INTRODUCTION

Top Abstract Introduction Conclusions Perspective References

DURING THE LAST DECADE a number of in vivo and in vitro studies have been performed to characterize the physiological role of nitric oxide (NO) in the control of renal renin synthesis and secretion. These studies, conducted under a variety of different experimental conditions, have, however, produced conflicting results and have led to controversy about the role of NO in control of renin secretion (cf. 112, 118). Therefore, there is currently no clear concept about the physiological impact of NO in the regulation of renin secretion and synthesis nor in its mode of action in renal juxtaglomerular granular (JGG) cells (112). This contribution therefore aims to bring some order to this puzzling situation by sorting the findings with respect to comparable experimental conditions. Furthermore, we will attempt to develop a consensus about the possible action of NO in renal JGG cells.

Renin synthesis and secretion are controlled by a variety of systemic parameters. The renin-angiotensin-aldosterone (RAAS) cascade plays an important role in the blood pressure and electrolyte and fluid homeostasis of the organism. The activity of the renin-angiotensin system in the circulation is mainly dependent on the activity of the protease renin, which is considered as the key regulator of the system. Renin found in the circulation predominantly originates from the kidneys, where renin is primarily produced by the so-called JGG cells. These cells are located in the medial layer of the afferent arteriole adjacent to the vascular poles of the glomeruli (7, 147). JGG cells develop from vascular smooth cells by a reversible metaplastic transformation (7, 8, 147). This particular differentiation of smooth muscle cells is associated by a marked change in cell morphology such that numerous granular (renin storage) vesicles of various sizes and shape appear, whereas the number of myofilaments decreases (8, 147). The morphological appearance of the cells becomes more epithelioid rather than smooth muscle cell-like. The number of renal renin-producing cells is not constant. In general, the number decreases with increasing age but is at any time subject to rapid changes in response to an altered requirement for renin. Thus, in states of chronic renin stimulation, additional smooth muscle cells in the afferent arterioles switch to renin-producing cells, whereas in states of chronic renin suppression the opposite occurs. Which intracellular events trigger and control the transition of smooth muscle cells into JGG cells and back is not yet known. Similarly the intracellular regulations of renin gene expression and also of renin secretion are incompletely understood. There is, meanwhile, consensus that cAMP is a potent stimulatory signal for renin secretion and renin synthesis, whereas the cytosolic calcium activity and probably also protein kinase C (PKC) activity exert negative control functions (26, 27, 51, 76, 77). At the organ level, renin secretion and renin synthesis in JGG cells are uniformly controlled by systemic factors such as the rate of sodium intake, by the renal perfusion pressure, and the rate of sympathetic outflow and renal nerve activity, as well as by circulating levels of ANG II (26, 51, 76, 77).

Renin-producing cells are encircled by cells with a high capacity for NO formation. The renin system in JGG cells is also influenced by local factors generated by adjacent cells. JGG cells are directly surrounded by four different cell types, namely, smooth cells of the afferent arterioles, endothelial cells covering the interior of the afferent arterioles, mesangial cells of the glomeruli, and the tubular macula densa cells. It is well known that the tubular macula densa cells influence JGG cells by an as yet undefined "macula densa signal," which has an inhibitory influence on renin secretion and renin synthesis (15). This rather elusive signal is generated in response to the salt transport activity of the macula densa cells, such that renin secretion changes inversely with the concentration of NaCl in the tubular fluid (and consequently salt transport) at the macula densa site (143).

NO has attracted considerable interest as a possible controller of renin secretion, since both macula densa cells and endothelial cells are sites of substantial NO formation (5) (Fig. 1). There is a high-level expression of NO synthase type I (nNOS) in the macula densa cells (93, 152, 156) and of NO synthase type III (eNOS) in endothelial cells (4, 154). It is already well established that NO formed within the kidney acts as a potent vasodilator that determines the basal tone of the renal arterioles independently of the myogenic autoregulation of renal blood flow (cf. 74, 99). Evidence for a functional role for NO produced in the juxtaglomerular region has been elaborated by studying the tubuloglomerular feedback system. Endogenous NO significantly modulates the feedback response by exerting a specific buffer function (24, 149, 151). Thus inhibition of NO formation exaggerates the afferent vasoconstriction in response to delivery of increased salt to the macula densa, whereas addition of NO attenuates the afferent vasoconstriction. In view of the high capacity of macula densa cells for NO formation, it is likely that NO is involved in the macula densa mechanism mediating tubuloglomerular feedback. Since the macula densa controls both afferent arteriolar tone and also renin secretion (15), it was reasonable to consider a role for NO in the control of renin secretion from renal JGG cells. Another reason to consider the influence of NO on the renin system resulted from findings that the endothelium effectively modulates the function of neighboring smooth muscle cells and that JGG cells are modified smooth muscle cells. A role of NO in the control of the renin system is furthermore suggested by the sequential appearance of NO formation and of renin expression in the kidney during ontogeny (35).

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Renal juxtaglomerular granular cells are encircled by endothelial, mesangial, and tubular macula densa cells, which have a high capacity for nitric oxide (NO) formation. eNOS, NO synthase type III; nNOS, NO synthase type I.

	INFLUENCE OF NITRIC OXIDE ON RENIN SECRETION IN VIVO

The physiological influence of NO on the renin system in vivo has been addressed by the administration of L-arginine, as substrate for NO formation, and by the use of substances considered as general or selective inhibitors of NO synthases.

The influence of L-arginine administration on plasma renin activity (PRA) has been examined in humans (58, 59, 106) and rats (61, 132). In these studies, no significant effect of acute L-arginine administration on plasma renin was observed, probably because L-arginine substrate is not rate limiting for NO formation under normal conditions.

