Effects of Ca2+ channel activity on renal hemodynamics during acute attenuation of NO synthesis in the rat

R. A. Kramp, P. Fourmanoir, L. Ladrière, E. Joly, C. Gerbaux, A. el Hajjam, and N. Caron

Service de Physiologie et Pharmacologie, Faculté de Médecine et de Pharmacie, Université de Mons-Hainaut, 7000 Mons, Belgium


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In cultured vascular muscle cells, nitric oxide (NO) has been shown to inhibit voltage-dependent Ca2+ channels, which are involved in renal blood flow (RBF) autoregulation. Therefore, our purpose was to specify in vivo the effects of this interaction on RBF autoregulation. To do so, hemodynamics were investigated in anesthetized rats during Ca2+ channel blockade before or after acute NO synthesis inhibition. Rats were treated intravenously with vehicle (n = 10), 0.3 mg/kg body wt NG-nitro-L-arginine-methyl ester (L-NAME; n = 7), 4.5 µg · kg body wt-1 · min-1 nifedipine (n = 8) alone, or with nifedipine infused before (n = 8), after (n = 8), or coadministered with L-NAME (n = 10). Baseline renal vascular resistance (RVR) averaged 14.0 ± 1.2 resistance units and did not change after vehicle. RVR increased or decreased significantly by 27 and 29% after L-NAME or nifedipine, respectively. Nifedipine reversed, but did not prevent, RVR increase after or coadministered with L-NAME. RBF autoregulation was maintained after L-NAME, but the autoregulatory pressure limit (PA) was significantly lowered by 15 mmHg. Nifedipine pretreatment or coadministration with L-NAME limited PA resetting or suppressed autoregulation at higher doses. Results were similar with verapamil. Intrarenal blockade of Ca2+-activated K+ channels also prevented autoregulatory resetting by L-NAME (n = 8). These findings suggest NO inhibits voltage-dependent Ca2+ channels and thereby modulates RBF autoregulatory efficiency.

autoregulation; renal vascular resistance; nifedipine; iberiotoxin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE ENDOTHELIUM-DERIVED RELAXING factor or nitric oxide (NO) induces a continuous relaxation of vascular smooth muscle cells. To do so, NO is released from endothelial cells and diffuses very rapidly through the interstitial space to activate the soluble guanylate cyclase of the adjacent smooth muscle cell, maintaining high levels of cGMP (19). cGMP activates, in turn, a protein kinase G that maintains intracellular Ca2+ low by reducing Ca2+ transport into the cytosol (18). In this regard, it has been shown in vitro that the NO-cGMP pathway markedly attenuates the activity of voltage-dependent Ca2+ channels and that Ca2+-activated K+ channels may be involved (1, 8, 24). Several studies in cultured smooth muscle cells that originated from different vascular beds have also provided evidence for a direct inhibitory effect of NO on the activity of voltage-dependent Ca2+ channels (7, 8).

In the kidney, voltage-dependent Ca2+ channels are selectively located in the preglomerular microvasculature, in particular in the afferent arteriole (10). The afferent arteriole is the main site for regulation of renal vascular resistance, which is rapidly adjusted when renal perfusion pressure (RPP) changes (26). Renal hemodynamics are therefore characterized by a powerful intrinsic capacity of the kidney to autoregulate renal blood flow (RBF) and glomerular filtration rate (GFR). Although NO does not seem to play a major role in the autoregulatory phenomenon of RBF, its in vitro inhibitory effect, either direct or indirect, on voltage-dependent Ca2+ channels of vascular smooth muscle cells nevertheless suggests some implication of NO in autoregulation of RBF. Therefore, the purpose of our study was to specify in vivo the impact of the interaction between NO and Ca2+ channel activity on renal hemodynamics, in particular on RBF autoregulation. To address this point, experiments on renal hemodynamics were undertaken in anesthetized euvolemic rats during the acute inhibition of NO synthesis induced with a low or a high dose of NG-nitro-L-arginine-methyl ester (L-NAME) injected intravenously. Moreover, to evaluate in vivo the involvement of NO in the activity of voltage-dependent Ca2+ channels in the kidney, the effects of Ca2+ channel blockade on renal hemodynamics were investigated before or after inhibition of NO synthase, as well as during the simultaneous inactivation of Ca2+ channels and inhibition of NO synthase. RBF autoregulation was also evaluated during intrarenal blockade of Ca2+-activated K+ channels, which have been localized in the preglomerular arterioles in the rat and may be implicated in NO-cGMP pathway and voltage-dependent Ca2+ channel interactions (1, 14), as well as during the intravenous infusion of angiotensin II because voltage-dependent Ca2+ channels are then activated (31).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal preparation. Male Wistar rats were maintained on a rat food diet (25 g daily of Muracon G. Ster.) containing 1.8 g/kg sodium and 9.6 g/kg potassium as stated by the manufacturer (Trouw, Gent, Belgium). Before experimentation, the rats, weighing ~300 g, were deprived of food overnight but had free access to tap water. They were anesthetized with Inactin (10 mg/100 g body wt ip; Byk-Gulden). The animals were placed on a heated table to maintain rectal temperature between 37 and 38°C. The left femoral artery was first catheterized to determine the initial hematocrit and to measure blood pressure. Blood pressure was measured by using a Statham P23 ID pressure transducer connected to a pressure monitor (Mennen Medical) and a recorder. The right femoral artery, for subsequent blood sampling, and vein were then rapidly catheterized. To avoid fluid shifts during further surgery, a 0.85% saline solution containing 2.5% albumin was infused immediately into the right femoral vein at a rate of 150 µl/min for 5 min and of 83 µl/min for the next 30 min. This infusion was maintained throughout experimentation at a rate of 8 µl/min. After tracheostomy, the right jugular vein was catheterized for subsequent infusions. The left kidney was exposed through a midline and subcostal abdominal incision as previously described (21). The segment of the aorta located between the two renal arteries as well as the left renal artery were then carefully dissected from surrounding tissues, avoiding as much as possible interference with nerves bundles (21). Finally, each ureter was cannulated for urine collection. In some rats, a tapered and curved thin catheter was introduced in the left iliac artery and pushed upward until its tip was positioned in the left renal artery. Thereafter, isotonic saline was continuously infused intrarenally at a rate of 5 µl/min. After completion of surgery and after a suitable prime, a 0.85% NaCl solution containing 3% inulin (Laevosan, Innsbruck, Austria), except if otherwise stated, was infused at a rate of 48 µl/min to measure GFR. The kidneys were removed, decapsulated, blotted dry, and weighed at the end of the experiment.

