Measurement of Active Renin by the 4G1 Anti-Human Renin Monoclonal Antibody

Three monoclonal human renin antibodies have been selected to settle two immunoradiometric assays of human plasma rénin (IRMA). The first pair of antibodies 3E8-4G1* recognizes exclusively active renin (AR), as demonstrated by the lack of increase either of the number of AR molecules or the renin enzymatic activity, when plasma is set free of inactive renin (IR) by immunoaffinity chromatography with human renin prosegment monoclonal antibody. The second pair of monoclonal antibodies 4E1-3E8* gives results which are very significantly correlated to those obtained with the 3E8-4G1* pair after trypsin activation.

The standardization of renin measurements with use of monoclonal antibodies, and the standardization of renin will allow the exchange of comparable informations between the various laboratories involved in the investigation of the renin-angiotensin system in humans.     

Cosegregation of the Renin Gene with an Increase in Mean Arterial Blood Pressure in the F2 Rats of SHR-Wky Cross

Using the restriction endonuclease, Bgl I, Samani et al. found a restriction fragment length polymorphism (RFLP) for the renin gene in spontaneously hypertensive rats (SHR) and its normotensive control Wistar-Kyoto (WKY) rats (1). This RFLP was confirmed in our laboratory in SHR and WKY rats using a rat renin cDNA probe. The correlation of blood pressure and the renin RFLP was examined in 106 F₂ rats produced from F₁ rats, the offspring of a cross between SHR males and WKY females. Systolic blood pressure was measured by the tail cuff method at 12 weeks of age. Mean arterial blood pressure of anesthetized rats was measured by cannulation of the femoral artery prior to sacrifice. The frequency of ranin genotype showed a typical 1:2:1 Mendelian ratio in F₂ rats of SHR and WKY cross. The mean arterial blood pressure of F₂ rats homozygous with the SHR allele was significantly higher than F₂ rats that were heterozygous or homozygous for the WKY allele. No significant difference in systolic blood pressure was observed in F₂ rats with different genotypes. Thus, the renin gene RFLP cosegregates with an increase in mean arterial blood pressure in the F₂ rats of SHR and WKY cross.     

Cosegregation of the Renin Gene With an Increase in Mean Arterial Blood Pressure in the F2 Rats of SHR-WKY Cross1

Using the restriction endonuclease, Bgl I, Samani et al. found a restriction fragment length polymorphism (RFLP) for the renin gene in spontaneously hypertensive rats (SHR) and its normotensive control Wistar-Kyoto (WKY) rats (1). This RFLP was confirmed in our laboratory in SHR and WKY rats using a rat renin cDNA probe. The correlation of blood pressure and the renin RFLP was examined in 106 F₂rats produced from F₁ rats, the offspring of a cross between SHR males and WKY females. Systolic blood pressure was measured by the tail cuff method at 12 weeks of age. Mean arterial blood pressure of anesthetized rats was measured by cannuiation of the fomoral artery prior to sacrifice. The frequency of renin genotype showed a typical 1:2:1 Mendelian ratio in F₂ rats of SHR and WKY cross. The mean arterial blood pressure of F₂ rats homozygous with the SHR allele was significantly higher than F₂ rats that were heterozygous or homozygous for the WKY allele. No significant difference in systolic blood pressure was observed in these F₂ rats. Thus, the renin gene RFLP cosegregates with an increase in mean arterial blood pressure in the F₂ rats of SHR and WKY cross.     

Immunocytochemistry of Renin in Renal Tumours

We used a panel of two polyclonal antisera and two monoclonal antibodies to human renin to assess the tissue distribution of immunoreactive renin in a range of tumours and normal human tissues. The only tissue showing positive staining for renin was kidney and all four antisera stained the myoepithelioid cells in the renal cortex. In the survey of tumours we found immunoreactive renin only in renal tumours, namely, renal cell carcinoma, and nephroblastoma (Wilms' tumour). The renin-positive cells were sparse and distributed mainly along the course of the tumour blood vessels. They stained positively with all four antibodies and, in pairs of serial sections, we showed that the same cell reacted with two different antisera. This suggests that renal cell carcinoma and nephroblastoma have within them cells which contain renin.     

