Earliest renin containing cell differentiation during ontogenesis in the rat. An immunocytochemical study

The differentiation of renin containing cells was studied by immunocytochemistry in normal rat fetuses by the use of highly specific renin, angiotensin I and II antisera. Renin synthesizing cells were detectable as early as the 15th day of gestation outside the nephrogen territories within the walls of mesonephrotic-gonadic and renal arteries. Intrarenal differentiation began at the 17th day and progressed along the intrarenal arterial tree. AII immunostaining appeared concomitantly in the renin containing cells and developed considerably during ontogenesis, suggesting intracellular biosynthesis. It can be suggested that in the fetus newly synthesized AII may contribute to the early systemic and renal blood pressure regulation.     

Cyclooxygenase-2 contributes to elevated renin in the early postnatal period in rats

We asked whether cyclooxygenase (COX) activity controls the renin-angiotensin system in the postnatal period. During kidney development, renin peaked at postnatal days 0-1 at the mRNA, tissue protein [renal renin concentration (RRC)], and plasma renin concentration (PRC) levels and was widely expressed along preglomerular vessels. PRC and renin mRNA expression was elevated until weaning in the 4th postnatal week compared with adult rats. Renocortical COX-2 was restricted to Tamm-Horsfall protein-positive cells in the thick ascending limb of Henle's loop, and cortical COX-2 mRNA and protein expression were elevated along with PRC in the 2nd and 3rd postnatal weeks. In contrast, cortical COX-1 expression was constant, but medullary COX-1 expression increased eightfold from the 1st to 4th postnatal week. A COX-2-selective blocker, parecoxib, and a nonselective blocker, indomethacin, given in a period with COX-2 induction from postnatal day 6 to day 12, markedly decreased PRC, but not renin mRNA or RRC. Inhibition of angiotensin AT(1) receptors by candesartan from postnatal day 1 to day 5 increased COX-2 mRNA (2.5-fold), protein, and distribution, renin mRNA (7-fold) and PRC (20- to 70-fold), but had no influence on COX-1 mRNA. Thus, due to very low levels of expression, COX-2 is unlikely to be responsible for the birth peak of renin, but COX-2 activity supports renin secretion later in the suckling period. ANG II negatively feeds back on renocortical COX-2 expression in the 1st postnatal days with high activity of the renin system. We suggest that suckling in the rat is correlated to an enhanced, COX-2-mediated, secretory activity of renin-producing juxtaglomerular cells.     

The renin-angiotensin system in the perinatal period in rats

Plasma concentrations of renin, angiotensinogen, kininogen, total protein, and renal renin concentration were measured in rats before spontaneous birth, immediately after vaginal delivery, during the subsequent 48 h, as well as at the ages of 10, 20 and 80 days. Preterm rats had a plasma renin concentration about 15 times higher than adults, which increased further in 1 h-old vaginally-delivered rats. Thereafter renin fell to very low levels within 2 h, rose again during the first day and remained at 4 times the adults level until day 10. Renal renin content and concentration increased over the whole observation period, except for a slight fall of renin concentration in the first 3 h after birth. In pre- and full-terms rats, angiotensinogen concentration was only 20% that of adults, reaching even lower values immediately after delivery, due to excessive consumption by renin. Thereafter, angiotensinogen increased more than 10 fold within 48 h. Kininogen concentration in plasma was higher than in adults and stable up to the 10th postnatal day. We conclude that vaginal delivery is a strong stimulus for renin release, the resulting high concentration of renin being responsible both for the increased turnover of angiotensinogen and the subsequent inhibition of renin release. The cause and biological significance of the dramatic increase of angiotensinogen during the first 48 h of life remains obscure.     

