Functional role of sodium-calcium exchange in the regulation of renal vascular resistance

Frank Schweda1, Helga Seebauer1, Bernhard K. Krämer2, and Armin Kurtz1

1 Institut für Physiologie I and 2 Klinik und Poliklinik für Innere Medizin II, Universität Regensburg, D-93040 Regensburg, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our study aimed to assess a possible functional role of the Na+/Ca2+ exchanger in the regulation of renal vascular resistance (RVR). Therefore, we investigated the effects of an inhibition of the Na+/Ca2+ exchanger either by lowering the extracellular sodium concentration ([Na+]e) or, pharmacologically on RVR, by using isolated perfused rat kidneys. Graded decreases in [Na+]e led to dose-dependent increases in RVR to 4.3-fold (35 mM Na+). This vasoconstriction was markedly attenuated by lowering the extracellular calcium concentration, by the L-type calcium channel blocker amlodipine or by the chloride channel blocker niflumic acid. Further lowering of [Na+]e to 7 mM led to an increase in RVR to 7.5-fold. In this setting, amlodipine did not influence the magnitude but did influence the velocity of vasoconstriction. Pharmacological blockade of the Na+/Ca2+ exchanger with KB-R7943, benzamil, or nickel resulted in significant vasoconstriction (RVR 2.5-, 1.8-, and 4.2-fold of control, respectively). Our data suggest a functional role of the Na+/Ca2+ exchanger in the renal vascular bed. In conditions of partial replacement of [Na+]e, vasoconstriction is dependent on chloride and L-type calcium channels. A total replacement of [Na+]e leads to a vasoconstriction that is nearly independent of L-type calcium channels. This might be due to an active calcium transport into the cell by the Na+/Ca2+ exchanger.

vasoconstriction; benzamil; KB-R7943; nickel


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CYTOSOLIC FREE CALCIUM concentration ([Ca2+]i) is the key regulator of the contractility of vascular smooth muscle cells (VSMC). As intracellular calcium levels result from calcium influx and efflux mechanisms, these appear to be main regulators of vasoconstriction. Calcium influx pathways in VSMC include L-type calcium channels and, to a lesser extent, also store-operated calcium channels. Extrusion mechanisms have been identified as sarcoplasmatic Ca2+-ATPase (uptake of calcium into intracellular calcium stores), plasma membrane Ca2+-ATPase (extrusion into the extracellular space), and the Na+/Ca2+ exchanger. Two structurally and functionally different forms of the Na+/Ca2+ exchanger have been identified so far: the cardiac Na+/Ca2+ exchanger, detectable in cardiac cells, VSMC, and most other cells (6, 8, 14), can be distinguished from another form, which was demonstrated in retinal rod outer segments (3). In kidneys, different isoforms of the "cardiac" Na+/Ca2+ exchanger have been identified (7).

Several studies provided evidence that under physiological conditions the Na+/Ca2+ exchanger acts as a calcium extrusion mechanism, transferring calcium from the intracellular to the extracellular space, the so-called "forward mode" (2, 21). The activity of this calcium extrusion mechanism has been shown to be dependent on extracellular sodium concentration ([Na+]e) and [Ca2+]i. Lowering of [Na+]e results in an increase in [Ca2+]i (2, 12, 13).

