(Hypertension. 1996;28:669-677.)
© 1996 American Heart Association, Inc.
Articles |
Receptor-Mediated Intrarenal Angiotensin II Augmentation in Angiotensin II–Infused Rats
the Department of Physiology, Tulane University School of Medicine, New Orleans, La.
Correspondence to Dr Li-Xian Zou, Department of Physiology SL39, Tulane University School of Medicine, 1430 Tulane Ave, New Orleans, LA 70112.
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Abstract |
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Chronic low-dose angiotensin II (Ang II) infusion for 13 days mimics two-kidney, one clip Goldblatt hypertension and increases intrarenal Ang II levels. We performed studies to determine the time course for the enhancement of intrarenal Ang II levels and whether the increased intrarenal Ang II is a tissue-specific event and requires a receptor-mediated step. Male Sprague-Dawley rats were uninephrectomized, and either vehicle or Ang II (40 ng/min) was infused via a subcutaneous osmotic minipump. Plasma and renal Ang II levels were measured 3, 7, 10, and 13 days after minipump implantation. Compared with controls (126±2 mm Hg), systolic pressure in Ang II–infused rats exhibited a detectable increase by day 6 (146±2 mm Hg) and continued to increase to 189±5 mm Hg by day 12. Plasma Ang II levels were elevated by day 3, whereas intrarenal Ang II levels were not significantly elevated until 10 days of Ang II infusion. Renal injury characterized by focal and segmental glomerulosclerosis was evident after 13 days of Ang II infusion. Losartan (30 mg/kg per day) prevented the development of hypertension in the Ang II–infused rats for the duration of the infusion period (125±1 mm Hg) and reduced the degree of glomerular injury. Plasma renin activity was suppressed in the Ang II–infused group but was elevated markedly in both losartan-treated groups. Plasma Ang II levels were elevated in the Ang II–infused rats and were even higher during losartan treatment. Intrarenal Ang II levels were enhanced significantly (354±60 versus 164±23 fmol/g) in the Ang II–infused rats. However, losartan treatment prevented the augmentation of intrarenal Ang II caused by Ang II infusion. Heart and adrenal Ang II levels were not significantly increased in the Ang II–infused rats but were significantly elevated during losartan treatment. These results suggest that the tissue-specific elevations of intrarenal Ang II levels caused by chronic Ang II infusion are mediated by angiotensin type 1 receptor activation, which leads to either receptor-mediated internalization of Ang II, enhancement of intrarenal Ang II formation, or both.
Key Words: angiotensin II • receptors, angiotensin II • renin-angiotensin system
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Introduction |
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Enhanced activity of the intrarenal renin-angiotensin system has been proposed to play a major role in the pathophysiology of 2K1C hypertension.1 2 3 4 5 Previous studies have shown maintained or elevated intrarenal Ang II levels in the contralateral nonclipped kidney even though renal renin is depleted and renal renin gene expression is markedly suppressed.6 7 8 9 Similar findings of elevated intrarenal Ang II levels with marked renal renin depletion have been observed in uninephrectomized rats infused chronically with low doses of Ang II for 2 weeks.7 Such elevated intrarenal Ang II levels could lead to reductions in renal function and sodium excretion and thus contribute to the development and maintenance of hypertension.1 2 10 This concept has received support from studies using Ang II antagonists in 2K1C hypertensive rats showing that the hypertensive response is prevented and that hemodynamic and excretory functions in the nonclipped kidney are restored or improved.2 7 10 11 12 13 The findings of renal vascular and glomerular injury in the nonclipped kidney of 2K1C rats and in Ang II–infused rats further support the potential role of elevated intrarenal Ang II in hypertension-associated renal injury.14 15 Intrarenal Ang II levels may also play an important role in the pathogenesis of hypertension in the spontaneously hypertensive rat. In a study by Lu et al,16 renal interstitial infusion of captopril selectively inhibited renal ACE activity, increased medullary blood flow and sodium excretion, and lowered blood pressure in spontaneously hypertensive rats.
Although it has been difficult to dissociate the effects of circulating versus intrarenal Ang II, Siragy et al17 reported significant increases in urine flow, urinary sodium excretion, glomerular filtration rate, and renal plasma flow when a combination of inhibitors of renin, ACE, and Ang II receptors was infused intrarenally, but these changes did not occur when the inhibitors were infused systemically. The hemodynamic and excretory changes that occurred during intrarenal administration of renin and ACE inhibitors were reversed by coadministration of Ang II intrarenally. It has also been demonstrated that intrarenal levels of Ang II in tubular fluid, peritubular capillaries, and interstitial fluid are greater than can be explained from the circulating levels.18 19 20 These results support the concept that intrarenally produced Ang II exerts a unique role in the control of renal function.
