Large scale purification of hog renin. Physicochemical characterization.

Corvol, Pierre; Devaux, C; Ito, Tsubasa; Sicard, P. J.; Ducloux, J; Ménard, Joël

doi:10.1161/01.res.41.5.616

Cited by 53 publications

(20 citation statements)

References 26 publications

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“…Although the apparent molecular weight of renin by gel filtration or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has been reported to be in the 38 to 42 kDa range, [35][36][37] it has also been reported that renin in kidney tissue has variable degrees of glycosylation, which may account for the reported variations in molecular weight. 36,37 Inagami et al identified in rat and hog kidneys a renin form responsible for all renin activity with a molecular weight between 55 to 60 kDa when they avoided the acidification step during the purification procedure.…”

Section: Discussionmentioning

confidence: 99%

“…36,37 Inagami et al identified in rat and hog kidneys a renin form responsible for all renin activity with a molecular weight between 55 to 60 kDa when they avoided the acidification step during the purification procedure. 38,39 These investigators reasoned that the native form of renin in the kidney was a 60 kDa molecule, which was converted during extraction to 40 kDa renin by a sulfhydryl-dependent protease.…”

Section: Discussionmentioning

confidence: 99%

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Enhancement of Collecting Duct Renin in Angiotensin II–Dependent Hypertensive Rats

et al. 2004

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Abstract-Distal nephron renin may provide a possible pathway for angiotensin (Ang) I generation from proximally delivered angiotensinogen. To examine the effects of Ang II on distal nephron renin, we compared renin protein and mRNA expression in control and Ang II-infused rats. Kidneys from sham (nϭ9) and Ang II-infused (80 ng/kg per minute, 13 days, nϭ10) Sprague-Dawley rats were processed by immunohistochemistry, Western blot, reverse transcriptase-polymerase chain reaction (RT-PCR), and quantitative real-time RT-PCR. Ang II infusion increased systolic blood pressure (181Ϯ4 versus 115Ϯ5 mm Hg) and suppressed plasma and kidney cortex renin activity. Renin immunoreactivity was suppressed in juxtaglomerular apparatus (JGA) cells in Ang II-infused rats compared with sham (0.1Ϯ0.1 versus 1.0Ϯ0.1 relative ratio) but increased in distal nephron segments (6.4Ϯ1.4 versus 1.0Ϯ0.1 cortex; 2.5Ϯ0.3 versus 1.0Ϯ0.2 medulla). Tubular renin immunostaining was apically distributed in principal cells colocalizing with aquaporin-2 in connecting tubules and cortical and medullary collecting ducts. Renin protein levels were decreased in the kidney cortex of Ang II-infused rats compared with that of sham (0.4Ϯ0.2 versus 1.0Ϯ0.4) rats but higher in the kidney medulla (1.2Ϯ0.4 versus 1.0Ϯ0.1). In kidney medulla, RT-PCR and quantitative real-time PCR showed similar levels of renin transcript in both groups. In summary, the detection of renin mRNA in the renal medulla, which is devoid of JGA, indicates local synthesis rather than an uptake of JGA renin. In contrast to the inhibitory effect of Ang II on JGA renin, Ang II infusion stimulates renin protein expression in collecting ducts and maintains renin transcriptional levels in the medulla, which may contribute to the increased intrarenal Ang II levels in Ang II-dependent hypertension. Key Words: renin Ⅲ angiotensin II Ⅲ angiotensinogen Ⅲ immunohistochemistry Ⅲ Western blot R enin is synthesized primarily by the juxtaglomerular apparatus (JGA). 1 However, renin mRNA and protein have been detected in proximal, connecting tubule and collecting duct cells of human and mouse kidneys as well as in extrarenal tissues. [2][3][4][5][6][7] Although regulation of renin synthesis and secretion from JGA cells has been extensively studied, 1,8 -13 very little is known about the regulation of tubular renin. 7,14 -16 Renin formation in tubular segments may have greater importance than previously thought in view of evidence of high concentrations of angiotensinogen (AGT), as well as angiotensin (Ang) I and Ang II in proximal tubule fluid, 17,18 and in view of the enhancement of renal AGT mRNA and protein levels in Ang II-dependent hypertension. 19,20 Recent studies in Ang II-infused hypertensive rats have shown that there is an increased urinary excretion of AGT, 21 which is closely correlated with intrarenal Ang II content. 21 Enhancement of urinary AGT excretion suggests increased distal nephron AGT delivery and subsequent Ang I and Ang II formation as long as there is availability of an adequate...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Enhancement of Collecting Duct Renin in Angiotensin II–Dependent Hypertensive Rats

et al. 2004

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“…Because the complex of renin and the binding protein easily dissociates into their components at pH. 3.0 or pH 10. 5.…”

