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There is accumulating evidence that local renin-angiotensin systems (RASs) influence cell growth and organ function in a variety of tissues including the ovary. The first aim of this study was to characterise the cellular location of RAS components in the rat ovary. This was facilitated by the use of the hypertensive transgenic (mRen-2)27 rat which overexpresses renin and angiotensin in extra-renal tissues. Comparisons were made with normal Sprague-Dawley (SD) rats. The second aim was to determine if the upregulated RAS of the transgenic (mRen-2)27 rat and infusion of angiotensin II (ANG II) in SD rats influences follicle number and litter size. Gene expression, immunohistochemical and autoradiographic techniques were used to identify a discrete RAS including ANG II receptors in the ovarian stroma, follicles (particularly atretic) and to a lesser extent corpora lutea. The RAS at these sites was most abundant in homozygous (HMZ) followed by heterozygous (HTZ) (mRen-2)27 rats and then SD rats. Large antral and preovulatory follicles and litter size were reduced in (mRen-2)27 rats. In HMZ (mRen-2)27 rats and SD rats infused with ANG II, angiotensin 1a (AT(1a)) receptor mRNA in the ovarian stroma was lower than control SD rats and was associated with a reduction in large antral and preovulatory follicles. These findings indicate that upregulation of the ovarian RAS in the rat influences follicular development and, potentially, reproductive capacity.

Background: We have previously reported that severe glomerulosclerosis progressively develops in the streptozotocin (STZ) diabetic transgenic (mRen-2)27 rat. In this diabetic model, monotherapy with either angiotensin converting enzyme inhibition (ACEI) or angiotensin type 1 (AT(1)) receptor blockade is largely renoprotective. The objective of the present study was to determine if a combination therapy at lower doses than monotherapy would confer greater renoprotection., Methods: At 6 weeks of age, non-diabetic control and STZ diabetic female heterozygous Ren-2 rats were randomized to receive vehicle, the AT(1) receptor blocker valsartan (V, 20 mg/kg/day), the ACEI perindopril (P, 6 mg/kg/day), or a combination of low-dose V+P (V, 3 mg/kg/day plus P, 0.5 mg/kg/day) for 12 weeks., Results: Systolic blood pressure was lowered with all treatments, but the greatest reductions were observed with V monotherapy and combination V+P therapy. All treatments reduced albuminuria, the decline in glomerular filtration rate, and cortical collagen staining, to the same extent. The glomerulosclerotic index was increased with diabetes and reduced with V and P monotherapy. However, the low-dose combination therapy was more effective than single therapy and reduced severe glomerulosclerosis to levels observed in non-diabetic controls., Conclusions: Monotherapy with either V or P reduced blood pressure and retarded the decline in renal function and glomerulosclerosis in the diabetic Ren-2 rat. Combination therapy has the additional benefit of requiring only low doses of AT(1) receptor blockade and ACEI to achieve superior renoprotective effects in this diabetic nephropathy model.

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.

Background: Endothelin (ET) and angiotensin II (Ang II) are vasoactive/trophic peptides that may contribute to the progression of diabetic nephropathy. The transgenic (mRen-2)27 rat exhibits overexpression of Ang II at sites of normal physiological expression. Unlike other rat strains, the streptozotocin-induced diabetic Ren-2 rat develops progressive renal pathology associated with a declining glomerular filtration rate (GFR) and provides a convenient model to evaluate the role of these vasoactive peptides in the nephropathic process., Methods and Results: Oral administration of either the endothelin A (ETA) and ETB receptor antagonist bosentan or the angiotensin type 1 (AT1) receptor antagonist valsartan for 12 weeks reduced systolic blood pressure (SBP) of nondiabetic and diabetic Ren-2 rats to normotensive levels. Diabetic renal pathology was associated with intense renin mRNA and protein in the proximal tubules and juxtaglomerular cells along with overexpression of transforming growth factor-beta1 (TGF-beta1) and collagen IV mRNA in glomeruli and tubules. With valsartan but not bosentan, renin mRNA and protein in the proximal tubules were not detected. Valsartan but not bosentan reduced TGF-beta1 and collagen IV mRNA and the severity of diabetic renal pathology. A declining GFR with diabetes was attenuated by both treatments. Albuminuria in diabetic rats rose further with bosentan but was reduced with valsartan., Conclusions: Despite producing normotension, severe diabetic renal pathology was not prevented by bosentan, suggesting dissociation of ET, albuminuria, and hypertension from the structural injury in this diabetic model. The beneficial effects afforded by valsartan therapy strengthen the importance of the local renin-angiotensin system in mediating progressive diabetic renal injury.

Thymic ablation and assay of organ renin revealed that one-third of the increasing plasma level of active renin after removal of kidneys and adrenals from Ren-2 rats originates from the thymus. Splanchnic arteriovenous difference and renin content indicate that gut can account for the remainder. Secretion of active renin from these sites correlated significantly with increasing plasma potassium. Prorenin was not secreted from these sites or from hindlimb in amounts sufficient to raise the plasma level, and yet plasma prorenin remained higher than active renin throughout the 12-h protocol. The source of prorenin that accounts for the high plasma prorenin phenotype of the intact conscious Ren-2 rat was not specifically identified. When sensitive assays were used, a low level of active renin secretion from thymus and gut was also apparent 12 h after removal of kidneys and adrenals in normal Sprague-Dawley rats, and plasma prorenin was at this time higher than active renin. A likely source of this extrarenal, extra-adrenal renin is the macrophage.

