MEASUREMENT AND IDENTIFICATION OF PRORENIN AND RENIN IN OVARIAN FOLLICULAR FLUID FROM CATTLE AND PIG

1. In previous studies we have demonstrated and solved several methodological problems in relation to the measurement of prorenin by trypsin activation in rat, bovine, hog and horse plasma. 2. The aim of the present study was to develop a method for the measurement of prorenin in bovine and porcine ovarian follicular fluid. 3. Trypsin activation of follicular fluid generated angiotensin I immunoreactive material (AI IM) in both species. 4. The AI IM interfered with the renin assay, but could be completely removed by a cation exchange resin in a batch-wise technique. 5. The enzymatic activity of trypsin-activated prorenin and pre-existing active renin was completely inhibited by a specific inhibitor of renin. 6. The reactions were optimized and an accurate measurement of prorenin in ovarian follicular fluid was developed. 7. The existence of prorenin and renin in bovine ovarian follicular fluid was established. Prorenin and renin in porcine ovarian follicular fluid was demonstrated for the first time. 8. The ratio between ovarian follicular fluid and plasma was 43 for prorenin and 19 for active renin in cattle. The same ratios in pigs were 1.3 and 0.4, respectively. These findings indicate a species difference with respect to the amount of prorenin or active renin present in ovarian follicular fluid.     

RENIN AND PRORENIN IN REPRODUCTIVE TISSUES DURING GESTATION IN PIGS AND CATTLE

1. High concentrations of prorenin and active renin were previously found in ovarian follicular fluid from cattle but not from pigs. In the present study female reproductive tissues and fluids from cattle and pigs during gestation were investigated to clarify a possible species difference in active renin and prorenin concentrations. 2. Very high concentrations of active renin but no prorenin were found in corpus luteum from both species. 3. Relatively low concentrations of active renin, in the same order as in maternal blood plasma, were found in myometrium, endometrium, placenta and fetal membranes from both species. Prorenin was undetectable in these tissues except for bovine myometrium and porcine endometrium in some animals. 4. The concentrations of active renin and prorenin in amnionic fluid from both species were below the maternal plasma values. In allantoic fluid the concentrations were higher than in amnionic fluid. 5. The plasma concentrations of active renin and prorenin did not change during gestation in pigs. This finding is in contrast to the observations in humans and does not support a systemic effect of prorenin during gestation. 6. The presence of renin in the reproductive tissues, especially the very high concentrations in the corpus luteum, indicates a local function of the renin-angiotensin system during gestation.     

RENIN, PRORENIN, AND RENIN GENE EXPRESSION IN RATS WITH ACUTE NEPHROTIC SYNDROME

1. The concentration of total, active and inactive renin was analysed in plasma, urine and kidney from control (C), pair-fed (PF) and nephrotic (NS) rats, as well as renin mRNA levels in kidney, liver and brain. 2. Nephrotic syndrome were induced by a single subcutaneous injection of puromycin aminonucleoside (PAN) and determinations were made 6 days after PAN injection. 3. Plasma total renin did not change, active renin increased in NS rats with respect to PF and C groups and in PF rats with respect to C. In contrast, the inactive renin percentage decreased in NS rats with respect to PF and C groups and in PF animals with respect to C. Total, active and inactive renal renin content did not change and active and inactive renin were significantly excreted by urine with no changes in the prorenin percentage with respect to C and PF groups. 4. In both NS and PF groups, renin mRNA levels did not change in any of the tissues studied. In another group of rats, kidney renin mRNA levels were measured on days 1, 3, 5 and 7 after PAN injection and no time-course changes in its expression were found. 5. These results suggest that renin gene expression is not altered in acute nephrotic syndrome and that plasma renin concentration is regulated at the translational or post-translational level in this experimental model.     