Localization of components of the renin-angiotensin system within the kidney

Evidence accumulates that intrarenal angiotensin II (AngII) plays important roles in the regulation of renal functions. To determine the mechanism and site of the intrarenal formation of AngII, we employed histochemical and cell biological methods. Immunohistochemical studies have revealed the coexistence of renin and AngII in juxtaglomerular (JG) cells, and electron microscopic studies and subcellular organelle fractionation have demonstrated the colocalization of renin and angiotensin in renin granules. The mechanism of this AngII accumulation has been investigated. Immunoreactive angiotensin I (AngI) appeared slowly in JG cells after prolonged administration of angiotensin-converting enzyme (ACE) inhibitors. Cloned and cultured renin-containing cells derived from rat kidney were also found to contain renin, ACE, and AngI and AngII. The subcellular fractionation of renin granules from rat kidney homogenate demonstrated AngI and AngII in the renin granule fractions. These findings suggest the formation of both angiotensins in JG cells. To study the release of AngII, we determined the presence of the angiotensins in renal lymph. Renin was found in renal lymph at a high concentration. Both AngI and AngII were also present in renal lymph in moderate concentrations. It is possible that AngII in the interstitial fluid may play a role in the regulation of renal functions. From these results it has been concluded that AngII is formed in JG cells in the kidney and is secreted with renin into interstitial fluid and plasma, and that AngII formed in the kidney cells may participate in various renal functions.     

孕酮对人早孕子宫蜕膜细胞活性肾素分泌的调节

子宫蜕膜是肾素产生的主要部位,肾素包括活性与非活性肾素,活性肾素可使血管紧张素原水解生成血管紧张素Ⅰ,继而调节血管紧张素Ⅱ的表达。实验表明：（１）妊娠早期（孕５～９周）,人子宫蜕膜活性肾素的含量随妊娠周龄增加而升高,孕８周时可达６３３７±１２８４ＡⅠｎｇ／ｇｗｗ·ｈ－１;（２）早孕子宫蜕膜组织活性肾素占总肾素量的１／４;（３）孕酮可调节蜕膜细胞活性肾素的合成与分泌。人早孕子宫蜕膜组织中存在高水平的活性肾素,性类固醇激素可调节蜕膜细胞活性肾素的表达,可以认为,子宫局部肾素血管紧张素系统在妊娠过程中发挥了重要作用。     

Increased renin excretion is associated with augmented urinary angiotensin II levels in chronic angiotensin II-infused hypertensive rats

Renin expression in principal cells of collecting ducts (CD) is upregulated in angiotensin II (ANG II)-dependent hypertensive rats; however, it remains unclear whether increased CD-derived renin undergoes tubular secretion. Accordingly, urinary levels of renin (uRen), angiotensinogen (uAGT), and ANG II (uANG II) were measured in chronic ANG II-infused Sprague-Dawley rats (80 ng/min for 14 days, n = 10) and sham-operated rats (n = 10). Systolic blood pressure increased in the ANG II rats by day 5 and continued to increase throughout the study (day 13; ANG II: 175 ± 10 vs. sham: 116 ± 2 mmHg; P < 0.05). ANG II infusion increased renal cortical and medullary ANG II levels (cortical ANG II: 606 ± 72 vs. 247 ± 43 fmol/g; P < 0.05; medullary ANG II: 2,066 ± 116 vs. 646 ± 36 fmol/g; P < 0.05). Although plasma renin activity (PRA) was suppressed in the ANG II-infused rats (0.3 ± 0.2 vs. 5.5 ± 1.8 ng ANG I·ml(-1)·h(-1); P < 0.05), renin content in renal medulla was increased (12,605 ± 1,343 vs. 7,956 ± 765 ng ANG I·h(-1)·mg(-1); P < 0.05). Excretion of uAGT and uANG II increased in the ANG II rats [uAGT: 1,107 ± 106 vs. 60 ± 26 ng/day; P < 0.0001; uANG II: 3,813 ± 431 vs. 2,080 ± 361 fmol/day; P < 0.05]. By day 13, despite suppression of PRA, urinary prorenin content increased in ANG II rats [15.7 ± 3 vs. 2.6 ± 1 × 10(-3) enzyme units excreted (EUE)/day, P < 0.01] as was the excretion rate of renin (8.6 ± 2 × 10(-6) EUE/day) compared with sham (2.8 ± 1 × 10(-6) EUE/day; P < 0.05). Urinary renin and prorenin protein levels examined by Western blot were augmented ～10-fold in the ANG II-infused rats. Concomitant AT(1) receptor blockade with candesartan prevented the increase. Thus, in ANG II-dependent hypertensive rats with marked PRA suppression, increased urinary levels of renin and prorenin reflect their augmented secretion by CD cells into the luminal fluid. The greater availability of renin and AGT in the urine reflects the capability for intratubular ANG II formation which stimulates sodium reabsorption in distal nephron segments.     