Plasma active renin and prorenin were followed for 12 h after bilateral, unilateral, and sham nephrectomy (BNx, UNx, and SNx) in anesthetized transgenic (mRen-2)27 rats to compare them with Sprague-Dawley and spontaneously hypertensive rats (SDR and SHR). In Ren-2 rats, active renin and prorenin increased with plasma potassium post-BNx and were augmented by potassium infusion. The increase in prorenin but not active renin was abolished by bilateral adrenalectomy (BADRx). However, this did not reduce prorenin below normal, indicating that the high plasma prorenin Ren-2 phenotype is not only of adrenal origin. SNx and UNx also raised plasma active renin and prorenin in Ren-2 rats, with positive correlations to plasma potassium. In SDR and SHR, active renin fell below prorenin post-BNx, and adrenal ablation and potassium loading (in SDR) modified the decreasing active renin profile consistent with low levels of regulated extrarenal secretion. In Ren-2 rats, adrenal but not extra-adrenal prorenin secretion is potassium sensitive and stress related. The unidentified source of active renin in BNx+BADRx Ren-2 rats is also potassium and stress related.

Background: The transgenic (mRen-2)27 rat (TGR) is a high tissue renin, high angiotensin (Ang) II model of hypertension. When administered streptozotocin (STZ), TGRs develop a rapidly progressive diabetic nephropathy with renal failure over 12 weeks. Bradykinin (BK) and Ang II are potent vasoactive peptides that may participate in the vascular and metabolic abnormalities of diabetes., Methods: TGR and Sprague-Dawley (SD) rats were administered STZ (diabetic) or citrate buffer (nondiabetic) at six weeks of age. Diabetic rats received daily ultralente insulin to maintain moderate hyperglycemia ( approximately 18 mM). Rats were sacrificed four- and eight-weeks post-STZ or vehicle., Results: Diabetes did not modify the blood pressure of either SD rats or TGRs. Diabetes increased levels of BK-(1-9) and its metabolite BK-(1-7) in kidney, aorta, and heart of both SD rats and TGRs. Diabetes did not influence Ang II levels in plasma, kidney, aorta, heart, or adrenal gland of SD rats, but reduced to normal the elevated Ang II levels in plasma, kidney, aorta, and adrenal gland of TGRs., Conclusions: STZ-induced diabetes was associated with elevated tissue levels of BK-(1-9) and "normal" circulating and tissue levels of Ang II. The increased BK-(1-9) levels were consistent with the participation of this peptide in the vascular and metabolic abnormalities of diabetes. However, the rapidly progressive nephropathy of diabetic TGRs was not associated with BK-(1-9) and Ang II levels in target organs that differed from those of diabetic SD rats.

Background: The tissue renin-angiotensin system (RAS) may modulate the structural and functional changes that occur in the diabetic kidney., Methods: Hypertensive transgenic (mREN-2)27 rat (TGR) that exhibit increased tissue renin expression were administered streptozotocin (STZ, diabetic) or citrate buffer (non-diabetic) at six weeks of age, and sacrificed 4 and 12 weeks later. Further groups were treated for 12 weeks post-STZ or vehicle with the angiotensin converting enzyme inhibitor, perindopril. Comparisons were made with 18-week-old non-diabetic and diabetic spontaneously hypertensive rats (SHR)., Results: In diabetic TGR, the most florid lesion was seen after 12 weeks of STZ, with kidneys exhibiting vacuolated tubules, hylanized arterioles, medullary fibrosis and necrosis and severe glomerulosclerosis. In contrast, only mild glomerulosclerosis was seen in non-diabetic TGR and diabetic SHR. Glomerular filtration rate was increased after four weeks of diabetes in TGR and 12 weeks of diabetes in SHR, but declined by greater than 50% after 12 weeks of diabetes in TGR. In both TGR and SHR, diabetes increased albuminuria but did not modify systolic blood pressure. Renal renin content increased progressively in diabetic TGR, and this was associated with increased renin immunolabeling in the juxtaglomerular apparatus (JGA) and the appearance of renin in proximal convoluted tubules. In contrast, renal renin content and JGA renin immunolabeling were unchanged in diabetic SHR. Perindopril attenuated renal pathology, improved renal function and abolished proximal tubular renin immunolabeling in diabetic TGR., Conclusions: This is the first report of a diabetic rodent model developing rapid onset renal impairment. Furthermore, this study suggests a role for an activated renal RAS in the acceleration of diabetic renal disease and confirms the benefit of drugs that inhibit this system.

The distribution and content of renin in Sprague-Dawley (SD) and transgenic (mREN-2)27 rats (TG) were compared to further define the cellular basis and function of the adrenal renin-angiotensin system. Antibody binding (to rat and mouse renin protein and prosequence) was visualised in serial paraffin sections using an avidin-biotin peroxidase technique. Chromaffin and adrenaline cells were identified by tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase immunoreactivity, respectively. In SD zona glomerulosa (ZG), renin and its prosequence localised to small steroid cells while in homozygous (receiving lisinopril) and heterozygous (untreated) TG, steroid cells labelled in all cortical zones. In addition, throughout the cortex of each strain, large polyhedral adrenaline chromaffin cells occurring singly or in small groups and occasionally in rays labelled for renin and prosequence. Similar large adrenaline cells immunolabelled for all antisera in medulla while other cells were only TH-positive. Total adrenal renin content was 53 times higher in heterozygous transgenics than SD rats and was mainly (74%) prorenin. In SD, 37% of cortical renin was prorenin but in adrenal medulla only active renin was detected. Thus, from present and previous work both renin and prorenin occur not only in mitochondrial dense bodies of the ZG, but also in secretory granules of adrenaline chromaffin cells in both cortex and medulla implying in situ synthesis and paracrine functions.

