Enhancement of angiotensinogen expression in angiotensin II-dependent hypertension

Abstract

Chronic infusion of angiotensin (Ang) II leads to the development of hypertension and enhances intrarenal Ang II content to levels greater than can be explained from the circulating concentrations of the peptide. We previously reported that renal angiotensinogen (Ao) mRNA is enhanced in Ang II-dependent hypertension and may contribute to augmented intrarenal Ang II levels, but the Ao protein levels were not significantly increased. Because a high-salt diet (H/S) has been shown to suppress renal expression of Ao mRNA, we examined the effects of chronic Ang II infusion on kidney and liver Ao mRNA and protein levels in male Sprague-Dawley rats (n=12) maintained on an 8% salt diet. Ang II was administered via osmotic minipumps (40 ng/min) to 1 group (n=6) while the remaining rats were sham-operated. A H/S diet alone did not alter systolic blood pressure in sham animals (109+/-6 mm Hg at day 12); however, Ang II infusions to the H/S rats significantly increased systolic blood pressure (167+/-7 at day 12) and intrarenal Ang II content (459+/-107 fmol/g versus 270+/-42) despite a marked suppression of plasma renin activity (0.9+/-0.2 ng Ang I. mL(-1). h(-1) versus 2.8+/-1.3). Ang II infusions significantly increased kidney Ao mRNA compared with the H/S diet alone by 1.9+/-0.1-fold. Western blot analysis of kidney protein extracts showed that the Ang II-infused rats had increased kidney Ao protein levels compared with the H/S diet alone (1.9+/-0.1-fold). Liver Ao mRNA and protein and plasma Ao protein were also significantly increased by Ang II infusions. These data demonstrate the effects of Ang II infusion to stimulate Ao mRNA and protein. Thus, the augmented intrarenal Ang II in Ang II-dependent hypertension may result, in part, by a positive amplification mechanism to activate renal expression of AO:

Figure 1

The relationship between the amount of kidney total RNA template (62.5 to 500 ng) and RT-PCR products was evaluated (A). A linear relationship was found between the amount of total RNA template and the amount of PCR product in the range of 62.5 to 500 ng for renal angiotensinogen at 28 cycles of PCR (B). On the basis of these data, the amount of renal total RNA template was selected as 125 ng for the following analysis.

Figure 2

Western blot analysis of plasma protein using the specific Ao polyclonal antibody. A, Representative autoradiograph of plasma Ao Western blot from Ang II–infused and sham-operated rats on a H/S diet. B, Densitometric analysis of the immunoreactive bands showed that Ang II infusion significantly increased both forms of plasma Ao protein. Similar observations were obtained from 2 other experiments. *P,0.05 vs H/S+Sham.

Figure 3

A, Representative gels showing Ao and corresponding GAPDH mRNA levels in the kidney (lanes A, Ang II–infused rats fed H/S diet; lanes S, sham-operated rats fed H/S diet). B, Densitometric analysis of the bands shows that Ang II infusion significantly increased renal Ao mRNA levels by 86%. Similar observations were obtained from 2 other experiments. *P<0.05 vs H/S+Sham.

Figure 4

Western blot analysis of renal protein using the specific Ao polyclonal antibody. A, Representative autoradiograph of renal Ao Western blot from Ang II–infused and sham-operated rats on a H/S diet. B, Densitometric analysis of the immunoreactive bands showed that Ang II infusion significantly increased renal Ao protein levels by 88%. Similar observations were obtained from 2 other experiments. *P<0.05 vs H/S+Sham.

Figure 5

A, Representative gels showing Ao and corresponding GAPDH mRNA levels in the liver (lanes A, Ang II–infused rats fed H/S diet; lanes S, sham-operated rats fed H/S diet). B, Densitometric analysis of the bands shows that Ang II infusion significantly increased hepatic Ao mRNA expression by 2.9-fold. Similar observations were obtained from 2 other experiments. *P<0.05 vs H/S+Sham.

Figure 6

Western blot analysis of hepatic protein using the specific Ao polyclonal antibody. A, Representative autoradiograph of hepatic Ao Western blot from Ang II–infused and sham-operated rats on a H/S diet. B, Densitometric analysis of the immunoreactive bands showed that Ang II infusion significantly increased hepatic Ao protein levels for 64-kDa protein; however, hepatic Ao protein levels for 52-kDa protein were not significantly increased. Similar observations were obtained from 2 other experiments. *P<0.05 vs H/S+Sham.