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【摘要】
Our study aimed to characterize the influence of changes in extracellular osmolarity on renin secretion from the whole kidney. For this purpose, the osmolarity of the perfusion medium of isolated rat or mouse kidneys was either decreased by lowering the NaCl concentration by 20% or was increased up to 133% by the addition of various salts or sugars. It turned out that changes in osmolarity led to instantaneous transient changes followed by a plateau of renin secretion, in that increases in osmolarity stimulated renin secretion, whereas decreases attenuated renin secretion. The peak amplitude of changes in renin secretion was related to steady-state renin secretion rates before the osmotic challenge but was independent of the maneuver used to modulate steady-state renin secretion. Osmolarity-induced changes in renin secretion were more related to relative rather than to absolute changes in osmolarity and were not dependent on the formation of nitric oxide or of prostanoids and did not require Na-K-2Cl cotransport function or swelling-activated chloride channels. Moreover, we obtained evidence that the pool of renin secretion excitable by hyperosmolarity is exhaustible and that its complete refilling takes at least 2 min. The observed behavior of renin secretion fits the concept about exocytosis proposing the existence of different pools of committed secretory vesicles, which have not yet undergone the final modification for initiation of exocytosis. Probably, a pool of readily releasable vesicles determines steady-state secretion rates from kidneys.
isolated, perfused kidney; renin release; exocytosis
RENIN - SECRETING JUXTAGLOMERULAR epithelioid cells have been described as behaving like sensitive osmometers, with small changes in osmolality eliciting rather prominent changes in renin secretion ( 3 ). Extensive studies with kidney slices, superfused isolated glomeruli ( 3, 5 - 7 ), isolated afferent arterioles ( 26 ), and cultured juxtaglomerular cells ( 4, 17 ) or chorionic cells ( 2 ) have shown that renin secretion changes inversely with extracellular osmolarity.
Because similar data were obtained with permeabilized glomeruli ( 6 ), it was assumed that osmolarity modulates renin secretion through direct effects on the secretory vesicles rather than through changes in cell volume, in the way that hyposmolarity favors renin secretion whereas hyperosmolarity inhibits renin secretion ( 6, 21 ).
This conclusion is in good accordance with a series of other studies reporting an inhibitory effect of osmolarity on secretion from a number of endo- and exocrine cells even if the cells are permeabilized ( 24 ). Paradoxically, calcium, which normally triggers exocytosis in the majority of endocrine cells, attenuates secretion evoked by hyposmolarity ( 24 ). The same holds true for the renin-secreting juxtaglomerular cells, in which calcium is a physiological inhibitor of secretion ( 18 ). Thus removal of calcium further increases the stimulation of renin secretion by hyposmolarity ( 7 ). Notably, however, the regulatory effect of calcium but not of osmolarity is lost in permeabilized cells ( 6, 7 ).
The underlying mechanisms for the inverse relationship between renin secretion and osmolarity in renin-secreting cells and other secretory cells are still subject to discussion.
It should be noted in this context that the findings mentioned above were mainly obtained with isolated, nonperfused preparations ( 3, 5 - 7 ). A very recent study on isolated, perfused juxtaglomerular apparatuses reports that hyperosmolarity induced by sugars stimulates rather than inhibits exocytosis of renin vesicles ( 25 ). Information about the effect of osmolarity on secretion from whole kidneys is rare. Almost 30 years ago, it was reported that acute increases in osmolarity in the renal artery increase renal blood flow and renin secretion in anesthetized dogs ( 28, 29 ). Another investigation reported that in intact rats moderate increases in NaCl concentration in the renal artery do not change renin secretion ( 9 ) despite the expected activation of the macula densa mechanism, which should inhibit renin secretion, suggesting that hyperosmolar NaCl might also exert a direct stimulatory effect on renin secretion.
In view of this rather unclear effect of osmolarity with regard to our understanding of renin secretion at the organ level, we were therefore interested in investigating the effect of changes in osmolarity on renin secretion from isolated, perfused rat and mouse kidneys, in which all physiologically relevant regulations of renin secretion are still preserved. Much to our surprise, already our first experiments revealed a highly reproducible stimulatory effect of osmolarity on renin secretion, quite in contrast to what is known from experiments with isolated glomeruli or cells. Following this, we therefore studied the effects of osmolarity changes on renin secretion from whole kidneys in more detail.
The sum of our findings leads to the assumption of different pools of renin secretory vesicles, which are regulated by cAMP and calcium-dependent pathways.
【关键词】 Osmolarityinduced secretion kidneys evidence releasable
MATERIALS AND METHODS
Isolated perfused kidney. All experiments were performed using isolated, perfused kidneys of rats, with the exception of the experiments shown in Fig. 1 ( bottom ), which were done using isolated, perfused kidneys of mice to exclude a species-specific effect of osmolarity on renin secretion.
Fig. 1. A : effects of adding NaCl, Na-isethionate (iseth), or choline-Cl at concentrations of 30 mmol/l to the normal perfusate on renin secretion from isolated, perfused rat kidneys. Values are means ± SE of 5 experiments. # P < 0.001 vs. control or preceding recovery period. B : effects of adding glucose, sucrose, or NaCl at concentrations of 20 and 10 mmol/l, respectively, to the normal perfusate on renin secretion from isolated, perfused mouse kidneys. Values are means ± SE of 5 experiments. # P < 0.001 vs. control or preceding recovery period. * P < 0.05 vs. preceding recovery period.