In previous studies, it has been shown that although the nonclipped kidney of 2K1C hypertensive rats and kidneys from Ang II–infused rats are renin depleted and have reduced renin mRNA, they are not depleted of Ang II, and the angiotensinogen mRNA levels are well maintained.6 7 8 9 21 22 Furthermore, the renal Ang II levels in the remaining kidney of Ang II–infused rats and the nonclipped kidney of 2K1C rats increased to levels above those that can be readily explained by passive accumulation of circulating Ang II.7 In view of the marked reductions in renin content and renin mRNA levels, the data suggest that there is an alternate mechanism or reninlike enzyme responsible for maintaining or augmenting intrarenal Ang II levels in response to increases in circulating Ang II.23
To investigate the mechanism of this augmentation process and its possible role in the pathogenesis of hypertension, we performed further studies to determine (1) whether the development of hypertension is associated with time-dependent augmentation of intrarenal Ang II levels, (2) whether augmentation of Ang II is a renal-specific event, and (3) whether the augmentation of intrarenal Ang II levels requires activation of Ang II receptors. By measuring SBP and intrarenal Ang II levels at various time points, we compared alterations of plasma and intrarenal Ang II levels with the associated increases in arterial pressure. By comparing Ang II levels in the kidney, heart, and adrenal gland, we could determine whether the augmentation of Ang II was specific to the kidney. Because the predominant angiotensin receptor in the kidney is the AT1 receptor,24 25 26 we used the AT1 receptor antagonist losartan to determine whether AT1 receptor blockade prevents the augmentation of intrarenal Ang II and the development of Ang II–induced hypertension and associated renal injury.
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Methods |
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Experimental Design
Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, Mass) were housed in wire cages and maintained in a temperature- and light-controlled room. Throughout the experiments, rats had free access to standard rat chow (Ralston Purina). All experiments were approved by the Tulane University Animal Care and Use Committee.
Surgical procedures were performed with rats under pentobarbital anesthesia (50 mg/kg IP) and with sterile conditions. Rats weighing 180 to 200 g were subjected to a right nephrectomy, and either 0.9% NaCl vehicle (12 µL/d) or Ang II was infused via an osmotic minipump (model 2002, Alza Corp) implanted subcutaneously at the dorsum of the neck.7 Ang II (Sigma Chemical Co) was delivered continuously at a rate of 40 ng/min.
Protocol 1: Intrarenal Ang II Levels and Alterations of Renal Structure During the Development of Hypertension
Rats were divided into two groups: group 1 rats (control, n=33) were subjected to vehicle infusion; group 2 rats (n=40) were subjected to Ang II infusion. We used subsets of these respective groups to determine plasma and intrarenal hormonal levels and to perform renal histological studies on tissues harvested 3, 7, 10, and 13 days after initiation of Ang II infusion.
Protocol 2: Effects of Losartan on Intrarenal Ang II Levels
Rats were divided into four experimental groups: group 1 rats (control, n=12) were subjected to vehicle infusion; group 2 rats (n=6) were subjected to vehicle infusion and treated with losartan; group 3 rats (n=13) were subjected to Ang II infusion; and group 4 rats (n=14) were subjected to Ang II infusion and treated with losartan. Losartan (DuPont-Merck Pharmaceutical Co) was administered in the drinking water at a dose of 30 mg/kg per day.12 27 We used subsets of these respective groups to determine plasma and intrarenal hormonal levels and to perform renal morphological studies after 13 days of Ang II infusion.
To monitor the development and progression of hypertension, we measured SBP in conscious rats by tail-cuff plethysmography (Harvard Apparatus) 2 to 3 days before surgery and on days 3, 6, 9, and 12 after surgical manipulations. We measured SBP between 9 AM and noon to control for circadian variation of this parameter. Daily average values for each rat were based on at least three determinations. On the day samples were collected, the rats were decapitated between 10 AM and noon. Trunk blood was collected for measurement of PRA and plasma angiotensinogen, Ang I, and Ang II levels. The kidney was removed quickly and one half immediately homogenized for measurement of angiotensinogen or Ang I and Ang II contents. The other half was placed in fixative for histological studies. The time delay between decapitation and homogenization of the kidney was approximately 60 seconds. To compare the changes in Ang II levels between kidney and extrarenal organs during Ang II infusion and losartan treatment, heart and adrenal samples were also immediately harvested and homogenized for Ang II determination.