Renal hemodynamics. An adjustable constriction clamp was placed around the aorta above the left and just beneath the right renal artery to reduce RPP in a stepwise manner. A small-diameter, noncannulating, and factory-precalibrated electromagnetic flow transducer (0.60 or 0.62 mm ID), connected to a square-wave electromagnetic flowmeter (MDL 1401 compact; Skalar Medical, Delft, The Netherlands) and a recorder, was vertically fitted around the left renal artery to continuously measure RBF. Calibration and use of these flow sensors as well as the final tests to check the accuracy of RBF measurements at the end of the experiment were previously described in detail (21). No marked changes in RBF measurements before or after these final tests were found. They averaged <3%.

RBF autoregulatory efficiency was investigated by stepwise aortic constrictions inducing 5-mmHg decrements in RPP from spontaneous mean arterial pressure (MAP) down to 60 mmHg and by measurement of RBF during 30-s periods. No measurements during increased MAP were undertaken.

Experimental protocol. After 60- to 90-min equilibration, baseline measurements of hemodynamics and renal function were carried out during three control periods of 20-min duration each. Control autoregulatory maneuvers (A1) were undertaken during the second control period. After the control periods, vehicle, L-NAME (Sigma Chemical), nifedipine (Bayer), or verapamil (Isoptin, Knoll) was administered intravenously. Moreover, iberiotoxin (Sigma Chemical), a specific inhibitor of Ca2+-activated K+ channels, was infused in the renal artery of some rats. After a brief equilibration period, six experimental periods of 20-min duration each were then undertaken. Autoregulatory maneuvers were carried out during the second (A2) and fifth (A3) experimental periods. After the first three experimental periods, an intravenous infusion of nifedipine was started in some of the rats previously injected with L-NAME, whereas L-NAME was injected intravenously in some of the rats infused with nifedipine since 1 h. The two drugs were simultaneously coadministered in other rats after the last control period. In each rat, blood was periodically sampled from the femoral artery, and urine was separately collected from each kidney at the end of each period.

The following experimental groups were considered.

1) Eight rats were treated with vehicle (Vehicle; 0.85% NaCl in bidistilled water injected iv as 200 µl/kg body wt or infused iv, at a rate of 5 µl/min).

2) Seven rats were injected intravenously with L-NAME at a dose of 0.3 mg/kg body wt (L-NAME).

3) Seven rats were infused intravenously with nifedipine at a dose of 4.5 µg · kg body wt-1 · min-1 (Nifedipine).

4) Eight rats were first injected intravenously with L-NAME at a dose of 0.3 mg/kg body wt, and, after 1 h, were infused intravenously with nifedipine at a dose of 4.5 µg · kg body wt-1 · min-1 (L-NAME/Nifedipine).

5) Eight rats were infused intravenously with nifedipine at a dose of 4.5 µg · kg body wt-1 · min-1, and, after 1 h, were injected intravenously with L-NAME at a dose of 0.3 mg/kg body wt (Nifedipine/L-NAME).

6) Five rats were treated concomitantly at the start of the first experimental period with nifedipine infused intravenously at a dose of 4.5 µg · kg body wt-1 · min-1, and with L-NAME injected intravenously at a dose of 0.3 mg/kg body wt. Five other rats, subjected to a similar protocol, were infused intravenously with verapamil at a dose of 10 or 15 µg · kg body wt-1 · min-1 instead of nifedipine [Nifedipine (Verapamil)+ L-NAME coadministered]. The results were combined because there were no marked differences between hemodynamic effects of the two Ca2+ channel blockers.

