of interlobular arteries for dimensional analysis

Animals were anesthesized with oxyfluorane, then both kidneys were excised and placed in ice cold physiological saline solution as previously described (11, 12). Renal interlobar arteries [121.5 ± 3.3 μm passive (Ca²⁺-free) inner diameter at 75 mmHg] were studied in a vessel chamber (CH/1/SH, Living Systems, Burlington, VT) and analyzed using MetaMorph software (Universal Imaging, Downingtown, PA) as described previously (11, 12). Following an initial incubation period (30 min at 75 mmHg), a pressure-diameter curve was generated to determine the effect of ENaC inhibition on myogenic constriction as previously described (12). Arteries were equilibrated for 30 min with benzamil (1 μM, ENaC inhibitor) extraluminally before repeating the pressure-diameter curve. The pressure-diameter curve was repeated in the presence of Ca²⁺-free PSS [same as above PSS plus 2 mmol/L EGTA and omit 1.8 mmol/L CaCl₂] to determine the passive pressure-diameter curve. Myogenic constriction was calculated as the percent difference between the active (PSS) and passive (Ca²⁺-free PSS) inner diameter at each pressure.

Effect of Ang II-infusion on phenylephrine-, KCl-, and acetylcholine-induced vasoreactivity

To determine the effect of Ang II on agonist-induced vascular reactivity, we assessed vasoconstriction to the depolarizing stimulus KCl (80 mM) and the α₁-adrenergic receptor agonist, phenylephrine (PE; 10⁻⁹–10⁻⁵ M). We also evaluated endothelium-dependent vasodilation with acetylcholine (ACh; 10⁻⁹–10⁻⁵ M) following pre-constriction with 10⁻⁶ M PE. Data are presented as percent vasoconstriction and vasodilation from control.

Statistics

All data are expressed as mean ± SE. A t-test or two-way ANOVA with repeated measures was used to make comparisons where appropriate. Differences among groups were compared using the Student-Newman-Keuls test. Statistical significance was considered at p<0.05.

Results

Slow-pressor response to Ang II

The average 24-hour MAP during the 12 day recording period in vehicle and Ang II infused animals are shown in Figure 1. In control animals (n=5), MAP was 115 ±2 and 113 ± 2 mm Hg during the 6-day control and infusion periods. In Ang II infused animals (n=9), MAP increased significantly from 109 ± 2 mm Hg during the control period to 114 ± 2 mm Hg (p = 0.009) during Ang II infusion. MAP was not significantly different between vehicle and Ang II infused groups (p =0.77).

Figure 1.

Chronic Ang II infusion on mean arterial pressure (MAP). Although MAP increased slightly following the onset of Ang II infusion, however MAP was not different between vehicle (□, n=5) and Ang II infused animals during the infusion period (△, n=9).

Effect of Ang II on renal VSMC ENaC expression

Representative images of α, β and γENaC and α-smooth muscle actin immuno-localization in renal VSMCs isolated from vehicle and Ang II treated animals are shown in Figure 2A, B and C, respectively. Quantitation of α, β and γENaC labeling, normalized to α smooth muscle actin are shown in Figure 2D. In renal VSMC isolated from vehicle treated animals, αENaC fluorescence was undetectable above background. Although Ang II infusion significantly increased the expression of αENaC (Figure 2A, p<0.001),αENaC expression was still only weakly detected. In contrast,β- and γENaC were robustly expressed in isolated VSMCs near the membrane from vehicle treated animals, and their expression diminished following Ang II infusion by 18.5 (p<0.001) and 18.2 % (p=0.001), respectively (Figure 2D). The near membrane localization pattern of β and γENaC were unaltered by Ang II infusion (Fig 2B and C).

Figure 2.

Chronic Ang II infusion on renal VSMC ENaC expression. Representative images (AC) and quantitative data (D) are shown. α, β or γENaC (red) and α-actin (white) staining are shown. Chronic Ang II increases αENaC, but decreases β and γENaC expression. Representative images and quantitative data are shown. ENaC expression was normalized to α actin staining and cell size and presented as relative fluorescence units (RFU). n= number of cells examined, each from n=2–3 animals per group. *Significant different from HS control, p<0.05.

