Rat Renal Renin Research Articles

Female mRen2.Lewis Exhibit Higher Intrarenal Renin But Lower Renal and Circulating Angiotensin II Than Male Littermates

The mRen2.Lewis rat (mRen), a congenic hypertensive model, exhibits marked gender differences in blood pressure and tissue injury. Male mRen have 2‐fold higher plasma and renal levels of Angiotensin II (Ang II) compared to female mRen or Lewis controls. Plasma Ang I was also higher in male mRen, but renal Ang I content was significantly reduced. Cortical ACE activity was similar for male and females, however, renal neprilysin protein was 10 fold higher in female mRen possibly contributing to the metabolism of intrarenal Ang II. The present studies further characterized gender differences in the mRen focusing on the expression of renin. Rat renal renin levels (pH 6.5) were 55% [M: 3.6±1 vs. F: 8.0±2 μg AngI/mg/hr; p≤0.01] and 80% [M: 0.5±0.1 vs. F: 2.9±0.3 μg AngI/mg/hr; p≤0.01] lower in males versus females for Lewis and mRen rats, respectively. Female rats also displayed higher mouse renin levels (pH 8.5). Correlation analysis of all groups revealed a negative association between renal renin and plasma Ang II [r2=0.4, p≤0.05], but not renal Ang II [p≥0.05]. Negative feedback of renal renin is evident in the mRen strain, although the mechanism for gender differences in renin regulation is not known. Moreover, the greater Ang II content in the male mRen kidneys may arise from a nonrenal source. Supported by NIH grants HL56973 & HL51952.

Additive effects of enalapril and losartan in (mREN-2)27 transgenic rats.

Blockade of angiotensin II AT1 receptors combined with angiotensin I-converting enzyme inhibition might amplify the potency of the renin-angiotensin system blockade. We studied whether chronic and simultaneous administration of enalapril and losartan would result in additive or synergistic effects in the (mREN-2)27 transgenic rat (TGR), the investigated targets being blood pressure, cardiac hypertrophy, renin-angiotensin system blockade achieved, and plasma active renin concentration. In addition, the origin (renal or extrarenal, rat or mouse) of the induced renin release was determined. Adult TGRs were treated orally and daily for 5 to 7 weeks with 1 mg/kg (E1) or 3 mg/kg (E3) enalapril or 1 mg/kg (L1) or 3 mg/kg (L3) losartan, or their combinations (E1L1 and E3L3). At the end of the treatment period, enalapril and losartan exerted dose-dependent and, when combined, additive effects in terms of blood pressure fall and cardiac hypertrophy limitation, and synergistic effects in terms of plasma active renin stimulation and blockade of exogenous angiotensin I pressor effects, with E3L3>E3>L3, E1L1>E1> or =L1, and E1L1=E3>L3). This indicates that in the TGR, (1) the greater the renin-angiotensin system blockade achieved, the greater are the reduction in blood pressure, the limitation of cardiac hypertrophy, and the reactive rise in plasma renin concentration elicited, and (2) the enalapril-losartan combinations are more potent at achieving these goals than any of their constituents individually. In contrast, there was no interaction between the two drugs regarding aldosteronuria reduction. Measurement of plasma renin concentration and renal renin at pH 6.5 and 8.5, ie, the optimal pH values for rat and mouse renin activities, respectively, indicates that in TGRs the counterregulatory process for renin release elicited by enalapril, losartan, or their combination involves primarily rat renin of renal origin, a finding supported further by the observed increase in the rat renal renin hybridization index.

Progression of renal failure after subtotal nephrectomy in transgenic rats carrying an additional renin gene [TGR(mREN2)27