7) In addition, seven rats were infused intravenously with nifedipine at a dose of 18 µg · kg body wt-1 · min-1 (n = 4) or with verapamil at a dose of 40 µg · kg body wt-1 · min-1 (n = 3), and were injected intravenously with L-NAME at a dose of 10 mg/kg body wt at the start of the first experimental period. Results were again combined. Five rats were injected intravenously only with L-NAME at a dose of 10 mg/kg body wt.

8) Eight other rats were infused intrarenally with iberiotoxin at a dose of 3.4 µg/min at the start of the first experimental period and, after 1 h, they were injected intravenously with L-NAME at a dose of 0.3 mg/kg body wt.

9) Finally, five rats, also prepared as described above and submitted to a similar experimental protocol, were infused intravenously with angiotensin II (Sigma Chemical) at a dose of 150 ng · kg body wt-1 · min-1 during the six experimental periods.

Only renal hemodynamics were studied in the animals of groups 7, 8, and 9.

Analytic methods. Urine volume from each kidney was estimated by gravimetry assuming a specific activity for water of 1.0. Inulin in plasma and urine samples was determined by using the anthrone method.

Calculations and statistics. Renal hemodynamic data were obtained from the left kidney, henceforth referred to as the experimental kidney. Mean values of MAP and RBF were obtained by averaging measurements carried out every 5 min on the recordings, except during autoregulatory maneuvers. Renal plasma flow (RPF) was estimated by using the following equation, RPF = RBF(1 - Hct), where Hct represents the hematocrit. Renal vascular resistance (RVR) was calculated as MAP/RBF, where MAP refers to the pressure in the femoral artery. RPP was arbitrarily equated to the femoral MAP. The relationship between RBF and MAP was assessed independently by two investigators in each experiment by applying progressively linear regressions by using the least squares method, as modified from the method described by Persson et al. (29). The magnitude of the correlation coefficient, the slope, and the intercept of the best-fit regression lines, as well as the amplitude of the change in RBF, were taken as criteria to attribute a data point with a precision of 5 mmHg to one of the following components of the RBF-MAP relationship: 1) the autoregulatory plateau, which corresponds to the pressure range in which autoregulatory efficiency was maximal; 2) a subautoregulatory zone, which corresponds to the pressure range in which some autoregulatory efficiency persisted; 3) the absence of autoregulation, which corresponds to the pressure range in which RBF changes were fully pressure dependent. The perfusion pressure, corresponding to the lower limit of the autoregulatory plateau, was defined as the pressure limit of efficient RBF autoregulation (PA). The perfusion pressure, corresponding to disappearance of RBF autoregulation, was defined as the pressure limit of no RBF autoregulation (Po). GFR and filtration fraction (FF) were calculated as usually described. GFR and FF were determined during two control and four experimental periods, which respectively corresponded to the pre- and postautoregulatory periods as indicated in the experimental protocol. Data obtained during autoregulatory periods (A1, A2, A3) were not considered because autoregulatory maneuvers transiently reduced GFR from the experimental kidney.

One-way ANOVA was applied for multiple intergroup comparisons, and one-way ANOVA for repeated measurements was applied for multiple intragroup comparisons, followed by a post hoc t-test. Two-way ANOVA for repeated measurements on one factor was used to compare the effects of different treatments on RVR. Significant differences were identified with the Newman-Keuls test for multiple pairwise comparisons (37). The paired t-test was used for a single comparison within a group. All results are presented as means ± SE. P < 0.05 was considered to be statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General observations. Body weight and left and right kidney weights averaged 292 ± 1, 1.156 ± 0.015, and 1.141 ± 0.012 g, respectively, in 73 rats. Plasma sodium, potassium, and osmolality were in the normal range for rats (data not shown). They did not differ from baseline at the end of the experiment. Table 1 presents the mean baseline values for Hct, MAP, RBF, RPF, RVR, GFR, and FF in rats treated with vehicle (n = 8), L-NAME (n = 7), Nifedipine (n = 7), L-NAME/Nifedipine (n = 8), Nifedipine/L-NAME (n = 8), or Nifedipine (Verapamil)+L-NAME coadministered (n = 10). The baseline values did not differ statistically among these experimental groups [not significant (NS), ANOVA]. At the end of the experiment, Hct averaged 44 ± 0.7, 44 ± 0.7, 45 ± 0.7, 46 ± 0.6, 45 ± 0.8, and 45 ± 0.9% in the respective experimental groups (NS from baseline, paired t-test).