The isolated, perfused rat kidney was prepared as previously described using a modified Krebs-Henseleit solution supplemented with bovine serum albumin (6% wt/vol) and human erythrocytes (Hct 10%) as a perfusate ( 16 ). The osmolarity of the perfusate was 296.9 ± 1.2 mosmol/l, as determined by freezing-point depression (Knauer Osmometer).
In brief, the perfusion of the kidneys of male Sprague-Dawley rats (280-330 g body wt) was performed in a recycling system. The animals were anesthetized with 100 mg/kg of 5-ethyl-5-(1-methylbutyl)-2-thiobarbituric acid (trapanal, Altana Pharma). After opening of the abdominal cavity, the right kidney was exposed and placed in a thermoregulated metal chamber. 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 into 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. 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). Renal flow rate and perfusion pressure were continuously monitored by a potentiometric recorder. Stock solutions of the drugs [isoproterenol, bumetanide, angiotensin II, amlodipine, N -nitro- L -arginine methyl ester ( L -NAME), acetylcholine, DIDS, indomethacin] or of osmolytes 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.
For determination of perfusate renin activity, aliquots ( 0.1 ml) were drawn, if not otherwise indicated, in intervals of 2 min from the arterial limb of the circulation and the renal venous effluent, respectively. The samples were centrifuged at 1,500 g for 15 min, and the supernatants were stored at -20°C until assayed for renin activity. For determination of renin activity, the perfusate samples were incubated for 1.5 h at 37°C with plasma from bilaterally nephrectomized male rats as a renin substrate. The generated ANG I (ng·ml -1 ·h -1 ) was determined by radioimmunoassay (Byk and DiaSorin). Renin secretion rates were calculated as the product of the arteriovenous differences of renin activity (ng ANG I·h -1 ·ml -1 ) and the perfusate flow rate (ml·min -1 ·g kidney wt -1 ).
For a demonstration of sufficient blockade of prostanoid formation by indomethacin, urinary PGE 2 excretion was determined. To this end, the ureter of the isolated kidney was cannulated with a small polypropylene tube (PP-10) and urine was collected for two 5-min periods during the control period as well as after the administration of the cyclooxygenase inhibitor. Urine samples were stored on ice, and PGE 2 concentrations were determined by PGE 2 monoclonal enzyme immunoassay (Cayman Chemical, Ann Arbor, MI) immediately after the end of each experiment. PGE 2 excretion was calculated from the urine flow and the PGE 2 concentration.
The isolated, perfused mouse kidney was prepared essentially as described above for rat kidneys, with the exception that the perfusate was not recirculated. A more detailed description of the model is given elsewhere ( 19 ).
All drugs used were purchased from Sigma.
All experiments were conducted in accordance with the Institute of Laboratory Research Guide for the Care and Use of Laboratory Animals (Washington, DC: National Academy Press, 1996) and German laws on the protection of animals.
Statistics. Values are given as means ± SE. For statistical analysis, all values obtained within an experimental period were averaged, if not indicated otherwise. Differences between the experimental maneuvers were analyzed by ANOVA and Bonferroni's adjustment for multiple comparisons. P values <0.05 were considered statistically significant.
RESULTS
To determine whether changes in osmolarity have any effect on renin secretion from isolated, perfused rat kidneys, we tested the effects of increasing the osmolarity of the perfusate by the addition of 30 mmol/l NaCl, Na-isethionate, choline-Cl, or sugars (not shown). As shown in Fig. 1 A, increasing the osmolarity by any maneuver led to an almost instantaneous strong and rapidly reversible enhancement of renin secretion of similar amplitude, irrespective of the agent used to increase the osmolarity. Thus renin secretion rates were stimulated by 30 mmol/l NaCl from 10.5 ± 0.35 (control period) to 30.9 ± 4.1 ( P < 0.001), by 30 mmol/l Na-isethionate from 14.7 ± 1.2 (preceding recovery period) to 33.2 ± 2.3 ( P < 0.001), and by choline-Cl from 10.3 ± 0.77 (preceding recovery period) to 33.7 ± 2.9 ng ANG I·h -1 ·min -1 ·g -1 ( P < 0.001, n = 5). To exclude the possibility that the effects of osmolarity on renin secretion are specific for rats, we performed one additional set of experiments using isolated mouse kidneys. Similar to the results obtained in rats, increasing the osmolarity of the perfusate by the addition of NaCl or of sugars stimulated renin secretion significantly ( Fig. 1 B, n = 5).
The stimulation of renin release by increases in NaCl concentration was concentration dependent ( Fig. 2 ). Thus addition of increasing concentrations of NaCl (, n = 5) enhanced renin secretion rates from 10.8 ± 0.29 (control) to 24.1 ± 2.8 (15 mmol/l NaCl; P < 0.05 vs. control), to 35.2 ± 3.7 (30 mmol/l NaCl, not significant vs. 15 mmol/l), and 49.3 ± 5.1 ng ANG I·h -1 ·min -1 ·g -1 (50 mmol/l NaCl, P < 0.05 vs. 30 mmol/l NaCl). This concentration dependency was preserved in the presence of 10 nmol/l isoproterenol ( Fig. 2,, n = 5), which itself is a well-known stimulator of renin secretion ( 18 ). Moreover, the amplitudes of changes in renin secretion elicited by graded increases in NaCl concentration were enhanced in the presence of isoproterenol (15 mmol/l NaCl: without isoproterenol 13.3 ± 2.9, with isoproterenol 55.6 ± 12.2, P < 0.05; 30 mmol/l NaCl: 21.9 ± 3.3 vs. 123.1 ± 27.0, P < 0.01; 50 mmol/l: 33.7 ± 4.8 vs. 202+31.9 ng ANG I·h -1 ·min -1 ·g -1, P < 0.001).
