Production of rat renin fusion protein in Escherichia coli and the preparation of renin-specific antisera
Abstract
Rat renin fused at the N-terminus with Sj26, a 26,000 Da glutathione S-transferase of Schistosomajaponicum, was expressed in Escherichia coli. The fusion protein was soluble and easily purified from crude bacterial lysates by affinity chromatography on immobilised glutathione. The fusion protein possessed no detectable renin activity. Antisera raised in rabbits against the fusion protein were specific for renin. These antisera did not bind soluble renin but bound immobilized renin. By immunoblotting, these antisera demonstrated rat renin to migrate on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as two broad bands of 33,000–34,000 and 35,000–37,000 Da. By immunocytochemistry of rat tissues, these antisera stained renin containing cells in the afferent arteriole of the glomerulus of the kidney, the zona glomerulosa of the adrenal and the corpus luteum of the ovary. However, apart from the afferent arteriole of the kidney, no immunoreactive renin was identified in blood vessels of the kidney, adrenal or ovary. These studies demonstrate that a recombinant renin fusion protein is a valuable alternative approach for the preparation of renin-specific antisera.
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Cited by (4)
Characterisation of a thymic renin-angiotensin system in the transgenic m(Ren-2)27 rat
2002, Molecular and Cellular EndocrinologyWe previously showed the rat thymus contains and secretes active renin. However, the cellular location of the thymic renin–angiotensin system (RAS) is unknown. To more easily study the thymic RAS we used the hypertensive transgenic (mRen-2)27 rat which overexpresses renin and angiotensin in extra-renal tissues. Comparisons were made with normotensive Sprague Dawley (SD) rats. All rats exhibited intense immunolabeling for renin protein and angiotensin in macrophages and thymic epithelial cells, however renin prosequence was not detected. In each rat strain, thymic renin was predominately active and highest in Ren-2 rats (Ren-2, 22.6±4.2, SD 0.8±0.1 mGoldblatt Units/g, mean±SEM). Renin mRNA was identified in Ren-2 and SD rat thymus by RT-PCR. Thymic angiotensin II concentrations/wet weight in Ren-2 (20.1±1.1 fmol/g) and SD (15.8±2.3 fmol/g) rats were similar to plasma. In conclusion, macrophages and epithelial cells are the source of active renin in the rat thymus. The thymic RAS may have actions systemically and may also influence local processes such as blood flow and cell growth.
A new model of diabetic nephropathy with progressive renal impairment in the transgenic (mRen-2)27 rat (TGR)
1998, Kidney InternationalA new model of progressive diabetic renal disease in the transgenic (mRen-2)27 rat (TGR).
The tissue renin-angiotensin system (RAS) may modulate the structural and functional changes that occur in the diabetic kidney.
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).
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.
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.
Adrenaline cells of the rat adrenal cortex and medulla contain renin and prorenin
1996, Molecular and Cellular EndocrinologyThe 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.
Renin processing and secretion in adrenal and retina of transgenic (mREN-2)27 rats
1994, Kidney InternationalRenin processing and secretion in adrenal and retina of transgenic (mREN-2)27 rats. 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 Muller 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.