                              
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Table 1.   Baseline values for hematocrit, mean arterial pressure, renal blood and plasma flow, renal vascular resistance, glomerular filtration rate and filtration fraction in rats treated with vehicle, L-NAME, or nifedipine alone, with L-NAME and nifedipine or nifedipine and L-NAME combined during the second experimental hour, or with nifedipine (or verapamil) and L-NAME coadministered

Temporal evolution of MAP and RBF. Figure 1 illustrates the temporal variations of MAP (A) and RBF (B) starting before each treatment until the end of the experiment. As shown in Fig. 1A, MAP did not change after injection of vehicle, nor with time. MAP increased slightly, but transiently, by 4 mmHg in the rats injected with L-NAME alone and returned to baseline after 1-h treatment without further changes. In contrast, MAP decreased progressively and significantly in the rats infused only with nifedipine, and averaged 94 ± 4 mmHg at the end of the experiment. In the L-NAME/Nifedipine group, MAP increased again slightly and rapidly during the first hour after injection of L-NAME but decreased progressively and significantly to 98 ± 6 mmHg after superimposition of the infusion of nifedipine. In the Nifedipine/L-NAME group, MAP decreased significantly to 105 ± 4 mmHg during the first hour of infusion of nifedipine but increased rapidly and significantly after the superimposed injection of L-NAME, averaging 111 ± 4 mmHg at the end of the experiment. Finally, MAP did not change during coadministration of nifedipine (or verapamil) and L-NAME. As shown in Fig.1B, RBF did not vary after injection of vehicle, nor with time. However, RBF decreased rapidly and significantly after the injection of L-NAME alone, and, thereafter, remained relatively stable, averaging 6.65 ± 0.43 ml/min at the end of the experiment. Conversely, RBF increased rapidly, but not significantly, in the rats infused only with nifedipine, averaging 8.47 ± 0.76 ml/min at the end of the experiment. In the L-NAME/Nifedipine group, RBF decreased again rapidly and significantly after the injection of L-NAME but increased significantly to 7.00 ± 0.30 ml/min after superimposition of the infusion of nifedipine. In the Nifedipine/L-NAME group, RBF increased slightly during the infusion of nifedipine and then decreased rapidly and significantly to 6.84 ± 0.68 ml/min after the superimposed injection of L-NAME. After coadministration of nifedipine (or verapamil) and L-NAME, RBF decreased more slowly and stabilized at 6.28 ± 0.36 ml/min within 1 h.


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Fig. 1.   Temporal variations of mean arterial pressure (MAP; in mmHg; A) and renal blood flow (RBF; in ml/min; B) in rats treated intravenously with vehicle (; n = 8), 0.3 mg/kg body wt NG-nitro-L-arginine-methyl ester (L-NAME; open circle ; n = 7), 4.5 µg · kg body wt-1 · min-1 nifedipine (black-triangle; n = 7), 0.3 mg/kg body wt L-NAME and, during second experimental hour, with 4.5 µg · kg body wt-1 · min-1 nifedipine (triangle ; n = 8), 4.5 µg · kg body wt-1 · min-1 nifedipine and, at the start of the second experimental h, with 0.3 mg/kg body wt L-NAME (; n = 8), or 4.5 µg · kg body wt-1 · min-1 nifedipine (or 10-15 µg · kg body wt-1 · min-1 verapamil) coadministered with 0.3 mg/kg body wt L-NAME at the start of the experimental periods (; n = 10). Values are means ± SE and are illustrated for last control and 6 experimental periods. They were arbitrarily set in the middle of each 20-min period. Some SE are too small to be shown. Differences in temporal variations of MAP and RBF were evaluated by ANOVA for repeated measurements followed by Newman-Keuls test between control and experimental values (statistical significance: * P < 0.05) and between experimental value at the end of the first hour treatment and last 3 experimental values (statistical significance: + P < 0.05).

In each of the six experimental groups, RPF followed a similar temporal evolution as RBF, whereas GFR did not differ significantly from baseline shown in Table 1, except that GFR increased to 1.235 ± 0.194 ml/min in the rats infused with nifedipine alone (NS). On the other hand, FF was significantly enhanced to 0.29 ± 0.02 (P < 0.025) and to 0.41 ± 0.03 (P = 0.01) in the rats treated with L-NAME alone or with nifedipine (or verapamil) and L-NAME coadministered, respectively.

Changes in RVR. Table 2 presents RVR before and 1 and 2 h after treatment was started. Control values of RVR did not differ statistically among the six experimental groups. There were no changes in RVR after treatment with vehicle. In contrast, RVR was significantly increased 1 and 2 h after injection of L-NAME and decreased progressively and significantly during the infusion of nifedipine. RVR was significantly enhanced during the first experimental hour in the L-NAME/Nifedipine group but was decreased to baseline levels after the superimposed infusion of nifedipine. RVR was reduced below baseline during the first experimental hour in the Nifedipine/L-NAME group and was increased significantly above baseline levels after the superimposed injection of L-NAME. Finally, RVR was significantly enhanced 1 and 2 h after starting coadministration of nifedipine (or verapamil) and L-NAME. Figure 2 summarizes the effects of the different treatments on RVR, expressed in percentage of baseline values. Changes in RVR differed significantly between each group, except between Vehicle and L-NAME/Nifedipine, and between L-NAME alone and Nifedipine/L-NAME or Nifedipine (Verapamil)+L-NAME coadministered. These two last groups also did not differ between each other. Similar statistical results were obtained when the changes in RVR were expressed in absolute values.