Purpose: An ocular renin-angiotensin system has been implicated in the proliferation of retinal blood vessels and blindness in diabetes mellitus. Its cellular basis has not been established. The objective was to identify sites of renin synthesis, secretion, and processing in eyes from humans, BALB/c mice, Sprague-Dawley rats, and a hypertensive transgenic rat model (mREN-2) that displays amplified extrarenal renin synthesis., Methods: Paraffin sections of eyes were incubated with antisera to renin protein, prorenin, vimentin, and Müller cells. Enzyme kinetic renin assay was performed on extracts of whole eyes (excluding lens and vitreous) and comparisons made with adrenal glands and kidneys. For detection of renin mRNA, retinas were separately pooled from BALB/c and Swiss mice., Results: In normal rodent and autopsy human eyes, labeling for renin, vimentin, and Müller cell protein was observed in the cytoplasm of all macroglial Müller cells, with renin labeling most obvious in endfeet closely apposed to retinal blood vessels. Prorenin labeling was not detected. Less intense renin labeling, again without prorenin, was seen in nonpigmented ciliary epithelium of rodents. In transgenic (mREN-2) rat eyes, renin and prorenin labeling of Müller cells and nonpigmented ciliary epithelium were intense. Prorenin was localized to the posterior region of Müller cells but only sparsely to endfeet in rodent retinas, and renin was present only in an active form in amounts one third that of one adrenal. Renin mRNA was readily detected. In human retina, renin was present in active and pro-forms, and the total amount was approximately one fiftieth that of adrenal., Conclusion: Renin is synthesized in the retina and is specifically localized to the macroglial Müller cells. Nonpigmented ciliary epithelium also contains renin. The presence of prorenin in the posterior part of the Müller cell, with active renin throughout but notably in endfeet in apposition to retinal capillaries, suggests directional processing of renin. These findings are consistent with earlier suggestions that retinal neovascularization may be associated with Müller cell dysfunction.

The transgenic TGR(mRen-2)27 rat, in which the Ren-2 mouse renin gene is transfected into the genome of the Sprague-Dawley rat, develops severe hypertension at a young age that responds to inhibitors of angiotensin-converting enzyme and to antagonists of the type 1 angiotensin II (Ang II) receptor. Despite this evidence that the hypertension is Ang II dependent, TGR(mRen-2)27 rats have suppressed renal renin and renin mRNA content, and there is controversy concerning the plasma levels of renin and Ang II in these rats. We investigated the effect of the transgene on circulating and tissue levels of angiotensin and bradykinin peptides in 6-week-old male homozygous TGR(mRen-2)27 rats. Systolic blood pressure of TGR(mRen-2)27 rats was 212 +/- 4 mm Hg (mean +/- SEM, n = 25) compared with 108 +/- 2 mm Hg (n = 29) for age- and sex-matched Sprague-Dawley rats. Compared with control rats, TGR(mRen-2)27 rats had increased plasma levels of active renin (4.5-fold), prorenin (300-fold), and Ang II (fourfold) as well as tissue levels of Ang II (twofold to fourfold in kidney, adrenal, heart, aorta, brown adipose tissue, and lung and 18-fold in brain). Plasma angiotensinogen levels were reduced to 73% of control, and plasma aldosterone levels were increased fourfold. Plasma angiotensin-converting enzyme was reduced to 64% of control. Compared with control rats, TGR(mRen-2)27 rats had increased bradykinin levels in brown adipose tissue (1.9-fold) and lung (1.6-fold).(ABSTRACT TRUNCATED AT 250 WORDS)

Two extrarenal tissue sources of renin were studied using quantitative assays and immunocytochemical methods during 12 hours following binephrectomy (BNx) in anesthetized hypertensive homozygous Ren-2 transgenic (TG) rats maintained off hypotensive drugs for three weeks. Compared to normal rats, circulating active renin was depressed 50% in conscious TG rats and prorenin was 5- to 10-fold higher. Post-BNx, arterial active and prorenin increased progressively to 10-fold, at which time adrenal venous outputs were 0.1 and 20 mGU/min, respectively. The ratio of active to prorenin (3.1 +/- 0.6%) remained unchanged with increasing plasma levels. Thus, either intrinsic enzyme activity of the transgenic prorenin contributed a constant proportion to the measured active renin, or processing to mature renin was coupled to prorenin synthesis and secretion in extrarenal tissues. In the TG rat eye, renin protein labeling was localized throughout retinal Müller cells with prosequence more obvious posteriorly, consistent with directional processing. Immunogold studies are in progress. In adrenal following BNx, labeling for renin and prosequence increased uniformaly in all zones of the cortex and in scattered medullary chromaffin cells. In cortex, both renin and prosequence were strongly located in intramitochondrial dense bodies. In chromaffin cells, renin labeling was present in both cytoplasmic vesicles and electron-dense granules, while prosequence was predominantly in cytoplasmic vesicles, consistent with processing of prorenin prior to storage in chromaffin granules.