Recently, the presence of the Na+/Ca2+ exchanger in isolated renal afferent arterioles has been demonstrated as lowering extracellular sodium, led to a marked increase in [Ca2+]i independently of L-type calcium channels (4). Also, a possible role of this exchanger in the pathogenesis of arterial hypertension in spontaneously hypertensive and salt-sensitive rats has been suggested (15, 16). Despite these evidences for a functional role of the Na+/Ca2+ exchanger in the regulation of intracellular calcium levels in VSMC, an impact of the exchanger on vascular contractility has not been demonstrated as yet. As the Na+/Ca2+ exchanger might be a regulator of renal blood flow besides hormonal regulation of vascular resistance, we examined the influence of the Na+/Ca2+ exchanger on renal vascular tone by using the isolated perfused rat kidney model. This experimental model provides the opportunity to examine renal vascular resistance (RVR) without hormonal or neural influence. Especially, the systemic renin-angiotensin system, which regulates the sodium balance of the body, is not effective in the isolated perfused rat kidney model as the perfusion medium lacks angiotensinogen, the substrate of renin.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolated perfused rat kidney. Male Sprague-Dawley rats (280-330 g body wt), having free access to commercial pellet chow and tap water were obtained from the local animal house and used throughout. Kidney perfusion was performed in a recycling system (19). In brief, the animals were anesthetized with 100 mg/kg of 5-ethyl-5-(1-methylbutyl)-2-thiobarbituric acid (Trapanal, Byk Gulden, Germany). Volume loss during the preparation was substituted by intermittent injections of physiological saline via a catheter inserted into the jugular vein. After opening of the abdominal cavity by a midline incision, the right kidney was exposed and placed in a thermoregulated metal chamber. The right ureter was cannulated with a small polypropylene tube (PP-10) that was connected to a larger polyethylene catheter (PE-50). After intravenous heparin injection (2 U/g), the aorta was clamped distal to the right renal artery so that the perfusion of the right kidney was not disturbed during the following insertion of the perfusion cannula in the aorta distal to the clamp. After ligation of the large vessels branching off the abdominal aorta, a double-barreled perfusion cannula was inserted into the abdominal aorta and placed close to the aortic clamp distal to the origin of the right renal artery. After ligation of the aorta proximal to the right renal artery the aortic clamp was quickly removed and perfusion was started in situ with an initial flow rate of 8 ml/min. By using this technique, a significant ischemic period of the right kidney was avoided. The right kidney was excised, and perfusion at constant pressure (100 mmHg) was established. To this end, the renal artery pressure was monitored through the inner part of the perfusion cannula (Statham transducer P 10 EZ), and the pressure signal was used for feedback control of a peristaltic pump. The perfusion circuit was closed by draining the venous effluent via a metal cannula back into a reservoir (200-220 ml). The basic perfusion medium, which was taken from a thermostated (37°C) reservoir, consisted of a modified Krebs-Henseleit solution containing (in mM) all physiological amino acids in concentrations between 0.2 and 2.0 mM, 8.7 glucose, 0.3 pyruvate, 2.0 L-lactate, 1.0 alpha -ketoglutarate, 1.0 L-malate, and 6.0 urea. The perfusate was supplemented with 6 g/100 ml bovine serum albumin, 1 mU/100 ml vasopressin 8-lysine, and with freshly washed human red blood cells (10% hematocrit). Ampicillin (3 mg/100 ml) and flucloxacillin (3 mg/100 ml) were added to inhibit possible bacterial growth in the medium. To improve the functional preservation of the preparation, the perfusate was continuously dialyzed against a 25-fold volume of the same composition but lacking erythrocytes and albumin. For oxygenation of the perfusion medium the dialysate was gassed with a 94% O2- 6% CO2 mixture. Under these conditions both glomerular filtration and filtration fraction remain stable for at least 90 min at values of ~1 ml · min-1 · g-1 and 7%, respectively (19). Perfusate flow rates were obtained from the revolutions of the peristaltic pump, which was calibrated before and after each experiment. Renal flow rate and perfusion pressure were continuously monitored by a potentiometric recorder. After the perfusate reperfusion loop was established, flow rates usually stabilized within 15 min. Stock solutions of the drugs to be tested were dissolved in freshly prepared perfusate and infused into the arterial limb of the perfusion circuit directly before the kidneys at 3% of the rate of perfusate flow.

RVR was calculated as RVR = perfusion pressure/perfusate flow.

Experimental maneuvers and chemicals. Different [Na+]e in the perfusate were achieved by replacing NaCl in the dialysate by choline-chloride (Sigma). A nearly complete exchange of sodium was achieved by additional replacement of Na-bicarbonate by choline-bicarbonate (Sigma). Resulting [Na+]e in the perfusate was determined by using a flame-photometer (Jenway PFP7).

For lowering the calcium concentration into the submicromolar range, we added the calcium-chelator EGTA (3.12 mM) to the perfusate.

Benzamil, nickel chloride, amlodipine, EGTA, and niflumic acid were obtained from Sigma. KB-R7943 was obtained from Tocris Cookson.

Statistics. Five kidneys were used for each experimental protocol. For evaluation of significance of a certain experimental maneuver on RVR, RVR values were calculated for at least four time points within this experimental period (2, 5, 10, 15 min after change of perfusate/drug-administration). These RVR values were averaged and compared with the average values of an adjoining experimental period or control values. Student's paired t-test was used to calculate levels of significance within individual kidneys. P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To asses the influence of [Na+]e on RVR, we lowered [Na+]e by replacing increasing amounts of sodium by choline. As shown in the original tracing (Fig. 1) and schematically in Fig. 2, this procedure led to a progressive vasoconstriction, with a significant increase in RVR already after replacement of 10 mM Na+ (1.25-fold of control, P < 0.05) (Fig. 2). Further reduction of [Na+]e to 35 mM led to a 4.3-fold increase in RVR (P < 0.001) (Fig. 2). Lowering of [Na+]e to 7 mM, and therefore below the intracellular sodium concentration, resulted in a further significant vasoconstriction. Time controls at constant [Na+]e did not change vascular resistance over a period of at least 60 min (Fig. 2).


