Is renin secreted by exocytotic mechanism through mature renin granules from juxtaglomerular cells?

Mature renin granules were isolated by the combination of discontinuous and continuous Percoll density gradient centrifugation. Stored renin in the renin granules was found to consist of isoelectrically seven different forms. The seven different isoelectric points (pIs) were 5.6, 5.35, 5.2, 5.0, 4.8, 4.6 and 4.4. Approximately 70% of the stored renin as the total enzymatic activities from all isoelectric peaks was found in a peak which pI corresponded to be 5.35. Renin secreted from isolated glomeruli was also focused into seven peaks possessing identical values. However, the distribution pattern of renin peaks was quite different from that of stored renin. In the secreted renin, peaks of 5.35 (pI) and 5.2 (pI) showed high renin activity and each had approximately 30% of released renin as the total recovered. These results indicate multiple forms of renin are stored and secreted by rat kidney. As the distribution pattern of enzymatic activities in renin peaks between stored renin and secreted renin are different, it is probable that renin may not secreted through mature renin granules by exocytotic mechanism.     

ATP-dependent mechanism for coordination of intercellular Ca2+ signaling and renin secretion in rat juxtaglomerular cells

A change in intracellular Ca2+ is considered to be the common final signaling pathway through which renin secretion is governed. Therefore, information relating to the generation, control, and processing of Ca2+ signaling in juxtaglomerular cells (JG) will be critical for understanding JG cell behavior. In this study, we investigated the means by which JG cells harmonize their intracellular Ca2+ signals and explored the potential role of these mechanisms in renin secretion. Mechanical stimulation of a single JG cell initiated propagation of an intercellular Ca2+ wave to up to 11.9+/-4.1 surrounding cells, and this was prevented in the presence of the ATP-degrading enzyme, apyrase (1.7+/-0.7 cells), or by desensitization of purinergic receptors via pretreatment of cells with ATP (1.8+/-0.9 cells), thus implicating ATP as a mediator responsible for the propagation of intercellular Ca2+ signaling. Consistent with this, JG cells were demonstrated not to express the gap junction protein connexin43, and neither did they possess functional gap junction communication. Furthermore, massive mechanical stretching of JG cells elicited a 3-fold increase in ATP release. Administration of ATP into isolated perfused rat kidneys induced a rapid, potent, and persistent inhibition of renin secretion, together with a transient elevation of renal vascular resistance. ATP (1 mmol/L) caused up to 79% reduction of the renin secretion activated by lowering the renal perfusion flow (P<0.01). Taken together, our results indicate that under mechanical stimulation, ATP functions as a paracellular mediator to regulate renin secretion, possibly through modulating intra- and intercellular Ca2+ signals.     

Adenylyl cyclase isoform v mediates renin release from juxtaglomerular cells

We have shown previously that decreasing intracellular calcium in the juxtaglomerular cells increases both cAMP formation and renin release. We hypothesized that this is because of an interaction between intracellular calcium and the calcium-inhibitable isoform of adenylyl cyclase, type-V. We used primary cultures of juxtaglomerular cells isolated from C-57/B6 mice at 70% to 80% confluence. Western blots were performed on isolated juxtaglomerular cells using antibodies against either of the 2 calcium inhibitable isoforms of adenylyl cyclase, types-V and -VI. Only the antibody against adenylyl cyclase-V gave us a strong band at 120 kDa as expected. Immunolabeling in juxtaglomerular cells with confocal microscopy found immunofluorescence for the adenylyl cyclase-V-specific antibody compared with either negative controls or cells stained with the adenylyl cyclase-VI antibody. Reducing isolated juxtaglomerular intracellular calcium with 100 micromol/L of the cytosolic calcium chelator BAPTA-AM stimulated both cAMP (3.49+/-0.70 to 10.09+/-0.81 pmol/mL per milligram of protein; P<0.002) and renin release (1001.8+/-81.5 to 1648.0+/-139.1 ng of angiotensin I per milliliter per hour per milligram of protein; P<0.01). The selective adenylyl cyclase-V inhibitor NKY80 completely blocked both BAPTA-AM-stimulated cAMP formation and renin release. We conclude that lowering intracellular calcium is permissive, allowing an increased activity of the calcium-inhibitable isoform adenylyl cyclase-V (but not adenylyl cyclase-VI) in the juxtaglomerular cell, producing cAMP, which stimulates renin secretion.     

Atrial natriuretic peptide inhibits renin release from juxtaglomerular cells by a cGMP-mediated process.