Effects of NOS inhibitors on basal renin secretion in vivo depends on dosage and duration of treatment. A substantial number of reports describe the influence of NOS inhibitors on PRA in vivo. At first glance, these experiments in rats, rabbits, dogs, sheep, pigs, and humans have produced conflicting results, since increases, decreases, or even no changes of PRA have been reported with NOS inhibitors. A more systematic analysis of the data, however, reveals that the overall effect of NOS inhibitors on the renin system in vivo is determined by the duration of treatment and the dosage of the NOS inhibitors (Table 1). Thus short-term applications including bolus injections, infusions, or oral application lasting from a few hours to 1 or 2 wk have frequently been reported to lower PRA, once the dose exceeded a certain threshold. Above this threshold, N-monomethyl-L-arginine (L-NMMA) (48, 66), nitro-L-arginine (L-NAG) (23, 120, 138), and nitro-L-arginine methyl ester (L-NAME) (3, 20, 21, 38, 46, 66-68, 73, 85, 95, 100, 107, 129, 131, 138, 140) reduce basal PRA in rats, dogs, and rabbits. Lower doses of NOS inhibitors were reported not to change (25, 33) or to slightly increase basal PRA values (61, 121, 124). A novel approach to inhibit NO-mediated actions is by the use of cross-linked hemoglobin, which scavenges NO. Infusion of cross-linked hemoglobin in dogs exerts hemodynamic actions similar to NOS inhibitors and produces a significant decrease of basal PRA values (17). In humans, sheep, or pigs, only single reports are available. Short-term intrarenal infusion of an extrapolated daily dose of 4 mg/kg was reported to increase PRA in piglets by ~50% but not to change PRA in adult pigs (144). Similarly, a daily dose of 10 mg/kg N-nitro-L-arginine (NOLA) did not change PRA in adult sheep, despite an increased mean arterial pressure (153). In humans, a trend to decrease basal PRA values was found for a single dose of 3 mg/kg of L-NAME (54). Taken together, the majority of findings suggest that acute and subchronic inhibition of NO action in vivo has an inhibitory rather than stimulatory effect on basal renin secretion. Apparently, the acute inhibitory effect of NOS blockers on renin secretion is rather dose dependent, as has clearly been demonstrated in several studies (3, 28, 100). Time course studies have revealed that inhibition of renin secretion switches to stimulation of renin secretion during chronic treatment with NOS inhibitors (110, 156). Consequently, PRA values are frequently found to be elevated if the treatment with NOS inhibitors is extended over several weeks in rats (2, 38, 60, 84, 92, 115, 145, 158). This elevation of PRA is associated with severe hypertension, leading to changes in organ morphology including ventricular hypertrophy and/or structural changes in the kidney. Thus the question arises as to whether the marked increase of PRA is directly due to the chronic inhibition of NOS activity or is secondary to hypertension-induced kidney damage. There are several observations supporting the latter assumption. L-NAME treatment of rats for 2 wk decreased PRA, whereas prolongation of the treatment over 4 wk led to enhanced PRA in association with the appearance of structural changes in the kidney (38). A chronic study with two different doses of L-NAME showed that the lower dose causing no obvious organ damage was associated with decreased PRA values, whereas the higher dose led to organ alterations and increased PRA levels (157). In accordance with these findings are other observations that PRA values in animals with chronic NOS inhibition were particularly elevated in those animals which displayed clear secondary signs of hypertension (2, 69). The increases of PRA during chronic treatment with NOS inhibitors are most prominent in spontaneously hypertensive rats (SHR) rats, which develop malignant hypertension with characteristic renal lesions (84, 145). Finally, PRA values remain elevated in the severely hypertensive animals for weeks after withdrawal of the NOS inhibitors (92). All together, it appears that during chronic treatment with NOS inhibitors, changes of renin secretion are probably determined by events secondary to chronic NOS inhibition such as hypertension rather than by events directly related to changes in NO formation. Furthermore, the enhancement of renin secretion during hypertension induced by NOS inhibition is part of a vicious circle maintaining or aggravating hypertension. Thus a number of studies suggest that the renin-angiotensin system is relevant for the hypertension during chronic NOS blockade (38, 60, 69, 84, 89, 92, 97, 98, 108, 158) and that hypertension and renal damage can be prevented by ANG II antagonists.

The acute pressor effect of NOS inhibition, however, appears to be independent of the systemic RAAS (44, 52, 96, 109, 139, 159), which is compatible with the notion that acute NOS inhibition inhibits, rather than stimulates, renin secretion.

The availability of genetically manipulated mice lacking NOS synthases provides another approach by which to gain information about the potential role of NO for renin secretion. A first study done with eNOS knockout mice reported a twofold increase of basal PRA (136). Such an elevation of PRA in animals chronically lacking eNOS would be in line with those experiments using chronic administration of NOS inhibitors. However, too little is known about the regulation of the renin system in animals lacking eNOS activity to allow clear physiological conclusions from these preliminary data.

