Influence of the Renal Endothelin A System on the Autoregulation of Renal Hemodynamics in SHRs and WKY Rats

Abstract

In this study, we investigated the influence of a short-term blockade of the renal endothelin A system on the autoregulation of total renal blood flow, cortical renal blood flow, and pressure-dependent plasma renin activity in spontaneously hypertensive rats (SHRs) and normotensive controls [Wistar-Kyoto (WKY) rats]. In anesthetized rats, renal blood flow was measured by a transit-time flow probe and cortical blood flow by a laser flow probe. Blood samples were taken for measurement of plasma renin activity. Renal perfusion pressure was reduced in 5-mm Hg steps by means of a servocontrolled electropneumatic device by an inflatable suprarenal cuff. During the experiments, the rats (n = 6, each group) received an intrarenal infusion of either the selective endothelin A-receptor antagonist BQ123 (3 mg/kg/h) or vehicle. We observed an improvement of total and cortical blood flow autoregulation as indicated by a shift of lower limits of autoregulation to lower threshold pressures [103 ± 2 vs. 132 ± 4 mm Hg compared with 98 ± 3 vs. 120 ± 4 mm Hg (mean ± SEM); p < 0.01 resp. p < 0.05] in BQ123-treated SHRs, whereas BQ123 had no influence on breakpoints of autoregulation in WKY rats (p > 0.05). Pressure-dependent plasma renin activity in SHRs was not influenced by BQ123. Renal blood flow autoregulation is improved in SHRs after short-term blockade of the renal endothelin A system. This effect is independent of the renin-angiotensin system. The endothelin A system does not seem to play an important role in the autoregulation of renal blood flow in normotensive WKY rats.

The kidney plays an important pathogenetic role in the development and/or maintenance of high blood pressure in primary hypertension (essential hypertension), as well as in several forms of the so-called secondary hypertension ^(1-4). Investigations in spontaneously hypertensive rats (SHRs) suggest that the kidney is genetically programmed for the development of high blood pressure. Furthermore it is probable that changes in the renal hemodynamics, particularly in the renal microcirculation, are of vital importance in the pathogenesis of essential hypertension ^(3,5,6). Studies on autoregulation of renal blood flow (RBF) and glomerular filtration rate (GFR) showed a disturbance of the regulatory behavior of these parameters in the form of a resetting to higher blood-pressure values ^(7-9). It has not yet been adequately clarified, however, what intrinsic factors are responsible for these changes in the renal hemodynamics in primary hypertension ^(6,10,11).

Several hormones, such as angiotensin II (Ang II), vasopressin, atrial natriuretic factor (ANF), bradykinin (BK), and nitric oxide (NO), are known to play an essential role in the regulation of renal function ⁽¹²⁾. These hormones, which occur mainly as paracrine/autocrine systems in the kidney, regulate numerous aspects of kidney function, including the hemodynamics and GFR, as well as the tubular absorptive and secretory processes. However, it has not yet been possible to attribute a decisive role in the pathogenesis of essential hypertension to one or more of these systems.

Since the first description of the endothelin (ET) system ⁽¹³⁾, it has become increasingly clear that the ET peptides influence the regulation of renal function by different mechanisms. Striking are, on the one hand, the marked vasoconstrictor properties and, on the other hand, the effects on tubular sodium and water transport ^(14-16). ET-1 was furthermore shown to inhibit the renal release of renin ^(17,18). Based on these multiple renal effects, it appears entirely possible that the renal ET system plays a vital role in the long-term control of the RBF, the GFR, and the renal sodium and water handling ^{(14,15,19,20)}, either directly or indirectly by influencing other regulatory systems, such as the renin-angiotensin system. In view of the pronounced vasoconstrictor effect of ET, it seems furthermore justified to assume that the ET system may play a role in the pathogenesis or maintenance or both of high blood pressure in essential hypertension. In conformity with such an assumption is the finding that the increased blood pressure in SHRs can be normalized by the intravenous administration of an ET antagonist, as we and others have shown ^(21,22). We recently demonstrated a marked overexpression of vascular ET-A receptors in kidneys of spontaneously hypertensive rats (SHRs) compared with normotensive Wistar-Kyoto (WKY) rats ⁽²¹⁾. However, the functional significance of this finding needs to be investigated further, given the lack of systematic studies on the role of the renal ET system in the autoregulation of RBF and renal microcirculation in essential hypertension.