                              
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Table 2.   RVR before and after 1- and 2-h treatment



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Fig. 2.   Percent changes (Delta ) from baseline in renal vascular resistance (RVR) occurring 2 h after intravenous treatment with vehicle (filled bar), 0.3 mg/kg body wt L-NAME (open bar), 4.5 µg · kg body wt-1 · min-1 nifedipine (hatched bar), 0.3 mg/kg body wt L-NAME and 4.5 µg · kg body wt-1 · min-1 nifedipine (vertically striped bar), 4.5 µg · kg body wt-1 · min-1 nifedipine and 0.3 mg/kg body wt L-NAME (crosshatched bar), or 4.5 µg · kg body wt-1 · min-1 nifedipine (or 10-15 µg · kg body wt-1 · min-1 verapamil) coadministered with 0.3 mg/kg body wt L-NAME (horizontally striped bar). Values are means ± SE. RVR changes averaged 2 ± 2, 27 ± 4, -19 ± 4, 2 ± 6, 24 ± 9, and 22 ± 5%, respectively, and differed significantly among groups (P < 0.001, ANOVA). All RVR changes differed significantly between each other, except between Vehicle and L-NAME/Nifepidine, between L-NAME alone and Nifedipine/L-NAME as well as Nifedipine (Verapamil)+L-NAME coadministered, and between Nifedipine/L-NAME and Nifedipine (Verapamil)+L-NAME coadministered (* P < 0.05, Newman-Keuls test).

RBF autoregulation. Figure 3 illustrates the RBF-MAP relationship 30 min before (A1) and 30 (A2) and 90 min (A3) after treatment with vehicle (A), L-NAME (B), or nifedipine (C) alone was started. As shown in Fig. 3A, the three autoregulatory curves were superimposed and were essentially similar in the vehicle-treated rats. The lower pressure limit of the RBF autoregulatory plateau, as well as the pressure limit of no RBF autoregulation, did not change after vehicle treatment, or with time (Table 3). After injection of L-NAME (Fig. 3B), the RBF autoregulatory plateau was maintained but was shifted downward from control (A2 and A3). Moreover, the autoregulatory plateau extended into the subautoregulatory zone so that the autoregulatory pressure limit was significantly lowered by >10 mmHg (Table 3). As illustrated in Fig. 3C, changes in RBF autoregulation occurred progressively in the rats continuously infused with nifedipine. After 30 min, the RBF-MAP relationship was slightly shifted upward from control (A2). RBF autoregulation was maintained in five of the seven animals. Thereafter, the autoregulatory plateau was abolished and the RBF-MAP relationship was shifted upward and leftward from control (A3). Figure 4, A and B, illustrates, respectively, the RBF-MAP relationship in the rats pretreated with L-NAME or with nifedipine during the first experimental hour (A2), and, thereafter, submitted to a combined treatment (A3). The RBF-MAP relationship and the autoregulatory pressure limits, 30 min before (A1) and after (A2) pretreatment, were as described above (Table 3). As shown in Fig. 4A, the autoregulatory plateau was maintained during the superimposed infusion of nifedipine but was shifted toward control (A3). The autoregulatory pressure limit remained significantly lowered (Table 3). As shown in Fig. 4B, RBF remained autoregulated after the superimposed injection of L-NAME, but the autoregulatory plateau was shifted downward (A3). The autoregulatory pressure limit was now less lowered (Table 3). In rats submitted to the simultaneous coadministration of nifedipine (verapamil) and L-NAME, the RBF autoregulatory plateau was again maintained but was only slightly shifted downward from control (Fig. 4C). It did not extend into the subautoregulatory zone (A2 and A3), so that pressure limits of the autoregulatory and subautoregulatory zones did not vary significantly from baseline (Table 3). Note that coadministration of 9 µg · kg body wt-1 · min-1 nifedipine with the low dose of L-NAME (n = 4) markedly attenuated RBF autoregulation (data not shown). The autoregulatory pattern (A2 and A3) was as illustrated below in Fig. 5B.


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Fig. 3.   RBF-MAP relationships during stepwise decrements by 5 mmHg in MAP in rats treated intravenously with vehicle (n = 8; A), 0.3 mg/kg body wt L-NAME (n = 7; B), or 4.5 µg · kg body wt-1 · min-1 nifedipine (n = 7; C). Autoregulatory curves were determined 30 min before (A1; ), and 30 (A2; open circle ) and 90 min (A3; down-triangle) after treatment was started. Values are means ± SE.


                              
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Table 3.   Pressure limits of efficient and no RBF autoregulation in rats 30 min before and 30 and 90 min after vehicle, L-NAME, or nifedipine treatment alone or combined during the 2nd experimental hour, or after a concomitant administration of nifedipine (or verapamil) and L-NAME, was started



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Fig. 4.   RBF-MAP relationships during stepwise decrements by 5 mmHg in MAP in rats treated intravenously at 1-h interval with 0.3 mg/kg body wt L-NAME and 4.5 µg · kg body wt-1 · min-1 nifedipine (n = 8; A) or with 4.5 µg · kg body wt -1 · min-1 nifedipine and 0.3 mg/kg body wt L-NAME (n = 8; B), or treated concomitantly with 4.5 µg · kg body wt-1 · min-1 nifedipine (or 10-15 µg · kg body wt-1 · min-1 verapamil) and 0.3 mg/kg body wt L-NAME (n = 10; C). Autoregulatory curves were determined 30 min before (A1; ), and 30 (A2; open circle ) and 90 min (A3; down-triangle) after treatment was started. Values are means ± SE.