Analytic Procedures of Samples
Ang I and Ang II levels were measured by radioimmunoassays as recently modified and validated in our laboratory.28 For assessment of plasma Ang I and Ang II levels, trunk blood was collected in chilled tubes containing a mixed inhibitor solution (5 mmol/L EDTA, 10 µmol/L pepstatin, 20 µmol/L enalaprilat, and 1.25 mmol/L 1,10-phenanthroline). To minimize in vitro generation of the peptides, blood samples were immediately centrifuged at 4°C for 10 minutes at 1000g, and plasma for analysis of angiotensin peptides was separated and immediately extracted by adsorption to and elution with 100% methanol from a phenyl-bonded solid-phase extraction column (Bond-Elut, Varian). The eluates were collected and stored at -20°C. Before radioimmunoassay, the eluates were evaporated to dryness under vacuum and reconstituted in assay diluent.
For analysis of renal Ang I and Ang II levels, one half of each kidney was weighed, immersed in cold methanol (100%), and homogenized with a glass homogenizer immediately after harvesting. The supernatants from the kidney homogenates were dried overnight in a vacuum centrifuge. The dried residue was reconstituted in 4 mL of 50 mmol/L sodium phosphate buffer, pH 7.4, containing 0.1 mg/mL human serum albumin. These samples were purified and stored as described above for plasma. For analysis of heart and adrenal Ang II levels, heart and adrenal gland were harvested, homogenized, and extracted as described for kidney. The reconstituted plasma, kidney, heart, and adrenal extracts were incubated with rabbit anti–Ang I or anti–Ang II antisera (Arnel) and 125I-labeled Ang I or II (Sigma) for 48 hours at 4°C. Bound and free angiotensin peptides were separated by dextran-coated charcoal, and the supernatants were counted by a computer-linked gamma counter for 3 minutes. Results are reported as femtomoles per gram kidney weight or femtomoles per milliliter plasma. The sensitivity of the Ang I and Ang II assays were 1.7±0.5 and 1.7±0.3 fmol, respectively, during 90% of maximal binding. For the Ang I and Ang II assays, the specific binding was 29.7±0.5% and 36.7±0.8%, respectively, with a nonspecific binding of 2.4±0.5% and 1.5±0.1%, respectively. As previously reported,28 cross-reactivity for antisera demonstrated that Ang-(1-10) and Ang-(2-10) were equipotent for the Ang I antisera, whereas Ang-(1-8) and its COOH-terminal fragments were more than 10 000-fold less effective in displacing 125I–Ang I. Ang-(1-8) and Ang-(2-8) exhibited similar potencies for the Ang II antisera. Ang-(3-8) was also detected by Ang II antisera, but the displacement of 125I–Ang II was not parallel to that elicited by Ang II, and Ang-(1-10), Ang-(2-10), and short COOH-terminal fragments of Ang II were only detected at concentrations more than 100-fold higher than those for Ang II. The recoveries of angiotensin peptides through the extraction and purification procedures were determined by direct addition of 125I–Ang I and II into the plasma samples immediately before application to the solid-phase extraction columns and into the homogenization solutions immediately before collection of the tissues. The recoveries of Ang I and II from plasma were 90% to 95% and from tissues were both 80%.
For renin determination, trunk blood was collected in chilled tubes containing EDTA (5 mmol/L) and then was centrifuged at 4°C for 10 minutes at 1000g. Plasma was separated, immediately frozen, and stored at -20°C until assayed. PRA was measured by the radioimmunoassay of Ang I generation with a standard commercial kit (Incstar) as described previously.7 Plasma and renal angiotensinogen assays were performed as previously described by Campbell et al.29 Angiotensinogen levels were indirectly measured by incubation with excess renin and the radioimmunoassay of Ang I generation. One hundred microliters of plasma (serially diluted in 100 mmol/L sodium phosphate, 10 mmol/L EDTA, 150 mmol/L sodium chloride, 1 g/L casein, 1 g/L sodium azide, pH 8.0) was incubated in a total volume of 505 µL, containing 100 µL of porcine kidney renin (Sigma); 300 µL of a 200-mmol/L sodium phosphate buffer containing 100 mmol/L citric acid, 50 mmol/L EDTA, and 1 g/L sodium azide (pH 5.7); and 5 µL of 144 mmol/L phenylmethylsulfonyl fluoride (PMSF) (in ethanol). After incubation at 37°C for 60 minutes, Ang I generation was quantified by radioimmunoassay. For renal angiotensinogen measurements, half of each kidney was weighed and immediately homogenized in 5 mL of a cold solution containing 1.5 mol/L ammonium sulfate, 100 mmol/L sodium phosphate, 10 mmol/L EDTA, 12.5 mmol/L N-ethylmaleimide, and 2 mmol/L benzamidine (pH 6.5), to which were added 0.5 mL of 1.5 mmol/L pepstatin (in ethanol) and 50 µL of 144 mmol/L PMSF and 10 mmol/L phenylmercuric acetate (in ethanol). The homogenate was centrifuged at 5000g for 15 minutes, and the supernatant was decanted. For precipitation of angiotensinogen, the ammonium sulfate concentration of the supernatant was adjusted to 2.5 mol/L by addition of 4 mol/L ammonium sulfate, and the mixture was centrifuged as before. The ammonium sulfate precipitate was resuspended in 4 mL water, and a 100-µL aliquot was incubated with renin, phosphate buffer, PMSF, and phenylmercuric acetate for a final volume of 505 µL as described above for plasma. After incubation at 37°C for 60 minutes, 0.5 mL of 1% trifluoroacetic acid was added for termination of Ang I generation, and the samples were extracted for purification. The eluates were dried under vacuum and reconstituted in assay diluent. Ang I was quantified by radioimmunoassay as previously described.