Fig. 2. Effects of adding graded concentrations of NaCl to the normal perfusate on renin secretion from isolated, perfused rat kidneys in the absence ( ) and presence ( ) of isoproterenol (10 nmol/l). Values are means ± SE of 5 experiments. Statistical analysis was performed within the groups with and without isoproterenol, respectively. * P < 0.05 vs. preceding period without NaCl addition. # P < 0.05 vs. 15 mmol/l NaCl. P < 0.05 vs. 30 mmol/l NaCl.
To determine the temporal association between changes in osmolarity and in renin secretion more closely, the venous effluent from isolated rat kidneys was sampled in 20-s intervals after the addition of 30 mmol/l NaCl to the perfusate and renin activity and sodium concentration (flame photometry) were assayed in parallel. To make changes in renin secretion more prominent, the experiments were performed in the presence of isoproterenol. As shown in Fig. 3 ( top ), there was no apparent time gap between the increase in sodium concentration and the increase in renin secretion, and it took 1 min to reach the maximal (peak) secretion rate. Whereas sodium concentration remained constantly elevated, renin secretion declined again to reach a new plateau after 4 min. After the addition of NaCl was stopped, sodium concentration and renin fell in parallel. Whereas sodium concentration leveled off at its prevalue, renin secretion fell below its prevalue and recovered thereafter with a time course of 5 min ( Fig. 3, top ).
Fig. 3. Higher resolution time course of the response of renin secretion ( top ) or perfusate flow ( bottom ) to hyperosmolar NaCl (+30 mmol/l) in isolated, perfused rat kidneys in the presence of isoproterenol (10 nmol/l). Values are means ± SE of 3 experiments.
The increase in osmolarity also led to an instantaneous increase in perfusate flow at a constant perfusion pressure from 16.3 ± 0.25 to 18.1 ± 0.7 ml·min -1 ·g -1 (peak value, P < 0.05 vs. control) ( Fig. 3, bottom ). After NaCl infusion was stopped, flow rates fell below normal and rapidly recovered thereafter ( Fig. 3, bottom ).
Apart from cAMP, renin secretion is known to be regulated by cytosolic calcium concentration in an unusual, inverse fashion ( 18 ). We therefore examined the effect of osmolarity on renin secretion in states of increased and of low cytosolic calcium concentrations. For this purpose, we used ANG II (300 pmol/l) to increase, and nominally calcium-free perfusate supplemented with 0.5 mmol/l EGTA to lower, cytosolic calcium concentration in renin-producing cells ( 10 ), respectively. Also under these conditions hyperosmolarity induced by adding 30 mmol/l NaCl to the perfusate stimulated peak renin secretion rates (normal calcium: from 7.8 ± 0.89 to 40.0 ± 2.2, P < 0.001; low calcium: from 28.4 ± 3.4 to 137.2 ± 10.3, P < 0.001; ANG II: from 5.8 ± 0.25 to 22.2 ± 1.7 ng ANG I·h -1 ·min -1 ·g -1, P < 0.05) ( Fig. 4, top ). However, the peak of renin secretion induced by +30 mmol/l NaCl was markedly lower in the presence of ANG II ( P < 0.05 vs. normal calcium) and was higher when extracellular calcium was low ( P < 0.001 vs. normal calcium). These effects were mirrored at a higher level in the presence of isoproterenol (10 nmol/l) ( Fig. 4, bottom ).
Fig. 4. Top : effects of prolonged administration of hyperosmolar NaCl (+30 mmol/l) on renin secretion from isolated, perfused rat kidneys at low ( ) and normal ( ) extracellular calcium concentration or in the presence of ANG II (300 pmol/l, ). Values are means ± SE of 5 experiments. * P < 0.05 peak value vs. period preceding NaCl addition. # P < 0.001 peak value vs. period preceding NaCl addition. Bottom : effects of prolonged administration of hyperosmolar NaCl (+30 mmol/l) on renin secretion from isolated, perfused rat kidneys at low ( ) and normal ( ) extracellular calcium concentration or in the presence of ANG II (300 pmol/l, ) during stimulation of renin secretion by isoproterenol (10 nmol/l). Values are means ± SE of 5 experiments. * P < 0.05 peak value vs. period preceding NaCl addition. # P < 0.001 peak value vs. period preceding NaCl addition. P < 0.05 average of last 3 values of NaCl period vs. period preceding NaCl addition.
Typically, the response of renin secretion to hyperosmolarity displayed two phases, namely, a shorter lasting peak and a following plateau phase, in which renin secretion only slowly declined but still remained elevated over control values ( Fig. 4, bottom,, average of last 3 values of NaCl period vs. control without NaCl, P < 0.001). In the presence of isoproterenol (10 nmol/l) at low extracellular calcium, renin secretion in response to 30 mmol/l NaCl behaved like the sum of the individual responses during isoproterenol at ambient calcium concentration and during low extracellular calcium in the absence of isoproterenol, i.e., a brief peak followed by a longer lasting sustained elevation of renin secretion (average of last 3 values of NaCl period vs. preceding period without NaCl, P < 0.001). Just the opposite was seen in the combined presence of isoproterenol (10 nmol/l) and ANG II (300 pmol/l), where 30 mmol/l NaCl produced a brief peak followed by a rather rapid normalization of renin secretion (average of last 3 values of NaCl period vs. preceding period without NaCl, not significant) ( Fig. 4, bottom ).