Prestimulation of the renin system unmasks a stimulatory role of NO for renin secretion. An inhibitory effect of acute NOS blockade on renin secretion in vivo becomes more clear in situations with prestimulated renin secretion. Thus NOS inhibitors substantially attenuate the rise of renin secretion induced by a reduction in renal perfusion pressure in dogs and rats (73, 95, 107, 128). NOS inhibitors also attenuate the increase in renin secretion in response to low sodium intake in rats and dogs (10, 28, 86, 87, 132, 157) and blunt the increased renin secretion induced by ANG II antagonists in rats (131, 148). Furthermore, NOS inhibitors impair the stimulation of renin secretion by furosemide in humans, rabbits, and rats (12, 83, 113, 130, 148) suggesting that macula densa stimulation of renin secretion is also attenuated by NOS inhibition. Finally, NOS inhibitors have been found to blunt the stimulation of renin secretion induced by -adrenoceptor activation in rabbits (21, 111). NOS inhibitors also further attenuate PRA in states of high salt intake (28), and also the effects are more marked during low salt intake, although the percent changes are quite similar during low and high salt intake (28, 42). Again, a few exceptions have been reported, since NOS inhibitors were reported not to change PRA in response to a fall in renal artery pressure in rats (68), in response to hemorrhage in rabbits (20), and in response to macula densa inhibition in dogs (124). In no instance, however, has an enhancement of prestimulated PRA values been reported with either acute or subchronic treatment with NOS inhibitors.

NOS inhibitors lower renal renin mRNA abundance in vivo. Data on the influence of NO on renin synthesis in vivo have been obtained with the measurement of renin mRNA abundance in the kidneys of rats under subchronic treatment with NOS inhibitors. NOS inhibition moderately decreased basal renin mRNA levels and renal renin content (128, 148). Accordingly, basal renin mRNA levels are also moderately reduced in mice with a disrupted eNOS gene (136) and renal renin content was reported to be reduced in mice lacking nNOS activity in the macula densa (53). In situations with an activated renin system accompanied by elevated renin mRNA levels, clear inhibitory effects of NOS blockers become apparent. Thus NOS inhibitors blunt the rise of renin mRNA in response to a fall of renal artery pressure (128), to ANG II antagonists (131, 148), to low sodium intake (132), and to furosemide (130, 148) in rats. For mice with disrupted NOS genes no data about renin mRNA levels during stimulation have been reported. NOS inhibitors have also been shown to prevent the characteristic retrograde recruitment of renin-expressing cells along the afferent arteriole during stimulation by ANG II antagonists (131).

Direct vs. indirect effects of NO on the renin system in vivo. Considering the marked systemic side effects of prolonged administration of NOS inhibitors, in vivo experiments with acute or subchronic administration of NOS inhibitors are likely to be more informative about the physiological influence of NO on renin secretion. The consensus from such studies is that the overall effect of NO on renin secretion and renin synthesis in vivo is stimulatory rather than inhibitory and that this effect becomes more obvious in situations of a stimulated renin system. Thus NO appears to act more as a general enhancer than a specific stimulator of renin secretion and synthesis, since NOS inhibition interferes with all classic regulators of the renin system, as outlined above. However, it is difficult to separate direct and indirect effects of NO on the renin system from these in vivo experiments. For example, an effect of NOS inhibitors in vivo is the well-known elevation in blood pressure, and since renal renin secretion and renin gene expression are inversely related to the renal perfusion pressure (26, 51), increases in blood pressure could account for the effect of NOS inhibitors on the renin system. Attempts have therefore been made to distinguish between pressure-dependent and pressure-independent effects on renin release in vivo. These experiments suggest that changes in blood pressure may contribute to, but do not essentially mediate, the effects of NOS inhibitors on the renin system. It was found in this context that in animals with servocontrolled renal perfusion pressure, NOS inhibitors attenuate renal renin release (68). Moreover, there is no clear correlation between the changes in blood pressure induced by NOS inhibitors and the changes of the renin system (132).

Another effect of NOS inhibitors in vivo that might indirectly impact on the renin system is a change of sympathetic nerve activity. It is well established that renal sympathetic nerve activity as well as circulating catecholamines are physiologically relevant stimulatory determinants for renin secretion and renin gene expression (26, 51). Direct measurements have revealed a stimulation of renal nerve activity upon application of NOS inhibitors in vivo (119), an effect that has been considered to be relevant for the development of hypertension by NOS inhibitors (88) and that would be expected to stimulate renin secretion. This may explain those studies reporting a stimulation of renin secretion upon acute systemic administration of NOS inhibitors and could also account for the stimulation of renin secretion caused by intracerebral administration of NOS inhibitors (32). When NOS inhibition itself leads to a net fall in PRA, renal denervation is reported to have no impact on PRA (68). The observation that blockade of -adrenoceptors during acute NOS inhibition converts an inhibition to a stimulation of renin release (138) may be related more to central effects of -blockade rather than to direct effects on the level of JGG cells.

Several studies have considered the involvement of endothelins in the increases of vascular resistance and of blood pressure during acute and subchronic administration of NOS inhibitors in vivo (6, 37, 44, 96), which is relevant in this context because endothelins inhibit renin secretion and renin synthesis (75). It has been recently reported that the inhibitory effects of subchronic NOS inhibition on renin secretion and renin gene expression in vivo are attenuated by concomitant treatment with an endothelin antagonist (148). However, additional experiments will be required to determine whether an interaction occurs between NO and endothelins in the control of the renin system.

Direct intrarenal administration of drugs that influence the NO system should have less secondary actions on renin secretion, but the results obtained are conflicting. Thus, in conscious rats treated with L-NAME for several days, intrarenal infusion of the NO donor sodium nitroprusside (SNP) stimulated renin secretion (73). In anesthetized dogs, intrarenal administration of NOS inhibitors moderately decreased basal renin secretion and markedly blunted the stimulation of renin secretion in response to a fall of renal artery pressure (95). Intrarenal infusion of 7-nitroindazole (7-NI), a preferential nNOS blocker, was reported not to change basal renin secretion but to attenuate renin secretion stimulated by macula densa blockade in anesthetized rats (12). In contrast to these studies, it was reported that in anesthetized dogs, intrarenal administration of a low dose of NOS inhibitor increases basal renin secretion and does not attenuate renin secretion stimulated by macula densa blockade (124). In anesthetized pigs, low-dose intrarenal administration of NOS inhibitors did not change basal renin secretion in adult animals but stimulated renin secretion in young animals (144). Another study suggested that NOS inhibition may alter proximal reabsorption and delivery of sodium to the macula densa and that this was a prominent factor in the change in renin secretion observed (124). At present, the available data are too conflicting to allow a conclusion about the directional effect of local, intrarenal NOS inhibition on renin secretion.

