Expression of angiotensinogen mRNA and protein in angiotensin II-dependent hypertension

Abstract

Chronic elevations in circulating angiotensin II (AngII) levels produce sustained hypertension and increased intrarenal AngII contents through multiple mechanisms, which may include sustained or increased local production of AngII. This study was designed to test the hypothesis that chronic AngII infusion increases renal angiotensinogen mRNA and protein levels, thus contributing to the increase in intrarenal AngII levels. AngII (80 ng/min) was infused subcutaneously for 13 d into Sprague-Dawley rats, using osmotic minipumps. Control rats underwent sham operations. By day 12, systolic arterial BP increased to 184 +/- 3 mmHg in AngII-treated rats, whereas values for sham-treated rats remained at control levels (125 +/- 1 mmHg). Plasma renin activity was markedly suppressed (0.2 +/- 0.1 versus 5.3 +/- 1.2 ng AngI/ml per h); however, renal AngII contents were significantly increased in AngII-treated rats (273 +/- 29 versus 99 +/- 18 fmol/g). Western blot analyses of plasma and liver protein using a polyclonal anti-angiotensinogen antibody demonstrated two specific immunoreactive bands, at 52 and 64 kD, whereas kidney tissue exhibited one band, at 52 kD. Densitometric analyses demonstrated that AngII infusion did not alter plasma (52- or 64-kD), renal (52-kD), or hepatic (52-kD) angiotensinogen protein levels; however, there was a significant increase in hepatic expression of the highly glycosylated 64-kD angiotensinogen protein, of almost fourfold (densitometric value/control value ratios of 3.79 +/- 1.16 versus 1.00 +/- 0.35). Renal and hepatic expression of angiotensinogen mRNA, which was examined by semiquantitative reverse transcription-PCR, was significantly increased in AngII-treated rats, compared with shamtreated rats (kidney, densitometric value/glyceraldehyde-3-phosphate dehydrogenase mRNA value ratios of 0.82 +/- 0.11 versus 0.58 +/- 0.04; liver, densitometric value/glyceraldehyde-3-phosphate dehydrogenase mRNA value ratios of 2.34 +/- 0.07 versus 1.32 +/- 0.15). These results indicate that increases in circulating AngII levels increase intrarenal angiotensinogen mRNA levels, which may contribute to the sustained renal AngII-generating capacity that paradoxically occurs in AngII-treated hypertensive rats.

Figure 1

(A) Immunohistochemical distribution of angiotensinogen in the rat renal cortex, illustrating prominent proximal tubular (PT) localization. Distal tubules (DT) exhibited negative staining. (B) Detection of strong positive immunostaining in proximal convoluted tubules (PCT) and weak positive immunostaining in glomerular endothelial cells (GEC). Distal tubules and the renal vasculature (RV) exhibited negative results. (C) Control experiment, demonstrating that no positive immunostaining was observed when primary antibody was omitted. Bars, 10 μm (A) or 40 μm (B and C).

Figure 2

Western blot analysis of plasma (5 μg), kidney (25 μg), and liver (25 μg) protein, using a specific anti-angiotensinogen polyclonal antibody. (A) Figure showing specific bands at 52 and 64 kD for plasma (left lane). Incubation of plasma protein with peptide N-glycosidase F yielded only one band, at 50 kD (right lane). (B) Densitometric analysis of plasma protein obtained 24 h after bilateral nephrectomy (lane P_Nx), showing that 64-kD angiotensinogen protein levels were elevated approximately 50%, compared with control values (lane P_C). Western blot analysis of kidney protein revealed one specific immunoreactive band, at 52 kD (lane K), whereas liver exhibited bands at 52 and 64 kD (lane L). (C) Representative autoradiograph of a plasma angiotensinogen Western blot of samples from angiotensin II (AngII)-treated (A) (n = 8) and sham-treated (S) (n = 7) rats. (D) Densitometric analysis of the immunoreactive bands, showing that AngII infusion did not alter plasma 52- or 64-kD angiotensinogen protein levels. Similar observations were obtained in two other experiments. Ao, angiotensinogen.

Figure 3

Western blot analysis of kidney (A and B) and liver (C and D) protein, using the angiotensinogen-specific polyclonal antibody. (A) Representative autoradiograph of a renal angiotensinogen Western blot of samples from AngII-treated (n = 6) and sham-treated (n = 7) rats. (B) Densitometric analysis of the immunoreactive bands, showing that AngII infusion did not alter renal angiotensinogen protein levels. (C) Representative autoradiograph of a hepatic angiotensinogen Western blot of samples from AngII-treated (n = 7) and sham-treated (n = 7) rats. Similar observations were obtained in two other experiments. (D) Densitometric analysis of the immunoreactive bands, showing that AngII infusion increased hepatic 64-kD angiotensinogen protein levels more than threefold; however, 52-kD protein levels were not altered. Similar observations were obtained in two other experiments. *P < 0.05.

Figure 4

(A and C) Representative ethidium bromide-stained gels, showing angiotensinogen and corresponding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels in the kidney (n = 6/group) (A) and in the liver (n = 6/group) (C). (B and D) Densito-metric analyses of the bands indicated that AngII infusion significantly increased renal angiotensinogen mRNA levels by 42% (B) and hepatic angiotensinogen mRNA levels by 78% (D). Similar observations were obtained in two other experiments. M, DNA size marker (HaeIII fragments of ϕX174 RF DNA). *P < 0.05.