Although the peaks of renin secretion elicited by hyperosmolar NaCl (30 mmol/l) were smaller in the presence of ANG II and higher when extracellular calcium was low, a correlation between peak secretion rates and steady-state secretion rates revealed a common relationship for all the experimental maneuvers used to modulate steady-state renin secretion ( Fig. 5 ). Thus the peak secretion rates varied in parallel with steady-state renin secretion rates before the hyperosmotic challenge. The values in Fig. 5 are shown in a log-log plot to allow a display of renin secretion rates over a broad range. In addition, the log-log plot makes evident that the correlation between peak secretion and steady-state secretion follows a curve that is nearly parallel to the unity line, indicating that peak secretion rates and steady-state secretion rates are in constant proportion. On average, peak renin secretion rates are 3.5-fold higher than steady-state renin secretion rates.
Fig. 5. Relationship between steady-state renin secretion and maximum of renin secretion (peak secretion) in response to hyperosmolar NaCl. Steady-state renin secretion rates were varied by activation of the cAMP pathway (3, 10, and 30 nmol/l isoproterenol) by lowering the extracellular calcium concentration in the absence and in the presence of isoproterenol (10 nmol/l) or by adding ANG II (300 pmol/l) in the absence and presence of isoproterenol. Subsequently, 30 mmol/l NaCl was added to the perfusate, and renin secretion rates were determined. Solid line indicates unity values.
To distinguish whether it was the absolute osmolarity or rather changes in osmolarity that influence renin secretion, the NaCl concentration in the perfusate was increased stepwise, by 10 mmol/l up to 50 mmol/l. As shown in Fig. 6, the peak increments of renin secretion on the stepwise addition of NaCl became continuously smaller, and there was no further increase in renin secretion after the last steps, from +30 to +40 and to +50 mmol/l NaCl. On the return of osmolarity to normal, renin secretion fell rapidly. A next increase in osmolarity by 50 mmol/l NaCl stimulated renin secretion to higher values than the stepwise increase to +50 mmol/l ( P < 0.001), suggesting that it was primarily the change in osmolarity rather than the absolute osmolarity that influenced renin secretion.
Fig. 6. Comparison of the effects of graded increases (+10 mmol/l steps) in hyperosmolar NaCl to a final concentration of 50 mmol/l with a single step increase in osmolarity. Values are means ± SE of 5 experiments. * P < 0.05 peak value vs. last value of preceding period. # P < 0.001 peak value vs. last value of preceding period. P < 0.001 peak value vs. peak value of stepwise increase to 50 mmol/l NaCl.
A similar conclusion was reached from experiments in which a reduction of osmolarity was achieved by switching to a perfusate containing 30 mmol/l NaCl less than normal. Because renin secretion rates are rather low under control conditions and a reduction in osmolarity was supposed to suppress renin release, renin secretion was prestimulated by isoproterenol (10 nmol/l) ( Fig. 7 ). Indeed, the reduction in osmolarity reduced steady-state renin secretion in the presence of isoproterenol from 45.7 ± 2.9 to 24.7 ± 1.8 ng ANG I·h -1 ·min -1 ·g -1 ( Fig. 7 ) ( P < 0.05). Readdition of 30 mmol/l NaCl, thereby normalizing perfusate osmolarity (osmolarity of perfusate after readdition of NaCl 297.6 ± 2.1 mosmol/l; normal perfusate 296.9 ± 1.2 mosmol/l), stimulated renin secretion rates to 124.0 ± 10.4 ng ANG I·h -1 ·min -1 ·g -1 and therefore above steady-state renin secretion rates achieved with isoproterenol in normal perfusate (45.7 ± 2.9 ng ANG I·h -1 ·min -1 ·g -1, P < 0.001).
Fig. 7. Effects of reducing osmolarity by lowering the NaCl concentration by 30 mmol/l and subsequent normalization of the osmolarity by addition of 30 mmol/l NaCl on renin secretion from isolated, perfused rat kidneys in the presence of isoproterenol (10 nmol/l). Values are means ± SE of 5 experiments. * P < 0.05 vs. normal osmolarity with isoproterenol. # P < 0.001 vs. normal osmolarity with isoproterenol.
Altogether, these findings suggested that relative changes in osmolarity are more effective in determining renin secretion than the absolute value of osmolarity.
To assess the relevance of flow changes in the stimulatory effect of hyperosmolarity on renin secretion, hyperosmolarity was achieved by +30 mmol/l KCl. This concentration of KCl usually causes a complete depolarization-induced contraction of the renal vascular bed and a cessation of perfusion. To avoid a dramatic fall in perfusion, kidneys were preinfused with the calcium channel blocker amlodipine (10 µmol/l) ( Fig. 8 ). In this setting, 30 mmol/l KCl stimulated renin secretion from 60.4 ± 2.0 (preceding period) to 128.4 ± 11.3 ng ANG I·h -1 ·min -1 ·g -1 ( P < 0.001) ( Fig. 8, top ) and markedly lowered flow from 12.71 ± 0.85 to 8.31 ± 0.37 ml·min -1 ·g -1 ( P < 0.001) ( Fig. 8, bottom ), whereas 30 mmol/l NaCl increased renin secretion to 134.5 ± 11.4 ng ANG I·h -1 ·min -1 ·g -1 ( P < 0.001 vs. preceding recovery period, not significant. vs. 30 mmol/l KCl) and tended to increase flow from 11.49 ± 0.57 (preceding recovery period) to 12.53 ± 0.61 ml·min -1 ·g -1 (not significant) ( Fig. 8 ), clearly suggesting that directed changes in perfusate flow are not essential for the stimulatory effect of hyperosmolarity on renin secretion.