Taken together, these studies indicate that the influence of NO on the renin system in vivo is probably determined by the net result of indirect and direct effects on renal JGG cells.

	INFLUENCE OF NITRIC OXIDE ON RENIN SECRETION IN VITRO

Since it is difficult to delineate the relative contributions of direct and indirect effects to the overall action of NO on renin release, it is necessary to further consider the effect of NO in vitro. The influence of NO on renin secretion in vitro has already been investigated in a number of experimental models ranging from isolated perfused kidneys to isolated JGG cells. Since the different experimental models have produced variable effects of NO on renin secretion, we will separately consider the findings with regard to the experimental model used.

NO appears as a stimulator of renin secretion from isolated kidneys. The role of NO on renin secretion from isolated perfused rat kidneys has been assessed by using native stimulators of NO formation such as acetylcholine, NO donors such as SNP or morpholino-sydnonimin-hydrochloride (SIN-1), and NOS inhibitors such as L-NAME, L-NMMA, or N-nitro-L-arginine (L-NNA).

Acetylcholine is known to evoke the release of NO from the endothelium (39). Addition of acetylcholine to the perfusate of isolated perfused kidneys was found to stimulate renin secretion (50, 94, 127), and this stimulation was blunted by adding NOS inhibitors to the perfusate (125), suggesting that the stimulation of renin secretion by acetylcholine may have involved the formation of NO. Also addition of the NO donors SNP or SIN-1 to the perfusate causes prompt and dose-dependent stimulation of renin secretion (50, 79, 127). At normal perfusion pressure, the amplitude of stimulation of renin secretion by physiological or pharmacological agents that release NO is relatively small compared with classic stimuli such as a reduction of renal perfusion pressure or -adrenoceptor agonists. The stimulatory effect of the NO releasers on renin secretion is dependent on the perfusion pressure such that secretion is enhanced at low perfusion pressure and is impaired at high perfusion pressures (127). The inhibitory effect of perfusion pressure on renin release is calcium dependent (127). A recent report that renin secretion stimulated by NO donors is attenuated at higher calcium activities and is enhanced by lower calcium activities (79) may explain the calcium-related effect of the perfusion pressure on the stimulatory role of NO on renin secretion from isolated kidneys.

Inhibitors of NO formation such as L-NAME, L-NMMA, or L-NAG have been found to decrease the basal release of renin from isolated rat kidneys (42, 43, 50, 127). They also decrease renin secretion from kidneys taken from animals kept on a low- or high-salt diet (42), which have a high and low basal renin secretion rate, respectively. This observation fits well with in vivo studies reporting that NOS inhibitors lower renin secretion during both low and high sodium intake (28, 158). Both these in vivo and in vitro studies showed that NOS inhibitors cause a decrease of renin secretion during different rates of sodium intake, with the relative changes being similar, whereas the absolute changes are dependent on the degree of prestimulation of the renin system. Moreover, it has been found that inhibitors of NO formation markedly attenuate the stimulation of renin secretion evoked by a fall in renal perfusion pressure, which is attributed to the so-called "baroreceptor" control of renin secretion (127). Notably, NOS inhibitors did not inhibit renin secretion at perfusion pressures above normal in the isolated perfused kidney, which is in agreement with the in vivo observations (95, 107). Finally NOS inhibitors also attenuated renin secretion stimulated by -adrenoceptors (80) or by macula densa blockade (80).

Whether this obvious stimulatory influence of NO on renin secretion from whole kidneys reflects a direct influence on renal JGG cells cannot be determined from these experiments. It should be kept in mind that in isolated perfused kidneys the macula densa mechanism for the control of renin secretion is still active, and it has been suggested that the macula densa is important for the influence of NO on renin secretion (124). In view of the potential relevance of macula densa-derived NO, it cannot be ruled out that the overall stimulatory effect of NO on renin secretion may have resulted from interference with the macula densa mechanism. However, perfusion experiments with isolated hydronephrotic rat kidneys devoid of functioning macula densa structures have also shown that inhibitors of NOS lowered renin secretion, whereas NO stimulated renin secretion (50). It is reasonable to assume therefore that NO may exert a direct, macula densa-independent effect on renin secretion in the whole kidney.

Taken together, these findings suggest that in the isolated perfused kidney, NO is a stimulator of renin secretion. There are striking parallelisms between findings with isolated perfused kidneys and with in vivo observations in that the role of NO under basal conditions appears to be moderate, whereas it becomes more apparent during situations of stimulation. In addition, NO appears to be relevant in virtually all conditions of altered renin secretion and appears to somehow define the "gain" of the response of renin secretion to challenge, very similar to the in vivo situation, where NO acts as a general enhancer of the renin system as outlined above.

Effect of NO on renin secretion on the level of JGG cells appears to be complex. Other in vitro experiments to study the influence of NO on the renin system were done with kidney slices. The results obtained with native stimulators of NO formation were conflicting in that acetylcholine was reported to inhibit renin secretion (155), whereas purinoreceptor activation was suggested to stimulate renin secretion (22), and both effects were suggested to be mediated by NO. More clearly, the NO donor SNP was found to inhibit renin secretion from kidney slices (13, 56), whereas inhibitors of NO synthases were reported to stimulate basal renin secretion from kidney slices (11, 13). Thus taking these findings with short-term application of NO donors and NOS inhibitors, one can see that the overall effect of short-term changes in NO on renin secretion in incubated kidney slices appears to be inhibitory.