PRORENIN AND ACTIVE RENIN CONCENTRATIONS IN OVARIAN FOLLICULAR FLUID INCREASE AFTER THE LH PEAK IN SUPEROVULATED HEIFERS

1. The aim was to analyse the in vivo variations with time of prorenin and active renin and their relationship to steroid hormones in ovarian follicular fluid during follicular growth in heifers. 2. Thirty one beef heifers were assigned to two groups after oestrous synchronization: an unstimulated and a follicle-stimulating hormone (FSH)-treated (superovulated) group. Within each group, animals were slaughtered at different times of the follicular phase of the oestrous cycle. Ovarian follicular fluids were aspirated and analysed for the concentrations of active renin, prorenin, oestradiol-17β (E₂) and progesterone (P₄). 3. Prorenin and active renin concentrations in follicular fluid remained constant until the luteinizing hormone (LH) peak, after which time they increased four- and two-fold, respectively, in superovulated heifers. 4. In follicular fluid, prorenin and active renin correlated negatively with oestradiol and E₂/P₄ ratio but positively with progesterone during follicular growth in superovulated heifers. Prorenin also correlated negatively with oestradiol and E₂/P₄ ratio in unstimulated heifers. 5. The increase of renin concentrations in ovarian follicles after the LH peak and the correlations to steroid hormones suggest an important role of the ovarian renin-angiotensin system in bovine follicular growth and maturation.     

CIRCULATING LEVELS OF ACTIVE, TOTAL AND INACTIVE RENIN (PRORENIN), ANGIOTENSIN I AND ANGIOTENSINOGEN IN CARBON TETRACHLORIDE-TREATED RATS

1. Plasma renin activity (PRA), plasma angiotensin I concentration (ANG I), plasma angiotensinogen concentration (PAC) and the plasma levels of active, total and inactive renin (prorenin) were measured in rats with carbon tetrachloride (CCl₄)-induced acute renal failure. Rats were treated with a single oral dose of CCl₄ (2.5 mL/kg) and killed 1, 2, 3 and 7 days later. 2. On days 1–3 PRA, ANG I and PAC decreased and increased on day 7. Active renin fell on days 2 and 3, total renin (trypsin treatment) augmented on day 1 and diminished on day 3, prorenin and per cent prorenin increased on days 1 and 2. Angiotensin I concentration paralleled PRA and PAC. The CCl₄-induced decrease in PRA was secondary to the fall in active renin and in PAC. Total renin augmented as a consequence of the elevation of prorenin. Renal function, evaluated by serum urea, serum creatinine and creatinine clearance, decreased on days 1 and 2 when PRA was low and plasma prorenin was high. 3. These data do not support the involvement of the circulating active renin-angiotensin system (RAS) in the pathophysiology of acute renal failure induced by CCl₄, however, increased prorenin levels were associated with the decrease in renal function.     

HAEMORRHAGE-INDUCED SECRETION OF ACTIVE AND INACTIVE RENIN IN CONSCIOUS AND PENTOBARBITONE-ANAESTHETIZED SHEEP

The response of plasma levels of active and inactive renin to haemorrhage was investigated in sheep with indwelling artery and vein catheters. In conscious animals, loss of 10% of estimated blood volume over a 5 min period increased plasma active renin by a mean of 59%, a surprisingly small change. Plasma inactive renin also increased, but only by 86%. Maximum increases in both forms of renin occurred within 1 h of the haemorrhage. The effects of an equivalent blood loss were investigated in pentobarbitone-anaesthetized sheep maintained in an upright posture using padded slings. Anaesthesia per se had no effect on plasma active or inactive renin. In anaesthetized sheep, 3 h after haemorrhage, plasma active renin had increased by 403% and inactive renin by 299% above control values, but a plateau (maximum) response was not reached during this time. In both conscious and anaesthetized animals the haemorrhage-induced increases in active and inactive renin occurred in parallel. It appears that haemorrhage of this intensity is a comparatively mild stimulus to increase plasma renin concentration in conscious sheep but is much more effective in anaesthetized animals. This may be linked to anaesthetic-induced increases in prostaglandin synthesis within the kidney.     

VERAPAMIL-INDUCED SECRETION OF ACTIVE AND INACTIVE RENIN IN CONSCIOUS SHEEP

1. Regulation of plasma active and inactive renin was investigated using conscious sheep with indwelling artery, vein and bladder catheters. Control and experimental studies were carried out in the same animals on different days. 2. The calcium antagonist drug verapamil was given as an initial bolus injection (0.5 mg/kg) followed by a continuous infusion (0.1 mg/kg per h) over a 2.5 h period. 3. Plasma active and inactive renin changed in parallel. Both were significantly increased within 15 min of the initial drug dose and both attained a peak increase after 45 min. Thereafter, the two forms of renin returned to basal levels despite continued infusion of the drug. 4. Effective renal plasma flow (C_PAH) was also transiently increased by verapamil and followed a similar time course to changes in plasma active and inactive renin concentration. Arterial blood pressure, however, remained suppressed by verapamil for the duration of the study. 5. Verapamil did not alter urine flow or sodium and potassium excretion rates. 6. These results are discussed in relation to the possible link between intrarenal haemodynamics and renin secretion in conscious and in anaesthetized animals and also in relation to the concept that variation in the relative amounts of active and inactive renin secreted in differing physiological situations represents a mechanism for regulating the renin-angiotensin system.     