Fig. 8. Effects of hyperosmolar KCl or NaCl (+30 mmol/l) on renin secretion ( top ) or perfusate flow ( bottom ) in isolated, perfused rat kidneys in the presence of amlodipine and isoproterenol. Values are means ± SE of 5 experiments. # P < 0.001 vs. preceding period.
To address the question of whether the effects of osmolarity on renin secretion might be mediated by renal autacoids, we first inhibited prostaglandin formation by the cyclooxygenase inhibitor indomethacin (10 µmol/l). Although this concentration of indomethacin sufficiently blocked renal prostaglandin formation, indicated by significantly reduced urinary PGE 2 excretion (from control 29.2 ± 5.1 to 3.8 ± 0.7 pg/min, n = 4, P < 0.01), it did not affect renin release either at control conditions or in response to 30 mmol/l NaCl ( Fig. 9, ). Thus renin secretion rates increased from 8.2 ± 0.81 to 24.7 ± 2.9 ng ANG I·h -1 ·min -1 ·g -1 ( P < 0.001) in kidneys treated with indomethacin and from 9.0 ± 0.93 to 27.8 ± 1.8 ng ANG I·h -1 ·min -1 ·g -1 in untreated control kidneys ( P < 0.001; Fig. 9, ) without any significant differences between untreated and treated kidneys. In the next set of experiments, the application of acetylcholine in its maximum effective concentration of 10 µmol/l, hereby stimulating the liberation of nitric oxide and endothelium-derived hyperpolarizing factor, induced a significant vasodilation (not shown) and stimulated renin secretion from 7.4 ± 1.1 to 15.2 ± 1.0 ng ANG I·h -1 ·min -1 ·g -1 ( P < 0.05, Fig. 9, ). Subsequent elevation of osmolarity by application of 30 mmol/l NaCl in the presence of acetylcholine further increased renin secretion rates to 42.2 ± 4.3 ng ANG I·h -1 ·min -1 ·g -1 ( P < 0.001). Finally, the effect of a blockade of nitric oxide synthesis on the stimulation of renin secretion by a hyperosmolar stimulus was investigated. L -NAME (1 mmol/l) significantly lowered perfusate flow from 12.5 ± 0.56 to 6.1 ± 0.76 ml·min -1 ·g -1 ( P < 0.001, not shown) and tended to decrease renin secretion from 8.9 ± 0.8 to 5.7 ± 0.3 ng ANG I·h -1 ·min -1 ·g -1 (not significant) but did not prevent the stimulation of renin secretion by the addition of 30 mmol/l NaCl (17.1 ± 1.4 ng ANG I·h -1 ·min -1 ·g -1, P < 0.001). Although the absolute values of renin secretion in response to NaCl were higher in the presence of acetylcholine ( P < 0.001) and lower under L -NAME treatment ( P < 0.001) compared with control kidneys without pretreatment, the relative increase in renin secretion did not differ between the treatments (control kidneys 3.1-fold, acetylcholine 2.85-fold, L -NAME 3.0-fold of baseline without NaCl), confirming that renin secretion in response to hyperosmolarity changes in parallel with steady-state renin secretion before stimulation, according to the curve shown in Fig. 5. As a consequence of these data, it appeared likely that the effect of osmolarity on renin secretion does not essentially involve local intercellular signaling pathways and is therefore more direct at the level of the renin-secreting cells.
Fig. 9. Effects of hyperosmolar NaCl (+30 mmol/l) under control conditions ( ) or in the presence of indomethacin (10 µmol/l, ), acetylcholine (10 µmol/l, ), or N -nitro- L -arginine methyl ester ( L -NAME; 1 mmol/l, ). Values are means ± SE of 5 experiments, respectively. For analysis, the 4 values of each experimental period were averaged. * P < 0.05 vs. preceding period. # P < 0.001 vs. preceding period.
A consideration of the decline in renin secretion after the initial peak raised the question as to whether this decline in renin secretion reflected a kind of cellular adaptation to prolonged exposure to hyperosmolarity, such as a regulatory volume increase after a hyperosmotic shock. The regulatory increase is mainly mediated by ion influx through the Na-K-2Cl cotransport system ( 11 ), which is abundantly expressed in renin-secreting juxtaglomerular cells ( 14 ), or might involve swelling-activated chloride channels ( 27 ). Furthermore, these channels might be involved in the rapid decline in renin secretion observed after the stopping of hyperosmolar NaCl. We therefore examined the effect of the Na-K-2Cl cotransport inhibitor bumetanide (100 µmol/l) or the chloride channel blocker DIDS (300 µmol/l) on the response of renin secretion to hyperosmolarity. As shown in Fig. 10, bumetanide as well as DIDS both significantly stimulated renin secretion rates to twofold of control ( P < 0.05 vs. control respectively), but they did not influence the decline of renin secretion after the initial peak because the renin secretion rates reached plateau phases (6 and 8 min after the start of NaCl addition) that remained 2.1-fold elevated (for both bumetanide and DIDS) compared with the renin secretion rates before NaCl addition. The plateau phase of untreated control kidneys remained at 2.4-fold of control (not significant vs. DIDS- or bumetanide-treated kidneys). Furthermore, neither bumetanide nor DIDS prevented the rapid normalization of renin secretion rates after the stopping of NaCl administration.