Objective: To determine the effects of hydronephrosis on glomerular number and juxtaglomerular cell synthetic activity and the protective influence of angiotensin converting enzyme inhibition., Design: A comparison of sham and contralateral kidneys with 8-week ipsilateral ureteral ligated hydronephrotic kidneys in BALB/c mice. Enalapril was administered from 5 weeks in additional sham and hydronephrotic kidney groups., Methods: Renin and prorenin immunohistochemistry was applied to sections of perfusion-fixed kidneys at the light and electron microscope level. Glomerular number was estimated by a physical disector-fractionator stereological method. An enzyme kinetic renin assay was performed in kidney tissue and plasma., Results: Glomerular number in hydronephrotic kidneys decreased significantly compared with sham and contralateral kidneys. Renin content in hydronephrotic kidneys did not change compared with sham or contralateral kidneys, but the renin content in the glomerulus was significantly greater in hydronephrotic than in contralateral kidneys and similar to in sham kidneys. Contralateral kidneys enlarged significantly and their total renin content decreased significantly compared with hydronephrotic and sham kidneys. Plasma renin was unchanged. Fewer juxtaglomerular cells were labelled for renin and prorenin in contralateral than in hydronephrotic or sham kidneys. Granulopoiesis and exocytotic profiles were markedly greater in hydronephrotic than in contralateral or sham kidneys. Following enalapril, glomerular number was significantly higher in hydronephrotic kidneys and renin content increased proportionally more in contralateral than in hydronephrotic or sham kidneys., Conclusion: Hydronephrosis for 8 weeks results in atrophy of 50% of glomeruli and exerts an inhibitory influence on contralateral juxtaglomerular cells while augmenting ipsilateral renin production per remaining glomerulus with maintenance of plasma renin. Enalapril preserves glomeruli and reverses the contralateral inhibitory influence, suggesting an angiotensin-related mechanism.

Xeric-adaptation was studied during 28 days of total water deprivation (TWD) in Notomys alexis. Beyond 7 days, the initial reductions in body weight and increases in haematocrit, plasma renin and juxtaglomerular (JG) cell morphological activity returned to normal. Mus musculus showed similar changes at 7 days but could not be maintained thereafter. TWD decreased the blood pressure of Notomys but endogenous angiotensin and vasopressin did not support pressure to a greater extent than controls, as revealed by selective antagonists. The normal morphology of the JG apparatus in Notomys was similar to other rodents. Fluid volume and blood pressure maintenance during TWD in Notomys do not depend upon enhanced activities of the renin-angiotensin and antidiuretic hormonal systems.

We examined renin processing in cultured juxtaglomerular (JG) cells of the hydronephrotic mouse kidney with immunocytochemical and biochemical techniques. Compared with JG cells in normal kidneys, there was less intense labeling for renin protein in mature granules of cultured JG cells. However, pro-renin labeling of transport vesicles and juvenile granules was maintained, suggesting incomplete passage of pro-renin through intermediate and mature granules. Immunogold evidence of exocytosis of mature granules containing renin protein was present at all stages. Labeling of transport vesicles for pro-renin, together with the absence of exocytosis of pro-renin from juvenile granules, indicated that pro-renin was exclusively released by a constitutive process. Active renin release into supernatants decreased with time, whereas the ratio of total renin to active renin increased, indicating that pro-renin synthesis and release were maintained but that the processing of pro-renin to active renin was interrupted. Angiotensin II inhibited and verapamil stimulated active renin release in culture; neither substance affected pro-renin release. Application of secretagogues that act via intracellular calcium or cAMP resulted in depletion of mature granules and their deformation by myelin figures and vacuoles, findings consistent with an exocytosis from mature granules. The absence of effect of any secretagogues on pro-renin release suggests that these stimulatory mechanisms are exclusively post-Golgi. In cultured JG cells in renal explants, renin vesicular transport and granular exocytosis are maintained but a defect in pro-renin passage from juvenile to intermediate granules is apparent.

Immunogold techniques were used to investigate renin processing within granular juxtaglomerular cells following short-term (6 h and 1 day) and long-term (4 weeks) enalapril treatment in female BALB/c mice. In control animals, renin protein labelling was localized to all types of granules (proto-, polymorphous, intermediate and mature) and to transport vesicles, whilst prorenin labelling was found in all these sites except mature granules, confirming that active renin is localized to mature granules only. Following short-term enalapril treatment, the exocytosis of renin protein from mature granules was increased. Long-term enalapril treatment resulted in increased numbers of transport vesicles and all types of granules, consistent with increased synthesis and storage of renin. More large intermediate granules contained discrete regions labelled for prorenin. Renin protein was exocytosed from individual and multiple granules, whilst prorenin was exocytosed from proto- and intermediate granules. It is concluded that under normal conditions prorenin is secreted constitutively by bulk flow from transport vesicles. On the other hand, active renin is secreted regulatively from mature granules. In conditions of intense stimulation (angiotensin-converting enzyme inhibition treatment), increased synthesis of prorenin leads to enhanced secretion of prorenin by both constitutive and regulative pathways. Under these conditions, the conversion of prorenin to active renin is increased, with increased secretion of active renin occurring in a regulative manner. Furthermore, the localization of prorenin to one discrete region of large intermediate granules leads us to conclude, that cleavage of the prosegment of renin occurs with the transition of intermediate to mature granules.