EFFECT OF MINERALOCORTICOIDS AND SALT LOADING ON RENIN RELEASE, RENAL RENIN CONTENT AND RENAL RENIN mRNA IN MICE

1. DOCA and 9 alpha-fludrocortisone were given to mice on a high-sodium diet for periods of up to 20 weeks, resulting in decreases in plasma renin concentration, renal renin concentration and renal renin mRNA with both treatments. 2. Plasma renin concentration was suppressed prior to suppression of renin mRNA and renal renin levels, indicating that suppression of synthesis and secretion of renin occur separately. 3. The decrease in renal renin concentration that occurred with DOCA was greater and more rapid than the decrease that occurred with 9 alpha-fludrocortisone, suggesting that DOCA caused intra-renal breakdown of renin. 4. When DOCA was given to mice on a low-sodium diet, plasma renin concentration and renal renin concentration increased, indicating that the effects of DOCA on renin levels were dependent on dietary sodium. 5. Renin secretion and synthesis appeared to be controlled by different mechanisms and sodium balance has an important effect on both processes.     

THE EFFECT OF PREGNANCY ON MINERALO- AND GLUCO-CORTICOID SECRETION IN THE SHEEP

1. The peripheral blood concentrations of aldosterone, corticosterone and cortisol were measured during pregnancy in conscious, undisturbed sheep. 2. Aldosterone levels did not change during pregnancy and the mean pregnant value, 1·2 s.d. 1·4 ng/100 ml (n= 12) was not significantly different from the non-pregnant value, 2·1 s.d. 1·7 (n= 16). 3. Cortisol levels likewise were unchanged by pregnancy–non-pregnant values were 0·56 s.d. 0·50 μg/100 mi (n= 12) compared with 0·46 s.d. 0·40 μg/100 ml (n= 16) in pregnant sheep. 4. Sheep of 110–140 days gestation had a 400 mmol greater total exchangeable sodium than non-pregnant sheep. Plasma volume and plasma renin concentration tended to be elevated near to term. 5. Very high aldosterone secretion rates and peripheral blood levels could be produced in pregnant sheep by stress, intravenous ACTH or angiotensin II infusions, and by sodium deficiency. It is suggested that the pregnant sheep may show increased sensitivity in contrast to non-pregnant sheep to these stimuli and the enlarged size of their adrenals may be a contributing factor.     

DISAPPEARANCE RATE OF CIRCULATING RENIN AFTER BILATERAL NEPHRECTOMY IN THE RAT

SUMMARY 1. Plasma renin concentration (PRC) was measured in eight male rats at intervals during a 6 h period after bilateral nephrectomy.
2. PRC fell rapidly, reaching 51% of the mean control level by 10 min, 29% by 30 min, and 2% by 4 h.
3. Under the conditions of the experiment, the half-life of circulating endogenous renin in the rat was about 10 min.     

EFFECT OF ACUTE ADMINISTRATION OF PRAZOSIN ON BLOOD PRESSURE, HEART RATE AND PLASMA RENIN LEVEL IN THE CONSCIOUS NORMOTENSIVE RAT

This study investigated whether the specific alpha-antagonist, prazosin, stimulated basal plasma renin levels and heart rate. Furthermore the beta-adrenergic nervous system was also investigated to ascertain whether it was involved in this effect. Prazosin (0.1 or 1 mg/kg) was injected subcutaneously (s.c.) to conscious normotensive rats, either alone or in combination with the beta-adrenoceptor antagonist, DL-propranolol (1 or 3 mg/kg). Rats bore chronically implanted dorsal aorta cannula for measurement of blood pressure and heart rate and blood sampling for renin determinations. Acute administration of prazosin (1 mg/kg, s.c.) produced a fall in mean arterial pressure accompanied by renin release and tachycardia. A tenfold lower dose of prazosin did not alter blood pressure or heart rate but did stimulate renin release. Acute administration of DL-propranolol, (1 or 3 mg/kg, s.c.) produced falls in blood pressure and heart rate but did not affect plasma renin level. Combinations of prazosin with propranolol gave falls in blood pressure similar to those predicted on the basis of a simple addition of the effects of the two drugs given separately. Prazosin-induced tachycardia and renin release were attenuated by propranolol. It appears that prazosin produces renin release and tachycardia via stimulation of the beta-adrenergic adrenoceptor.     

