INTRODUCTION

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IT HAS BEEN PROPOSED FOR DECADES that calcium plays an unusual role in the control of renin secretion from renal juxtaglomerular cells in the way that an increase in the calcium concentration inhibits the exocytosis of renin, whereas in other secretory cells calcium supports secretion. The assumption of inhibitory effects on renin secretion is based on the evidence that lowering of the extracellular concentration of calcium stimulates renin secretion (1, 4, 9, 14, 24, 27, 29, 39) and that calcium-mobilizing hormones inhibit renin secretion (9, 18, 27), dependent on the availability of extracellular calcium (4, 24, 27, 29, 39). Whether it is the internal calcium release or the transmembrane calcium influx that is relevant for the control of renin secretion cannot be distinguished from previous experiments because external calcium is required for both filling of internal stores and for influx into the cytosol. Moreover, also the routes along which calcium could enter juxtaglomerular cells, in particular the role of voltage-gated calcium channels, are subject to controversy (3, 7, 8, 12, 15, 17, 19, 23, 30). Although we could not find electrophysiological evidence for a functional role of voltage-gated calcium channels in renal juxtaglomerular cells (19), we found that angiotensin II, which is a classic inhibitor of renin secretion, mobilized calcium from internal stores and transiently increased the calcium permeability of juxtaglomerular cells (17). The nature of this angiotensin II-triggered calcium influx pathway and its relevance for renin secretion, however, is yet unknown.

During the last decade evidence has been elaborated that release of calcium from internal stores can trigger transmembrane calcium influx through a signal related to the filling state of internal calcium stores (2, 26). The resulting calcium-release-activated calcium current (I_crac) flowing across the plasma membrane is detectable by using the patch-clamp technique (11). Such a store-operated calcium influx is seen in a broad variety of cells (25), but its existence in renal juxtaglomerular cells has not been demonstrated till now. We therefore wondered whether such a calcium store-regulated calcium influx also exists in renal juxtaglomerular cells and what its impact for renin secretion may be.

Store-triggered calcium influx can experimentally be activated by inhibition of calcium uptake into internal stores through the inhibition of endoplasmatic Ca²⁺-ATPases (33, 34). We therefore characterized the effects of the Ca²⁺-ATPase inhibitors thapsigargin and cyclopiazonic acid on renin secretion from isolated perfused rat kidneys. Our results indicate that store-triggered calcium influx acts as a powerful inhibitor mechanism for renin secretion.

	MATERIALS AND METHODS

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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 (30). In brief, the animals were anesthetized with 100 mg/kg of 5-ethyl-5-(1-methylbutyl)-2-thiobarbituric acid (Trapanal, Byk Gulden, Konstanz, 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 -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 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 · min¹ · g¹ and 7%, respectively (30). 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 reperfusion loop perfusate flow was established, 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.

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Chemicals. Isoproterenol, thapsigargin, cyclopiazonic acid, gadolinium (III)-chloride, lanthanum chloride, and flufenamic acid were purchased from Sigma Chemical (Deisenhofen, Germany).

Statistics. For evaluation of significance of a certain experimental maneuver on renin secretion, all renin secretion rates obtained within this experimental period (4 values) were averaged and compared with the average values of renin secretion of an adjoining experimental period. Student's paired t-test was used to calculate levels of significance within individual kidneys. P < 0.05 was considered significant.

	RESULTS

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Effects of Ca²+-ATPase inhibition on renin secretion and perfusate flow. Basal flow rates and renin secretion rates from the isolated rat kidneys reached stable plateaus 10-15 min after onset of artificial perfusion (Fig. 1). To deplete internal calcium stores we used the Ca²⁺-ATPase inhibitor thapsigargin (300 nM). Thapsigargin produced a marked fall in renin secretion (P < 0.01) (Fig. 1, bottom), whereas perfusate flow rates at constant pressure declined only moderately (not significant) (Fig. 1, top). Addition of isoproterenol (10 nM) to the perfusate in the presence of thapsigargin increased flow rates (not significant) (Fig. 1, top) and restored basal renin secretion rates (Fig. 1, bottom). If isoproterenol was administered in the absence of thapsigargin, renin secretion increased eightfold over basal (P < 0.01) (Fig. 1, bottom). Flow changes were not statistically significant. Apparently, thapsigargin not only inhibited basal renin secretion but also markedly attenuated the stimulability by -adrenoreceptor activation.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Effects of thapsigargin (300 nM) and isoproterenol (isoprot; 10 nM) on renin secretion rate and perfusate flow (). , Time control without thapsigargin. Values are means ± SE.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effects of isoproterenol (10 nM) and cyclopiazonic acid (20 µM) on perfusate flow and renin secretion rate. Values are means ± SE. For statistics, see text.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Effects of bumetanide (100 µM; ) or reduction of perfusion pressure to 40 mmHg () and thapsigargin (300 nM) on perfusate flow and renin secretion rate. Values are means ± SE. For statistics, see text.