For histological studies, kidneys of Ang II–infused rats (n=12) and Ang II–infused losartan-treated rats (n=6) were weighed and immediately fixed in 10% buffered formalin solution and embedded in paraffin for light microscopic studies. Sections were cut at thicknesses of 2 to 3 µm and stained with hematoxylin-eosin, periodic acid–Schiff reagent, and periodic acid–methenamine-silver (PAM). Histological examination was performed by two observers in a blind fashion. For semiquantitative evaluation, glomerular injury scores were obtained by use of the sections stained with periodic acid–Schiff and examination of approximately 50 glomeruli for each specimen. Each glomerulus was graded from 1 to 4 as previously described by Komatsu et al30 : grade 1, normal glomerulus identified by light microscope; grade 2, involvement of up to one third of the glomerular areas; grade 3, involvement of one third to two thirds of the glomerulus; and grade 4, two thirds involvement or global sclerosis. Each score was calculated according to the following formula: Glomerular Injury Score=[(0xNo. of Grade 1 Glomeruli)+(1xNo. of Grade 2)+(2xNo. of Grade 3)+(3xNo. of Grade 4)]x100/(No. of Glomeruli Graded).
Data Analysis
All data are presented as mean±SE. The statistical analyses for tissue hormone and histological studies were performed with one-way ANOVA and Fisher's least significant difference post hoc test. Variances for plasma hormonal data were found to be heterogeneous by the F maximum test for homogeneity of variances and necessitated the logarithmic transformation of the plasma hormone data before analysis by one-factor ANOVA. Differences between and within groups for SBP measurements were analyzed by two-way ANOVA with repeated measures on one factor and Fisher's least significant difference post hoc test. A value of P<.05 was considered statistically significant.
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Results |
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Influence of Losartan on the Development of Hypertension During Ang II Infusions
Before surgical manipulations, SBP values in all the experimental groups were normotensive and were not significantly different among groups, having an overall average of 126±1 mm Hg (Fig 1
). In agreement with previous studies,7 the Ang II–infused rats exhibited progressive increases in SBP. SBP increased significantly above control values by day 6 (146±2 mm Hg). By day 9, SBP continued to increase to 164±2 mm Hg and reached 189±5 mm Hg at 12 days. Losartan treatment prevented the development of hypertension in the Ang II–infused group, and SBP remained at normal levels (125±1 mm Hg) for the duration of the study. SBP in the losartan-treated rats infused with saline decreased slightly (127±1 to 112±2 mm Hg, P<.05). SBP in the control rats remained stable and normotensive throughout the duration of the experiment (127±2 to 126±2 mm Hg).
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Effects of Ang II and Losartan on Plasma Renin
PRA in control rats averaged 4.35±0.62 ng Ang I/mL per hour but was markedly suppressed in the Ang II–infused rats by day 3; this suppression was maintained throughout the infusion period (Table 1). In contrast, both losartan-treated groups exhibited a marked 12-fold increase in PRA but were not different from each other at 13 days.
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Effects of Ang II and Losartan on Plasma and Kidney Angiotensinogen
As shown in Fig 2, control values of plasma and kidney angiotensinogen levels averaged 497±34 pmol/mL and 121±27 pmol/g, respectively. Plasma angiotensinogen levels were increased by 58% in the Ang II–infused group and were decreased by 35% and 41%, respectively, in the losartan-treated groups infused with saline or Ang II. Although similar trends were observed in renal angiotensinogen contents, the differences did not achieve statistical significance.