Further experiments to study the role of NO in control of renin secretion in vitro were done in isolated renal afferent arterioles. In arterioles with intact juxtaglomerular apparatus including macula densa and glomerulus, NO exerted a dual effect on renin secretion. If an NOS inhibitor was administered via microperfusion to the macula densa, then the stimulation of renin secretion by low NaCl concentration at the macula densa site was attenuated (55). Conversely, L-arginine, the natural substrate for NO formation enhanced renin secretion via the macula densa site (55). These findings fit well with data obtained in vivo (12, 130) or in isolated kidneys (79). However, macula densa-induced stimulation of renin secretion in this isolated juxtaglomerular apparatus in vitro preparation was markedly inhibited if the NO donor SNP or the NO precursor L-arginine were applied directly onto the JGG cell portion of the afferent arterioles (55).

Other experiments used afferent arterioles isolated from rats pretreated with NOS inhibitors in vivo. Those arterioles displayed reduced levels of renin mRNA and renin content and had a reduced spontaneous renin secretion in vitro (19). The stimulation of renin secretion by the adenylate cyclase stimulator forskolin was abolished in those arterioles but was restored by the addition of the NO donor SIN-1 in vitro (19).

Similar to the observations made with the microdissected juxtaglomerular apparatus, acute administration of NO donors to isolated JGG cells was also reported to inhibit renin secretion (40, 47, 78, 133), whereas either L-arginine or NOS inhibitors per se exerted no effect on renin secretion from isolated JGG cells (47, 81, 135). In freshly dispersed renal cortical cells, NO donors at higher concentrations also inhibit renin secretion, whereas at lower concentrations they stimulate renin secretion (101). In cultured JGG cells, the initial inhibition of renin secretion after addition of an NO switches to a significant stimulation of renin secretion with a delay of 1 to 2 h (40, 133). If isolated JGG cells are cocultured on endothelial cells, then NOS inhibitors or arginine deprivation not only prevent the formation of NO in the cocultures but also decrease basal renin secretion (81, 135). Together, these findings could indicate that prolonged exposure of isolated JGG cells to NO exerts a stimulatory effect on renin secretion, whereas short-term exposure to higher concentrations of NO inhibits renin secretion.

Does the macula densa determine the effect of NO on renin secretion? One possibility to explain the contradictory findings on the influence of NO on renin secretion is that the macula densa is the primary determinant of the effect of NO on renin secretion in the whole kidney and that a striking difference exists between the effect of NO from the macula densa and that originating from endothelial cells. This is supported by the findings discussed above that NO formation at the macula densa site has a positive effect on renin secretion, whereas direct administration of NO to JGG cells blocks renin secretion in the same preparation (55). Thus macula densa-derived NO may stimulate renin secretion, whereas endothelium-derived NO inhibits renin secretion. In agreement with this possibility, 7-NI (which is considered as a more specific blocker of nNOS activity and consequently of NOS activity in the macula densa; Ref. 91), was reported to attenuate the stimulation of renin secretion through the macula densa in a rather selective fashion in rats (10, 12). In contrast, in anesthetized dogs intrarenal application of a general NOS inhibitor was reported to stimulate basal renin secretion but not to change macula densa-stimulated renin secretion, and it was inferred from these data that the macula densa inhibits rather than stimulates renin secretion by an NO-dependent mechanism (124). In the isolated perfused hydronephrotic rat kidney devoid of macula structures, stimulation of NO formation was reported to stimulate renin secretion, whereas NOS inhibitors attenuated renin secretion (50), suggesting that in the absence of the macula densa, NO can stimulate renin secretion. Finally, in experiments with isolated perfused normal rat kidneys, it was found that the stimulation of renin secretion induced by blockade of macula densa transport was markedly attenuated by a general NOS inhibitor but that NO donors also stimulated renin secretion during blocked macula densa function (80).

All together, the findings presently available do not allow one to conclude that a major difference exists between the effects of NO generated in and outside of the macula densa cells.

	CONCLUSIONS

Top Abstract Introduction Conclusions Perspective References

The experiments on the overall effect of NO on renin secretion in vivo, in isolated kidneys, and in other in vitro preparations together suggest that the tonic effect of NO on renin secretion is primarily stimulatory. NO appears to act as a general enhancer of renin secretion and renin synthesis rather than as a specific activator. In in vitro preparations, acute administration of NO donors appears to inhibit renin secretion, whereas a more prolonged availability of NO exerts a stimulatory effect on renin secretion. Since the in vivo situation and the isolated perfused kidney probably represent conditions of continual availability of NO, the data obtained in vivo and in isolated kidneys, kidney slices, and isolated JGG cells are not necessarily contradictory. Apparently, the tonic stimulatory effect of NO becomes interrupted in the in vitro situation of kidney slices, microdissected juxtaglomerular apparatuses or isolated JGG cells, where the effect of NO switches to an inhibition of renin secretion.