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Fig. 5.   RBF-MAP relationships during stepwise decrements by 5 mmHg in MAP in rats injected intravenously with 10 mg/kg body wt L-NAME (n = 5; A) or treated concomitantly with 18 µg · kg body wt-1 · min-1 nifedipine (or 40 µg · kg body wt -1 · min-1 verapamil) and 10 mg/kg body wt L-NAME (n = 7; B). Autoregulatory curves were determined 30 min before (A1; ), and 30 (A2; open circle ) and 90 min (A3; down-triangle) after treatment was started. Values are means ± SE. MAP and RBF averaged, respectively, 113 ± 4 and 133 ± 3 mmHg (P = 0.017), and 7.59 ± 0.21 and 3.70 ± 0.17 ml/min (P < 0.001) before and after L-NAME alone, and 109 ± 3 and 103 ± 3 mmHg (not significant), and 7.00 ± 0.35 and 4.06 ± 0.48 ml/min (P < 0.001) before and after simultaneous coadministration.

For comparative purposes, autoregulatory experiments were also undertaken in rats injected intravenously with a high dose of L-NAME alone (n = 5), or coadministered with very high doses of nifedipine or verapamil (n = 7). As shown in Fig. 5A, the RBF autoregulatory plateau was maintained after injection of L-NAME alone but was now markedly shifted downward from control and extended into the subautoregulatory zone (A2 and A3). There were no changes with time. Pressure limits of the autoregulatory plateau (PA) and subautoregulatory zone (Po) averaged, respectively, 100 ± 4 (A1), 84 ± 4 (A2), and 81 ± 5 mmHg (A3) (P = 0.001, ANOVA and Bonferroni test), and 85 ± 4 (A1), 64 ± 2 (A2), and 64 ± 3 mmHg (A3) (P = 0.002). As illustrated in Fig. 5B, the RBF autoregulatory plateau was disrupted in the rats treated with high doses of either Ca2+channel blocker and L-NAME. RBF tended to become pressure dependent at each pressure step, and autoregulatory pressure limits could not be determined.

RBF autoregulation was also investigated in eight rats during intrarenal infusion of iberiotoxin followed by an intravenous injection of a low dose of L-NAME. As shown in Table 4, autoregulatory pressure limits, PA and Po, did not change from control under these conditions. The autoregulatory profile was indeed similar to control during the intrarenal infusion of iberiotoxin and was only shifted downward from control after L-NAME. Note that the increase in MAP and RVR was more marked when L-NAME was injected after pretreatment with iberiotoxin than after L-NAME alone (see Fig. 1A and Table 2).

                              
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Table 4.   Preautoregulatory values for Hct, MAP, RBF, RVR, and PA and Po in 8 rats before and during intrarenal infusion of iberiotoxin, followed by an injection of a low dose of L-NAME

Figure 6 illustrates the RBF-MAP relationship in five other rats infused intravenously with 150 ng · kg body wt-1 · min-1 of angiotensin II to increase MAP and to lower RBF. Under these conditions, the RBF-MAP relationship was markedly shifted downward from control at each pressure decrement, 30 (A2) and 90 min (A3) after the infusion of angiotensin II was started. The RBF autoregulatory plateau was maintained, but the subautoregulatory zone was markedly extended to the right. The lower autoregulatory pressure limit was now significantly reset from 99 ± 2 (A1) to 115 ± 5 (A2; P < 0.01), and to 111 ± 6 mmHg (A3; P < 0.025, ANOVA and Bonferroni t-test). In contrast, the lower pressure limit of the subautoregulatory zone did not vary, averaging 82 ± 1 (A1), 80 ± 5 (A2), and 83 ± 4 mmHg (A3), respectively (NS, ANOVA).