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Effects of Ang II and Losartan on Plasma and Kidney Ang I
As predicted from the PRA data, plasma Ang I levels in the Ang II–infused rats were markedly reduced by day 3, and these levels remained suppressed over the 13-day period (Fig 3). Plasma Ang I levels were significantly elevated in both losartan-treated groups to the same extent (Fig 4). Intrarenal Ang I contents showed a different pattern from that of plasma Ang I levels (Figs 3 and 4). In particular, the Ang II–infused rats did not exhibit significant reductions in intrarenal Ang I levels during the Ang II infusion in any of the groups despite marked plasma renin and Ang I suppression. Also, losartan administration did not increase intrarenal Ang I contents in the saline-infused group. Intrarenal Ang I levels in these two groups were similar to control levels. However, the rats that received losartan treatment combined with the Ang II infusion demonstrated a significant twofold elevation in renal Ang I contents compared with controls.
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Effects of Ang II and Losartan on Plasma and Kidney Ang II
As shown in Fig 5, in the Ang II–infused groups, plasma Ang II levels were elevated by day 3 of Ang II infusion and seemed to increase slightly during the duration of infusion. In both losartan-treated groups (Fig 6), plasma Ang II levels were increased markedly by day 13 regardless of whether Ang II was infused. Plasma Ang II levels in the losartan-treated groups were twofold higher than in the Ang II–infused group and sixfold higher than in the control group. Intrarenal Ang II contents, however, showed a different pattern (Figs 5 and 6). Unlike plasma Ang II levels, intrarenal Ang II contents in the Ang II–infused rats did not increase significantly until 10 days after the start of infusion. At 13 days, intrarenal Ang II contents were increased twofold above controls (354±60 versus 164±23 fmol/g). In contrast to plasma Ang II levels, intrarenal Ang II contents were not elevated in either of the losartan-treated rat groups, demonstrating that intrarenal Ang II levels are not simply a reflection of plasma concentrations caused by nonspecific uptake. This dissociation between plasma and intrarenal Ang II levels indicates that intrarenal Ang II levels are regulated independently of circulating Ang II levels. This pattern was not common to all the tissues tested. Rather, Ang II levels in heart and adrenal gland were not altered significantly during Ang II infusion but were markedly enhanced by losartan treatment in association with the increases in plasma Ang II levels (Fig 7).
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Effects of Ang II and Losartan on Renal Structure
The ratios of tissue weight (milligrams) to body weight (grams) were quantified in all groups. In the control group, tissue weight–to–body weight ratios in adrenal glands, heart, and kidney averaged 0.20±0.02, 3.18±0.14, and 5.23±0.08, respectively. Ratios of kidney and adrenal gland to body weight were similar in all groups despite the hypertension associated with the Ang II–infused group. Rats receiving losartan treatment had significantly (P<.05) decreased heart weight–to–body weight ratios compared with one-kidney controls and Ang II–infused hypertensive rats. Histological studies (Table 2) indicated that renal injury was not severe enough to document early in the development of hypertension using the glomerular injury score but was of a significant magnitude by day 13 compared with controls (glomerular injury score: 77±8 versus 14±3, P<.01). The severity of these lesions was significantly decreased in Ang II–infused rats receiving losartan. Moreover, the percent with an injury score of grade 3 (+2) was significantly decreased in the losartan-treated rats infused with Ang II. As shown in Fig 8, the arterioles, glomeruli, and interstitium remained histologically normal in one-kidney controls, but the Ang II–infused rats revealed prominent and widespread renal injury characterized by focal and segmental glomerulosclerosis, glomerular hypertrophy, increased mesangial matrix, and adhesion of the glomerular tuft to Bowman's capsule. The area of segmental sclerosis often contained vacuolated epithelial cells and hyaline deposition by the basement membrane and matrix material. In addition, Ang II–infused rats exhibited moderate to marked medial thickening, with hypertrophy of vascular smooth muscle cells and an increase in the endothelial cells of the interlobular arteries. The afferent arterioles showed slight hyalinosis with luminal encroachment. Losartan reduced this Ang II–associated renal injury, especially in the glomeruli; however, the increased interlobular artery thickness was not prevented to the same extent in the losartan-treated rats. These lesions were prominent only in the rats infused with Ang II for 12 to 13 days and were not sufficient to receive a positive score in tissue harvested from rats infused for 3, 6, or 10 days. Kidneys from rats infused with Ang II for 10 days demonstrated some evidence of transitional changes, but these changes were not clearly quantifiable at this point.