We have examined the effect of a synthetic analogue of human alpha-atrial natriuretic peptide (ANP), APII, on renin release in cultured renal juxtaglomerular cells (JGA cells). Using cell cultures containing 80-90% renal juxtaglomerular cells, we found that ANP (10(-13)-10(-9) M) strongly inhibited renin release from the cells in a dose-dependent fashion (ki, 10 pM) to about 10% of control. Inhibition of renin release by ANP was paralleled by an increase in cellular cGMP levels; while in the presence of the cGMP-phosphodiesterase inhibitor M&B 22948 (1 mM), concentrations of ANP lower by a factor of 100 were required to obtain the same effects on renin release and cGMP levels. The guanylate cyclase inhibitor methylene blue (10 microM), on the other hand, shifted the dose-response curves for renin release and cGMP levels to 100-fold higher concentrations of ANP. Neither the influx of 45Ca into the cells nor the intracellular quin-2 signal, which is a measure for changes of intracellular Ca concentration, was in any way altered by ANP. Our results suggest that ANP inhibits renin release from juxtaglomerular cells by a cGMP-dependent process that does not involve changes in intracellular calcium.     

Control of renin secretion.

The present study was designed to examine the interrelationship between the intrarenal vascular receptor and the sympathetic nerve, beta-adrenergic system, for renin secretion in the anesthetized dog. 1) A reduction in renal arterial pressure from a control pressure to 100 mmHg changed neither ther flow rates of all cortex zones nor renin secretion. Further reduction of renal arterial pressure to 75 mmHg resulted in a significant increase of renin secretion and a decrease of blood flow in the outer cortex. Intrarenal arterial infusion of norepinephrine at a control pressure increased a renin secretion. However, norepinephrine infusion at a reduced pressure suppressed the renin release with a recovery of the vascular resistance to the control level. These results suggest that the changes in the degree of blood flow and pressure in the renal afferent arterioles are not essential for the renin secretion,but renin secretion by the pressure reduction might be related to the autoregulatory capacity of afferent arterioles in the outer cortex. 2) At 5 min of hemorrhagic period (75 mmHg) arterial PRA elevated in control, and phenoxybenzamine and propranolol treated groups and any significant difference in responses was not observed among groups. However, at 60 min of hemorrhagic hypotensive period PRA in control and phenoxybenzamine treated groups further increased, but PRA in propranolol treated group was not alter from its 15 min value. These results indicated that the roles of vascular receptor and renal sympathetic nervous sytem in receptor and renal sympathetic nervous system in renin secretion might be separated, and that the renal sympathetic nervous system did not relate to the early response of renin release, but related to the late response. 3) Intrarenal arterial infusion of cAMP and DbcAMP resulted in a significant increase of renin release. In addition, CaC12 solution was infuesed into the renal artery and a significant rise in renal venous PRA was observed within 5 min of infusion.These data suggested that a beta-adrenergic receptor-adenyl cyclase-cAMP system was involved in the control of renin secretion, and that since the intracellular effect of cAMP was partly related to the change of intracellular Ca distribution, its change resulted in an increase in renin secretion.     

A biphasic effect of noradrenaline on renin release from rat juxtaglomerular cells in vitro is mediated by alpha 1- and beta-adrenoceptors.

The direct effect of noradrenaline on renin release from juxtaglomerular (JG) cells in vitro were investigated in a dynamic superfusion system of dispersed rat renal cortical cells. At low concentrations (1-100 nmol/l), noradrenaline stimulated renin release in a dose-dependent manner, while at higher concentrations (0.1-1 mmol/l) it inhibited renin release. The stimulatory effect of 0.1 mumol noradrenaline/l was completely blocked by a beta-adrenoceptor antagonist, propranolol (0.1 mumol/l). When applied at concentrations of 1 mumol/l or 10 mumol/l, noradrenaline had no consistent effect on renin release, although 10 mumol noradrenaline/l had an inhibitory effect in the presence of propranolol (0.1 mumol/l). The inhibitory effect of noradrenaline (0.1 mmol/l) was converted to a stimulatory effect by the addition of an alpha 1-adrenoceptor antagonist (bunazosin, 1 mumol/l), but was not altered by the addition of an alpha 2-adrenoceptor antagonist (yohimbine, 1 mumol/l). These results indicate that low concentrations of noradrenaline directly stimulate renin release from JG cells by the activation of beta-adrenoceptors, while high concentrations of noradrenaline inhibit renin release by the activation of alpha 1-adrenoceptors. Accordingly, a dynamic balance may exist between beta-adrenergic stimulation and alpha 1-adrenergic depression of renin release.     