To study the evolution of glomerulosclerosis after renal ablation in a model with abnormal regulation of the renin gene. Four-month-old female ovariectomized hypertensive heterozygous transgenic rats (TGR) harbouring the murine REN-2 gene were compared with pressure-matched, pair-fed, stroke-prone, spontaneously hypertensive rats (SHRsp). Both groups were followed for 6 weeks after 70% subtotal nephrectomy (SNX) or sham operation. Blood pressures in the SNX group at the end of the experiment were 193 +/- 3 mmHg in TGR and 199 +/- 5 mmHg in SHRsp. The final C(in) was 306 +/- 68 microliters/min per 100 g body weight in TGR that had undergone SNX and 550 +/- 93 microliters/min per 100 g body weight in SHR that had undergone SNX (P < 0.02), whereas inulin clearance (C(in)) in sham-operated pair-fed TGR and SHRsp controls did not differ from each other. The glomerulosclerosis index was 1.75 +/- 0.08 in perfusion-fixed TGR that had undergone SNX versus 1.21 +/- 0.03 in SHR that had undergone SNX (P < 0.005). In addition, the media thickness of preglomerular vessels was significantly greater in TGR that had undergone SNX (7.48 +/- 0.79 microns) than it was in SHRsp that had undergone SNX (5.27 +/- 1.38 microns, P < 0.02). Rat renal renin messenger RNA (mRNA) expression and, in parallel, mouse REN-2 gene expression were lower in TGR after SNX. Plasma renin and angiotensin II (ANG II) concentrations were reduced to a similar extent in both SNX groups, but plasma prorenin was higher in TGR that had undergone SNX than it was in SHRsp that had undergone SNX. The angiotensin II:I ratio in the kidney was significantly higher in TGR (P < 0.01). There was no significant difference between sham-operated or subtotally nephrectomized TGR and SHRsp with respect to angiotensin type 1 mRNA and angiotensinogen mRNA. The renal angiotensin converting enzyme activity, however, was significantly higher in sham operated and subtotally nephrectomized TGR than it was in sham operated SHRsp and in SHRsp that had undergone SNX. Deterioration of renal function is accelerated in subtotally nephrectomized transgenic rats [TGR(mREN2)27] compared with that in comparably hypertensive SHRsp despite suppressed circulating active mRNA and decreased renal renin mRNA. Although alternative explanations are possible, this observation is consistent with a role for local ANG II in the genesis of glomerulosclerosis.

Amino-terminal amino acid sequence and heterogeneity in glycosylation of rat renal renin.

We isolated 7.4 mg of pure renin from 2 kg of rat kidneys using affinity chromatography on pepstatin-aminohexyl-Sepharose and an octapeptide renin inhibitor, H-77-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that renin consists of two polypeptide chains linked by a disulfide bond, one of Mr = 36,000 (heavy chain) and the other of Mr = 3,000 (light chain). The amino-terminal 10-amino acid sequences of the heavy and the light chains were identical to the sequences beginning at Ser72 and Asp355, respectively, of the amino acid sequence of preprorenin deduced from the renin cDNA sequence. Amino acid sequencing of the carboxyl-terminal peptide of the heavy chain, generated by digestion with lysyl endopeptidase, showed that the carboxyl-terminal residue of the heavy chain is Phe. Thus, the propeptide of prorenin is cleaved after Thr71, followed by removal of two amino acids, Arg353 and Asn354, the result being formation of the heavy and light chains. Thus, the site of cleavage of rat prorenin is after a nonbasic amino acid, in contrast to the cleavage of the propeptide after a pair of basic amino acids in mouse submaxillary renin, human renal renin, and many secretory proteins. Treatment of renin with neuraminidase or glycopeptidase F had no apparent effect on the charge heterogeneity of renin. Glycosylation probably does not contribute to charge heterogeneity.

Cellular and subcellular distribution of exogenously administered renal renin in rat liver and kidney.

Using highly purified 125I-labeled rat renal renin, we have demonstrated that both the liver and kidney are responsible for clearance of renal renin. In the present study, light and electron microscope autoradiography of intravenously administered 125I-labeled rat renal renin was performed in rat liver and kidney to observe the cellular and subcellular distribution of the renin. Fifteen and 60 min after the injection, the liver and kidneys were removed and fixed with 0.1 M sodium phosphate buffer, pH 6.8, containing 2.5% glutaraldehyde and 0.5% tannic acid, which allowed for the removal of breakdown products of the labeled renin, and then autoradiography was performed. In the liver, silver grains were mainly localized in Kupffer cells and not in hepatocytes. In the kidney, silver grains were evident in the proximal tubule cells. In both liver and kidney cells, silver grains were mainly located over the lysosomes. Gel permeation high-performance liquid chromatography analysis of the liver and kidney extracts indicated two main fractions, including immunoreactive 125I-renin and the breakdown products (free 125I and 125I-tyrosine). In conclusion, circulating renal renin is mainly taken up by Kupffer cells and proximal tubule cells and is subsequently transported to the lysosomes to be degraded. These results, taken together with our previous finding that nonglycosylated submaxillary renin does not distribute in the liver, suggest that the carbohydrate moieties of renal renin are necessary for the recognition by Kupffer cells.

Metabolism of Circulating Renin by Liver and Kidney of Rats

Rat renal renin, highly purified and labeled with 125I, was intravenously given to conscious rats, to study the fate of the circulating renin. Rat antirenin anti-serum was used to identify the labeled renin. The disappearance of [125I]-renin from the plasma showed two exponential components and the metabolic clearance rate was 11.4 +/- 1.0 ml/min/kg. Both 70% hepatectomy and bilateral nephrectomy decreased the clearance rate by about 50%. [125I]-renin accumulated mainly in the liver and kidney, and high performance liquid chromatography (HPLC) analysis indicated the degradation of [125I]-renin by these organs. Biliary excretion of [125I]-renin was negligible and urinary excretion accounted for 2% of the injected dose. Light- and electron-microscopic autoradiography indicated that [125I]-renin is taken up mainly by Kupffer cells and proximal convoluted tubular cells in the liver and kidney, respectively, and thereafter distributes to the lysosomes. In conclusion, both the liver and kidney are responsible for the clearance of circulating renin.

