Determination of plasma and urinary angiotensinogen levels in rodents by newly developed ELISA

Abstract

We recently reported that urinary excretion rates of angiotensinogen provide a specific index of the intrarenal renin-angiotensin system status in angiotensin II-dependent hypertensive rats. Angiotensinogen concentrations in mouse plasma are thought to be much lower than those in rat plasma; however, detailed information is deficient due to lack of direct quantitative measurements of rodent angiotensinogen. To elucidate this issue, we have developed a quantitative method for measurement of rodent angiotensinogen using a sandwich-type ELISA. The standard curve for mouse and rat angiotensinogen exhibited a high linearity at 0.16-10 and 0.08-5 ng/ml, respectively, with correlation coefficients >0.99. While plasma angiotensinogen concentrations of male high serum IgA (HIGA) mice (IgA nephritis model animals, 1,308 +/- 47 ng/ml; n = 10) were lower than those of control BALB/c mice (1,620 +/- 384; n = 12), urinary angiotensinogen concentrations of HIGA mice (14.6 +/- 1.5 ng/ml; n = 34) were higher than those of BALB/c mice (4.6 +/- 0.1; n = 2). In a similar manner, while plasma angiotensinogen concentrations of Zucker diabetic fatty (ZDF) obese rats (type 2 diabetic model animals, 1,789 +/- 50 ng/ml; n = 5) were lower than those of control ZDF lean rats (2,296 +/- 47; n = 5), urinary angiotensinogen concentrations of ZDF obese rats (88.2 +/- 11.4 ng/ml; n = 15) were higher than those of ZDF lean rats (31.3 +/- 1.9; n = 15). These data indicate that plasma and urinary angiotensinogen concentrations are less in mice than rats. However, these data suggest that urinary angiotensinogen levels are different from plasma angiotensinogen levels in rodents. The development of rodent angiotensinogen ELISA allows quantitative comparisons in mouse and rat angiotensinogen levels in models of hypertension and cardiovascular and kidney diseases.

Augmentation of angiotensinogen expression in the proximal tubule by intracellular angiotensin II via AT1a/MAPK/NF-кB signaling pathways

“…Plasma AGT levels and 24-h urinary AGT excretion were measured by a specific AGT ELISA as previously described (18,20). Briefly, highly purified recombinant rodent AGT protein was used as the standard.…”

Multiphoton Imaging of the Glomerular Permeability of Angiotensinogen

“…10,37,38 Urinary albumin was measured by ELISA (Nephrat II; Exocell, Philadelphia, PA). Plasma samples or the whole-kidney tissue were homogenized in a buffer containing 20 mM TriszHCl, 1 mM EGTA, pH 7.0, and a protease inhibitor cocktail (BD Bioscience, San Jose, CA); 40 mg protein was separated on a 4%-20% SDS-PAGE and transferred onto polyvinylidene difluoride membrane.…”

Plasma and tissue concentrations of proangiotensin-12 in rats treated with inhibitors of the renin-angiotensin system

“…21,22 After decapitating the rats, tissues of interest were carefully resected and boiled for 10 min in 10 volumes of distilled H 2 O. Acetic acid was then added to the samples to a final concentration of 1.0 mol l À1 , and the samples were homogenized using a Polytron mixer and then centrifuged for 20 min (12 000 r.p.m. at 4 1C).…”

“…The angiotensinogen concentrations in plasma were determined using an ELISA purchased from Immuno-Biological Laboratories (Gunma, Japan). 21,22 Plasma renin activity was determined using a GammaCoat Plasma Renin Activity kit from Kyowa Medex (Tokyo, Japan). Estimates of renin activity were based on the plasma concentrations of generated Ang I following incubation at 37 1C.…”