cGMP likely mediates the effect of NO on renin secretion. Assuming that both the inhibition of renin secretion and the stimulation of renin secretion are specific events due to a direct influence of NO on juxtaglomerular cells, we raise the question concerning the nature of the intracellular pathways mediating those opposing effects. Although there is evidence for cGMP-independent intracellular actions of NO on secretion, such as stimulation of insulin secretion from pancreatic -cells (123) via calcium release from mitochondria (82), the best established intracellular effect of NO is the stimulation of soluble guanylate cyclase activity leading to the enhanced formation of cGMP (64, 90, 122). Native NO and NO donors stimulate cGMP formation in isolated JGG cells (78, 133, 135), suggesting that the NO-guanylate cyclase-cGMP pathway exists in native JGG cells. In fact, both the stimulatory effects of NO in isolated kidneys (80) and isolated JGG cells (133) in freshly dispersed renal cortical cells (102), as well as the acute inhibitory effect of NO in isolated JGG cells (47), were found to be abrogated by guanylate cyclase inhibitors, suggesting an involvement of cGMP in both stimulation and inhibition of renin secretion by NO. The role of cGMP in renin secretion has already been assessed by a number of studies using either membrane-permeable analogs of cGMP or atrial natriuretic peptide as an activator of particulate guanylate cyclase activity in JGG cells. Notably, the results of these studies on the effect of cGMP on renin secretion in vitro were as conflicting as those obtained for the effect of NO itself. Membrane-permeable cGMP analogs such as 8-bromo-cGMP or 8-para-chlorophenylthio-cGMP (8-pCPT-cGMP) at concentrations higher than 5 µmol/l were found to inhibit renin secretion from isolated perfused kidneys (80), from kidney slices (56), from isolated JGG cells (47, 56, 63, 78, 133), and from dispersed renal cortical cells (102). At lower concentrations, cGMP analogs had either no effect on renin secretion from kidneys slices (146) or were reported to slightly increase renin secretion from dispersed renal cortical cells (102, 146).

Atrial natriuretic peptide was found to inhibit renin secretion from isolated kidneys (103), from kidneys slices (1, 56, 57), from isolated glomeruli (70), from isolated JGG cells (47, 56, 78, 133), from freshly dispersed renal cortical cells (102), and from nephroblastoma cells (30). On the other hand, there are also reports of a stimulatory effect of atrial natriuretic peptide on renin secretion from isolated kidneys (49, 127) and from dispersed renal cells (102, 146). Similarly, intrarenal infusion of atrial natriuretic peptide in conscious dogs was found to stimulate renin secretion (31). Overall, the effect of cGMP on renin secretion in JGG cells in vitro is variable, ranging from inhibition to stimulation of renin secretion, similar to the effects of NO donors.

A dual effect of cGMP could explain variable effects of NO on renin secretion. When considering the temporal relationship between guanylate cyclase activation by NO and renin secretion in isolated JGG cells, it becomes obvious that maximal activation of cGMP formation is associated with inhibition of renin secretion, whereas renin secretion appears to increase when cGMP formation is more modest (133). Together with the findings discussed above on the inhibitory effect of cGMP analogs, this supports the concept of a direct inhibitory action of cGMP on renin secretion in JGG cells. The pathways through which cGMP influences intracellular functions include the activation of cGMP-dependent kinases, the activation of cyclic nucleotide-gated ion channels, and the activation or inhibition of cAMP-phosphodiesterases (cAMP-PDEs) (9, 122). Since membrane-permeable cGMP analogs (e.g., 8-bromo-cGMP or 8-pCPT-cGMP) have a very low affinity to cAMP-PDEs (16), it is unlikely that the inhibitory effects of these compounds on renin secretion are mediated by reductions in cellular cAMP levels. There is, moreover, yet no direct or indirect evidence for the existence of cyclic nucleotide-triggered currents in JGG cells (77). As a consequence, inhibition of renin secretion by cGMP-dependent protein kinase (G-kinase) activation appears to be the more likely explanation of the inhibitory effect of cGMP on renin secretion. This assumption is supported by the findings that infusion of G-kinase inhibitors into isolated kidneys stimulates renin secretion (79) and that membrane-permeable cGMP analogs that are specific high-affinity activators of G-kinase (16) inhibit renin secretion from a variety of preparations. Two different G-kinases have been characterized, named GK-I and GK-II (122), and both kinases have been demonstrated in JGG cells (41). This is notable since within the renal vascular tree, GK-II appears to be selectively expressed in the JGG cells (41). Given that cGMP can inhibit renin secretion via G-kinase activation, it is conceivable that NO activation of G-kinases could account for the inhibition of renin secretion observed in vitro, as mentioned before. The manner in which G-kinase activation could inhibit renin secretion is not yet known.

In experiments with isolated perfused kidneys, it was found that the stimulatory effect of an NO donor on renin secretion was enhanced during G-kinase inhibition but was attenuated during inhibition of the cAMP-dependent protein kinase (A-kinase) (79), suggesting that the stimulatory effect of NO on renin secretion could be related to the cAMP pathway. In comparison with the membrane-permeable cGMP analogs, native cGMP demonstrates a lower affinity to G-kinases but a higher affinity to cAMP-PDEs (16). The family of cAMP-PDEs includes two members that are regulated by cGMP, namely, PDE2, which is activated by cGMP, and PDE3, which is inhibited by cGMP (9). Since cAMP is the major established stimulator for renin secretion and renin gene expression (26, 27, 51, 76), modulation of cAMP levels by cGMP could, in principle, mediate the effects of NO on renin secretion and gene expression. Although no evidence has yet indicated a functional role of PDE2 on the effects of NO on renin secretion, there are accumulating data of a central involvement of PDE3. Thus it was shown in anesthetized rats that pharmacological inhibition of PDE3 virtually mimics the stimulatory effect of NO on renin secretion (21). Consequently, a concept was developed by Reid and associates (20, 21, 112) proposing that the stimulatory role of NO on the renin system is related to an increase of cAMP levels induced by inhibition of PDE3. This concept was recently supported by an extensive study in isolated perfused rat kidneys, which showed that PDE3 inhibitors, but not other PDE inhibitors, fully mimicked and at the same time completely abrogated additional stimulatory effects of endogenous or exogenous NO on renin secretion from this preparation (80). It is known that PDE3 is distributed over a variety of tissues including blood vessels, where it is thought to participate in the vasodilatory properties of NO (9). The existence of PDE in the renal vasculature has already been demonstrated (114), and more recently we found that afferent arterioles express PDE3 at a rather high level (unpublished data).