THE EFFECT OF PHENTOLAMINE ON ACTIVE AND INACTIVE RENIN RELEASE IN HUMAN RENOVASCULAR HYPERTENSION

1. Phentolamine was infused at low increasing doses (0.2, 0.3, 0.4 and 0.5 mg/min) in five patients with unilateral renal artery stenosis measuring active and inactive (cryoactivable) renin in the renal veins from the stenosed and nonstenosed kidney and in a peripheral vein. 2. PRA values from the stenosed kidney (11.59, s.e.m. = 5.79 pmol ang I/ml per h) were higher than those in the peripheral vein (5.19, s.e.m. =2.64) while these latter were similar to those from the contralateral kidney (5.09, s.e.m. =2.93). Phentolamine significantly increased PRA from the stenosed kidney and in the peripheral vein in a dose-related manner. PRA changes were unrelated both to blood pressure decrements and to heart rate increments induced by the drug. 3. Before phentolamine, inactive renin from the stenosed kidney (5.19, s.e.m. = 2.84 pmol ang I/ml per h) did not differ significantly from that on the contralateral side (3.15, s.e.m. = 1.96) and in the peripheral vein (4.40, s.e.m. = 1.96). Phentolamine induced significant (P < 0.005) increments of inactive renin only from the stenosed kidney at the doses of 0.3, 0.4 and 0.5 mg/min. Inactive renin from the contralateral kidney was unchanged and it tended to increase, but not to a significant extent, in the peripheral vein. A highly significant relationship was found between active and inactive renin from the stenosed kidney (r = 0.79, P < 0.001, n= 25) and in peripheral blood (r = 0.71, P < 0.001, n= 25) but not from the stenosed kidney (r = 0.29, n= 25). 4. These results suggest that phentolamine, infused at low increasing doses causes an increase of PRA only in the stenosed kidney, an action which does not seem to be wholly explained by either sympathetic nervous system activation or decrease of renal perfusion pressure, and which suggests an action on intrarenal a-adrenoreceptors. Furthermore, phentolamine stimulated inactive renin release only from the stenosed kidney without evidence of intrarenal conversion of the inactive into the active form.     

THE EFFECT OF DOCA AND 9α-FLUDROCORTISONE ON RENAL RENIN CONTENT AND PRODUCTION

1. DOCA and 9 alpha-fludrocortisone were given to rats. 2. Plasma renin fell rapidly with both treatments. 3. Renal renin fell slowly to a low level. 4. Renal renin fell to a lower level with DOCA than with 9 alpha-fludrocortisone. 5. When DOCA and 9 alpha-fludrocortisone were stopped plasma renin levels rose rapidly and the renal renin levels increased. 6. The data suggest that synthesis is altered rapidly but it takes a prolonged time for the kidney to become depleted of renin due to the high tissue stores and the associated inhibition of release.     

PLASMA RENIN RESPONSE TO ACUTE BLOCKADE OF ANGIOTENSIN II IN THE ANAESTHETIZED RAT

SUMMARY 1. Anaesthetized rats were infused intravenously for 1 h with a specific antagonist of angiotensin II, 1-Sar-8-Ala-angiotensin II (P-113), at a rate of 5 μ/kg per min, or with saline.
2. Blood samples were taken, before and after infusion, for measurement of plasma renin activity (PRA) and plasma renin concentration (PRC).
3. Saline infusion did not affect PRA or PRC.
4. Infusion of P-113 produced steep and highly significant increases in PRA (652%) and PRC (724%), despite a slight rise in mean arterial pressure.
5. Within 30 min of terminating P-113 infusion, PRA fell to 300%, and PRC to 278% of pre-infusion levels, and subsequently continued to fall.
6. It is suggested that the hypersecretion of renin produced by P-113 is due to blockade of the inhibitory control normally exerted by endogenous angiotensin II on renin release.     

