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Diabetes mellitus is one of the most prevalent diseases and is associated with increased incidence of structural and functional derangements in the kidneys, eventually leading to end-stage renal disease in a significant fraction of afflicted individuals. The renoprotective effects of renin-angiotensin system (RAS) blockade have been established; however, the mechanistic pathways have not been fully elucidated. In this review article, the cardinal role of an activated RAS in the pathogenesis of diabetic nephropathy (DN) is discussed with a focus on 4 themes: (1) introduction to RAS cascade, (2) intrarenal RAS in diabetes, (3) clinical outcomes of RAS blockade in DN, and (4) potential of urinary angiotensinogen as an early biomarker of intrarenal RAS status in DN. This review article provides a mechanistic rational supporting the hypothesis that an activated intrarenal RAS contributes to the pathogenesis of DN and that urinary angiotensinogen levels provide an index of intrarenal RAS activity.

Angiotensin II (AngII) infusions augment renal angiotensinogen mRNA and protein and urinary angiotensinogen excretion (U(AGT)). Further experiments were performed in 4 groups of rats: normal salt diet with sham operation, NS+Sham, n=6; NS with AngII infusion at 40 ng/min via osmotic minipump, NS+AngII(40), n=9; NS with AngII infusion at 80 ng/min, NS+AngII(80), n=9; high-salt diet with deoxycorticosterone acetate salt pellet (100 mg), HS+DOCA, n=4. These experiments sought to determine whether enhanced U(AGT) is specifically associated with increased kidney AngII levels or is a nonspecific consequence of the hypertension. Systolic BP (SBP) was significantly increased to 131+/-2 and 162+/-2 mm Hg at day 11 in NS+AngII(40) and NS+AngII(80), respectively, compared with NS+Sham (110+/-1). Regression analysis demonstrated a positive relationship (R=0.49) between SBP and U(AGT) for NS+Sham (1.1+/-0.3 nmol AngI/d), NS+AngII(40) (2.5+/-0.9), and NS+AngII(80) (5.5+/-1.5). U(AGT) was also highly correlated (R=0.70) with kidney AngII content for NS+Sham (49+/-6 fmol/g), NS+AngII(40) (215+/-49), and NS+AngII(80) (347+/-47); but not with plasma AngII (R=0.12). HS+DOCA rats also exhibited increased SBP to 134+/-1 mm Hg, but U(AGT) (1.4+/-0.4 nmol AngI/d) and intrarenal AngII content (13+/-2 fmol/g) were not increased despite the hypertension. Infused human angiotensinogen could not be detected in urine of sham-operated or AngII-infused rats (n=4 each). These data demonstrate that U(AGT) increases in AngII-dependent hypertension in a dose- and time-dependent manner, but not in hypertension elicited by HS+DOCA. The results support the hypothesis that AngII-dependent hypertension results in elevated intrarenal AngII and angiotensinogen levels, reflected by increased U(AGT), which does not occur in an AngII-independent hypertensive model.

Background: 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)., Methods: 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)., Results: 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)., Conclusions: 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.

Intrarenal angiotensin II (Ang II) is regulated by several complex processes involving formation from both systemically delivered and intrarenally formed substrate, as well as receptor-mediated internalization. There is substantial compartmentalization of intrarenal Ang II, with levels in the renal interstitial fluid and in proximal tubule fluid being much greater than can be explained from the circulating levels. In Ang II--dependent hypertension, elevated intrarenal Ang II levels occur even when intrarenal renin expression and content are suppressed. Studies in Ang II--infused rats have demonstrated that augmentation of intrarenal Ang II is due, in part, to uptake of circulating Ang II via an Ang II type 1 (AT(1)) receptor mechanism and also to sustained endogenous production of Ang II. Some of the internalized Ang II accumulates in the light and heavy endosomes and is therefore potentially available for intracellular actions. The enhanced intrarenal Ang II also exerts a positive feedback action to augment intrarenal levels of angiotensinogen (AGT) mRNA and protein, which contribute further to the increased intrarenal Ang II in hypertensive states. In addition, renal AT(1) receptor protein and mRNA levels are maintained, allowing increased Ang II levels to elicit progressive effects. The increased intrarenal Ang II activity and AGT production are associated with increased urinary AGT excretion rates. The urinary AGT excretion rates show a clear relationship to kidney Ang II content, suggesting that urinary AGT may serve as an index of Ang II--dependent hypertension. Collectively, the data support a powerful role for intrarenal Ang II in the pathogenesis of hypertension.

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.