Active and Inactive Renin in the Cat

Pentobarbitone-anesthetized cats underwent peritoneal dialysis and had blood samples removed and kidneys deep frozen at sacrifice. Inactive renin is easily measurable in cat plasma and peritoneal dialysate fluid. Only small amounts are found after acid activation at pH 4.0, but large amounts after trypsin 2mg/ml at 4°C for 10 minutes. Mean active renin in pentobarbitone-anesthetized cats was 1.8±0.4pmoles AI/ml/hr, while inactive renin was 2.3±0.5pmoles AI/ml/hr.

The increased angiotensin I producing activity after trypsin in peritoneal dialysate was most active at pH 7.0 (plasma and kidney active renin 7.25 and 7.85), and had an apparent molecular weight of 39-40, 000. (Plasma active renin had an apparent MW of 33, 500 and kidney active renin 36, 000. Plasma inactive renin had an apparent MW of 35, 500) Cat plasma after cibacron-blue affinity chromatography showed mainly active renin in the breakthrough buffer (30% of total renin eluted), and renin which is almost entirely inactive in the bound peak (70% of total renin eluted). Active renin from plasma and kidney, and activated inactive renin from concentrated peritoneal fluid, showed exactly similar inhibition by the renin inhibitor H77 (IC₅₀ 0.3μM). Cat plasma angiotensinogen had an apparent MW of 53, 000.     

Corticosteroid Modulation of the Renin System and Blood Pressure in the Spontaneously Hypertensive Rat

This study examines the role of gluco- and mineralcorticoids in the regulation of the renin-angiotensin system and blood pressure in the spontaneously hypertensive rat (SHR). Effects of adrenalectomy and selective treatment with either aldosterone (30 μg/kg/day) or dexamethasone (60 μg/kg/day) on plasma renin substrate, active renin (PRA), total renin and blood pressure were studied in 10 week old SHR and control WKY rats. Systolic blood pressure was moderately lower in adrenalectomized rats (129±2 mm Hg vs 137±4 mm Hg in control WKY and 145±4 mm Hg vs 160±3 mm Hg in control SHR) but could be restored to the control range by aldosterone. Dexamethasone repletion induced substantial increments of systolic blood pressure to comparable levels in both species (202±8 mm Hg in WKY and 192±6 mm Hg in SHR). Renin substrate was markedly lower in adrenalectomized, saline repleted rats. This could be reversed by dexamethasone in both species and by aldosterone in WKY rats only. Both PRA and total renin were higher (p<0.01) in the adrenalectomized, saline repleted state. This increase was not observed in aldosterone repleted rats. However, dexamethasone inhibited the adrenalectomy associated increase of PRA and total renin in SHR but not in WKY rats. Differences in blood pressure between SHR and WKY persist even in adrenalectomized state despite comparable stimulation of the renin system. Conversely, while blood pressure of both species responds similarly to selective corticosteroids therapy, the response of the renin-angiotensin system in SHR and WKY rats is distinct. Therefore factors other than the adrenal gland and the renin system must be involved in the determination of the high blood pressure in SHR.     

Inhibition of Renin in the Primate Callithrix Jacchus (Common Marmoset)

The effects of an antiserum against human kidney renin and of H-142, a peptide inhibitor of human renin, were studied in the primate Callithrix jacchus (common marmoset). In severely volume-depleted (low-salt diet and repetitive injections of furosemide), conscious marmosets, the antiserum reduced blood pressure to the same extent as the converting enzyme inhibitor teprotide (-31 ×s 7 SEM mmHg and -30 ×s 5 mmHg).