Control of renin synthesis and secretion

The aspartyl protease renin is the rate limiting activity of the renin-angiotensin-aldosterone system (RAAS). Renin is synthesized as an enzymatically inactive proenzyme which is constitutively secreted from several tissues. Only renin-expressing cells in the kidney are capable of generating active renin from prorenin, which is stored in prominent vesicles and which is released into the circulation upon demand. The acute release of renin is controlled by cyclic adenosine monophosphate (cAMP) and by calcium signaling pathways, which in turn are activated by a number of systemic and local factors. Longer lasting challenges of renin secretion lead to changes in the number of renin-producing cells, which occur by a metaplastic transformation of renin cell precursors such as preglomerular vascular smooth muscle or extraglomerular mesangial cells. This review aims to briefly address the state of knowledge of these various aspects of renin synthesis and secretion and attempts to relate them to the in vivo situation, in particular in men.     

Transmural pressure control of prorenin processing and secretion in diabetic rat juxtaglomerular cells.

In diabetic patients, the elevation of plasma prorenin levels or arterial pressure is correlated with the severity of diabetic nephropathy. This study was designed to assess the effects of transmural pressure on prorenin regulation in juxtaglomerular (JG) cells from diabetes rats. The JG cells, harvested from rats intraperitoneally injected with streptozotocin 7 (early-diabetic) or 28 (late-diabetic) days previously, were exposed to atmospheric pressure (AP) and AP+40 mmHg for 12 h, and the renin secretion rate (RSR), prorenin secretion rate (PRSR), active renin content (ARC), prorenin content (PRC), and total renin content (TRC) were determined. Exposure of control JG cells to AP+40-mmHg significantly decreased RSR, PRSR, and ARC and significantly increased PRC without affecting TRC, suggesting the occurrence of pressure-mediated inhibition of prorenin processing and secretion. Exposure of early-diabetic and late-diabetic cells to AP+40-mmHg significantly decreased ARC and significantly increased PRC without affecting RSR, PRSR, or TRC. The changes in ARC and PRC were similar in the control and early-diabetic cells, but greater changes were observed in late-diabetic cells. However, when streptozotocin-treated rats were continuously treated with insulin (9 U/kg/day), the transmural pressure control of prorenin in JG cells was similar to that observed in the JG cells from control rats. In late-diabetic cells, treatment with a phospholipase C inhibitor did not alter the pressure control of ARC or PRC; however, treatment with a phospholipase D inhibitor did inhibit the changes in ARC and PRC with transmural pressure. Thus, pressure-mediated inhibition of prorenin secretion from JG cells has already been impaired in early diabetes. Pressure-induced inhibition of prorenin processing in JG cells via phospholipase D-dependent pathways is enhanced in late diabetes.     

Endogenous or overexpressed cGMP-dependent protein kinases inhibit cAMP-dependent renin release from rat isolated perfused kidney, microdissected glomeruli, and isolated juxtaglomerular cells

An overactive renin-angiotensin-aldosterone system (RAAS) has a central role in the pathogenesis of hypertension and cardiac hypertrophy, precursors of cardiac failure. Natriuretic peptides and NO acting through their second messenger, cGMP, increase natriuresis and diuresis, and inhibit renin release; however the mechanism by which this inhibition of the RAAS system functions is obscure. We recently reported cloning of the cDNA for type II cGMP-dependent protein kinase (cGK II), elucidated its first known function of inhibiting the cystic fibrosis transmembrane conductance regulator in rat intestine, and initially described its location in rat kidney juxtaglomerular (JG) cells, the ascending thin limb, and the brush border of proximal tubules. Here, we demonstrate inhibition of isoproterenol- or forskolin-stimulated renin release by 8-para-chlorophenylthio-cGMP (8-pCPT-cGMP), a selective activator of cGK, and prevention of this inhibition by a selective inhibitor of cGK, Rp-8-pCPT-cGMPS. In systems of differing complexity, inhibition by 8-pCPT-cGMP was nearly complete in isolated perfused kidney and microdissected afferent arterioles but only ≈25% in isolated JG cells. Expression of either cGK II or cGK I in JG cells by using adenoviral vectors enhanced the inhibition of forskolin-stimulated renin release by 8-pCPT-cGMP to 50%. Our results indicate that cGK II, and possibly cGK I, can mediate cGMP inhibitory effects on renin release and are physiological components of the cGMP signal transduction system which opposes the RAAS.     