Fig. 10. Effect of hyperosmolar NaCl (+30 mmol/l) on renin secretion in isolated, perfused rat kidneys in the presence of bumetanide (100 µmol/l, ) or DIDS (300 µmol/l, ). For comparison, results of control kidneys (see Fig. 9 ) are depicted. Values are means ± SE of 5 experiments. # P < 0.001 last 2 values of NaCl ("plateau phase") vs. preceding period.
We next considered the possibility that the decline in renin secretion after the initial peak could reflect an exhaustion of renin storage granules. To prevent cellular adaptation to continuous hyperosmolarity, we exposed the kidneys to a series of hyperosmotic pulses lasting for 4 min followed by 2-min periods of normal osmolarity. As shown in Fig. 11 ( top, ), these repetitive hyperosmotic pulses produced a decline in renin secretion with a time course that was not different from that seen with continuous hyperosmotic NaCl infusion ( ), suggesting, again, that cellular adaptation to hyperosmolarity is not the major reason for the characteristic time course of renin secretion during prolonged hyperosmolarity.
Fig. 11. Top : time of course of renin secretion in response to a continuous elevation of osmolarity (+30 mmol/l NaCl,, n = 5) or by hyperosmolar pulses of 4 min followed by 2-min recovery periods (, n = 7) by isolated, perfused rat kidneys in the presence of isoproterenol (10 nmol/l). Values are means ± SE. Bottom : time of course of renin secretion in response to a continuous elevation of osmolarity (+30 mmol/l NaCl,, n = 5; same data as in top ) or in response to a series of 4-min hyperosmolar pulses (+30 mmol/l NaCl) followed by 4-min recovery periods by isolated, perfused rat kidneys in the presence of isoproterenol (10 nmol/l). Values are means ± SE. * P < 0.05 vs. continuous NaCl at same time point.
If a similar pulse protocol of hyperosmolarity with hyperosmotic pulses lasting for 4 min followed by 4-min periods of normal osmolarity was applied, then the decline of renin secretion was obviously protracted ( Fig. 11, bottom, ), because the peak renin secretion rates remained significantly elevated compared with the respective time controls of continuous hyperosmolar NaCl infusion, suggesting that a recovery period of somewhat more than 2 min is required to maintain a constant response of renin secretion to repetitive pulses of hyperosmolarity.
DISCUSSION
Our study shows that changes in extracellular osmolarity, regardless of whether they are brought about by ions or by sugars, are powerful modulators of renin secretion from whole kidneys, in the way that an increase in osmolarity increases renin secretion, whereas a decrease in osmolarity lowers renin secretion. We are aware that these highly reproducible and species-independent effects are at odds with a number of in vitro studies showing that a decrease rather than an increase in osmolarity stimulates renin secretion from kidney slices, isolated glomeruli ( 3, 5 - 7 ), and cultured juxtaglomerular cells ( 4, 17 ). However, our findings are in full accordance with a previous study performed in dogs showing that acute increases in renal plasma osmolarity enhance renin secretion ( 29 ). That study described an instantaneous about threefold increase in renin secretion due to increases of renal plasma osmolarity of 30-50 mosmol/l, independent of the osmolyte used.
All of these characteristics were also seen in our study in isolated perfused mouse and rat kidneys. Moreover, Young and Rostorfer ( 28 ) described a rebound of renin secretion on normalization of osmolarity, and they reported an increase in perfusion in response to hyperosmolarity, as also seen in our study. In addition, recently Toma and Peti-Peterdi ( 25 ) provided evidence that hyperosmotic sugars stimulate renin secretion from isolated perfused juxtaglomerular apparatuses. Thus the effects of osmolarity changes on renin secretion apparently depend on the experimental model used. The inhibitory influence of osmolarity on renin secretion is seen even in permeabilized cells, suggesting an osmotic effect on renin storage granules themselves. In fact, it has been reported that a decrease in osmolarity directly stimulates renin release from a renin vesicle preparation ( 20 ). One may speculate, therefore, that in isolated, nonperfused preparations, in which physiological interstitial pressure and wall tension are not defined, an osmotic effect on storage vesicles becomes predominant.
Under more physiological settings such as in the perfused kidney, in which renin-producing cells are under wall stress, increases in osmolarity stimulate renin secretion.
Our data show that renin secretion stimulated by hyperosmolarity is sensitive to changes in osmolarity rather than to absolute osmolarity itself, suggesting an involvement of cell volume changes. In fact, it is known that osmolarity influences renal vascular resistance secondarily to changes in cell volume, in the way that hyposmolarity-induced cell swelling increases electrical and mechanical activity of vascular smooth muscle and vice versa ( 8, 27 ), likely by the modulation of mechanosensitive cation channels. However, we could not find any evidence for the functional relevance of these channels in the regulation of renin release by changes in osmolarity, because blockade of the channels by DIDS attenuated neither the stimulation of renin secretion by high osmolarity nor normalization of renin release after the stopping of NaCl infusion. The characteristic increase in renal perfusion in response to hyperosmolarity could be relevant for the stimulation of renin secretion. However, our data for hypertonic KCl, which stimulates renin secretion but decreases perfusate flow through the kidneys, argue against a dependence of renin secretion on changes in flow. Moreover, we obtained no evidence that endothelial autacoids such as prostaglandins, endothelium-derived hyperpolarizing factor, or nitric oxide are relevant to the stimulation of renin secretion by hyperosmolar challenge. Finally, because the blockade of NaCl transport by the Na-K-2Cl - cotransporter in the thick ascending limb of Henle and the macula densa did not alter the effects of osmolarity on renin secretion, the observed effects appear not to be mediated by the macula densa mechanism. In sum, we infer that the mechanisms initiating the stimulatory effect of osmolarity on renin secretion reside in the renin-secreting cells themselves.