Renin immunohistochemistry in the mesonephros and metanephros of the pig embryo

Summary The occurrence and distribution of renin was investigated in meso- and metanephric kidneys of pig embryos in various gestational stages. The immunohistochemical peroxidase-antiperoxidase-method (PAP) was used on paraffin sections after application of an antiserum against mouse renin which cross reacts with pig renin. Renin immunoreactivity was already found in the mesonephros of 21 day pig embryos (crown-rump(CR)-length 12 mm) with the strongest reaction in the media of the juxtaglomerular afferent arteriole. Efferent vessels, mesonephric arteries, and the aortic wall also contained scattered renin-positive cells. In the definitive kidney, renin was not detected prior to the 25 mm CR-length-stage. In 45 mm embryos, immunocytochemical staining was observed not only in the media of kidney arteries and arterioles, but also in proximal tubules after pinocytic absorption of filtered renin. TEM-studies revealed that the media of both the mesonephric and the developing metanephric arteries and arterioles contains epithelioid cells whose ultrastructure is very similar to that of renin-producing cells in the adult organ. The observed distribution of renin-producing cells along the entire renal arterial tree points to the possibility that the major function of the renin-angiotensin system in the fetal animal is to participate in the stabilization of renal perfusion pressure.     

Renin immunohistochemistry in the mesonephros and metanephros of the pig embryo

The occurrence and distribution of renin was investigated in meso- and metanephric kidneys of pig embryos in various gestational stages. The immunohistochemical peroxidase-antiperoxidase-method (PAP) was used on paraffin sections after application of an antiserum against mouse renin which cross reacts with pig renin. Renin immunoreactivity was already found in the mesonephros of 21 day pig embryos (crown-rump(CR)-length 12 mm) with the strongest reaction in the media of the juxtaglomerular afferent arteriole. Efferent vessels, mesonephric arteries, and the aortic wall also contained scattered renin-positive cells. In the definitive kidney, renin was not detected prior to the 25 mm CR-length-stage. In 45 mm embryos, immunocytochemical staining was observed not only in the media of kidney arteries and arterioles, but also in proximal tubules after pinocytic absorption of filtered renin. TEM-studies revealed that the media of both the mesonephric and the developing metanephric arteries and arterioles contains epithelioid cells whose ultrastructure is very similar to that of renin-producing cells in the adult organ. The observed distribution of renin-producing cells along the entire renal arterial tree points to the possibility that the major function of the renin-angiotensin system in the fetal animal is to participate in the stabilization of renal perfusion pressure.     

Sta!le and transient expression of mouse submaxillary gland renin cDNA in AtT20 cells: proteolytic processing and secretory pathways

Apart from kidney, where renin synthesis takes place in all mammals, the submaxillary gland (SMG) of most mouse strains constitutes an important source of an isoenzyme, renin-2, that is highly homologous to renal renin, but unglycosylated [(1982) Nature 298, 90-92]. This unique phenotype is due to the presence of an extra copy of th renin gene. A puzzling observation is that (pro)renin-2 cannot be detected in the kidney of these animals, although both mRNAs accumulate at similar levels [(1985) Proc. Natl. Acad. Sci. USA 82, 6196-6200]. In order to investigate whether (pro)renin-2 expression is detectable in mouse heterologous cell lines we transfected the renin-2 cDNA into AtT20 (pituitary corticotrope) and BTG9A (hepatoma) cells. Stable clones expressing renin were obtained in both cases. BTG9A cells secreted only prorenin while AtT20 cells secreted prorenin and active renin. In addition, in AtT20 cells the secretion of active renin was stimulated by 8-Br cAMP. Our results show that unglycosylated (pro)renin-2 can be expressed and secreted in two murine cell lines. Moreover, it is correctly processed to active renin and secreted upon stimulation in AtT20 cells.     