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Discussion |
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It has been demonstrated that the contralateral nonclipped kidney of 2K1C hypertensive rats manifests an augmentation of intrarenal Ang II that may play an important role in the pathogenesis and maintenance of hypertension.6 These findings have received support from studies in which Ang II was infused into uninephrectomized rats for 14 days at doses sufficient to elicit a pattern and degree of hypertension comparable to that after unilateral renal arterial stenosis.7 In agreement with the changes observed in the contralateral nonclipped kidney of 2K1C rats, the renal Ang II levels in the remaining kidney of the Ang II–infused rats were also increased.7 The present study extends the previous results and demonstrates that the hypertension and enhancement of intrarenal Ang II levels induced with chronic low-dose Ang II infusion are prevented by losartan treatment despite increases of PRA and plasma Ang II. Since heart and adrenal Ang II levels did not demonstrate the same pattern of augmentation, it appears that the Ang II–induced enhancement of intrarenal Ang II and the blockade by losartan are tissue specific.
As expected, PRA levels were suppressed in the Ang II–infused rats and were markedly elevated in the losartan-treated rats. These results are consistent with an important role of AT1 receptor activation in the Ang II–induced negative feedback effect on renin release.31 32 33 Plasma angiotensinogen levels were increased in the Ang II–infused rats, which can be explained by the marked suppression of circulating renin and by the stimulation of hepatic angiotensinogen formation by Ang II.32 34 Losartan decreased plasma angiotensinogen levels, most likely because of the higher consumption of substrate by the elevated circulating renin activity and/or blockade of the Ang II stimulatory effect on hepatic angiotensinogen production.32 Chronic administration of losartan was also associated with elevated plasma Ang I and Ang II levels, which can be accounted for by the marked elevation of circulating renin that would lead to increased formation of Ang I and conversion to Ang II. Since the AT2 receptor subtype was not blocked by the AT1 receptor antagonist and plasma Ang II concentrations were markedly increased, any function mediated by this AT2 receptor might be enhanced in the losartan-treated rats. The present studies do not address possible AT2-mediated actions during chronic Ang II infusions, but it is possible that the failure of losartan to block the increases in tissue Ang II levels in adrenal and heart tissue was due to the continued activity of AT2 receptors. Both the heart and adrenal have been shown to have greater densities of AT2 receptors than kidneys.35 36 Ang II levels in the heart were relatively low, and the changes observed roughly paralleled the plasma Ang II levels. Thus, in this tissue, the changes in Ang II content could also be explained by interstitial accumulation of Ang II. In contrast, adrenal Ang II levels are several times higher than plasma levels, indicating substantial intracellular accumulation of Ang II in adrenal glands. The increases in adrenal and heart Ang II levels observed in the losartan-treated rats suggest that they could have occurred via AT2 receptors.
Unlike plasma Ang II levels, which were elevated by day 3 in the Ang II–infused rats, intrarenal Ang II contents showed an upward trend at 6 days and were significantly elevated at 10 days after Ang II infusion. Importantly, the tissue Ang II contents expressed as femtomoles per gram are greater than the plasma concentrations expressed as femtomoles per milliliter, suggesting that the intrarenal Ang II contents cannot be explained simply by nonspecific extracellular accumulation of circulating Ang II. In addition, intrarenal Ang I levels were not reduced to the same extent as plasma Ang I levels. This dissociation between plasma and renal levels of Ang I along with the elevated Ang II levels suggests that there is an alternate reninlike pathway responsible for regulating intrarenal Ang II levels under the conditions of renin depletion. In contrast to the markedly elevated plasma Ang II levels, intrarenal Ang II contents were not elevated in the losartan-treated rats. The finding that losartan prevents the Ang II–induced enhancement of intrarenal Ang II indicates that activation of AT1 receptors mediates the enhanced intrarenal Ang II levels and provides further evidence that the augmentation of intrarenal Ang II levels during chronic low-dose Ang II infusion is not due to nonspecific sequestration of circulating Ang II.
One mechanism to explain Ang II–induced enhancement in intrarenal Ang II levels is that circulating Ang II is internalized into a protected intracellular compartment subsequent to binding with the AT1 receptor. This possibility is supported by a recent study showing that Ang II is internalized via AT1 receptors and that losartan can effectively compete with Ang II to block Ang II internalization in cultured explant-derived rat aortic vascular smooth muscle cells.37 In a recent study,38 we infused [Val5]-Ang II for 14 days to determine the contribution of minipump-derived [Val5]-Ang II to the elevated intrarenal Ang II content. We demonstrated that 70% of total kidney Ang II at 14 days is derived from infused [Val5]-Ang II. Losartan treatment markedly diminished the increase in intrarenal [Val5]-Ang II.39 These observations provide further evidence that the kidney can accumulate circulating Ang II into intrarenal sites that protect against degradation and metabolism. Since losartan prevents enhancement of intrarenal Ang II, augmentation of intrarenal Ang II levels during Ang II infusion can be explained in part by AT1 receptor–mediated internalization of Ang II. To the extent that this occurs, the internalized Ang II might then be conveyed by an intracellular transporter protein to the nucleus, where the hormone may affect the expression of regulatory elements.37 40 41 Eggena et al41 have described Ang II–specific binding sites in nuclei from hepatic cells. Alternatively, the cytosolic protein may carry Ang II from an intracellular site of synthesis to the plasma membrane for release.37 40 41 In either case, the internalized Ang II could exert important functional effects.