The renin-angiotensin system in sub-genus Mus displays unique features including duplication of the renin gene in most strains, strong expression of the second gene in submandibular gland of males, and inhibited responses to injected renin. Our findings indicate that this inhibition results from a paucity of renin substrate and is consistent with first-order kinetics. We find substrate paucity to be a feature of both sexes and all sub-species and strains of Mus irrespective of gene duplication. Attempts to increase the level of substrate in blood by intravenous injection caused marked increases in blood pressure in Mus, suggesting that substrate paucity was a phenotypic prerequisite for successful emergence of enhanced renin expression in salivary gland. We propose that these phenomena are linked to salivary "lethal factor", possibly transferred by biting, in an evolutionary sequence that has provided a major selective advantage for Mus and influenced the ecology and evolution of rodents.

Recent evidence suggests that a considerable proportion of plasma angiotensin is generated not in blood but in peripheral tissues. Through the measurement of angiotensin peptides and renin in the plasma of 11 anephric subjects, we have investigated whether kidney-derived renin, or some other tissue mechanism for angiotensin generation, is the major determinant of plasma angiotensin. Particular care was taken to prevent inadvertent activation of inactive renin and possible generation, conversion and metabolism of angiotensin peptides during processing of blood samples. Initial experiments revealed that plasma from anephric subjects contains high amounts of material which interferes in radioimmunoassays for angiotensin, even after high-performance liquid chromatography (HPLC). Therefore, in order to obtain an unambiguous identification of angiotensin peptides, a dual HPLC method was developed in which angiotensin peptides were first separated by HPLC, then acetylated and run again on HPLC before radioimmunoassay for angiotensin I and II (detection limits, 0.25 and 0.2 fmol/ml, respectively). The levels of angiotensin I and II were 1.2 +/- 1.6 and 0.7 +/- 0.5 fmol/ml (mean +/- s.d., n = 9-10), respectively, being 6% of levels in normal subjects, and were consistent with the active renin levels (1.8 +/- 1.7 muIU/ml, n = 11) which were 7% of levels in normal subjects. Artefactual activation of prorenin and angiotensin generation during sample processing were excluded as significant causes of the low levels of active renin and angiotensin I and II in anephric plasma. These data indicate that kidney-derived renin is the major determinant of angiotensin levels in normal human plasma. However, the present demonstration of low levels of active renin and angiotensin I and II in plasma of anephric subjects provides unequivocal evidence for a functional extrarenal renin-angiotensin system in man.

Total renin protein concentration (TRC) was measured in stored follicular fluid (FF) samples from 42 women. Samples were selected according to their origin from follicles either without recovered ova ('empty', n = 38) or fertilized but with failed implantation ('failed', n = 36) or successful deliveries ('deliveries', n = 71). Ratios of number of embryos transferred to number of infants delivered were 2:1, 3:1 or 4:2 but 1:1 was not available. Non-parametric testing was applied to FF-TRC, volume and outcome. TRC was significantly higher in the delivery than the failed (P = 0.001) or empty (P = 0.002) categories. Assuming that the range of renin in failed follicles can identify the sub-population of unsuccessful follicles in the delivery category, then elevated FF-TRC was clearly associated with successful outcome. For individual women, the odds of infant delivery increased 17-fold as a function of average FF-TRC between 10,000 and 25,000 microIU/ml. For failed and delivery but not empty follicles, higher renin levels occurred in the smaller follicles, consistent with a burst of renin synthesis associated with the presence of an oocyte. The results suggest that FF-TRC relates to ovum viability with ovarian hyperstimulation and may have predictive use in IVF programmes.

The short-term and long-term effects (for up to 98 days) of the angiotensin converting enzyme inhibitor enalapril were investigated in male and female BALB/c mice. In control animals, separate antisera to renin and its prosequence produced an identical pattern of staining in granular cells of the juxtaglomerular apparatus (JGA) a short distance from the glomerulus. After 1 day of the enalapril treatment there was a decrease in the number of JGA granular cells immunostained with antisera to both renin and its prosequence. Electron microscopy revealed degranulation of mature granules from JGA granular cells. Fusion of granules with the cell membrane was not observed, but numerous membrane-like structures (myelin figures) were identified in the cytoplasm and extracellular space, indicating possible secretion. In addition, the volume proportion of granulated cells in relation to the glomerular volume was decreased, as was renal renin content. With continuing enalapril treatment, separate antisera to renin and its prosequence stained the same granulated JGA cells with equal intensity. The cells so stained increased in number, extending down the wall of the afferent arteriole to cortical radial arteries (interlobular arteries) upstream from the glomerulus. Ultrastructural studies revealed a progressive development of cytoplasmic granulation in JGA granular cells and in smooth muscle cells extending into cortical radial arteries. Furthermore, the volume proportion of granulated cells in relation to the glomerular volume was significantly increased, as was renal renin content. Thus, short-term enalapril treatment in mice provoked rapid secretion of renin via degranulation of mature granules from JGA granular cells. In contrast, long-term enalapril treatment produced a continuing stimulus for renin synthesis, secretion and storage, resulting in an increased thickness of the afferent arteriolar wall. The mechanism for this change appears to be hypertrophy and hypergranulation of granular JGA cells and neogranulation of smooth muscle cells upstream from the glomerulus. Identification of the intrarenal mediators that induce these phenotypic changes presents an interesting challenge.