In mildly volume-depleted (normal salt diet and single injection of furosemide), conscious marmosets, H-142 and teprotide induced a similar fall in blood pressure (-14 ×s 4 mmHg and -15 ×s 2 mmHg). When H-142 was infused after the injection of teprotide, it had no further effect on blood pressure. Conversely, teprotide was ineffective when injected during the infusion of H-142. These results show that in marmosets blood pressure can be lowered by specific inhibitors of human renin. A converting-enzyme inhibitor has similar effects. Hence the renin-angiotensin system appears to contribute significantly to the maintenance of blood pressure in conscious, volume-depleted marmosets.     

Studies on the Regulation of Renin Genes Using Transgenic Mice

ABSTRACT

Transgenic mice are being used as an assay to identify the DNA sequences that direct the tissue specificity and physiological regulation of murine renin gene expression. In initial studies, a DNA segment encoding the duplicated renin gene, Ren-2, has been injected into the pronuclei of fertilized eggs homozygous for the Ren-1^e allele. The 24 kb XhoI fragment utilized consists of 5.3 kb of upstream flanking sequences and 9.5 kb of 3′ flanking sequence in addition to the 10 kb segment encoding the 9 exons of the natural gene. Seventeen transgenic founder mice were identified of which at least 7 exhibited evidence of transgene expression. The 4 expressing lines that have been studied in detail integrated from 5 to 15 copies of the transgene but expression was not proportional to the number of copies integrated. Qualitatively, expression is detected in the correct spectrum of tissues, and submandibular gland expression of the transgene responds appropriately to known hormonal modulators. However, quantitatively, the levels of expression deviate considerably from normal.     

程新刘宝玲王海滨金壮张杰林CrumpackerClyde人刘宝玲王海滨金壮张杰林CrumpackerClyde巨细胞病毒临床分离株感染血管内皮细胞伴肾素基因表达

目的探讨巨细胞病毒（Cytomegalovirus,CMV）是否感染血管内皮细胞伴肾素表达。方法（1）用10^7pfu（空斑形成单位）／mlCMV临床分离株BI-5和实验室型CMVAD169分别与10^6腹主动脉内皮细胞和人脐静脉内皮细胞共同孵育,在23小时后、第3天、第7天、第10天和第14天分别收集培养上清200μl,第14天用PBS缓冲液洗细胞3次,收获细胞。每组实验均设培养液代替病毒液的无感染对照;（2）COBAS定量PCR检测培养上清中CMVDNA拷贝数;（3）PCR检测感染细胞中CMVpol基因;（4）RT—PCR、RealtimeRT—PCR和Westernblot检测肾素在感染细胞内的表达。结果（1）BI-5和AD169感染静脉和动脉细胞后,其形态学变化相似,无细胞裂解病理效应;（2）ADl69感染细胞不同时间培养上清中CMVDNA拷贝数无明显增加,BI-5呈增殖趋势;（3）BI-5感染动脉细胞CMVDNA拷贝数和肾素表达量均大于静脉细胞。结论临床分离株CMV以非裂解形式在血管内皮细胞持续存在并诱导肾素基因表达,血管内皮细胞分泌肾素可能是CMV感染引起心血管疾病的新机制。     

Quantitation of Renal Renin and Renin mRNA in Dahl Rats in Response to Provocative Stimuli

It is known that inbred Dahl salt-sensitive (S) rats carry a different renin allele than inbred Dahl salt-resistant (R) rats. The levels of, and responses of, renal renin and renal renin mRNA to sodium deficient diet and to sodium deficient diet plus Captopril treatment were compared in S and R rats. S rats had lower renal renin and lower renal renin mRNA than R rats on control (1% NaCl) diet. Sodium deficient diet caused a modest increase, and sodium deficient diet plus Captopril caused a very large increase in both renal renin and renal renin mRNA in both strains. In general, both S and R strains appeared to regulate renin similarly in response to provocative stimuli.