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Fig. 1.   Original tracing of perfusate flow at constant perfusion pressure of 100 mmHg. Stepwise reduction of extracellular sodium concentration ([Na+]e) results in a graded reduction of perfusate flow.



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Fig. 2.   Effects of lowering [Na+]e to 130, 121, 87, 35, or 7 mM on renal vascular resistance (RVR). Inset: RVR (mmHg · min · ml-1) in dependency of [Na+]e.

To test whether the observed vasoconstriction was dependent on the availability of extracellular calcium, we added the calcium chelator EGTA (3.12 mM) to the perfusate and subsequently lowered [Na+]e to 35 mM (Fig. 3). Administration of EGTA resulted in a significant vasodilation (RVR 0.92-fold of control). Lowering of [Na+]e in this situation tended to increase RVR (1.1-fold of EGTA); however, this change was not statistically significant (P = 0.09). However, after withdrawal of EGTA, vascular resistance almost instantaneously increased to 7.8-fold. After 15 min, a new plateau of RVR was reached at 3.9-fold of control (Fig. 3).


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Fig. 3.   Effects of the extracellular Ca2+ concentration ([Ca2+]e) on vasoconstriction induced by lowering [Na+]e to 35 mM. Lowering [Ca2+]e into the submicromolar range resulted in a significant vasodilation and prevented significant vasoconstriction when [Na+]e was lowered to 35 mM. Withdrawal of EGTA led to a peak increase in RVR, followed by a new plateau.

As calcium influx via L-type calcium channels is involved in the induction of the vasoconstrictive effects of a variety of vasoconstrictors (i.e., angiotensin II, endothelin-1, KCl), we were interested in the role of these channels in the mediation of vasoconstriction by lowering [Na+]e. Therefore, we administered the L-type calcium channel blocker amlodipine (5 µM) before or after switching to a low-sodium perfusate ([Na+]e 35 mM). Addition of amlodipine led to a slight but significant decrease in RVR from 6.2 ± 0.1 to 5.7 ± 0.1 mmHg · min · ml-1 (P < 0.05) (Fig. 4, top). Subsequent lowering of the [Na+]e mediated a significant vasoconstriction (1.26-fold vs. amlodipine, 1.16-fold vs. control), which was marginal, however, compared with the vasoconstriction observed without blockade of calcium channels (4.3-fold vs. control) (Fig. 4, top). Moreover, amlodipine reversed the existing vasoconstriction when added after a switch to low extracellular sodium from 4.3- to 1.6-fold of control (P < 0.05) (Fig. 4, bottom). In the control group without administration of amlodipine, RVR remained constantly on an elevated level (P < 0.05 vs. control) (Fig. 4, bottom).


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Fig. 4.   Effects of amlodipine (5 µM) and lowering of [Na+]e to 35 mM on RVR () compared with lowering of [Na+]e alone (open circle ). Amlodipine attenuated vasoconstriction when administered either before (top) or after (bottom) lowering of [Na+]e.

The next experiments were performed to examine a possible involvement of chloride channels in the mediation of the vasoconstriction by lowering [Na+]e. To this end, we added the chloride channel blocker niflumic acid (300 µM) to the perfusate and lowered [Na+]e to 35 mM subsequently. As shown in Fig. 5, top, administration of niflumic acid resulted in a vasodilation (RVR 5.7 ± 0.1 vs 6.2 ± 0.10 mmHg · min · ml-1 for control) and markedly attenuated vasoconstriction after the switch to the low-sodium perfusate (1.3- vs. 4.4-fold of control without niflumic acid), indicating an important role of chloride channels in this mechanism. Similar to the results obtained with amlodipine, niflumic acid was able to reverse a preexisting vasoconstriction induced by low sodium from 3.7- to 1.6-fold of control in additional experiments (Fig. 5, bottom).


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Fig. 5.   Effects of niflumic acid (300 µM) and lowering of [Na+]e to 35 mM on RVR () compared with lowering of [Na+]e alone (open circle ). Niflumic acid attenuated vasoconstriction when administered either before (top) or after (bottom) lowering of [Na+]e.