Effects of insulin and insulin-like growth factor-I on renin gene expression in the renal cortical cells of ovine fetuses

Renin gene expression and insulin-like growth factor-I (IGFI) gene expression are both developmentally upregulated in the renal cortex of ovine fetuses and decline after birth. The infusion of IGFI into ovine fetuses in late gestation increases plasma renin activity and concentration. In order to determine whether there are direct effects of IGFI or insulin on renin gene expression in the kidneys of ovine fetuses, we treated the renal cortical cells of ovine fetuses with IGFI or insulin. The results showed that the responses of renal renin mRNA to IGFI or insulin treatment in vitro were dependent on the culture conditions. Renin mRNA levels were significantly elevated by IGFI or insulin if the cells were cultured in medium devoid of serum (starved) for 16-18 h before treatment. In contrast, no obvious changes in renal renin mRNA expression were observed in the cells cultured in the presence of serum (non starved) before treatment with IGFI or insulin. IGFI and insulin also significantly enhanced cAMP concentrations in the medium of the cells starved in vitro. The data suggest that IGFI and insulin can act directly on the renal cortical cells from ovine fetuses to stimulate renin mRNA expression. It is possible that IGFI and insulin stimulate renin mRNA expression by increasing cAMP concentration in the cells.     

Effects of the renin-angiotensin-aldosterone system on the cardiovascular system during 20-days bed rest

Long term change of renin-angiotensin-aldosterone system (RAAS) induced by bed rest and its effects on cardiovascular system are still controversial. The purpose of this study was to obtain a general conclusion on these questions by analyzing our two 20-days horizontal bed rest experiments in past two years with 18 subjects. Plasma renin activity and aldosterone were consistently increased during the bed rest, but angiotensin II was increased only during the early days. Decrease in urinary sodium excretion and increase in urinary potassium excretion were observed during day 3-8 and day 7-12, respectively. Mean arterial pressure increased during day 3-8. Pulse pressure was returned to pre-bed rest level by day 10 after an initial decrease. All these results indicated an activated RAAS and its active effects on cardiovascular and overall fluid regulating systems during our horizontal bed rest studies. Direct effect of change in gravitational force on renal pressure-sensitive cells or effects related to physical inactivity may explain our results.     

Placental insufficiency results in temporal alterations in the renin angiotensin system in male hypertensive growth restricted offspring

Reduced uterine perfusion initiated in late gestation in the rat results in intrauterine growth restriction (IUGR) and development of hypertension by 4 wk of age. We hypothesize that the renin angiotensin system (RAS), a regulatory system important in the long-term control of blood pressure, may be programmed by placental insufficiency and may contribute to the etiology of IUGR hypertension. We previously reported that RAS blockade abolished hypertension in adult IUGR offspring; however, the mechanisms responsible for the early phase of hypertension are unresolved. Therefore, the purpose of this study was to examine RAS involvement in early programmed hypertension and to determine whether temporal changes in RAS expression are observed in IUGR offspring. Renal renin and angiotensinogen mRNA expression were significantly decreased at birth (80 and 60%, respectively); plasma and renal RAS did not differ in conjunction with hypertension (mean increase of 14 mmHg) in young IUGR offspring; however, hypertension (mean increase of 22 mmHg) in adult IUGR offspring was associated with marked increases in renal angiotensin-converting enzyme (ACE) activity (122%) and renal renin and angiotensinogen mRNA (7-fold and 7.4-fold, respectively), but no change in renal ANG II or angiotensin type 1 receptor. ACE inhibition (enalapril, 10 mg x kg(-1) x day(-1), administered from 2 to 4 wk of age) abolished hypertension in IUGR at 4 wk of age (decrease of 15 mmHg, respectively) with no significant depressor effect in control offspring. Therefore, temporal alterations in renal RAS are observed in IUGR offspring and may play a key role in the etiology of IUGR hypertension.     

Immunocytochemical localization of renin in the submandibular gland of the mouse during postnatal development.

The localization of renin in the developing mouse submandibular gland was studied immunocytochemically using the unlabelled antibody-enzyme method of Sternberger ('74). Bouin-fixed submandibular glands of mice of both sexes were examined at 5-day-intervals from birth (day 0) to 50 days of age. At all stages studied, only granular convoluted tubule (GCT) cells stained immunocytochemically for renin; such cells were first seen in glands of 30-day-old males and of 30-day-old females. The size and number of renin-containing GCT cells increased rapidly in males, attaining adult status by 50 days of age. In females, differentiation of GCT cells immunoreactive for renin was slower and less regular than in males, and at 50 days of age the GCT segment had not yet reached adult conditions with respect to the distribution of renin. Renin appears in GCT cells at later ages than other GCT cell products (e.g., EGF and amylase), suggesting the existence of independent developmental control for the expression of various biologically active substances in the GCTs.     