Another potential mechanism to explain the increased intrarenal Ang II is that the elevated circulating Ang II levels and associated hypertension stimulate local de novo Ang II formation by an alternate reninlike pathway. This possibility is consistent with our recent findings38 demonstrating that endogenous Ang II levels in the kidney of [Val5]-Ang II–infused rats are maintained even though renal renin is markedly suppressed. In addition, it has been documented that intrarenal ACE activity in the nonclipped kidney of 2K1C rats and in the remaining kidney of Ang II–infused rats is increased, thus allowing for an enhanced conversion of Ang I to Ang II.6 7 The unique contributions of the increased circulating Ang II levels and the associated increases in arterial pressure to the enhanced intrarenal Ang II contents and renal injury are difficult to delineate.15 It is possible that the augmentation of intrarenal Ang II and associated renal injury occur primarily in a setting of hypertension and reduced renal renin activity.
Previous studies8 9 and current data indicate that renal angiotensinogen mRNA and angiotensinogen contents are maintained in 2K1C and Ang II–infused hypertensive rats, and it has also been shown that Ang II stimulates hepatic angiotensinogen synthesis and release32 34 and induces angiotensinogen gene expression in both liver and kidney.32 33 34 Schunkert et al33 reported increases in mRNA expression of renal and hepatic angiotensinogen in response to Ang II administration. Ingelfinger et al42 demonstrated that Ang II directly increases angiotensinogen production and angiotensinogen mRNA levels in cultured proximal tubular cells, thus supporting the hypothesis that Ang II can augment the intrarenal Ang II formation rate. These in vitro studies indicate that the coexisting hypertension that develops in vivo is not essential for the Ang II–mediated enhancement of intrarenal Ang II production. These findings also help explain the ability of the kidney to provide a sustained source of substrate for continued intrarenal Ang II production. Collectively, the data suggest that elevated circulating Ang II levels stimulate local production of intrarenal Ang II by stimulating or maintaining local angiotensinogen production coupled with an increased ACE activity. Moreover, it has been found that the kidney contains reninlike enzymes, serine proteases, and sequential carboxypeptidase, all of which are capable of producing Ang I from angiotensinogen and possibly maintaining intrarenal Ang I and Ang II contents despite renal renin depletion.43
The present study demonstrates that rats infused with Ang II for 13 days exhibited substantial glomerular and vascular injury that was not observed in the one-kidney controls. Although glomerular injury was not readily quantifiable at earlier time points, elevated intrarenal Ang II levels were already apparent in the tissue harvested from rats receiving Ang II for 10 days. These data are consistent with the suggestion that increases in intrarenal Ang II can activate certain growth factors that are closely associated with renal injury.14 15 44 Furthermore, the observations that the Ang II–infused rats with losartan treatment had reduced intrarenal Ang II levels and a reduced degree of glomerular injury despite elevated plasma Ang II levels further suggest that the renal injury was due either to AT1 receptor–activated processes or to the associated increases in arterial pressure.
In summary, the results of the current study indicate that chronically increasing circulating Ang II leads to the development of hypertension not only via its direct vasoconstrictor actions but also by enhancing intrarenal Ang II contents in a tissue-specific manner that may augment Ang II–dependent influences on renal function and sodium excretion. Differences in the time-related changes in circulating and intrarenal Ang II levels suggest a slowly developing action of circulating Ang II to enhance intrarenal Ang II. The observation that losartan not only blocks the development of hypertension but also prevents the progressive increases in intrarenal Ang II levels indicates that Ang II–induced hypertension and enhancement of intrarenal Ang II levels require activation of AT1 receptors. Possible mechanisms for intrarenal Ang II augmentation include AT1 receptor–mediated binding and internalization of Ang II, AT1 receptor–dependent stimulation of intrarenal Ang II formation, or both.