Immunoelectron microscopic localization of renin in the juxtaglomerular cells of the amphibian Bufo bufo

The ultrastructural localization of renin in the juxtaglomerular apparatus of the kidney of the toad Bufo bufo has been examined using an immunogold staining method for electron microscopic immunocytochemistry and an antiserum to renin isolated from the submandibular gland of the mouse. Renin immunoreactivity was confined to lamellated granules in the cytoplasm of epitheloid or juxtaglomerular cells in the glomerular afferent arterioles and also in the media cells of larger arteries. Mouse kidney tissue, examined for purposes of comparison, showed immunolabeling limited to the granules of the juxtaglomerular cells. The presence of renin or a renin-like substance in the juxtaglomerular granules of the toad kidney is discussed in relation to the lysosomal nature of these granules. A model is presented linking the lysosomal function of the juxtaglomerular granules and the release of renin mediated by beta-adrenergic receptors present on the surface of the juxtaglomerular cells.     

Angiotensin II immunoreactivity coexists with renin in the juxtaglomerular granular cells of the kidney.

The multiple physiologic functions of angiotensin II(AII) are generally supposed to be mediated by the peptide generated in the blood circulation. In addition to this extracellular mechanism of AII formation, we have obtained immunohistochemical evidence for the intracellular synthesis of AII in the kidney. Rats were perfused with fixative, and paraffin sections of the kidneys were processed with antisera against renin (EC 3.4.99.19), AII, and other components of the renin--angiotensin system. Renin immunoreactivity was regularly observed in the epithelioid granular cells in the media of the afferent vessel of the glomerulus. AII immunoreactivity was found to coexist within the same cells. This observation points to an intracellular production of AII in the juxtaglomerular epitheloid granular cells. AII may then be released concomitantly with renin in the interstitial fluid and in the blood. The paracrine secretion of AII could exert a local regulatory influence on the tonus of the glomerular vessels.     

Epithelioid cells: membrane potential changes induced by substances influencing renin secretion

Microelectrode recordings were performed in renin-containing epithelioid (JG) and vascular smooth muscle (VSM) cells of the afferent arteriole in the isolated hydronephrotic mouse kidney. Both cell types had a membrane potential of about -75 mV and exhibited small, spontaneous depolarizing transients, probably resulting from random transmitter release by sympathetic axon terminals. Substances depressing renin secretion, such as angiotensin II, arginine-vasopressin, and alpha 1-adrenergic agents reversibly depolarized both JG and VSM cells. On a molar basis, the action of angiotensin II was strongest. Stimulators of renin release, e.g. isoproterenol, histamine, and prostaglandin E2 did not influence the membrane potential of both cell types. VIP and NPY, possible co-transmitters of norepinephrine, as well as AP II, were also without effect. It is proposed that suppression of renin secretion from JG cells is mediated by depolarization and Ca2+ influx, whereas stimulation is triggered independently from membrane potential changes, e.g. by adenylate cyclase activation.     

Control of renin secretion from adrenal gland in transgenic Ren-2 and normal rats

In Ren-2 rats, plasma active renin and prorenin increase following binephrectomy (BNx) related to increasing plasma potassium. Adrenal is the source of the increasing prorenin but active renin comes mainly from thymus and gut. Trophic influences other than potassium were tested in the present work. Angiotensin did not influence the post-BNx increases in plasma active or prorenin but suppressed resting plasma prorenin from non-adrenal, non-renal sources virtually to zero. ACTH and histamine had no discernible effects. Hexamethonium decreased by 50% the post BNx increase in prorenin but not active renin. In Sprague-Dawley and spontaneously hypertensive rats, low levels of active renin secretion were detected from adrenal but no prorenin. Thus, in anesthetized Ren-2 rats, secreted prorenin is from two sources, i.e. extrarenal and extra-adrenal sites readily suppressible with angiotensin and the adrenal that is partly suppressible by autonomic blockage. This may assist in identifying the origin of extra-renal prorenin secreted in man.     

Suppression of renin secretion in the rat kidney by a nonvascular alpha-adrenergic mechanism.

We studied the effect of alpha-adrenergic stimulation, using phenylephrine, on basal and isoproterenol-provoked renin secretion in the isolated perfused rat kidney. Infusion of phenylephrine increased renal perfusion pressure and prevented the response in renin secretion to isoproterenol. No suppression of basal secretion was observed. Renal vasoconstriction was abolished, and the response in renin secretion to isoproterenol was restored by alpha-adrenoceptor blockade with phenoxybenzamine. In contrast, when renal vasoconstriction was prevented by dihydralazine, suppression of renin release by phenylephrine still occurred. These observations support an inhibitory effect of a nonvascular alpha-adrenergic mechanism on renin release. We suggest that the alpha receptor mediating this effect is related directly to the juxtaglomerular cell.     