The aim of this study was therefore to investigate the influence of the selective ET-A-receptor antagonist BQ123 on autoregulation of RBF, cortical blood flow (CBF), and pressure-dependent renal renin release in anesthetized SHRs and WKY rats.

METHODS

The experiments were performed in male SHRs and WKY rats weighing 300-400 g. The rats were obtained from Møllegaard Breeding and Research Centre, Lille Skensved, Denmark. The environmental conditions were constant: temperature, 20°C; air humidity, 65%; and a day/night cycle of 12 h each. The rats had free access to drinking water and received a standard diet (Altromin 1324, Lage, Germany). Rats were anesthetized with an intraperitoneal injection of 100 mg/kg thiobutabarbital (Inactin). Animals were placed on a heating table, and a tracheotomy was performed. Arterial and venous catheters were inserted into the femoral vessels. Infusion of saline (500 μl/100 g/h) over the venous catheter was started. The tip of the right arterial catheter was positioned 2 mm below the left renal artery for measurement of renal perfusion pressure (RPP), and the tip of the left arterial catheter was placed in the orifice of the left renal artery for intrarenal infusion of vehicle or BQ123 by means of a high-precision infusion pump. The catheter intended for insertion in the renal artery consisted of a polyethylene tube (PP10; 610 μm OD, 280 μm ID) tapered at its curved tip to ∼150 μm (OD) over a length of 3 mm. The catheter was pushed in the aorta and inserted in the orifice of the left renal artery with aid of two glass hooks. The position of the tip was visualized under a high-magnification operating microscope. In pilot experiments, we demonstrated that a 3% Lissamine green solution was evenly distributed over the entire kidney both after bolus injection and during continuous intrarenal infusion. An inflatable silicon cuff was placed around the aorta between the superior mesenteric artery and the right renal artery. RBF was measured by a transit-time flow probe (1RB; Transonic Systems Inc., Ithaca, NY, U.S.A.) placed around the left renal artery. This flow probe measures absolute RBF with a precision of ±5% ^(23,24). The probes were precalibrated by the producer. CBF was measured by a prism laser flow probe (MBF 3D; Moor Instruments Ltd., Devon, U.K.) that was placed on the surface of the left kidney covered with body-temperature mineral oil ⁽²⁵⁾. RPP was reduced via a servocontrolled electropneumatic device (SCR 010, Rebsch Electronics, Mannheim, Germany) connected to the suprarenal cuff, which allowed reduction of RPP at any given level and kept it constant within a pressure range of maximally ±2 mm Hg ⁽²⁶⁾.

Renin measurement

Blood samples (100 μl) were taken for measurement of plasma renin activity (PRA; ng Ang I/ml/min) and replaced with 5% bovine albumin in isotonic saline. PRA was measured by radioimmunoassay (RIA), as previously described ⁽²⁷⁾.

Experimental protocols

Four groups of animals were studied: group 1 (WKY rats; n = 6) and group 2 (SHRs; n = 6) received an intrarenal (i.r.) bolus injection (over a 5-min period; injection volume, 600 μl/kg) of BQ123 (3 mg/kg) followed by a continuous i.r. infusion of BQ123 (3 mg/kg/h) via the renal catheter for the entire duration of the experiment. The concentration of the drug solution was adjusted to obtain the required dose by infusing 50 μl/kg/min. Group 3 (WKY rats; n = 6) and group 4 (SHRs; n = 6) served as controls and received vehicle (0.9% NaCl) instead of the ET-A-receptor antagonist (bolus injection of 600 μl/kg over a 5-min period followed by continuous infusion of 50 μl/kg/min). The dose of BQ123 was selected on the basis of preliminary experiments, demonstrating that this dose did not have any effect on the RBF or CBF, whereas an increase of the dose influenced these parameters. All groups underwent the same experimental protocol. At 30 min after the start of the i.r. infusion, RPP was reduced in steps of 5 mm Hg to a pressure of 50 mm Hg. Each pressure step was held for 5 min. RPP, RBF, and CBF were continuously recorded. After each 20- to 30-mm Hg reduction in RPP, a blood sample was obtained for determination of PRA. At the end of the studies, 200 ng of ET-1 dissolved in 100 μl saline was injected i.r. as a bolus over a 1-min period, and the effects on MAP, RBF, renal vascular resistance (RVR), CBF, and cortical vascular resistance (CVR) were recorded.