In the next set of experiments, we lowered [Na+]e below the intracellular concentration, thereby turning the direction of calcium transport to an inward direction (Fig. 6). A stepwise reduction of [Na+]e to 35 and 7 mM resulted in significant increases in RVR, as shown in Figs. 1 and 2. Performing this maneuver in the presence of amlodipine attenuated the increase in RVR at 35 mM [Na+]e (1.3- vs. 4.5-fold of control). However, lowering [Na+]e to 7 mM resulted in an increase in RVR to 6.6-fold of control, even under a blockade of L-type calcium channels (7.7-fold of control without amlodipine). Although the magnitude of these increases in RVR was not statistically different, the velocity of the increase was lowered in the kidneys exposed to amlodipine as the maximum vasoconstriction occurred 10-15 min later in this group (Fig. 6). These results were confirmed by additional experiments in which [Na+]e was lowered from 140 to 7 mM with or without amlodipine (5 µM; Fig. 6, inset). Again, the magnitude of vasoconstriction 20 min after lowering of [Na+]e was similar in both groups, whereas the velocity of vasoconstriction was different.


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Fig. 6.   Effects of amlodipine (5 µM) and lowering [Na+]e stepwise to 35 and to 7 mM on RVR (). open circle , Controls without amlodipine treatment. Inset: lowering of [Na+]e to 7 mM in 1 step with (open circle ) or without amlodipine ().

To strengthen our hypothesis that the vasoconstriction induced by the lowering of [Na+]e was attributable to an inhibition of the Na+/Ca2+ exchanger, we performed a pharmacological blockade of this transporter with the selective inhibitor KB-R7943. As maximum vasoconstriction was achieved at a concentration of 50 µM in preceding experiments, this dosage was used in the further experiments. As shown in Fig. 7, administration of KB-R7943 resulted in a significant increase in RVR to 2.5-fold of control. Subsequent addition of amlodipine (5 µM) reversed this vasoconstriction significantly (RVR 1.14-fold of control). Moreover, benzamil (100 µM) or nickel (3 mM), both unspecific blockers of the Na+/Ca2+ exchanger, induced significant vasoconstrictions (RVR 1.8- or 4.2-fold of control) (data not shown).


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Fig. 7.   Effects of pharmacological blockade of the Na+/Ca2+ exchanger with KB-R7943 (50 µM) and subsequent addition of amlodipine (5 µM) on RVR at normal [Na+]e.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our data demonstrate a functional role of the Na+/Ca2+ exchanger in the renal vasculature in mediating vasoconstriction due to decreases in extracellular sodium. As shown in Figs. 1 and 2, this effect already occurs in a physiological concentration range, as lowering of [Na+]e from 140 to 130 mM resulted in an increase in RVR to 1.25-fold. This marked effect at rather small changes in [Na+]e seems to be somewhat contradictory to cell culture studies, as in those investigations where an increase in [Ca2+]i was observed only at rather low levels of [Na+]e (2). However, as the Na+/Ca2+ exchanger acts as a calcium extrusion pathway, its activity depends, beside other regulators, on [Ca2+]i (20). As in our experimental model, in contrast to cell culture studies, the VSMC of the renal vasculature are exposed to a physiological perfusion pressure of 100 mmHg. L-type calcium channels are activated under control conditions to maintain vascular resistance. Therefore as shown in Fig. 3, elimination of extracellular calcium or blockade of L-type calcium channels (Fig. 4) results in a small but significant decrease in RVR. As activation of these channels leads to a calcium influx into the cell, calcium extrusion mechanisms are relevant for maintaining intracellular calcium homeostasis already under control conditions in our experimental model. Blockade of one of these extrusion mechanisms, as the Na+/Ca2+ exchanger, might therefore result in a more pronounced increase in [Ca2+]i as in cell culture preparations.

Furthermore our experiments demonstrate that vasoconstriction due to a partial replacement of sodium is dependent on L-type calcium channels and chloride channels as amlodipine and niflumic acid could prevent and reverse vasoconstriction. One possible explanation for this result is a depolarization cascade. According to this hypothesis, a slight increase in [Ca2+]i, as observed after inhibition of the Na+/Ca2+ exchanger, is capable of activating calcium-triggered chloride channels, causing depolarization. This depolarization finally activates L-type calcium channels, resulting in calcium influx into the cell, mediating vasoconstriction. Interruption of this cascade by blockade of calcium or chloride channels therefore should prevent vasoconstriction.