Another indirect mechanism by which native cGMP could stimulate renin secretion is via transactivation of the A-kinase by cGMP (18, 36). It was recently suggested that NO-induced stimulation of renin secretion may be mediated by activation of the A-kinase and a cytosolic calcium reduction in the JGG cells (124). Comparison of the relative affinities of native cGMP and of membrane-permeable cGMP analogs for G-kinase, A-kinase, and PDE3 (16), however, suggests that transactivation of A-kinase is a less likely explanation.

Although further experiments are required to firmly establish the particular intracellular pathways involved in the effects of NO on renin secretion and renin gene expression, one may summarize from the information presently available that NO could exert both an inhibitory and a stimulatory effect on the renin system (Fig. 2). The inhibitory effect is probably mediated by activation of G-kinase, whereas the stimulatory effect appears to be mediated by A-kinase via inhibition of cAMP degradation. Such a dual control of renin secretion by NO would not only be compatible with the divergent findings about the effect of NO on renin secretion as outlined above, but might also explain different overall effects of NO on renin secretion in the whole kidney compared with isolated preparations. A stimulation of renin secretion by NO through cAMP via inhibition of cAMP degradation requires a significant background formation of cAMP via adenylate cyclase activity. A tonic stimulation of adenylate cyclase in JGG cells in the whole kidney could be provided by local factors such as catecholamines, prostaglandins, adrenomedullin, and calcitonin gene-related peptide and others (65, 76). In isolated JGG cells, background stimulation of adenylate cyclase activity by local factors is likely to be low, resulting in a predominance of the inhibitory G-kinase pathway upon elevation of endogenous cGMP by either NO or atrial natriuretic peptide.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Comprehensive demonstration of the possible pathways along which NO could influence renin secretion from renal juxtaglomerular cells. AC, adenylate cyclase; GC, guanylate cyclase; PDE3, cAMP phosphodiesterase 3; A-kinase, cAMP-dependent protein kinase; G-kinase, cGMP-dependent protein kinase; PKC, protein kinase C; PGE2, prostaglandin E2; PGI2, prostacyclin; CGRP, calcitonin gene-related peptide; RG, renin granule.

The putative existence of such a dual effect of NO on the renin system raises the question about the determinants for the overall effect of NO. As outlined in detail before, the overall effect of NO both in vivo and in the isolated kidneys is normally stimulatory, suggesting that the stimulatory A-kinase effect predominates over the inhibitory G-kinase effect. Since the stimulation of renin secretion and renin gene expression via A-kinase in general is significantly impaired in states of enhanced cytosolic calcium and PKC activity in JGG cells (51, 116, 117), it may be that in those instances of reduced cAMP efficacy, an inhibitory effect of NO via G-kinase could become visible. In fact, experiments with isolated perfused kidneys have provided evidence that the renin stimulatory effect of NO donors is greatly enhanced at low extracellular calcium concentrations, whereas in the presence of the hormone ANG II, which mobilizes calcium and activates PKC in JGG cells (76) and which per se inhibits renin secretion (51), NO donors cause a further inhibition of renin secretion (79). This is in agreement with findings obtained with kidney slice experiments in which stimulation of endogenous cGMP formation by atrial natriuretic peptide did not change basal renin secretion, but enhanced the inhibition of renin secretion by ANG II (57). Thus, in addition to the background activity of adenylate cyclase, cytosolic calcium activity and/or PKC activity in JGG cells could be major determinants of the overall effect of NO on renin secretion and renin gene expression (Fig. 2).

Evidence for altered NO formation in states of altered renin secretion. Considering the high-level expression of NOS in the macula densa and the important influence of the macula densa on JGG cells, it is possible that macula densa-derived NO could be directly involved in the macula densa control of renin secretion. Although the stimulation of renin secretion by low rates of macula densa salt transport is attenuated by general NOS inhibition (80, 129) and by more specific inhibition of NO production by the macula densa (12, 57), there is no direct information as to whether changes of macula densa NO production directly mediate macula densa-triggered changes in renin secretion.

An altered release of NO from the macula densa is suggested by changes in macula densa nNOS gene and protein expression. Prolonged NOS inhibition was found to downregulate both nNOS gene and protein expression in the macula densa and renin expression in JGG cells (14). Similarly, in the contralaterals of hypoperfused kidneys, both macula densa nNOS and renin were reported to be downregulated, whereas in the stenosed kidneys, nNOS expression and renin synthesis and secretion were upregulated (14, 130), indicating parallel changes of renin synthesis and nNOS expression in these instances. Further information about a parallel regulation of the renin system and renal nNOS was obtained in conditions of altered sodium intake, with nNOS expression in the macula densa increasing during low sodium intake and decreasing during high sodium intake (14, 134, 140). These changes would fit well with a report about an enhanced renal excretion of nitrates in rats during dietary sodium restriction (101). However, it must be mentioned in this context that sodium restriction has also been found to diminish urinary nitrite and nitrate levels (28, 137). Furthermore, it was found that macula densa NOS expression is markedly upregulated in angiotensinogen knock-out mice (71), which are also expected to be sodium deficient. Additional sodium restriction in these animals causes a further increase of NOS expression in the macula densa (72).