Relevance of extracellular calcium for the inhibitory effects of Ca²+-ATPase inhibitors. The next set of experiments was designed to evaluate the relevance of extracellular calcium for the effects of Ca²⁺-ATPase inhibitors on renin secretion. For this purpose renin secretion was prestimulated by isoproterenol, which then was significantly attenuated by thapsigargin (P < 0.05) (Fig. 4). In the presence of isoproterenol plus thapsigargin, the concentration of extracellular calcium was then rapidly lowered from 3 mM into the lower submicromolar range by the addition of 3.12 mM EGTA to the perfusate. This maneuver almost instantaneously reversed the inhibition of renin secretion by thapsigargin (Fig. 4) and tended to increase perfusate flow (not significant). The effect of EGTA was completely reversible, because, after withdrawal of EGTA, renin secretion values rapidly fell to below control (Fig. 4).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Effects of lowering extracellular calcium by addition of EGTA on the effects of thapsigargin. Stimulation of renin secretion with isoproterenol (10 nM) and concomitant administration of thapsigargin (300 nM). Extracellular calcium concentration was lowered into the submicromolar range by adding EGTA (3.12 mM) to the perfusate over a period of 10 min, following recovery period by stopping EGTA infusion. Values are means ± SE. * P < 0.05.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Effects of calcium-free perfusate on renin secretion and flow rate and on the effects of isoproterenol (10 nM) and thapsigargin (300 nM; ). , Time control with physiological extracellular calcium concentrations and isoproterenol and thapsigargin perfusion. Values are means ± SE. For statistics, see text.

Effects of blockers of store-operated calcium channels and L-type calcium channels on thapsigargin-induced inhibition of renin secretion. Assuming that the inhibitory effect of Ca²⁺-ATPase blockade on renin secretion was causally related to calcium influx from the extracellular space, we tested the effects of drugs that were previously reported to block store- operated calcium entry, such as lanthanum, gadolinium, or flufenamic acid (13, 32). As shown in Fig. 6, however, neither lanthanum nor flufenamic acid or gadolinium was capable of reversing the inhibitory effect of thapsigargin on renin secretion. Moreover, they exerted no significant effect on perfusate flow. Also blocking L-type calcium channels by amlodipine (5 µM) had no effect on the renin suppressing effects of thapsigargin (Fig. 6). In contrast, lowering extracellular calcium into the submicromolar range by adding EGTA (3.12 mM) led to an increase in renin secretion to isoproterenol-stimulated values, indicating a reversal of the thapsigargin effect (Fig. 6).

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Renin secretion rates as percent of values after isoproterenol stimulation. Values are means ± SE. Effect of lanthanum (lant; 50 µM), flufenamic acid (flufen; 100 µM), gadolinium (gado; 50 µM), amlodipine (amlo; 5 µM), and EGTA (3.12 mM) on renin secretion rate and on thapsigargin (thap; 300 nM) effect on renin secretion. Iso, isoproterenol (10 nM). n.s., Not significant. # P < 0.001 vs. isoproterenol alone.

	DISCUSSION

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The purpose of our study was to examine the effect of calcium influx triggered by depletion of internal stores on renin secretion from renal juxtaglomerular cells. To empty internal calcium stores we used a widely accepted maneuver, namely, inhibition of calcium uptake into internal stores via the inhibition of endoplasmatic Ca²⁺-ATPases by using thapsigargin and cyclopiazonic acid. Both drugs have been shown to increase intracellular calcium concentration primarily by inhibition of endoplasmatic Ca²⁺-ATPase and thereby blocking the calcium transport from the cytosol into the intracellular calcium stores. The resulting calcium depletion of the stores activates a calcium influx from the extracellular space via store-operated calcium channels (6, 33, 34, 36, 37). The activation of store-operated calcium channels seems restricted to stores sensitive to inositol triphosphate (10), a known second messenger of angiotensin II.