1. Biochemical characteristics of a renin-like enzyme secreted by a pulmonary adenocarcinoma have been studied. A very high renin content was revealed by both enzymatic (10.4 Goldblatt units/g of tissue) and direct radioimmunoassay of immunoreactive renin (23 Goldblatt units/g of tissue). 2. The higher value by direct radioimmunoassay suggested the presence of an inactive form of the enzyme. Indeed 40--100% activation occurred with treatment by trypsin or pepsin or prolonged dialysis at pH 7.4 with or without prior acid dialysis. This neutral activation was completely abolished by a serine proteinase inhibitor. 3. A large fraction of the renin in this tumour is inactive. In comparison with other prohormones produced in tumours the findings support strongly the proposition that renin passes through a proenzyme step in synthesis.

Active (A) and inactive (I) plasma renin concentrations (PRC) were examined during haemodialysis in relation to blood pressure and body weight changes. Patients had either one (n = 15) or two (n = 51) remnant kidneys in situ, or were anephric (n = 4). 6 of the two remnant kidney patients had non-functioning renal allografts. Inactive renin was activated by acidification. Renin assay was by enzyme kinetic technique using sheep substrate and angiotensin I radioimmunoassay. Cryoactivation during handling was excluded. For the whole patient group the proportion of IPRC was inversely related to APRC in pre-dialysis plasma (p less than 0.001). APRC and IPRC did not change with heparinisation nor did activation of renin occur across the dialyser. Dialysis with up to 3 kg reduction in body weight did not consistently raise APRC or IPRC. Blood pressure was not significantly correlated with IPRC or APRC during dialysis. In all situations a high APRC was associated with a low IPRC:APRC ratio consistent with preferential secretion of the active form and/or consumption of the inactive renin zymogen.

The relationship between active (A) and inactive (I) plasma renin concentrations (PRC) was examined in critically ill patients to test for intravascular renin activation in states of shock and tissue damage. Critically ill patients had significantly elevated APRC and lowered IPRC:APRC ratio compared with age and sex matched healthy subjects. IPRC in the critically ill was similar to the control group. During blood donation normal volunteers showed a twofold increase in APRC. The rise in APRC was proportionately greater than for IPRC, with a subsequent fall in IPRC:APRC ratio. In both critically ill patients and blood donors elevated APRC was associated with decreased IPRC:APRC ratio, consistent with either consumption of the inactive renin zymogen or preferential secretion of the active form. Individual critically ill patients displayed markedly depressed ratios but with only moderately elevated APRC, a pattern suggestive of intravascular renin activation. Consistent evidence for intravascular or extravascular activation of renin was not apparent.

Arterial plasma prorenin underwent 58% extraction during one passage through the coronary circulation in eleven patients undergoing myocardial metabolic studies. Active renin and renin substrate were unaffected. Changes in renin and renin substrate concentrations were not detected across the enterohepatic, femoral, or renal vascular beds. This high degree of selective extraction of prorenin by the heart accounts for its proposed half-life. Prorenin could be a cardiotropic hormone.

The effects of indomethacin (I), a blocker of prostaglandin (PG)-synthetase, was studied in rats in an attempt to elucidate the physiological role of renal PGE. Plasma-I-concentrations of 13-14 mug/ml reduced renal venous plasma PGE-concentration significantly from 216 to 85 pg/ml within 45 min. Urine flow and solute excretion decreased by 42% and 20%, respectively, while urine osmolality increased 450 mOsm. Inulin clearance (CIN) and proximal reabsorption rate was unaffected, while renal plasma flow (RPF) decreased by 18%. Plasma renin concentration decreased slightly but significantly. An i.v. saline load equal to 1% b.wt. given to I-treated rats failed to elevate significantly either urine flow, solute excretion, CIN, RPF or proximal reabsorption rate, but plasma renin decreased further. The normal inverse relationship between plasma renin and proximal reabsorption rate was absent. The data are consistent with the concept that intrarenal PGE plays in adjustment of renal vascular resistance, and support the concept of a physiological role of intrarenal PGE in regulating salt and water excretion. The data do not support any major physiological role of PGE in regulating proximal tubular function.

1. The properties of inactive and active renin in human plasma and amniotic fluid were studied chromatographically. Activation was achieved at pH 3.3 with and without added pepsin. 2. Acid activation of renin was time- and temperature-dependent but was inhibited by dilution of the sample. The dilution effect was corrected by adding pepsin. Such characteristics indicate that activation at low pH is catalysed by intrinsic enzymes. 3. Separation and/or dilution of the activating enzyme during ion-exchange chromatography concealed the eluted position of inactive renin and reduced the amount recovered. Only after full activation of the eluted renin was achieved with added pepsin was a distinct peak of inactive renin exposed. 4. At pH 7.5 inactive renin carried a lower negative charge than the active enzyme. This charge difference was lost after activation. 5. No molecular-weight differences between active, inactive renin or the International Renin Standard were detected by gel filtration. No renin of larger molecular weight was present. 6. These findings will be helpful in purification studies of human inactive renin.