Fate of circulating renin in conscious rats.

Highly purified 125I-labeled rat renal renin (125I-renin) was given intravenously to conscious rats to study the fate of circulating renin. Specific antirat renin antiserum was used to identify the labeled renin molecules. In sham-operated rats, the disappearance of 125I-renin from the plasma showed two exponential components with a half-life of 6.7 +/- 0.4 min for the rapid component and 65.1 +/- 5.7 min for the slow component. The metabolic clearance rate was 11.4 +/- 1.0 ml X min-1 X kg-1. In bilaterally nephrectomized rats, the metabolic clearance rate of 125I-renin was reduced by 55%, but the half-life of the slow component remained unchanged. Seventy percent hepatectomy caused a 54% decrement in the metabolic clearance and prolonged the half-life of the slow component. Five minutes after injection of 125I-renin, approximately 59 and 11% of the administered 125I-renin had accumulated in the liver and the kidneys, respectively, and at later time points the 125I-renin was highly concentrated in these organs. High-performance liquid chromatographic analysis of the liver and kidney extracts demonstrated that 125I-renin was catabolized by these organs. Biliary excretion of 125I-renin was negligible. Urinary excretion of 125I-renin up to 120 min was approximately 2% of the injected dose. We conclude that both the liver and the kidney are responsible for the clearance of circulating renin, with participation of the liver being predominant.

O-196 - Plasma disappearance rate of 125I-labelled rat renal renin

O-196 - Plasma disappearance rate of 125I-labelled rat renal renin

Generation of angiotensins in cultured pheochromocytoma cells

Cultured rat pheochromocytoma cells, PC-12, were found to contain renin-like activity. The activity which was inhibited by monospecific antibodies to rat renal renin showed a pH optimum between 6 and 7, indicating that it was a specific renin. The cells also contained angiotensin I and angiotensin I converting enzyme. Angiotensin II III were also detected in lysate. These findings indicated an intrinsic pathway of angiotensin formation in pheochromocytoma. The formation of angiotensins in pheochromocytoma cells suggests modulating functions of angiotensin on neurotransmitter release from adrenal medulla.

Renin in the arterial wall.

Homogenates of rat aortic wall can generate angiotensin I when incubated with nephrectomised rat plasma. This renin-like activity is due to a mixture of proteolytic enzymes. Thus the capacity to generate angiotensin I is greater at pH 5.3 than pH 6.5, although the latter is the pH optimum for rat renal renin. The present work addresses itself to two questions. Is this activity derived from plasma renin? Secondly, does vascular renin-like activity play a role in blood pressure control? Plasma and aortic renin were altered by bilateral nephrectomy and modulation of salt intake. In addition four models of hypertension were studied (early and chronic Goldblatt 2-kidney 1-clip, DOC-salt and spontaneous hypertension). The results indicated that in steady state conditions, aortic and plasma renin-like activity (measured with an incubation pH of 6.5) changed in parallel. When plasma renin was altered acutely however by intravenous injection of renin into nephrectomised rats the half-life of plasma renin was much shorter than the half life of aortic renin. Under these circumstances the pressor response to renin correlated much better with aortic than with plasma renin-like activity. Whilst these studies suggest therefore that renin taken up by the arterial wall is an important determinant of blood pressure, they provide no evidence that accumulation of renin locally produces hypertension in the presence of normal or low plasma renin activity.

Inhibition of rat renin by antibody elicited against pure mouse renin and the effect on blood pressure.

Antibodies raised against mouse submaxillary renin showed affinity for rat renal renin. The affinity was characterized by enzyme kinetics in vitro. The antibodies and the purified Fab fragments showed total inhibition of rat renin, with a Ki of 6.5 +/- 0.7 x 10(-8)mol/l for the Fab fragments. The effect of enzymatic inhibition on blood pressure in anaesthetized rats was compared with converting enzyme inhibition. The antibodies showed a significant effect on blood pressure, with a depressor effect corresponding to that of the converting enzyme inhibitor [SQ 20 881]. The reduction of mean arterial pressure was from 116 +/- 3 to 108 +/- 3 mmHg for the antibodies and to 107 +/- 4 mmHg for the converting enzyme inhibitor. The pressor response of infused rat renal renin was totally blocked by the antibodies while pressor responses of angiotensin II were unchanged.

Enzyme inhibitors are not helpful in preserving big renin in the rat kidney.