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Fig. 6.   RBF-MAP relationships during stepwise decrements by 5 mmHg in MAP in 5 rats infused intravenously with 150 ng · kg body wt-1 · min-1 angiotensin II. Autoregulatory curves were determined 30 min before (A1; ), and 30 (A2; open circle ) and 90 min (A3; down-triangle) after infusion of angiotensin II was started. Values are means ± SE. Baseline and end MAP and RBF averaged, respectively, 116 ± 3 and 130 ± 2 mmHg (P < 0.005), and 8.68 ± 0.85 and 4.63 ± 0.15 ml/min (P < 0.005).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Treatment with the low dose of L-NAME selected after preliminary dose-response tests diminished renal blood circulation by 25% in our rats, as previously found by Walder et al. (36). The renal vasoconstriction was essentially due to a reduction in RBF because blood pressure was only mildly and transiently elevated. The decrease in RBF was stabilized within 15 min after injection of L-NAME and remained so for more than 2 h, underlining the effectiveness of the inhibition of the synthesis and release of intrarenal NO throughout the time period of the experiment. The concomitant increase in FF indicated that the vasomotor tone of the pre- as well as of the postglomerular vessels was affected. There were, however, no marked changes in GFR, nor in salt and water excretion (data not shown). These conditions were considered to reflect an acute attenuation of NO synthesis and to be of interest because they evoked some of the pathophysiological consequences encountered during the early stages of vascular and renal endothelial dysfunction in humans (12, 30). In comparison, the renal vasoconstriction was more pronounced after the acute and systemic administration of a high dose of L-NAME (4, 6). MAP was then elevated by 20-30 mmHg, and RBF was reduced by 50%. These conditions were assimilated to an acute blockade of NO synthesis (5). To test the influence of voltage-dependent Ca2+ channels on the hemodynamic consequences of the attenuated synthesis of NO, dihydropyridine-sensitive Ca2+ channels were blocked with nifedipine. The dose of nifedipine was sufficiently effective to enhance slightly and rapidly RBF until the end of the experiment and to reduce blood pressure near the autoregulatory pressure range, thus producing a true renal vasodilation. It was slightly higher than the dose range of nifedipine that normalized blood pressure and abolished RBF autoregulation in anesthetized, spontaneously hypertensive rats (22). Higher doses of nifedipine reduced blood pressure below autoregulatory limits and diminished RBF (unpublished observations). Verapamil, another type of Ca2+ channel blocker, was infused in some rats to test the reproducibility of the nifedipine findings. It should be pointed out that very high doses of Ca2+ channel blockers may interfere with other Ca2+ transport systems (15).

An important feature of our study concerned the action of nifedipine on the preestablished renal vasoconstriction induced by the attenuated synthesis of NO. The renal vasoconstriction was, indeed, completely reversed by the Ca2+ channel antagonist. Interestingly, the effect of nifedipine was not limited to the systemic circulation, as displayed by the decrease in blood pressure, but involved also the renal vasculature because the reduction in RBF was reversed, at least in part, by the Ca2+ channel antagonist. These findings are in agreement with recent observations in human volunteers infused intravenously with NG-monomethyl-L-arginine, in whom nifedipine completely restored systemic hemodynamics and reversed partially renal vasoconstriction (13). Pretreatment with nifedipine or simultaneous coadministration of nifedipine and the low dose of L-NAME, however, did not hinder the increase in RVR. In conscious rats, Baylis et al. (5) found that a high dose of verapamil rapidly administered after the injection of a high dose of L-NAME did not affect renal vasoconstriction. In contrast, these authors observed that blood pressure and RVR did not change after concomitant administration of the two drugs. In our anesthetized rats, blood pressure also did not increase after coadministration of high doses of nifedipine, or verapamil, and of L-NAME. However, RBF decreased by 42% so that RVR was significantly enhanced. Differences between the two studies could, at least in part, relate to different experimental conditions, such as the use of conscious or anesthetized rats.

An interesting finding of our study concerned the RBF-MAP relationship during inhibition of NO synthesis. The autoregulatory pattern was then maintained, but the pressure limit of the autoregulatory plateau was significantly lowered by ~15 mmHg, whether a low or a high dose of L-NAME was injected. This observation contrasts with previous studies undertaken in conscious and anesthetized dogs as well as in anesthetized rats indicating that the autoregulatory pressure limit after treatment with L-arginine competitors was either unchanged or slightly reset to a higher pressure (3, 6, 23, 34). Nevertheless, some evidence for improved autoregulation has been reported in vivo, for example, in juxtamedullary glomeruli of the hydronephrotic kidney in anesthetized female rats and in the kidney of normal and two-kidney, one-clip, hypertensive anesthetized rats (16, 35). Also, a pressure-dependent increase in active wall tension of interlobular arteries of in vitro perfused juxtamedullary nephrons was found to be amplified during inhibition of NO synthesis (20). Furthermore, NO donors have been shown to modulate autoregulatory responses of arcuate arteries and afferent arterioles in the in vitro blood-perfused juxtamedullary nephron preparation (9). However, it could be argued that resetting of the autoregulatory pressure limit to a lower pressure was artifactual in the presence of a reduced RBF. This possibility is unlikely because resetting to a higher pressure limit of the autoregulatory plateau, by 15 mmHg, could also be disclosed in our rats infused with angiotensin II. RBF was then decreased by 50%, and blood pressure was markedly increased. Thus our autoregulatory methodology was appropriate to detect variations of autoregulatory pressure limits in the presence of a reduced renal blood circulation.