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Acknowledgments |
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These studies were supported by Grant HL-26371 from the National Heart, Lung, and Blood Institute and by an Education Grant from Merck and Co, Inc. Li-Xian Zou is a postdoctoral fellow and was supported by a Training Award from the International Society of Nephrology. Dr John Imig was supported by a National Research Service Award from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-08676). Annette Von Thun was an MD-PhD predoctoral trainee supported by a fellowship from the Louisiana Board of Regents. Dr Hidehiko Ono, a visiting scientist from the Division of Hypertension and Cardiorenal Disease, Dokkyo University School of Medicine, Mibu, Tochigi, Japan, was under the direction of Dr Edward Frohlich and supported by the Hypertension Research Trust Fund of The Alton Ochsner Medical Foundation. The authors express their gratitude to Dr Igor V. Yosipiv, Dr Yuko Ono, Lynn Gauthier-Lewis, and Anthony Cook for their excellent assistance with these studies. Losartan was generously provided by Dr R. Smith of DuPont-Merck Pharmaceutical.
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Footnotes |
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Portions of this study were presented at the 27th Annual Meeting of the American Society of Nephrology, Orlando, Florida, October 26-29, 1994.
Received January 23, 1996; first decision March 11, 1996; accepted June 10, 1996.
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References |
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O. Baltatu, J. A. Silva Jr, D. Ganten, and M. Bader The Brain Renin-Angiotensin System Modulates Angiotensin II-Induced Hypertension and Cardiac Hypertrophy Hypertension, January 1, 2000; 35(1): 409 - 412. [Abstract] [Full Text] [PDF]
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S. Y. Chin, K. N. Pandey, S.-J. Shi, H. Kobori, C. Moreno, and L. G. Navar Increased activity and expression of Ca2+-dependent NOS in renal cortex of ANG II-infused hypertensive rats Am J Physiol Renal Physiol, November 1, 1999; 277(5): F797 - F804. [Abstract] [Full Text] [PDF]
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H. M. Siragy and R. M. Carey Protective Role of the Angiotensin AT2 Receptor in a Renal Wrap Hypertension Model Hypertension, May 1, 1999; 33(5): 1237 - 1242. [Abstract] [Full Text] [PDF]
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D. Lombardi, K. L. Gordon, P. Polinsky, S. Suga, S. M. Schwartz, and R. J. Johnson Salt-Sensitive Hypertension Develops After Short-Term Exposure to Angiotensin II Hypertension, April 1, 1999; 33(4): 1013 - 1019. [Abstract] [Full Text] [PDF]
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L. Song and D. P. Healy Kidney Aminopeptidase A and Hypertension, Part II : Effects of Angiotensin II Hypertension, February 1, 1999; 33(2): 746 - 752. [Abstract] [Full Text] [PDF]
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L. M. Harrison-Bernard, S. S. El-Dahr, D. F. O'Leary, and L. G. Navar Regulation of Angiotensin II Type 1 Receptor mRNA and Protein in Angiotensin II–Induced Hypertension Hypertension, January 1, 1999; 33(1): 340 - 346. [Abstract] [Full Text] [PDF]
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 L. G. Navar, L. Zou, A. Von Thun, C. Tarng Wang, J. D. Imig, and K. D. Mitchell Unraveling the Mystery of Goldblatt Hypertension Physiology, August 1, 1998; 13(4): 170 - 176. [Abstract] [Full Text] [PDF]
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S. Y. Chin, C.-T. Wang, D. S. A. Majid, and L. G. Navar Renoprotective effects of nitric oxide in angiotensin II-induced hypertension in the rat Am J Physiol Renal Physiol, May 1, 1998; 274(5): F876 - F882. [Abstract] [Full Text] [PDF]
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A. Herizi, B. Jover, N. Bouriquet, and A. Mimran Prevention of the Cardiovascular and Renal Effects of Angiotensin II by Endothelin Blockade Hypertension, January 1, 1998; 31(1): 10 - 14. [Abstract] [Full Text] [PDF]
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D. Casellas, N. Bouriquet, and A. Herizi Bosentan Prevents Preglomerular Alterations During Angiotensin II Hypertension Hypertension, December 1, 1997; 30(6): 1613 - 1620. [Abstract] [Full Text]
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J. P. van Kats, L. M. de Lannoy, A. H. J. Danser, J. R. van Meegen, P. D. Verdouw, and M. A. D. H. Schalekamp Angiotensin II Type 1 (AT1) Receptor–Mediated Accumulation of Angiotensin II in Tissues and Its Intracellular Half-life In Vivo Hypertension, July 1, 1997; 30(1): 42 - 49. [Abstract] [Full Text]
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L. M. Harrison-Bernard, J. Zhuo, H. Kobori, M. Ohishi, and L. G. Navar Intrarenal AT1 receptor and ACE binding in ANG II-induced hypertensive rats Am J Physiol Renal Physiol, January 1, 2002; 282(1): F19 - F25. [Abstract] [Full Text] [PDF]
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