The effect of four different homogenization methods, proteolytic enzymes and enzyme inhibitors on rat renal renin was studied. Rat kidney homogenate was treated with trypsin or pepsin. Neither of these enzymes had any effect on the molecular weight pattern. Enzyme inhibitors, 10 mM N-ethylmaleimide, 5.4 mM EDTA, 1.0 mM toluenesulfonyl fluoride, 2.3 mM o-phenanthroline, 2.3 mM p-hydroxymercuribenzoate, which was added to the homogenization medium, all or some of them, as well as 1/20 to the buffer solution used throughout the experiments for dialysis and column chromatography, did not alter the molecular weight pattern. These results suggest that renins, with molecular weights from above 60 000 Dalton to 37 000 Dalton were preformed in the rat kidney and extracted in a native form.

Immunohistochemical localization of renin in luteinizing hormone-producing cells of rat pituitary.

The location of renin (EC 3.4.99.19) in rat pituitary was determined by the peroxidase-antiperoxidase immunohistochemical technique. By using antisera prepared with purified rat renal renin, an immunoreactive substance was localized within ovoid cells scattered throughout the anterior pituitary. These cells were shown to be luteinizing hormone-producing cells by staining with anti-luteinizing hormone antisera in adjacent sections. By using the double staining method, the renin-containing cells were differentiated from cells containing corticotropin, thyrotropin, growth hormone (somatotropin), and prolactin (mammotropin). These results suggest a possible local role for renin in the anterior pituitary.

Subcellular distribution and storage form of rat renal renin.

The subcellular distribution and nature of rat renal renin has been investigated by means of analytical subcellular fractionation and gel filtration on Sephadex G-100. During differential centrifugation, renin activity was recovered mainly in soluble and heavy mitochondrial fractions. On sucrose gradient centrifugation in either a conventional or in a B XIV zonal rotor, renin activity equilibrated at 1.54 M sucrose and was partially resolved from marker enzymes for mitochondria (succinate dehydrogenase), lysosomes (acid phosphatase), plasma membranes (alkaline phosphatase), and peroxisomes (catalase). On gel filtration of the soluble or extracts of the renin-granular fractions on Sephadex G-100, renin activity eluted as a single peak with an apparent molecular weight (MW) of 42,000; no change in activity was found when these fractions were acidified to pH 3.0. When kidney homogenates were prepared in the presence of the proteolytic inhibitor N-ethylmaleimide (NEM, 10 mM), whereas the renin from the granular fractions displayed a MW of 44,000, that from the soluble fraction was apparently higher (69,000). Addition of NEM (10 mM) to the soluble fraction previously shown to contain only the low MW form of renin also resulted in an apparently high MW form of renin. These results indicate that rat renal renin is associated with a mechanically fragile, distinct type of subcellular organelle. Renin within this structure is of the low MW form and is not acid activatable. The soluble fraction, however, contains a factor(s) that, in the presence of NEM, combines with the low MW renin to form a complex of apparently high MW.

Purification of rat renal renin from crude kidney extracts by diaminohexamethylene-Sepharose chromatography

Renin from rat kidney extracts was adsorbed to diaminohexamethylene-sepharose columns at extremely low ionic strength and neutral pH. Renin was retarded while the column was developed in 1 mM sodiumpyrophosphate and extraneous proteins were removed. Elution of renin was performed using a linear gradient of sodiumpyrophosphate, 1 – 17 mM at pH 6.8. Renin was purified in a yield up to approx. 60 per cent of the applied activity and a purification factor between 5 – 122 depending on the specific activity of the applied sample. The specific activity after this single chromatography of crude rat kidney homogenate on diaminohexamethylene-sepharose showed a median of 11.3 Goldblatt units per mg protein in a range of 5.3 – 42.0 Goldblatt units per mg protein. The renin binding capacity of the column was 1 Goldblatt unit per ml wet gel. The purified renin was subjected to G-100 Sephadex chromatography demonstrating two molecular weight forms of 44000 and 50000 dalton. Polyacrylamide gel electrophoresis demonstrated three separate fractions of renin.

Column chromatographic analysis of relationship between hypertension-inducing potency and renin content of kidney extract.

The relationship between hypertension-inducing potency and renin content of kidney extract was analyzed in rats using Sephadex G-100, CM-Sephadex C-50, DEAE-Cellulose or Concanavalin A-Sepharose column chromatography. Hypertension-inducing potency of each fraction obtained was evaluated in the basis of the final blood pressure level attained by repeated injections into the test animals for 10 days. Hypertension-inducing potency was found mainly in the renin-contaning fractions. And it seems reasonable to conclude that renin is implicated in the pathogenesis of hypertension produced by kidney extract. However, there were significant discrepancies between hypertension-inducing potency and renin content of subdivided fractions of these renin-containing eluates. Possible explantations for the discrepancies disclosed--including the possibility of the involvement of an unknown substance(s)--have been discussed. In addition, it was suggested from the results of Concanavalin A-Sepharose chromatography that rat renal renin has a glycoprotein nature.