Ang II on renal interlobar artery myogenic vasoconstriction

Vessel pressure (mm Hg) –diameter (μm) responses in vehicle and Ang II infused rats under control, 1 μM benzamil and Ca²⁺ free conditions are shown in Figure 3A and B, respectively. Passive vessel diameter responses (Ca²⁺ free) were similar between groups. Renal interlobar ateries from vehicle infused rats maintained internal diameter with increases in intraluminal pressure. In contrast, renal interlobar arteries from Ang II treated animals dilated with increases in intraluminal pressure. The effect of Ang II infusion on the development of myogenic tone is shown in Figure 3C. Renal interlobar arteries from vehicle treated rats developed significantly more myogenic tone at 100, 125 and 150 mm Hg (32 ± 3, 38 ± 3 and 34 ±2%), while Ang II infusion reduced myogenic tone (27 ±2, 25 ± 1 and 24 ±1 at 100, 125 and 150 mm Hg, respectively). Nearly all myogenic tone was inhibited by ENaC blockade with benzamil (Figure 3C). The amount of myogenic tone developed following benzamil treatment was not different between vehicle and Ang II treated arteries.

Figure 3.

Chronic Ang II infusion on renal myogenic constriction in renal interlobar arteries. Pressure-diameter relationship under control, ENaC inhibition (1 μM benzamil) and Ca²⁺ free conditions in control (A) and chronic Ang II infused animals (B). C. Total myogenic with and without ENaC inhibition in control (□, n=5) and chronic Ang II infusion (△, n=5). Total myogenic tone is reduced following one week of Ang II infusion.

*Significant different from HS control, p<0.05.

Ang II on renal interlobar artery agonist-induced vascular reactivity

Interlobar artery vascular reactivity in vehicle and Ang II treated animals are shown in Figure 4. Normalized vasoconstrictor responses to 80 mM KCl and PE (10⁻⁹ –10⁻⁵ M) were not different between groups (Figure 4A and B). Furthermore, vasodilation to ACh (10⁻⁹ – 10⁻⁵ M) was not different in vehicle versus Ang II infused animals (Figure 4C).

Figure 4.

Chronic Ang II infusion on agonist-induced vasoconstriction and vasodilation in renal interlobar arteries. Renal interlobar artery vasoconstricion to 80 mM KCl (A) and 10⁻⁹ – 10⁻⁵ M PE (B) was not different between control (□, n=5) and Ang II infused (△, n=5) animals. Vasodilation to 10⁻⁹ – 10⁻⁵ M Ach was not different between control (□, n=4) and Ang II infused (△, n=5) animals.

Discussion

Recently, our laboratory demonstrated the importance of ENaC proteins in renal myogenic vasoconstriction (11, 12). We have shown that β and γ, are expressed in renal artery VSMCs. ENaC proteins are closely related to a family of proteins in the nematode, Caenhorhbditis elegans, that form the pore of a mechanosensory ion channel complex in muscle and neurons (14). Because of the evolutionary link to the nematode proteins, ENaC proteins have been considered as potential mammalian mechanosensors. Empirical evidence supports such a link; ENaC protein and activity are required for mechanosensitive events such as arterial baroreception, touch sensation, and myogenic constriction (3, 4). While regulation of tubular ENaC by cardiovascular relevant hormones has been addressed, regulation of vascular ENaCs has never been determined. Therefore, the goal of this study was to determine the importance of one of these many ENaC regulators, Ang II, on vascular ENaC expression and function. The major findings from this study are 1) Ang II decreases β- and γENaC and leads to weak expression of αENaC and 2), significantly inhibits ENaC-dependent myogenic responsiveness in renal interlobar arteries. Our results suggest Ang II differentially regulates ENaC proteins in VSMCs.

