Urinary Angiotensinogen as an Indicator of Intrarenal Angiotensin Status in Hypertension

In rats maintained on a high salt diet (H/S) to suppress basal renal angiotensinogen levels, angiotensin II (Ang II) infusion for 13 days increased renal angiotensinogen mRNA and protein, thus providing a mechanism for further augmentation of intrarenal Ang II levels. The present study tested the hypothesis that enhanced intrarenal angiotensinogen formation during Ang II infusion is reflected by secretion into the tubular fluid leading to increased urinary excretion of angiotensinogen (UAGT). The effects of chronic Ang II infusion were examined on kidney and plasma Ang II levels and UAGT in male Sprague-Dawley rats maintained on an 8% salt diet for three weeks (N=10). Following one week on the H/S diet, Ang II (40 ng/min) was administered for two weeks via an osmotic minipump to one group (H/S + Ang II, N=5), while the remaining rats were sham-operated (H/S + Sham, N=5). Additionally, a control group was prepared with normal salt diet and sham-operation (N/S + Sham, N=5). H/S alone did not alter systolic blood pressure (BP) (103 +/- 2 vs. 104 +/- 2 mm Hg), while Ang II infusion to H/S rats significantly increased systolic BP from 103 +/- 2 to 154 +/- 2 after two weeks. Intrarenal Ang II content in H/S + Ang II was significantly greater than H/S + Sham (435 +/- 153 vs. 65 +/- 14 fmol/g). Ang II infusion significantly increased UAGT (4.0 +/- 0.5 vs. 1.0 +/- 0.2 nmol Ang I/day by radioimmunoassay of generated Ang I; 57 +/- 15 vs. 14 +/- 2 densitometric units by Western blotting analysis) compared to Sham. UAGT by radioimmunoassay was highly correlated with kidney Ang II content (r=0.79); but not with plasma Ang II concentration (r=0.20). These data demonstrate that chronic Ang II infusion increases urinary excretion rate of angiotensinogen, and suggest that UAGT provides a specific index of intrarenal angiotensinogen production in Ang II-dependent hypertension.

We examined angiotensinogen gene expression in rat kidney by in situ hybridization histochemistry. Using a rat cRNA probe to angiotensinogen, we demonstrated angiotensinogen mRNA to be localized predominantly in the proximal renal tubule, with considerably lesser amounts in distal tubular segments and glomerular tufts. Previous studies have localized renin immunoreactivity to the juxtaglomerular cells, glomerular tufts, and proximal tubules. Such findings provide further evidence for a local tissue renin angiotensin system within the kidney which may influence regional function. Based on our data, we hypothesize that a major site of angiotensin production is the proximal tubule. We postulate that angiotensin synthesized in and/or around the proximal tubule may directly modulate tubular transport of sodium, bicarbonate, and water. In addition to the proximal tubule, the specific localization of the renin angiotensin components elsewhere in the kidney would also support the other proposed regional functions of the intrarenal system, including modulation of tubuloglomerular balance.

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.