1. A two-site monoclonal immunoradiometric assay (IRMA) for total renin protein was established to cover the range 0.3-300 microIU. The limit of sensitivity was 2 microIU/ml plasma. 2. IRMA after acid activation of plasma prorenin gave the highest and most consistent values for total plasma renin (TPRC = 197 microIU/ml, s.e.m. = 22, in 17 normal adults). In untreated plasma, TPRC measured by IRMA was lower than expected, but in amniotic fluid expected values were obtained. 3. Human angiotensinogen at normal concentrations did not interfere with binding of renin to the first antibody, but ovine angiotensinogen displaced the standard curve significantly. 4. TPRC measured by IRMA is convenient and highly sensitive, but either the state of activation of the enzyme or another effect of acidification of plasma influences the estimated value. 5. Active renal renin may not be the appropriate reference standard for this IRMA unless plasma renin is previously activated.

1. A method is described for studying renin release from superfused rat glomeruli following their rapid isolation by a magnetic iron-oxide technique. 2. Microscopically selected glomeruli were free of tubular components. Some possessed vascular pole protrusions of up to 20 mum, unrelated to renin content. 3. Renin content of 102 batches, each of 400 glomeruli, was 1.34 plus or minus 0.08 times 10-4 Goldblatt hog units per 100 glomeruli (plus or minus S.E. of mean). Different osmolarities (305, 355 and 400 m-osmole/1.), sodium concentrations (110 and 135 mM) and buffer compositions of the preparation solution did not alter this value. Renin content per glomerulus in intact kidney was 100-fold higher. 4. At 30 degrees C the contained juxtaglomerular cells released renin at consistent but decreasing rates over 4-6 hr. Initial release rate in 110 mM sodium, 305 m-osmole/1. solutions were 0.86 plus or minus 0.068 times 10-6 units per 100 glomeruli per 30 min (plus or minus S.E. of mean, n = 42) or 0.546 plus or minus 0.046 percent of content per 30 min. In 135 mM sodium, 305 m-osmole/1. solutions, release was 2.4-fold higher (P less than 0.001) and remained elevated for at least 3 hr. When related to renin content per glomerulus resting release rate in vitro was higher by at most one order of magnitude than calculated in vivo values. 5. Release was augmented by gentle physical agitation of the glomeruli. 6. Release rate was inversely ralated to temperature. On reducing temperature from 30 degrees C, release increased 2.6-fold at 20 degrees C and 6.7-fold at 10 degrees C (P less than 0.001, n = 11). The response was reversible. 7. 3 mM sodium cyanide plus 3 mM sodium iodoacetate caused a variable release of renin associated with depletion of content within 4 hr. The response was progressive and reached a peak after 60 min. 8. Sensitivity of renin release to temperature and metabolic blockade indicates that energy is required for retention of renin by the cell. This, together with the release observed with increased sodium concentration at constant osmolarity, suggests a dependence of renin release upon the mechanism controlling the volume of the juxtaglomerular cell or its organelles.

1. The effects of infusions of frusemide at low (0.05-0.1 mg.kg-1.min-1) and high (0.5-2.0 mg.kg-1.min-1) rates were studied on renin secretion and urinary outputs of sodium and potassium in anaesthetized dogs in which one kidney was removed and the remaining kidney was either innervated or denervated. 2. When the kidney was innervated, low rates of infusion of frusemide did not significantly affect renin secretion if urinary volume and sodium losses were replaced. Without replacement of urinary losses, renin secretion increased at sodium deficits of 0.7-0.9 mmol.kg-1 in the presence of elevated rates of sodium and potassium excretion. 3. High rates of infusion of frusemide caused an immediate increase in renin secretion from innervated kidneys which was not related to urinary losses. 4. Denervation of the kidney increased the urinary outputs of sodium and potassium while it decreased the rate of renin secretion to one-tenth of the resting value. 5. Denervation of the kidney abolished the renin-releasing action of frusemide at both low and high infusion rates even when the sodium deficit amounted to 4.3 mmol.kg-1. 6. Constriction of the aorta producing a fall of 10-30 mmHg in perfusion pressure raised the rate of renin secretion from denervated kidneys to control levels and partially restored the renin-releasing action of frusemide at high infusion rates. 7. The findings indicate that frusemide has a site of action apart from the macula densa in mediating renin release.

1. An experimental technique utilizing 'denervation diuresis' from one kidney with measurement of renin release from the contralateral innervated kidney was developed to study the sensitivity of renin secretion to volume depletion. 2. With urine excretion, release of renin increased progressively from the innervated kidney. The increase was significant at a sodium deficit of 0-23 mole.kg-1. At a sodium deficit of 0-6 m-mole.kg-1 renin release had doubled. 3. Bilateral vagotomy did not alter this response. 4. Precise replacement of sodium loss with isotonic saline but without replacement of other urinary components returned renin release to control levels. 5. Slow haemorrhage causing a rate of volume and sodium loss equivalent to urinary drainage did not alter the rate of renin release. 6. With a single denervated kidney and contralateral nephrectomy, renin release fell progressively to minimal levels despite sodium deficits up to 2-6 m-mole.kg-1. 7. It is concluded that renin secretion is sensitive to at most a 0-5% change in body fluid volume and should be considered a primary response to volume depletion. The sensitivity of the response depends upon normal renal innervation but is not mediated via vascular volume receptors nor via receptors innervated by the vagus. 8. It is proposed that the extreme sensitivity of the renin-secreting system in these experiments results from the combination of volume depletion and slight hypotonicity of extracellular fluid acting on the renal afferent arteriole without the mediation of the macula densa,