Our data now show that inhibitors of Ca²⁺-ATPase in fact are highly effective on renin secretion, in the way that they strongly inhibit basal and stimulated renin secretion. This inhibitory effect is not restricted to a specific pathway of stimulation because it not only affected renin secretion stimulated by -adrenoreceptor activation but also attenuated stimulation by inhibition of macula densa salt transport with bumetanide and by a drop in the renal artery pressure. This inhibitory effect of the Ca²⁺-ATPases was calcium dependent because thapsigargin failed to suppress isoproterenol-stimulated renin secretion when extracellular calcium concentrations were low. In addition, lowering extracellular calcium into the submicromolar range by adding EGTA immediately reversed thapsigargin-induced renin suppression. Given a specific effect of the Ca²⁺-ATPase inhibitors on renin secretion that is related to calcium, then their effects on renin secretion could be due either to an accumulation of cytosolic calcium, which cannot flow off into internal stores, or to an activation of calcium influx from outside, triggered by a signal related to the filling state of internal calcium stores. The observation that the inhibitory action of Ca²⁺-ATPase inhibitors on renin secretion was almost instantaneously abolished by a lowering of the extracellular calcium concentration supports the conclusion that it is the calcium influx from outside that primarily mediates the inhibitory action of Ca²⁺- ATPase blockers on renin secretion.

We infer from these findings, that a significant store-operated calcium influx exists in renal juxtaglomerular cells and that activation of this influx represents a powerful inhibitory signal for renin secretion. Both conclusions are in good accordance with previous findings. Thus we have reported previously that angiotensin II mobilizes calcium from internal stores in juxtaglomerular cells and transiently increases the calcium permeability of the plasma membrane, what would be compatible with store-regulated calcium influx (17). Furthermore, it is well known that the inhibitory action of angiotensin II on renin secretion is strongly dependent on extracellular calcium, compatible with an inhibition of renin secretion by store-operated calcium influx (1, 39).

The results of our study, moreover, suggest that the store-regulated calcium influx into renal juxtaglomerular cells does not follow the well-characterized pathway of store-operated calcium influx, which can be blocked by lanthanum, gadolinium, or flufenamic acid (13, 28, 32). L-type calcium channels do not appear to play a role in mediating the effects of thapsigargin on the renin-secretion either, as amlodipine failed to reverse its suppression mediated by thapsigargin. Alternative ways for calcium influx include the recently identified more unspecific cation channels that are also activated by depletion of internal calcium stores (21, 38, 41) and calcium-activated calcium channels that are store independent and have been detected in a large variety of cells (16, 22, 40).

A further interesting result of our study is that the only moderate attenuation of perfusate flow by thapsigargin and cyclopiazonic acid indicating that inhibition of endoplasmatic reticulum Ca²⁺-ATPase has only minor effects on the contractility of renal resistance vessels in our experimental model. Although the renal vascular resistance is set on a rather low level in the isolated perfused kidney model compared with the in vivo situation and the interpretation of the perfusate flow changes might therefore be limited, it has been shown several times that vasoconstrictors like angiotensin II mediate a dose-dependent vasoconstriction in our model (30, 35). Therefore, it can be speculated from our results that internal calcium stores and calcium release-activated calcium channels are not the main regulatory step in the control of vascular tone of renal resistance vessels. Our results are in good accordance with a recent study showing thapsigargin not altering diameters of afferent and efferent arterioles of isolated perfused hydronephrotic kidneys under basal conditions (35). Furthermore, our data support previous findings that angiotensin II-induced calcium influx mediating vasoconstriction of resistance vessels and suppression of renin secretion in juxtaglomerular cells follows to different pathways. Although we and others found a marked effect of L-type Ca²⁺-channel blockers on regulation of vascular tone (5, 20, 30), we found no attenuation of renin secretion (30). As mentioned above, we could not find evidence for a role for voltage-operated calcium channels in juxtaglomerular cells (19) but in more proximal smooth muscle cells of afferent arterioles (31).

In conclusion, our data show a marked inhibitory effect on renin secretion by inhibition of the endoplasmatic Ca²⁺-ATPase. Instantaneous reversal of this inhibition by lowering extracellular calcium concentration into the submicromolar range suggests that a calcium influx from the extracellular space is responsible for this inhibition. Store-operated calcium channels do not appear to be a main regulatory step for the contractility of renal resistance vessels, because inhibition of endoplasmatic Ca²⁺-ATPase attenuated perfusate flow only slightly. Together, these results emphasize our previous findings that calcium influx in juxtaglomerular cells and renal smooth muscle cells mediating suppression of renin secretion and vasoconstriction follow different pathways.