If these changes of nNOS expression are at all indicative of respective changes of macula densa NO formation, then one could imagine that the macula densa produces more NO in states of sodium deficiency and of renal hypoperfusion. This enhanced NO production might then contribute to the stimulation of renin secretion and of renin gene expression under these circumstances. The mechanisms that induce these changes in macula densa nNOS expression are currently unknown. One critical issue in this context is that changes in nNOS expression may be secondary to the changes of the renin system, rather than being the reason for the change of the renin system. This could be resolved by determination of the kinetics of changes of nNOS and of renin expression in response to low sodium intake or to renal hypoperfusion. Since stimulation of renin secretion and of renin gene expression by ANG II antagonists or by furosemide in rats that are in sodium balance is not accompanied by altered nNOS expression (132), changes in renin secretion do not obligatorily cause changes in nNOS expression in the juxtaglomerular apparatus. This suggests that the stimulations of nNOS expression in the macula densa are not secondary to the stimulation of the renin system. In view of the changes of macula densa nNOS expression and the sensitivity of renin secretion toward NOS inhibition during states of sodium deficiency, it is likely that in this situation, an enhanced NO release from the macula densa contributes importantly to the stimulation of the renin system. In contrast, there is only a moderate increase of nNOS expression during renal hypoperfusion despite strong stimulation of the renin system. In fact, NOS inhibitors only attenuate but cannot abrogate the stimulation of the renin system under this condition (107, 128). Therefore other factors apart from NO must contribute to the activation of the renin system during renal hypoperfusion. The stimulations of the renin system by ANG II antagonists (132) or by furosemide (in salt-supplemented rats) (132) are not associated with changes of nNOS expression. Nonetheless, the increases of renin secretion and of renin gene expression induced by these drugs can be substantially attenuated by NOS inhibitors (128, 129, 146), suggesting that in these situations a tonic release of NO from the macula densa may be relevant for the stimulation of the renin system. Such a general tonic stimulatory enhancer function of NO would be compatible with an effect of NO on cAMP levels, which would provide a stimulus-independent enhancer mechanism for renin secretion and renin gene expression. According to this hypothesis, locally produced NO would play a permissive, stimulatory role for the renin system, which is counteracted by inhibitors of the renin system such as the increased perfusion pressure, by ANG II, or by the macula densa signal. Once these inhibitory stimuli are "switched off," e.g., during a fall in renal perfusion pressure, during blockade of ANG II receptors, or during inhibition of the macula densa mechanism, the stimulatory effect of NO then becomes apparent. This inference drawn from in vivo experiments is in accordance with the idea that it is the balance between inhibitory and stimulatory effects of NO that determines the overall effect of NO on renin secretion and that this balance is influenced by the calcium/PKC activity within JGG cells.

Since the biological range of NO is locally restricted because of its very short half-life, it is difficult to imagine that macula densa-derived NO could account for the stimulation of renin expression distant from the glomerular vascular pole such as occurs during retrograde recruitment of renin-producing cells, which may occur at 100 µm or even more from the macula densa. It is reasonable therefore to also assume a major role of the eNOS in regulation of the renin system, in particular, for the recruitment of renin-producing cells. Such a role of endothelium-derived NO is also suggested by experiments with hydronephrotic kidneys devoid of macula densa structures (50). Furthermore, acetylcholine, a well-known stimulator of endothelial NO formation (5), enhances renin secretion from isolated kidneys (50, 125) and inhibits renin secretion from kidney slices (155) in an NO-dependent fashion, suggesting that hormonal modulation of endothelial NO production can regulate renin secretion. Shear stress is another determinant of endothelial NO formation (64, 90); presumably an enhanced endothelial NO formation occurs in states of increased shear stress in the afferent arterioles. Increased shear stress probably occurs during increased renal perfusion pressure secondary to increased blood flow velocity and decreased luminal diameter because of the myogenic autoregulation of renal blood flow. The role of flow-related NO formation on renin secretion has not yet been investigated.

Recent observations suggest that also acute increases of ANG II may favor the release of NO (34, 45, 105). Also, for whole kidneys (142) and for larger renal resistance arteries, a stimulation of NO release by ANG II was reported (150). More continuous elevations of the ANG II concentration, however, were found not to change or even to decrease NO formation (29). Whether ANG II also evokes the release of NO in renal afferent vessels at all is currently also not clear (104).

	SUMMARY AND FUTURE PERSPECTIVES

Top Abstract Introduction Conclusions Perspective References

Taking all of the available information, it seems likely that NO is normally a stimulator of renin secretion and renin synthesis on the level of the whole kidney. NO appears to be a general enhancer of renin release, and this effect may be mediated by a reduction in rate of cAMP degradation via inhibition of cAMP-PDE3. In parallel, NO might also exert an inhibitory effect via cGMP-stimulated protein kinase. This inhibitory effect is normally overridden by the stimulation via the cAMP pathway, but may become apparent under certain conditions such as an increase of the calcium activity in JGG cells. This could explain those findings that suggest an inhibitory effect of NO on renin secretion.

Future experiments will have to prove the validity of the concept about a dual control of renin secretion by NO via cGMP. In this context, the role of G-kinase for renin secretion and renin synthesis will have to be characterized in more detail. If the concept of a dual control of renin secretion by NO via A-kinase and G-kinase turns out to be correct, then characterization of the intracellular factors determining either the stimulatory effect via cAMP or the inhibitory effect via G-kinase will deserve attention. Finally, the cellular and molecular events underlying the changes of macula densa NOS expression, particularly during changes in salt intake, require elucidation.