On the other hand, we provide evidence that after nearly complete replacement of extracellular sodium, RVR increases independently of the activity of L-type calcium channels (Fig. 6). These findings are in good accordance with a previous study using isolated renal afferent arterioles showing an increase in [Ca2+]i due to a total replacement of [Na+]e, which was not preventable by L-type calcium channel blockers (4). The possible explanation for this L-type calcium channel-independent vasoconstriction might be the reverse mode action of the Na+/Ca2+ exchanger. As shown in several studies, the Na+/Ca2+ exchanger acts as an inward calcium pump in conditions with intracellular sodium concentrations exceeding [Na+]e (2, 4, 15). Lowering [Na+]e to 7 mM might therefore result in an active calcium transport into the cell. If this calcium influx exceeds calcium extrusion by the remaining extrusion mechanisms, an intracellular calcium accumulation leading to vasoconstriction will occur.

As replacing extracellular sodium not only effects the Na+/Ca2+ exchanger but also other sodium-driven exchange mechanisms, we performed a selective pharmacological blockade of the Na+/Ca2+ exchanger using KB-R7943. This novel compound has been shown to block the Na+/Ca2+ exchanger without affecting sodium channels or other sodium-driven transport systems (10). As shown in Fig. 7, pharmacological blockade of the Na+/Ca2+ exchanger resulted in a marked vasoconstriction, which was reversible by blockade of L-type calcium channels; behavior similar to this was observed after inhibition of the Na+/Ca2+ exchanger by partial replacement of sodium. Moreover, nickel and benzamil, both unspecific inhibitors of the exchanger, exerted marked vasoconstrictive effects. However, because benzamil, similar to lowering [Na+]e, attenuates the exchange capacity of the Na+/H+ exchanger (5), the vasoconstriction might be related in part to this inhibition. However, the inhibition of the Na+/H+ exchanger with ethylisopropylamiloride resulted in a decline of intracellular pH but did not change the [Ca2+]i in aortic VSMC (2). Moreover, several studies provided evidence for a vasodilating action of Na+/H+ exchange inhibition (1, 17, 18).

The comparison of the different maneuvers to block sodium-calcium exchange capacity shows that lowering of [Na+]e and nickel are more potent vasoconstrictors than KB-R7943 and benzamil. As we used the most effective dose of each drug in our experiments, we should expect a similar extent of vasoconstriction in all groups if vasoconstriction was related exclusively to the inhibition of the Na+/Ca2+ exchanger. We have to conclude from this discrepancy either that choline and nickel exert additional vasoconstrictory effects or that KB-R7943 and benzamil were not capable of blocking the Na+/Ca2+ exchanger completely. As demonstrated for the different isoforms (9, 11) even the different splice variants found in the kidney may confer different sensitivity on the blockers used.

Another possible problem of our experimental design lies in the replacement of sodium by choline. As, for example, the permeability of the cytoplasmic membrane is smaller for choline than for sodium, choline exerts a higher osmotic action and might thereby influence contractility. However, we did not examine an increase in vascular resistance after raising extracellular osmolarity with sucrose or after adding choline to the perfusate in additional experiments. Moreover, an inhibitory effect of choline itself on the Na+/Ca2+ exchanger has been described (5). As this was the desired effect of our procedures, this "side effect" did not influence the interpretation of our results.

In summary, our data are highly compatible with a functional role of the Na+/Ca2+ exchanger in the renal vasculature, mediating vasoconstriction due to a lowering of the [Na+]e. In the physiological concentration range, sodium mediation of vasoconstriction is dependent on L-type calcium channels and chloride channels, whereas the reverse-mode action of the Na+/Ca2+ exchanger mediates vasoconstriction only at [Na+]e, far below the physiological range.


    ACKNOWLEDGEMENTS

The expert technical assistance provided by Karl-Heinz Götz is gratefully acknowledged.


    FOOTNOTES

This study was financially supported by the Deutsche Forschungsgemeinschaft (Ku 859/2-3).

Address for reprint requests and other correspondence: F. Schweda, Institut für Physiologie I, Universität Regensburg, 93040 Regensburg, Germany (E-mail: frank.schweda{at}klinik.uni-regensburg.de).

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. Section 1734 solely to indicate this fact.

Received 10 April 2000; accepted in final form 27 September 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Fluid Electrolyte Physiol 280(1):F155-F161
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