Autoregulation of blood flow in the kidney depends on the interplay of the myogenic and tubuloglomerular feedback mechanisms (27). The myogenic mechanism allows rapid and appropriate adjustments of RVR, occurring in the preglomerular vasculature and predominantly in the afferent arteriole (2, 11, 26). These adjustments are regulated by the level of intracellular Ca2+, which depends, in part, on Ca2+ influx through voltage-dependent Ca2+ channels because their blockade markedly attenuates autoregulatory responses in vivo as well as in vitro, and as also shown by the present results (11, 17, 22, 28). In our study, the basic pattern of the RBF-MAP relationship was maintained whether voltage-dependent Ca2+ channel activity was inhibited before or after administration of the low dose of L-NAME. However, resetting of the pressure limit of the autoregulatory plateau was significantly reduced by pretreatment with nifedipine and was abolished by simultaneous coadministration of nifedipine (or verapamil) and the low dose of L-NAME. RBF autoregulation was even markedly attenuated by increasing the dose of nifedipine, and during coadministration of very high doses of nifedipine or verapamil and the high dose of L-NAME. These findings suggest thus that extension of the autoregulatory plateau occurring during inhibition of NO synthesis was dependent, at least in part, on influx of Ca2+ through dihydropyridine-sensitive Ca2+ channels. Although it cannot be excluded that our observations might be coincidental, it should be pointed out that intrarenal blockade of Ca2+-activated K+ channels also prevented the autoregulatory resetting induced by inhibition of NO synthesis. These observations do support interactions between the NO-cGMP pathway and voltage-dependent Ca2+ channels involving Ca2+-activated K+ channels (1). The role of angiotensin II must also be taken into consideration because angiotensin II interferes with NO and because renal vascular reactivity to angiotensin II depends, in part, on voltage-dependent Ca2+ channels (27, 31). Moreover, angiotensin II is implicated in the setting of the autoregulatory pressure limit as shown in our study. It is thus possible that extension of the autoregulatory plateau was facilitated during inhibition of NO synthesis, because mechanisms activating the synthesis of renin in afferent arteriolar cells are then ineffective (32).

The tubuloglomerular feedback mechanism that is "essential for maximum autoregulatory efficiency" could also be involved in the extension of the autoregulatory plateau detected under our experimental conditions. Indeed, sensitivity and reactivity of the tubuloglomerular mechanism are increased during inhibition of intrarenal NO synthesis (27, 33). However, it is not known at present to what extent the autoregulatory resetting occurring in our rats was influenced by enhancement of tubuloglomerular feedback activity. Interestingly, Ca2+ channel blockade decreases the sensitivity of this mechanism (25). Therefore, hindrance by Ca2+ channel blockade of the autoregulatory pressure limit resetting induced by L-NAME may implicate changes in myogenic as well as in tubuloglomerular feedback mechanisms. Their respective contribution remains to be further investigated.

In summary, attenuation of endothelial synthesis of NO with a low dose of L-NAME induced a significant renal vasoconstriction and an enhanced efficiency of RBF autoregulation. The renal vasoconstriction was reversed by inactivation of voltage-dependent Ca2+ channels with nifedipine, whereas autoregulatory resetting was hindered by prior or concomitant administration of nifedipine (or verapamil) and L-NAME. RBF autoregulation was also more efficient after blockade of NO synthesis with a high dose of L-NAME but was attenuated by coadministration of very high doses of nifedipine or verapamil. Moreover, intrarenal blockade of Ca2+-activated K+ channels prevented the autoregulatory resetting induced by L-NAME. In conclusion, these hemodynamic findings suggest that NO and/or the NO-cGMP pathway exerts inhibitory effects on the activity of voltage-dependent Ca2+ channels, which may be important to modulate RVR in the rat. Our pharmacological manipulations also suggest that the release of intrarenal NO may reduce the efficiency of RBF autoregulation at renal perfusion pressures below 100 mmHg.


    ACKNOWLEDGEMENTS

We appreciate the dedicated technical assistance of B. Blairon, F. Coulon, and V. Jenart and the secretarial assistance of M. Fontaine. We thank Bayer AG Belgium for the generous gift of nifedipine. This study is dedicated to the late Dr. C. W. Gottschalk.


    FOOTNOTES

This study was presented in part at the 2nd European Kidney Research Forum in Baveno, Italy, on May 24-26, 1996, and at the 29th Annual Meeting of the American Society of Nephrology in New Orleans, LA, on November 3-6, 1996. It appeared in abstract form in Kidney Int 50: 1785, 1996, and in J Am Soc Nephrol 7: 1566, 1996. E. Joly is a Collaborateur scientifique of the Fonds National de la Recherche Scientifique.

Present address of L. Ladrière: Laboratoire de Médecine Expérimentale, Faculté de Médecine de l'Université Libre de Bruxelles, route de Lennik, 808, 1070 Bruxelles, Belgium.

Present address of P. Fourmanoir: Baxter R&D Europe, 7 rue du Progrès, 1400 Nivelles, Belgium.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. A. Kramp, Service de Physiologie et Pharmacologie, Faculté de Médecine et de Pharmacie, Université de Mons-Hainaut, 7000 Mons, Belgium (E-mail: Ronald.Kramp{at}umh.ac.be).

Received 19 February 1999; accepted in final form 24 November 1999.


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