The fate of circulating inactive prorenin was examined in patients and volunteers. Prorenin was activated either by acid-dialysis with warming at pH 3.3 or with trypsin. The results were similar but omission of warming reduced the value by 13%. In 6 volunteers, 20 min forearm venous occlusion raised regional total (T) and inactive (I) plasma renin concentration (PRC) by 51% and 48% without change of active (A) renin. During intense forearm exercise the ratio APRC: IPRC did not change in muscle or skin venous blood. Body anaerobic exercise increased APRC 3.7-fold without change in IPRC. These procedures activate plasminogen but are without effect on prorenin. In 18 patient with stable angina, TPRC was lower in coronary sinus than arterial blood (p less than 0.001) but APRC was not affected. A-V differences were not detected across the leg. Prorenin is apparently stable in the circulation but extracted by the heart.

Biochemical and immunological characteristics of renin secreted by two malignant renin-secreting tumors [pulmonary (PT) and paraovarian (POT)] were studied. They both contain inactive renin (IR), as renin activity of tumoral extracts was able to be increased after acid activation or trypsin treatment (10.1 to 20.8 Goldblatt units/g tissue for PT and 1.4 to 3.71 for POT). Renin activity after activation reached the value obtained by direct RIA of human renin (23 and 3.4, respectively), as both forms are recognized by renin antiserum. Both enzymatic activities could be completely inhibited by renin antiserum. Displacement curves for the two tumoral renins paralleled the MRC renin in the direct RIA. After chromatography on affigel blue, active renin was not bound to the gel, and inactive renin eluted only with 1 M NaCl. On pepstatin A Sepharose and CBL-pepstatin Sepharose (an N-modified-pepstatin), a separation of the two forms of pulmonary renin was obtained; inactive renin eluted with breakthrough proteins, whereas active renin was strongly bound to the gel. After this affinity chromatography, the molecular weights of inactive and active renin, determined on Ultrogel, were very close (46,000 and 42,500). We conclude that 1) ectopic renin in these cases in similar to the renal enzyme; 2) renin can be secreted in an inactive form, supporting the hypothesis of an inactive initial state of renin; and 3) molecular weight differences between the two forms are very slight.

1. The effects of different energy substrates, of low temperature, of urea, and of ouabain and ethacrynic acid were studied on the rate of renin release from viable juxtaglomerular cells during superfusion of isolated rat glomeruli. 2. Neither lactate nor glutamate altered renin release rate from that observed using glucose as the sole energy substrate. Succinate 10 mM elevated release transiently but did not influence the release caused by reductions in osmolality through lowering sucrose concentration. 3. Peak renin release was more prolonged and returned more slowly to control following reductions in osmolality in phosphate-Ringer than in bicarbonate-Ringer. 4. At 37 degrees C, the peak of renin released induced by hypo-osmolality was smaller and delayed, and returned earlier to control than at 30 degrees C. Reduction in temperature from 30 to 4 degrees C resulted in a 32-fold increase in basal release rate. At 4 degrees C a 20 m-osmole/kg reduction in tonicity caused an additional 2-5-fold increase in release rate. 6. Increasing superfusate osmolality with urea did not affect basal renin release but 100 mM urea suppressed the releasing effect of a 15 mM reduction in NaCl concentration. 7. Ouabain (10(-4) M) caused a small (33 +/- 9%, P less than 0-025) transient increase in renin release. Ethacrynic acid (10(-3) M) provoked a progressive increase in release reaching 100 +/- 15% above control within 50 min. In the presence of both inhibitors the release provoked by hyposmolality was prolonged. 8. It is concluded that renin release in vitro is a function of actively regulated cell volume and it is proposed that a similar mechanism could underline both barorecptor and macula densa controls of renin secretion in vivo.

Sheep renin substrate was prepared 28% pure, labelled with 125I and then diluted in concentrated unlabelled sheep substrate. Ten percent of the 125I-label was released as a small fragment on incubation with excess human renin. Adequate separation of the labelled fragment from the parent protein was accomplished with charcoal precipitation or gel filtration. Renin concentration in human plasma was assayed by incubating the 125I-labelled substrate with plasma under zero-order kinetic conditions. Renin was assayed in plasma that had been either acidified or untreated. The acidification procedure was employed to activate inactive renin which accounted for more than half of the total plasma renin concentration. The results of this radiochemical assay correlated closely with the results of bioassay. Specificity of the method was established with antirenin. The assay was sufficiently sensitive to measure suppressed plasma renin levels. The iodinated substrate was stable on storage at -10 degrees C over a six-month period. Because of its high affinity for human renin, ease of purification and stability of the iodinated product, sheep substrate provides a convenient means for routine determination of plasma renin concentration by radiochemical assay.

Small reductions in renal perfusion pressure to levels still within a physiologic range, which did not reduce renal blood flow, caused the kidney to release renin. Renin appeared in much larger amounts in renal-vein blood than in renal lymph. Release of reruin appears to be mediated by a renal baroceptor rather than by ischemia.