Given that changes in cell volume are relevant in this context, this raises the question of how those changes could affect renin secretion. One possibility could be a change in ion channel activity in the plasma membrane. There is evidence that cation channels in vascular smooth muscle cells are more active in swollen cells ( 12 ), leading to depolarization. Conversely, membrane shrinkage hyperpolarizes cells. To counteract a possible effect of hyperpolarization, we used hyperosmolar KCl, which depolarizes cells, as indicated by its contractive effect on the renal vasculature. Because KCl and NaCl had very similar effects on renin secretion, we assume that a change in membrane potential is not an important mediator of the effect of hyperosmolarity on renin section. Moreover, as mentioned above, blockade of swelling-activated chloride channels by DIDS did not affect the regulation of renin release by changes in osmolarity. Also, changes in calcium channel activity appear not to be essential, because the stimulatory effect of hyperosmolarity was also preserved in a calcium-free perfusate.
Our data show that the effects of changes in osmolarity are of rapid onset and are almost instantaneously reversible. A characteristic feature of the effects of osmolarity changes on renin secretion from the isolated, perfused kidney is the appearance of two phases, an initial rapid transient followed by a more stable plateau. The rapid "on-off" kinetic of the effect of hyperosmolarity on renin secretion resembles the effect of hyperosmolarity on the release of synaptic vesicles from a so-called readily releasable pool of vesicles ( 23 ), which has also been defined for other secretory cells ( 1, 5, 13, 22 ). Readily releasable vesicles are ready for fusion with the plasma membrane and just await the final signal to open the fusion pore of secretory vesicles ( 1, 13, 15, 22 ). The plateau level of secretion following the peak could reflect the flux of committed vesicles through the pool of rapidly releasable vesicles.
We are aware that this comparison is somewhat hampered by the fact that in our experiments we measured the sum of the secretory behavior of tens of thousands of cells instead of a single or a small group of cells. In any case, the constant relationship between peak secretion rates and steady-state secretion rates suggests that only 30% of the vesicles or vesicle contents ready for secretion actually undergo exocytosis. It should be noted that this proportion remains constant regardless of whether cAMP or calcium is high or low in the renin-secreting cells.
A common feature of a readily releasable pool of vesicles in different secretory cells is that it can be exhausted and can be refilled ( 1, 13, 15, 22 ). Our data also suggest that renin secretion stimulated by hyperosmolarity can be exhausted and that its complete reestablishment takes a few minutes.
Given that the stimulation of renin secretion evoked by hyperosmolarity reflects the existence of a readily releasable pool of renin secretory vesicles, the constant proportion between steady-state renin secretion and the osmotic excitable renin secretion would then suggest that the pool size of committed vesicles itself rather than a final maturation of committed vesicles determines the secretion rate of renin from juxtaglomerular epithelioid cells.
If so, then the low renin secretion rates in the presence of ANG II should be attributed to a small pool of readily releasable vesicles, whereas in the presence of isoproterenol, which activates the cAMP signaling cascade, the pool is larger. It is thought that readily releasable vesicles are already functionally docked to the plasma membranes ( 1, 13, 15, 22 ). Transferring this concept to the physiology of renin-secreting cells would mean that the cAMP pathway favors, whereas a calcium-dependent process inhibits, the functional docking of renin vesicles to the plasma membrane.
In summary, our data clearly show that changes in osmolarity are powerful regulators of renin secretion from isolated, perfused mouse and rat kidneys, an increase in osmolarity stimulating and a decrease inhibiting, the exocytosis of renin. These effects occur most likely directly on the level of the juxtaglomerular cells because the stimulation of renin release by high osmolarity is very rapid in onset, is not attenuated by blockade of tubular salt transport or by blockade of prostaglandin or nitric oxide formation, and is not dependent on directed changes in perfusate flow. In view of the fact that <10% changes in osmolarity and probably of cell volume exert already strong effects on renin secretion, it appears conceivable that also changes in local osmolarity or acute changes in the cell volume of renin-secreting cells could be part of the physiological control of renin secretion. Moreover, our data fit into the concept of exocytosis comprising the existence of different pools of secretory vesicles. Whether the functional docking of renin vesicles to the plasma membrane is indeed controlled by cAMP or intracellular calcium concentration, thereby finally determining renin release, needs to be addressed in future studies.
GRANTS
This study was financially supported by grants from the Deutsche Forschungsgemeinschaft (KU859/13-1; SCHW778/2-1).
ACKNOWLEDGMENTS
The expert technical assistance provided by Marlies Hamann and Karlheinz Götz is gratefully acknowledged.
【参考文献】
Bratanova-Tochkova TK, Cheng H, Daniel S, Gunawardana S, Liu YJ, Mulvaney-Musa J, Schermerhorn T, Straub SG, Yajima H, and Sharp GW. Triggering and augmentation mechanisms, granule pools, and biphasic insulin secretion. Diabetes 51, Suppl 1: S83-S90, 2002.