Data calculation and statistical evaluation

Data are expressed as means ± SEM. During pressure reduction in steps from baseline pressure, the lower pressure limit of RBF autoregulation (i.e., the breakpoint of autoregulation) was defined by following the method of Iversen et al. ^(7,8) as the perfusion pressure that permanently reduced RBF with ≥0.2 ml/min from the baseline value. Reduced RBF autoregulatory capacity was defined to be present when the pressure range of maintained RBF during autoregulation was reduced compared with measurements in control animals (i.e., when the lower limit of the autoregulatory plateau was reset to higher pressure values). Similarly the breakpoint of CBF autoregulation was defined as the perfusion pressure that permanently reduced CBF with ≥15 U/min from the baseline value. Differences between groups were assessed by one-way analysis of variance (ANOVA). Two-tailed t test with Bonferroni's correction for adjustment for multiple testing was used after ANOVA for further pairwise comparison of the groups. The paired t test was used to examine values before and after intrarenal administration of BQ123. Statistical significance was defined as p < 0.05. Statistical analysis was performed by using the statistical analysis package SAS ⁽²⁸⁾. To allow a better comparison of the RBF and CBF autoregulatory studies, data are presented as percentage of control (baseline) values, as the laser Doppler method only allows measurement of arbitrary units of regional (i.e., cortical) tissue flow and not absolute blood flow.

RESULTS

The basal values of RBF and CBF did not differ between SHRs and WKY rats. Basal MAP (p < 0.01), basal RVR (p < 0.05), and basal CVR (p < 0.05) were higher in SHRs than in WKY rats, whereas the basal PRA was higher (p < 0.05) in WKY rats (Table 1).

TABLE 1: Basal values before and after administration of vehicle or BQ 123

BQ123 completely blocked renal and systemic ET-A receptors, as the intrarenal injection of ET-1 had no effects on MAP, RBF, RVR, CBF, and CVR in either WKY rats or SHRs (Table 2).

TABLE 2: Maximal effects of ET-1 after ET-A-receptor blockade with BQ123

BQ123 or vehicle did not influence the RBF, CBF, or PRA (Table 1). In contrast to vehicle, BQ123 induced a MAP decrease in both SHRs (−7 ± 1 mm Hg; p < 0.05) and WKY rats (−4 ± 1 mm Hg; p < 0.05; Table 1). The MAP decrease after BQ123 was greater in SHRs than in WKY rats (p < 0.05).

Control SHRs (vehicle) showed autoregulation of RBF down to a breakpoint of 132 ± 4 mm Hg (Fig. 1), whereas WKY control animals (vehicle) autoregulated down to a breakpoint of 84 ± 3 mm Hg (Fig. 1). The breakpoint of RBF autoregulation was higher (p < 0.01) in control SHRs than in WKY control animals.

FIG. 1: Autoregulation of renal blood flow (RBF: percentage of basal value) in vehicle- (circles) and BQ123-treated (triangles) spontaneously hypertensive rats (SHRs) (A) and in vehicle- (circles) and BQ123-treated (triangles) Wistar-Kyoto (WKY) rats (B).

Compared with the control SHR group, the BQ123-treated SHRs evidenced a leftward shift (p < 0.01) of the RBF autoregulation breakpoint (103 ± 2 mm Hg; Fig. 1). The RBF autoregulatory behavior was not affected by the i.r. infusion of BQ123 in WKY rats [i.e., the lower-pressure limit of RBF autoregulation was 86 ± 3 mm Hg (Fig. 1)].

WKY control animals showed an autoregulation of CBF down to a breakpoint of 84 ± 2 mm Hg (Fig. 2), whereas control SHRs had a CBF autoregulation breakpoint of 120 ± 4 mm Hg (p < 0.01) (Fig. 2). The lower-pressure limits of CBF and RBF autoregulation did not differ in SHRs and WKY control animals.

FIG. 2: Autoregulation of renal cortical blood flow (CBF: percentage of basal value) in vehicle- (circles) and BQ123-treated (triangles) spontaneously hypertensive rats (SHRs) (A) and in vehicle- (circles) and BQ123-treated (triangles) Wistar-Kyoto (WKY) rats (B).

