Characterization of inactive renin from human kidney and plasma. Evidence of a renal source of circulating inactive renin.

Abstract

A B S T R A C T An inactive form of renin has been isolated from human plasma. It has been suggested that this may represent renin precursor secreted from the kidney. However, early studies failed to isolate inactive renin from human renal tissue. In this investigation, rapid processing of human kidney cortex at temperatures below 40C in the presence of protease inhibitors followed by cibacron-blue affinity chromatography allowed us to extract a totally inactive form of renal renin. Furthermore, we found that in kidney inactive renin constituted from 10 to as much as 50% of the total renin concentration. Biochemical characterization of the inactive renin from plasma and from kidney indicates that they are structural homologues and, when activated, have enzymatic properties that resemble active renal renin. Renal and plasma inactive renin were found to have the following properties in common: (a) a pH optimum of activation of 3.3; (b) reversible activation by acid dialysis on return to pH 7.4, 370C; (c) pH optima of enzyme activity of 7.8 with sheep angiotensinogen and 5.5 and 6.7 (biphasic) with human angiotensinogen; (d) Michaelis-Menten constants, Km, of 0.29-0.34 AM with sheep angiotensinogen, and 0.99-1.25 AM with human angiotensinogen; (e) an antibody to human renal renin mean inhibitory titer of 1:30,000 with 1

Cardiovascular and Renal Phenotype in Mice With One or Two Renin Genes

“…PRA was determined by incubating the plasma sample solely with endogenous substrate. Alternatively, PRC samples were incubated with an excess of exogenous partially purified sheep angiotensinogen 15 and incubated at 37°C for 30 minutes. The supernatant was frozen for later analysis.…”

“…Tissue renin content (renal cortex, brain, submandibular gland) was determined similarly to PRC using an excess of partially purified sheep angiotensinogen 15 with supernatant from homogenized tissue. Tissue was blotted, weighed, and placed in cold bicarbonate buffer (10% wet tissue weight; 90% buffer) 16 pre-equilibrated with a mixture of 95% O 2 /5% CO 2 to a pH of 7.4 at 37°C, and incubated with an inhibitor cocktail of 3%PMSF and 3.8% disodium EDTA.…”

“…The next dose was not administered until 10 to 15 minutes after blood pressure and RBF had returned to baseline. We used sheep angiotensinogen because it is a universal renin substrate 15 and is routinely used in our laboratory for PRC. We hypothesized that if mice were deficient in circulating angiotensinogen, exogenous substrate should induce a pressor and renal vasoconstrictor response.…”

Pathophysiological Significance of Plasma Total Renin and Prorenin in Patients with Diabetes Mellitus.

“…Plasma inactive renin, viz. prorenin, has been of interest because of its significant increase in certain pathological states including renin secreting tumors [9,10] and diabetes mellitus [11,12] has been reported to be significantly increased [11,12] and to correlate with diabetic complications [12][13][14][15][16][17] Plasma prorenin has been determined by enzyme kinetic assay after activation by various pretreatments [1][2][3][4] in which the angiotensin I generated by plasma in vitro is measured by radioimmunoassay. The plasma renin activity, however, can be affected by the amount of plasma angiotensinogen.…”

Isometric exercise in patients with chronic advanced heart failure: hemodynamic and neurohumoral evaluation.

“…The determination of both epinephrine and norepineprhine was performed with an isotope radioenzymatic technique.8 Plasma renin concentration was measured at baseline and during exercise in 12 patients and in all 10 normal subjects by a radioimmunoassay of angiotensin I after addition of excess sheep angiotensinogen. 9 Isometric exercise. Isometric exercise was performed with a commercially available, calibrated hand dynamometer.…”