It is common for ENaC subunits to be differentially regulated. For example, in the distal nephron, aldosterone increases αENaC transcription, but not β- and γ^{Error! Reference source not found.}ENaC (1, 2). Ang II blockade, in rats fed a low salt diet, inhibits αENaC and stimulates β and γENaC (9). Similarly, AT1a knockout mice have reduced αENaC and elevated β and γENaC (15). Consistent with these findings, our current study demonstrates Ang II increases renal VSMC α^{Error! Reference source not found.}ENaC, but decreases β and γ^{Error! Reference source not found.}ENaC. The mechanism(s) of differential expression is unclear, but may involve effects on transcription, translation, and degradation (1, 2).

Several lines of evidence suggest that β and γENaC are components of a vascular mechanosensor in the kidney (11, 12). First, β and γ, but not αENaC, are detected in vascular tissues isolated from rat and mouse renal vessels (11, 12). Second, pharmacologic inhibition of ENaC channels with amiloride (5 μM) or benzamil (1 μM) abolishes pressure-induced, but does not alter agonist-induced constriction (11, 12). Third, gene specific silencing, using siRNA or expression of dominant-negative isoforms, specifically abolishes pressure-induced constriction (11, 12).

Consistent with the hypothesis that β and γENaC proteins may mediate pressure-induced constriction, we found that Ang II inhibited the myogenic tone (~25%) to a similar degree as β and γENaC (~18%) expression in the current study. Furthermore, myogenic tone in control and Ang II infused groups was abolished to the same level (5–8% tone) by ENaC inhibition with 1 μM benzamil, suggesting myogenic tone in renal interlobar arteries, regardless of Ang II, is predominantly dependent on ENaC signaling. Similar to our previous investigations, we were unable to detect αENaC in control renal VSMCs. In contrast, αENaC became detectable by immunofluorescence following chronic Ang II infusion. However, it should be noted that relative to β and γENaC, αENaC expression is extremely low and barely detectable above background. This finding suggests renal VSMC expression of αENaC may be suppressed under normal conditions and up-regulated under certain conditions.

Our finding that Ang II infusion inhibits pressure-induced constrictor responses in interlobar arteries is consistent with previous investigations. Animal models of Ang II dependent hypertension (two kidney, one-clip model, Ang II infusion) have suppressed pressure-induced regulation of afferent arteriolar tone and renal blood flow (16–21). In contrast to our current investigation, these studies used higher doses of Ang II and elicited substantial increases in pressure, which are associated with renal injury. In a subsequent study, Inscho et al. demonstrated that the prevention of increases in arterial pressure with Ang II receptor antagonists or triple therapy (hydralazine, hydrochlorothiazide, and reserpine) prevented the autoregulatory dysfunction suggesting that increased blood pressure, rather than Ang II, caused the loss of autoregulatory response (21). In the current study, MAP increased 7 mm Hg after one week of Ang II. However, MAP in Ang II infused animals was not different from control animals. We speculate that the low rate of infusion (0.5 μl/hr) of the miniosmotic pump likely lead to Ang II degradation and thus circulating levels of Ang II were not sufficient to produce hypertension. Thus, it is unknown whether the small change in pressure, increased Ang II, or both can lead to altered myogenic responsiveness. Additional studies are required to determine the importance of pressure, Ang II, and their interaction on ENaC expression and ENaC-dependent myogenic tone.

Our finding that chronic infusion of Ang II suppresses expression of vascular β and γENaC and renal myogenic constriction has implications for hypertension-induced renal injury. The myogenic response has been suggested to be an important mechanism protecting microvessels from injury caused by high systemic pressures, especially in hypertension (7, 8). Since Ang II is elevated in many forms of hypertension, our findings raise the possibility that Ang II suppression of vascular ENaC expression and myogenic constrictor responses may contribute to increase susceptibility to renal injury in hypertension. In summary, our data suggest chronic Ang II infusion suppresses expression of vascular ENaC proteins that contribute to myogenic constriction in renal interlobar arteries.