Cooke SF, Craven DJ, and Symonds EM. Osmolarity changes can enhance the release of renin from human chorion. Am J Obstet Gynecol 151: 819-821, 1985.
Frederiksen O, Leyssac PP, and Skinner SL. Sensitive osmometer function of juxtaglomerular cells in vitro. J Physiol 252: 669-679, 1975.
Friis UG, Jensen BL, Aas JK, and Skøtt O. Direct demonstration of exocytosis and endocytosis in single mouse juxtaglomerular cells. Circ Res 84: 929-936, 1999.
Holdsworth S, McLean A, Morris BJ, Dax E, and Johnston CI. Renin release from isolated rat glomeruli. Clin Sci Mol Med Suppl 3: 97s-99s, 1976.
Jensen BL and Skøtt O. Osmotically sensitive renin release from permeabilized juxtaglomerular cells. Am J Physiol Renal Fluid Electrolyte Physiol 265: F87-F95, 1993.
Jensen BL and Skøtt O. Renin release from permeabilized juxtaglomerular cells is stimulated by chloride but not by low calcium. Am J Physiol Renal Fluid Electrolyte Physiol 266: F604-F611, 1994.
Johansson B and Jonsson O. Cell volume as a factor influencing electrical and mechanical activity of vascular smooth muscle. Acta Physiol Scand 72: 456-468, 1968.
Kopp UC and DiBona GF. Effects of physiological increments in renal arterial plasma osmolality on renin secretion rate. J Lab Clin Med 102: 250-259, 1983.
Kurtz A and Penner R. Angiotensin II induces oscillations of intracellular calcium and blocks anomalous inward rectifying potassium current in mouse renal juxtaglomerular cells. Proc Natl Acad Sci USA 86: 3423-3427, 1989.
Lang F, Busch GL, Ritter M, Volkl H, Waldegger S, Gulbins E, and Haussinger D. Functional significance of cell volume regulatory mechanisms. Physiol Rev 78: 247-306, 1998.
Lang F, Busch GL, Zempel G, Ditlevsen J, Hoch M, Emerich U, Axel D, Fingerle J, Meierkord S, Apfel H, Krippeit-Drews P, and Heinle H. Ca 2+ entry and vasoconstriction during osmotic swelling of vascular smooth muscle cells. Pflügers Arch 431: 253-258, 1995.
Li J and Schwarz TL. Genetic evidence for an equilibrium between docked and undocked vesicles. Philos Trans R Soc Lond B Biol Sci 354: 299-306, 1999.
Plotkin MD, Kaplan MR, Peterson LN, Gullans SR, Hebert SC, and Delpire E. Expression of the Na + -K + -2Cl - cotransporter BSC2 in the nervous system. Am J Physiol Cell Physiol 272: C173-C183, 1997.
Schneggenburger R, Sakaba T, and Neher E. Vesicle pools and short-term synaptic depression: lessons from a large synapse. Trends Neurosci 25: 206-212, 2002.
Scholz H and Kurtz A. Disparate effects of calcium channel blockers on pressure dependence of renin secretion and flow in the isolated perfused rat kidney. Pflügers Arch 421: 155-162, 1992.
Schricker K and Kurtz A. Role of membrane-permeable ions in renin secretion by renal juxtaglomerular cells. Am J Physiol Renal Fluid Electrolyte Physiol 269: F64-F69, 1995.
Schweda F and Kurtz A. Cellular mechanism of renin release. Acta Physiol Scand 181: 383-390, 2004.
Schweda F, Wagner C, Kramer BK, Schnermann J, and Kurtz A. Preserved macula densa-dependent renin secretion in A1 adenosine receptor knockout mice. Am J Physiol Renal Physiol 284: F770-F777, 2003.
Sigmon DH and Fray JC. Chemiosmotic control of renin release from isolated renin granules of rat kidneys. J Physiol 436: 237-256, 1991.
Skøtt O and Taugner R. Effects of extracellular osmolality on renin release and on the ultrastructure of the juxtaglomerular epithelioid cell granules. Cell Tissue Res 249: 325-329, 1987.
Sorensen JB. Formation, stabilisation and fusion of the readily releasable pool of secretory vesicles. Pflügers Arch 448: 347-362, 2004.
Stevens CF and Williams JH. "Kiss and run" exocytosis at hippocampal synapses. Proc Natl Acad Sci USA 97: 12828-12833, 2000.
Strbak V and Greer MA. Regulation of hormone secretion by acute cell volume changes: Ca 2+ -independent hormone secretion. Cell Physiol Biochem 10: 393-402, 2000.
Toma IMK and Peti-Peterdi J. High glucose directly stimulates renin release from juxtaglomerular cells (Abstract). FASEB J 19: 669, 2005.
Twu BM, Cannon ME, and Hsu CH. The role of osmolarity in renin release from afferent arterioles. J Hypertens 7: 757-761, 1989.
Welsh DG, Nelson MT, Eckman DM, and Brayden JE. Swelling-activated cation channels mediate depolarization of rat cerebrovascular smooth muscle by hyposmolarity and intravascular pressure. J Physiol 527: 139-148, 2000.
Young DB and Rostorfer HH. Blood flow and filtration rate responses to alterations in renal arterial osmolarity. Am J Physiol 225: 1003-1008, 1973.
Young DB and Rostorfer HH. Renin release responses to acute alterations in renal arterial osmolarity. Am J Physiol 225: 1009-1014, 1973.
作者单位：Physiologisches Institut der Universität Regensburg, Regensburg, Germany