EFFECT OF RENAL AND ADRENAL DENERVATION ON THE RENIN RESPONSE TO SLOW HAEMORRHAGE IN DOGS

SUMMARY 1. The effect on plasma renin activity of moderate slow haemorrhage (7.5 ml. kg⁻¹. h⁻¹ for 2 h) was studied in intact dogs and in dogs whose adrenals and kidneys had been denervated surgically.
2. In dogs with intact renal and adrenal nerves, plasma renin activity rose during haemorrhage without any accompanying change in systemic blood pressure, but with marked decreases in renal function, an increased filtration fraction and an increased haematocrit. The latter effects can be attributed to sympathetic stimulation evoked by haemorrhage.
3. In dogs with denervated kidneys and adrenals, haemorrhage did not result in a significant elevation of plasma renin activity until the 2 h collection period, at which time mean systemic blood pressure had fallen 32 mmHg. Renal function changes were less marked and neither filtration fraction nor haematocrit rose, suggesting the absence of sympathetic stimulation.
4. It concluded that the sympathetic nervous system plays an important role in the renin response to slow haemorrhage until such time as blood pressure falls sufficiently to activate a second stimulatory mechanism, perhaps a renal baro-receptor.     

EFFECT OF SODIUM INTAKE AND SODIUM DELIVERY TO THE MACULA DENSA ON RENAL RENIN CONTENT AND JUXTAGLOMERULAR APPARATUS MORPHOLOGY

Active and inactive renin were measured in individual juxtaglomerular apparatus (JGA) and in whole kidney homogenates. The morphology of the JGA was examined in microbiopsy glomerular specimens and in kidneys fixed by arterial perfusion. In rats on high and normal salt intake the total renin content of a single JGA was 14 (s.e.m. = 3) and 29 (s.e.m. = 4) ng AI/h, respectively. The amount of cystoplasm occupied by renin granules was 20% and 27%. Crystalline cores were seen in 1.5% and 7% of the granules, respectively. Increased delivery of NaCl to the macula densa did not alter total renin, but decreased inactive renin from 30% to 0, crystalline core-containing cells from 33% to 14% and decreased the percentage of granules with crystalline cores from 12% to 2.2%. Increased sodium in the diet and increased delivery of NaCl to the macula densa decreased the proportion of renin present in the inactive form and decreased the proportion of crystalline cores. These coincidental alterations suggest that crystalline cores contain inactive renin and suggest that the delivery of sodium to the macula densa activates renin.     

STRUCTURE OF HUMAN RENIN AND EXPRESSION OF THE RENIN GENE

The amino acid sequence of human renin was identified for the first time. This was determined from the nucleotide sequence of exons in the human renin gene identified in a genomic library by recombinant DNA techniques. Examination of amino acid residues involved in the enzymatic hydrolysis by human renin of the unique Leu10-Val11 bond of human angiotensinogen revealed features peculiar to this highly specialized aspartyl protease. The expression of the renin gene was examined with a hybridization probe for renin mRNA in sections and extracts of tissues. In the submandibular gland of mice renin mRNA, like renin, increased during development and in response to testosterone in females; sodium depletion increased renin mRNA in kidney.     

ANGIOTENSINS II AND III PREVENT CAPTOPRIL-INDUCED RENIN RELEASE IN THE RAT

1. The effects of exogenous angiotensins II and III (50 pmol/min i.v.) on plasma renin release following captopril injection (5 mg/kg, i.v.) were studied in anaesthetized Sprague-Dawley rats, to determine whether angiotensin II blockade is the major mechanism by which captopril induces renin release. 2. Captopril produced a 12-fold increase in plasma renin concentration compared with saline-injected controls. This was completely reversed by pre-infusion of angiotensin II or III. 3. The fall in blood pressure following captopril treatment was also abolished by angiotensins II and III pre-infusion. Noradrenaline pre-infusion (200-800 ng/min, i.v.) also prevented the captopril-induced hypotension but did not alter the rise in plasma renin. 4. Ureteric ligation did not significantly reduce captopril-induced renin release suggesting that acute changes in sodium excretion or delivery of electrolyte to the macula densa were not involved in renin release. 5. These findings suggest that captopril induces renin release by inhibiting angiotensin II feedback control of renin secretion and that angiotensin III may also modulate renin release.     

THE ROLE OF THE RENAL NERVES IN RENIN SYNTHESIS

1. Renin synthesis and secretion were studied in Balb/c mice with a denervated left kidney. 2. Denervation inhibited renin secretion. 3. Denervation reduced the renal renin content. 4. Denervation reduced renal renin mRNA. 5. Renal denervation inhibits renin secretion by blocking the synthetic system prior to mRNA formation.