Control of thyroglobulin secretion from the rat thyroid gland

Serum thyroglobulin (Tg), measured by radioimmunoassay, was high in 6-propylthiouracil (PTU)-treated rats but low in thyroxine (T4)-treated animals compared with euthyroid controls. Thyroid-stimulating hormone (TSH) stimulated Tg release in vitro from enzymatically dispersed normal rat thyroid cells in a dose-dependent manner. Thyroid cells prepared from T4-treated animals behaved similarly to cells from control rats, whereas in vitro basal release of Tg from thyroid cells prepared from PTU-treated animals was high and the response to TSH was lost. Our data confirm the TSH dependency of Tg release in vivo and in vitro and our system provides a means of studying the control of Tg secretion in vitro.     

The physiology of renin secretion in essential hypertension. Estimation of renin secretion rate and renal plasma flow from peripheral and renal vein renin levels

From renin measurements made in blood collected simultaneously from renal veins, aorta and vena cava, an equation was developed for estimating renin secretion rates in patients with three renin subtypes of essential hypertension. From these data a second equation was derived for estimating differential renal plasma flow in patients with unequal kidney perfusion. The latter equation achieves maximum precision when there is no renin secretion from one side.Plasma renin activity was identical in blood collected from the aorta or the vena cava. It was also similar, but higher, in blood collected from either right or left renal veins. The ratio of renin from the two renal veins, an expression of the variability in renal vein renin measurements in essential hypertension, was 1.5 or less in 87 per cent of patients and less than 1.63 in 95 per cent.Renal vein renin content remained proportional to arterial renin over the range of peripheral renin levels found in essential hypertension, so that renal vein renin concentration from each kidney was consistently 124 per cent of arterial renin. The constancy of this relationship complements previous observations indicating that the metabolic clearance rate of renin is proportional to arterial renin levels. The observed equality of renin values between renal veins suggests that differential renal plasma flow is fairly equal and constant in patients with essential hypertension. Moreover, since renal plasma flow from each kidney is inversely related to the increment in renal vein renin concentration relative to arterial renin input [(V-A)/A], differential changes in (V-A)/A can be used to identify differential changes in renal plasma flow.These derived interrelationships are relevant to an analysis of renovascular hypertension since, with this approach, reductions in renal plasma flow can be estimated using only renal vein and arterial renin measurements and adequacy of sampling can be assessed from the sum of (V-A)/A from each kidney.There was no measurable difference in plasma renin substrate in the three renin subgroups of patients with essential hypertension so that observed differences in plasma renin activity levels appear entirely due to differences in renal renin secretion. Under conditions of this study renal renin secretion per minute was 144 times the arterial renin level.     

Progesterone secretion by primary cultures of rat luteal cells

Primary cultures were prepared from ovaries of immature rats that had been superovulated. The dispersed luteal cells attached to growth surfaces, formed monolayers and secreted progesterone. Progesterone accumulation in the medium was most pronounced in the first week of culturing. Removal of serum from the medium resulted in a progressive decline in progesterone concentration in the culture medium which reached basal levels by 5 hours. In medium without serum, the addition of hCG, FSH or prolactin stimulated an increase in progesterone secretion within 1 hour. Also cholera toxin stimulated a significant increase in progesterone levels in the medium. Prior exposure of cultures to estradiol for 3 days did not augment the response to hCG and inhibited the ability of cholera toxin to stimulate progesterone secretion. These results indicate that the steroidogenic function of rat luteal cells can be studied in culture and that a number of hormones rapidly stimulate the secretion of progesterone from these cells.     

The storage form of renin in renin granules from rat kidney cortex

Renin granules were partially purified from rat kidney cortex, and a storage form of renin in the granules was examined. Renin granules were isolated by discontinuous Percoll density gradient centrifugation followed by continuous Percoll density gradient centrifugation. The partially purified fraction was free from mitochondria and microsomes, as judged by the absence of marker enzymes of these organelles, but contained some lysosomal enzyme activities. The specific renin activity was 0.58 mg angiotensin I/hr/mg protein, 500 times as active as the original homogenate. Immunochemical staining with specific antisera against rat kidney renin revealed that about 10% of the granules recovered in the partially purified fractions were stained strongly. The stored renin was not activated either by acidification or by trypsin treatment, indicating that stored renin was in the fully active form. By sodium dodecyl sulfate gel electrophoresis, the stored renin had two different molecular weights, 38,000 and 36,000, and these molecular weights were not reduced by dithiothreitol or 2-mercaptoethanol, suggesting that these renins are single-chain types as opposed to the two-chain type found in male mouse submaxillary gland. These results suggest that active renins with two different molecular weights may be released from renin granules of juxtaglomerular cells.