This study was performed to determine the influence of neuronal nitric oxide synthase (nNOS) on renal arteriolar tone under conditions of normal, interrupted, and increased volume delivery to the macula densa segment and on the microvascular responses to angiotensin II (ANG II). Experiments were performed in vitro on afferent (21.2 ± 0.2 μm) and efferent (18.5 ± 0.2 μm) arterioles of kidneys harvested from male Sprague-Dawley rats, using the blood-perfused juxtamedullary nephron technique. Superfusion with the specific nNOS inhibitor, S-methyl-l-thiocitrulline (l-SMTC), decreased afferent and efferent arteriolar diameters, and these decreases in arteriolar diameters were prevented by interruption of distal volume delivery by papillectomy. When 10 mM acetazolamide was added to the blood perfusate to increase volume delivery to the macula densa segment, afferent arteriolar vasoconstrictor responses tol-SMTC were enhanced, but this effect was again completely prevented after papillectomy. In contrast, the arteriolar diameter responses to the nonselective NOS inhibitor, N ω-nitro-l-arginine (l-NNA) were only attenuated by papillectomy.l-SMTC (10 μM) enhanced the efferent arteriolar vasoconstrictor response to ANG II but did not alter the afferent arteriolar vasoconstrictor responsiveness to ANG II. In contrast, l-NNA (100 μM) enhanced both afferent and efferent arteriolar vasoconstrictor responses to ANG II. These results indicate that the modulating influence of nNOS on afferent arteriolar tone of juxtamedullary nephrons is dependent on distal tubular fluid flow. Furthermore, nNOS exerts a differential modulatory action on the juxtamedullary microvasculature by enhancing efferent, but not afferent, arteriolar responsiveness to ANG II.

Objective The present study was performed to examine in two-kidney, one clip (2K1C) Goldblatt hypertensive mice, first, the relative contribution of angiotensin II (ANG II) receptor subtypes 1A (AT1A) and 1B (AT1B); second, the role of ANG II type 2 (AT2) receptors in the development of hypertension in wild-type (AT1A+/+) and AT1A receptor knockout (AT1A−/−) mice and third, the role of increased nitric oxide synthase (NOS) activity in counteracting the hypertensinogenic action of ANG II in this model. Methods AT1A+/+ and AT1A−/− mice underwent clipping of one renal artery and were infused with either saline vehicle or with the selective AT2 receptor agonist CGP-42112A (CGP). Blood pressure (BP) was monitored by radiotelemetry. BP responses to the NOS inhibitor nitro-L-arginine-methyl-ester (L-NAME) were evaluated. Results AT1A+/+ mice responded to clipping by a rise in BP which was not modified by CGP infusion. Clip placement caused a slight increase in BP in AT1A−/− mice which remained significantly lower than in AT1A+/+ mice. Acute NOS inhibition caused greater increases in BP in 2K1C/AT1A+/+ than in AT1A+/+ mice. Conclusions The present data support the critical role AT1A receptors in the development of 2K1C hypertension, whereas AT1B receptors play only a minor role in BP regulation in this model of ANG II-dependent hypertension. Activation of AT2 receptors does not play an antagonistic role in the AT1 receptor-mediated hypertensinogenic actions of ANG II in this model. Finally, enhanced NOS activity plays a protective role by counteracting the vasoconstrictor influences of ANG II in 2K1C hypertensive mice.

The presence of receptors for angiotensin II (AII) on the luminal membranes of various nephron segments has been well established for many decades.1,2 Originally their function remained unclear because tubular fluid AII concentrations were thought to be quite low due to the presence of various degrading enzymes on the brush border of proximal tubular cells. However, a series of reports in the 1990s demonstrated that the proximal tubular concentrations of AI and AII are in the nanomolar range and much higher than can be explained by tubular fluid reabsorption or equilibration with the circulating levels. 3,4 These findings led to a paradigm shift in our concepts regarding the role of luminal AII receptors in various nephron segments, and it is now well accepted that intraluminal AII and other angiotensin peptides exert various actions on transport systems in essentially all nephron segments predominantly through activation of AT1 receptors. 5-10In further studies, proximal tubular fluid samples were incubated with excess renin to determine substrate availability. The resultant AI concentrations demonstrated that the proximal tubular fluid angiotensinogen (AGT) concentrations are also very high 11 and greater than the circulating concentrations, indicating that it is unlikely the tubular AGT concentrations are derived from filtered AGT, in particular considering the limited permeability of the AGT because of its large size.11 Using in situ perfusion of proximal tubules with artificial tubular fluid with the delivery of filtrate blocked, Braam et al.4 collected the tubular fluid from downstream segments and found these also had elevated AII concentrations in the nanomolar range, supporting tubular secretion of AII or its precursors. Studies on isolated perfused tubules from S2 segments indicated that AII is produced intracellularly and secreted preformed into the tubular lumen, supporting the presence of intact AGT in isolated proximal tubule segments.12 These findings, along with clear evidence for the presence of AGT mRNA and protein in proximal tubular cells, [13][14][15] provided the foundation for the concept that the intratubular AII concentrations are derived primarily from locally synthesized substrate. The local production of AGT in the proximal tubule was supported by Terada et al., 14 who demonstrated the presence of a large signal for AGTmRNA in microdissected proximal convoluted and straight tubules. In vitro studies have also consistently demonstrated mRNA encoding AGT in proximal tubular cell lines extracted from proximal tubule segments.15,16 However, Pohl et al. 17 recently reported predominant localization of mRNA encoding AGT to S3 segments.The findings of kidney tubular mRNA encoding AGT notwithstanding, the kidney's ability to produce AGT is dwarfed by that of the liver, the organ primarily responsible for the maintenance of circulating AGT concentrations. In addition, it is well recognized that filtered AGT can be taken up by proximal tubule scavenger receptors such as megalin and cubilin, ...

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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.