After BQ123, the CBF breakpoint did not significantly change in WKY rats (82 ± 3 mm Hg; Fig. 2) but shifted to the left in SHRs (98 ± 3 mm Hg; p < 0.05; Fig. 2).

Control SHRs exhibited a very weak, nonsignificant increase of PRA in response to a reduction in RPP, which was not altered after treatment with BQ123. In WKY control animals, an increase in PRA was observed after reduction of RPP at 60 ± 3 mm Hg (p < 0.05), whereas BQ123-treated WKY rats showed an immediate increase in PRA at 80 ± 3 mm Hg (p < 0.05; Fig. 3).

FIG. 3: Pressure-dependent plasma renin activity [PRA: ng angiotensin I (Ang I)/ml/min] in vehicle- (circles) and BQ123-treated (triangles) spontaneously hypertensive rats (SHRs) (A) and in vehicle- (circles) and BQ123-treated (triangles) Wistar-Kyoto (WKY) rats (B). Solid circles compared with triangles show basal values for PRA before injection of vehicle or BQ123. *p < 0.05 compared with baseline value.

DISCUSSION

This study demonstrated that the short-term administration of a selective ET-A-receptor antagonist leads to a resetting of the lower-pressure limits of RBF and CBF autoregulation in SHRs to lower values. No change in pressure-dependent renin release was observed in SHRs after administration of BQ123.

It seems improbable that this effect of the selective ET-A-receptor antagonist was caused by secondary hemodynamic changes, as BQ123 did not produce any significant changes in RBF, CBF, or PRA at a dose that completely blocked both pressor and renal vasoconstrictor response to an injection of ET-1. Furthermore the systemic effects of the ET-A-receptor antagonist (i.e., decrease in MAP) in SHRs were negligible and also were observed in WKY rats.

The observation that the administration of an ET-A-receptor antagonist leads to a leftward shift of the threshold value of RBF autoregulation indicates that the renal ET system exerts a tonic vasoconstrictive influence on one or more renal vascular segments in SHRs. We recently demonstrated a marked overexpression of vascular ET-A-receptors in kidneys of SHRs compared with WKY rats ⁽²¹⁾. This overexpression of ET-A receptors appears to cause an increase of the RVR in SHRs ⁽²¹⁾. There are several ways in which the ET system could influence the threshold value of RBF autoregulation in SHRs. A perfusion-pressure-dependent influence on renal ET secretion itself would theoretically be conceivable, especially because vascular shear forces are known to influence ET release ^(15,29). Evidence against this assumption, however, is the fact that the induction of ET secretion takes several hours, and no storage granules for ET have as yet been detected ⁽¹⁵⁾. The influence of the ET system on the threshold pressure of RBF autoregulation in SHRs is therefore likely to be mediated via the highly regulated ET-A receptors.

The renal vascular ET system could exert a vasoconstrictor influence on all preglomerular resistance vessels by uniformly highly regulated vascular ET-A receptors and thus lead to an uniform reduction of the pressure-dependent dilatation capacity of these vessels, which would result in a limited autoregulatory reserve. This assumption is in accordance with immunohistochemical studies showing a uniform distribution of the highly regulated vascular ET receptors over all renal vessel segments in SHRs ⁽²¹⁾. However, the methods used may not be sensitive enough to recognize segmental differences in the expression of ET-A receptors. ET could, on the other hand, cause a vasoconstriction of mainly the vascular segments proximal to the preglomerular resistance vessels (i.e., afferent arterioles). The consequence would be a restricted autoregulatory reserve, because the segments farther downstream would have to dilate compensatorily to maintain a constant glomerular pressure (descending myogenic autoregulation). In accordance with this hypothesis is the finding that the ET-like immunoreactivity is localized most consistently in the endothelium of small- and medium-sized vessels in all regions of rat kidneys ⁽³⁰⁾. Furthermore, it has been shown that ET constricts mainly the arcuate and interlobular arteries in SHRs and WKY rats ⁽³¹⁾. An in vivo study on the split hydronephrotic rat kidney demonstrated a marked constriction of larger preglomerular vessels, including arcuate and interlobular arteries, but not of afferent arteries during an intravenous infusion of ET-1 ⁽³²⁾. Accordingly, an ET-A-receptor blockade, by disrupting the physiologic balance between vasoconstriction and vasodilation, would result in an improved autoregulatory capability of the renal vasculature as a result of an enhanced myogenic vasodilator response of larger preglomerular vessels. This modulation of the RBF autoregulatory behavior in SHRs would be largely mediated by an influence on the cortical vessels, as our data show that the improvement of pressure-dependent regulation of CBF, which accounts for the largest part of total RBF, perfectly parallels the regulation of the total RBF.