Section: Discussionmentioning

confidence: 99%

<b>INTERMEDIATE MOLECULAR WEIGHT RENIN AND RENIN-BINDING PROTEIN(S) IN THE HOG </b><b>KIDNEY </b>

et al. 1980

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The pressor enzyme renin (EC 3.4.99.l9) exists in a high molecular weight (M 1. 60,000) and a low molecular weight form (M,2 40,000) in kidney extracts. However, the mechanism responsible for the conversion between the two forms of renin is unclear.We found a new form of renin which was intermediate between the high molecular and the low molecular weight renin in kidney extract. The incubation of hog kidney extract at 37°C for 60 min yielded a renin of intermediate size (Mr: 50,000) from low molecular weight renin. This new form was converted to the high molecular weight form by the addition of sodium tetrathionate, a sulfhydryl reagent. The reverse conversions, i.e., from the high molecular to the intermediate size and then from the intermediate to the low molecular weight form were mediated by dithiothreitol and acid or alkali treatment, respectively. We further demonstrated the presence of renin-binding substance(s) in the hog renal cortex and subsequently separated them completely from renin by a pepstatin-aminohexyl-agarose column. When incubated with a preparation of this binding substance, pure hog renal renin (M,:40,000) was converted to the high molecular weight form through the intermediate form. This conversion could also be reversed. These results indicate that the intermediate molecular and the high molecular weight renin is the reversible complex of lower molecular weight renin and the renin-binding substance(s). Furthermore sulfhydryl group(s) of renin and/or the binding substance(s) are a key factor in the dissociation and association of the complex.

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“…Standard curves were obtained with pure human renin (1 to 100 ng). In the same assay were run purified human renal inactive renin (I to 100 ng; see above, renin source V); pure mouse submaxillary gland renin (24) (10-10,000 ng); pure hog renin (25) (10- Enzymatic assay. In this assay we determined the ability of 4(1 and 4BI I monoclonal antibodies to inhibit the enzymatic activity of renin in various species.…”

Section: Inactive Renal Renin Inactive Renin Was Extracted Frommentioning

confidence: 99%

New monoclonal antibodies directed against human renin. Powerful tools for the investigation of the renin system.

Galen¹,

Devaux²,

Atlas³

et al. 1984

J. Clin. Invest.

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134

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A bstract. Monoclonal antibodies directed against human renin were obtained by the fusing of myeloma cells with spleen cells from Balb/c or highresponder Biozzi mice injected with pure tumoral or highly purified renal renin. These procedures resulted in the production of seven stable monoclonal antibodies to human renin. Antibodies in the hybridoma culture medium were screened by binding to pure iodinated renin or insolubilized renin in a solid phase assay. The concentration of purified antibodies that provided a 50% binding to iodinated renin varied from 1 X 10-10 to 1 X 10-7 M. Two monoclonal antibodies were found to be potent inhibitors of renin enzymatic activity in vitro, behaving as noncompetitive inhibitors (Ki, 1 to 4 X 10`s M). They were specific for primate renin. Three monoclonal antibodies provided suitable immunoadsorbants for renin purification. One of these immunoadsorbants was used for large-scale purification of the renal enzyme, resulting in an 825-fold renin enrichment in a single step. Two antibodies were able to distinguish between active and inactive renin and enabled concomitant separation and purification of the two enzyme forms in various biological fluids. Monoclonal antibodies also stained human and monkey renal renin when indirect immunofluorescence and peroxidase-antiperoxidase techniques were used. A highly sensitive radioimDr. Atlas is with the

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Large scale purification of hog renin. Physicochemical characterization.

Cited by 53 publications

References 26 publications

Enhancement of Collecting Duct Renin in Angiotensin II–Dependent Hypertensive Rats

Enhancement of Collecting Duct Renin in Angiotensin II–Dependent Hypertensive Rats

<b>INTERMEDIATE MOLECULAR WEIGHT RENIN AND RENIN-BINDING PROTEIN(S) IN THE HOG </b><b>KIDNEY </b>

New monoclonal antibodies directed against human renin. Powerful tools for the investigation of the renin system.

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