High salt intake attenuates the development of hypertension in two-kidney, one-clip Goldblatt rats.

Effects of high salt intake on the early onset of hypertension were examined in two-kidney, one-clip rats. They were divided into high salt and control groups which were supplied with 1.0% NaCl and tap water, respectively, as a drinking solution for 12 days after clipping the left renal artery. The high salt group showed a lower plasma renin concentration and a higher plasma atrial natriuretic peptide (ANP) along with an attenuation of the magnitude of early hypertension, as compared with the control group. A significant positive correlation between blood pressure and plasma renin concentration and an inverse correlation between plasma renin concentration and ANP were shown. Cortical renal renin content was comparable between the two groups. In another two groups of sham-clipped rats, the high salt group did not differ from the tap water-drinking group in any of the parameters examined, except that ANP was significantly higher. These results demonstrate that high salt intake attenuates the developmental phase of hypertension in two-kidney, one-clip rats by increasing the ANP and suppressing the release of renin.     

Effect of the administration of 5,6-dihydroxytryptamine to the amygdala and dorsal raphe nucleus on plasma renin activity

The effect of injections of 5,6-dihydroxytryptamine, a potent and selective neurotoxic of serotonin neurons, into amygdala and dorsal raphe mesencephalic nucleus on the plasma renin activity has been studied in male Wistar rats. Plasma renin activity was estimated on 2nd, 4th, Tth and 14th day after injections in both areas. The administration of 5,6-dihydroxytryptamine in amigdala produced a significant decrease in plasmatic renin activity between 2nd and 4th day, but the inverse effect between 7th and 14th day. Similar effects were found after injections in dorsal raphe nucleus. The contents of cerebral 5-HT were simultaneously evaluated in the entire brain when the drug was implanted in dorsal raphe, and only in amygdaloid tissue when the injection was restricted to this area. A significant decrease in serotonin content was produced 7th day in both places, while partial recuperation was found toward 14th day. The results, especially the ones related to the chemical lesion of dorsal raphe nucleus, suggest that serotoninergic brain systems are involved, as stimulators, in the control of the dynamics of renin-angiotensin system.     

Ablation of renin-expressing juxtaglomerular cells results in a distinct kidney phenotype

Renin-expressing cells are peculiar in that they act as differentiated cells, producing the hormone renin, while they also seem to act as progenitors for other renal cell types. As such, they may have functions independent of their ability to generate renin/angiotensin. To test this hypothesis, we ablated renin-expressing cells during development by placing diphtheria toxin A chain (DTA) under control of the Ren1d mouse renin promoter by homologous recombination in a two-renin gene strain (Ren2 and Ren1d). Renin-expressing cells are essentially absent from kidneys in homozygotes (DTA/DTA) which, unlike wild-type mice, are unable to recruit renin-expressing cells when homeostasis is threatened. In contrast, renin staining in the submandibular gland (SMG), which expresses mainly Ren2, is normal. Homozygous mice survive normally, but the kidneys are small and have morphological abnormalities: 25% of the glomeruli are hyperplastic or atrophic, tubules are dilated and atrophic, and areas of undifferentiated cells exist near the atrophic glomeruli and tubules. However, in contrast to the very abnormal renal vessels found when renin-angiotensin system genes are deleted, the kidney vessels in homozygotes have normal wall thickness and no decrease in lumen size. Homozygotes have severely reduced kidney and plasma renin concentrations and females have reduced blood pressure. Homozygotes have elevated blood urea nitrogen and potassium levels, which are suggestive of altered renal function. We conclude that renin cells per se are necessary for the morphological integrity of the kidney and may have a role in maintenance of normal kidney function.     

Tissue-specific antigen of chick adenohypophysis appearing at early stages of histotypic differentiation]

The tissue-specific water-soluble antigen characterized by alpha1-globulin electrophoretic mobility was revealed in chick adenohypophysis. The antigen was shown to appear in embryogenesis at early stages of histotypical differentiation of the adenohypophysis by indirect immunofluorescence. The first cells with specific fluorescence were found simultaneously in the cephalic and caudal lobes of 6-day embryo adenohypophysis. The bright patterned fluorescence was observed in all cellular cords of the adenohypophysis by the 8--10th day of the development. This antigen may be used as a common marker for pituitary cell differentiation.