In this context, it is of note that endogenous ET has been shown not to influence tubuloglomerular feedback (TGF) responsiveness by the ET-A receptor, because the TGF effect site of the afferent arteriole is not sensitive to ET or escapes the vasoconstrictor effect of ET through counterregulation by locally produced vasodilating factors ^(33,34). Endogenous NO was shown to participate actively in the control of glomerular arteriolar tone through TGF ⁽³⁴⁾. Assuming that ET-A blockade did not influence TGF responsiveness, one could argue that improvement of autoregulatory capability was the result of an enhanced action of local vasodilator factor(s) on renal vessels after a shift of vasoconstrictor/vasodilator balance after renal ET-A blockade. Modulation of the ET-B-receptor activation in this situation of selective ET-A blockade would account for an increased production of NO or a lack of ET-A-mediated counterregulation of vasodilatory effects of NO ⁽³⁵⁾.

The results of our study, however, do not contribute to a better understanding of the possible role of the ET system in the maintenance of hypertension in SHRs. Theoretically the renal ET-A system could be responsible for the observed resetting of autoregulation of renal hemodynamics on a long-term basis. Indirect evidence is provided by the overexpression of vascular ET-A receptors in kidneys of SHRs. Future studies will have to investigate the influence of long-term administration of a selective ET-A-receptor antagonist on behavior of renal autoregulation and arterial blood pressure in SHRs.

The renal ET-A system in WKY rats appears to differ from that in SHRs with respect to its functional importance for regulation of RBF. Intrarenal infusion of BQ123 had no effect on the lower-pressure limits of RBF and CBF autoregulation, making it probable that the ET-A blockade did not alter the presumed vasoconstrictor/vasodilator balance of renal vasculature in WKY rats. The lower expression of ET-A receptors in the kidneys of WKY rats ⁽²¹⁾ along with the lower vascular resistance ⁽²¹⁾ also observed in our study strongly argue against a major role of the ET-A system in the regulation of renal hemodynamics in WKY rats.

The pressure-dependent renin release in WKY rats increases immediately under BQ123 with reduction of the RPP, whereas WKY control animals show a threshold value of renin release. This threshold value corresponds more or less to that of RBF autoregulation. These findings argue against a significant role of the renin-angiotensin system in the control of RBF and CBF autoregulation, as changes in pressure-dependent renin release were not accompanied by changes in autoregulatory behavior. As could be shown in juxtaglomerular cells ⁽¹⁸⁾ and cortical slices ⁽¹⁷⁾, ET-1 can inhibit the renal renin release. The ET-A blockade with BQ123 would abolish the inhibitory effect of ET on renin release. We did not, however, observe any effect of BQ123 on basal PRA in SHRs and in WKY rats. Furthermore the ET-A antagonist did not influence the pressure-dependent renin release in SHRs, making a major role of the renin-angiotensin system in the regulation of RBF in SHRs, either directly or through the ET system, very improbable. We are aware that changes in PRA do not necessarily reflect changes in renal Ang II formation and receptor activation. We cannot exclude that the dose applied was too small to exert an influence on renin release in SHRs. However, because we were able to prevent both pressor and renal vasoconstrictor response to an injection of ET-1, we believe that our data provide strong evidence against an important role of the renin-angiotensin system in modulating the influence of the renal ET-A system on renal autoregulation in SHRs.

In summary, our data demonstrate that short-term blockade of the renal ET-A system in SHRs improves RBF and CBF autoregulation without affecting pressure-dependent renin release. In contrast, the ET-A system seems not to play an important role in the autoregulation of RBF and CBF in WKY rats. Future studies will have to investigate the influence of a long-term blockade of the renal ET-A system on the resetting of RBF autoregulation in SHRs.