Renal Accumulation of Circulating Angiotensin II in Angiotensin II–Infused Rats
Abstract Previous studies have demonstrated that low-dose angiotensin II (Ang II) infusion for 14 days mimics two-kidney, one clip Goldblatt hypertension and increases intrarenal Ang II levels. The objective of the present study was to determine whether the augmented intrarenal Ang II is due to intrarenal accumulation of the infused Ang II and/or to an increase in intrarenal formation of endogenous Ang II. Male Sprague-Dawley rats were uninephrectomized and divided into three groups: control (n=6), those infused with [Ile5]Ang II (endogenous form) (n=6), and those infused with [Val5]Ang II (n=8). [Ile5]Ang II or [Val5]Ang II was infused at 40 ng/min via an osmotic minipump implanted subcutaneously. By day 12, systolic blood pressure increased significantly in both [Val5]Ang II–infused rats (197±7 mm Hg) and [Ile5]Ang II–infused rats (173±3 mm Hg). Blood and kidney samples were harvested, subjected to high-performance liquid chromatography to separate [Val5]Ang II from [Ile5]Ang II, and then measured by radioimmunoassay. Plasma renin activity was markedly suppressed in both [Ile5]Ang II– and [Val5]Ang II–infused rats. Plasma Ang II levels were elevated in rats infused with both [Ile5]Ang II (121±24 fmol/mL) and [Val5]Ang II (119±14 fmol/mL) compared with controls (69±15 fmol/mL). Both [Ile5]Ang II– and [Val5]Ang II–infused rats exhibited an enhancement of total intrarenal Ang II. Only [Ile5]Ang II (358±53 fmol/g) was detected in the kidneys of rats infused with [Ile5]Ang II. In [Val5]Ang II–infused rats, a significant portion of total renal Ang II (371±57 fmol/g) was in the form of [Val5]Ang II (256±44 fmol/g). Renal [Ile5]Ang II levels were maintained in the [Val5]Ang II–infused rats (116±15 fmol/g) compared with control rats (116±11 fmol/g) despite marked suppression of renin release. These results support the hypothesis that infused circulating Ang II is bound to receptor or taken up intrarenally in a manner that protects against degradation.
Elevated intrarenal Ang II levels have been proposed to play a critical role in the pathophysiology of 2K1C hypertension.1 2 3 This concept has received support from studies demonstrating that chronic infusion of subpressor doses of Ang II by osmotic minipump into uninephrectomized rats for 14 days mimics the pattern of hypertension observed in 2K1C rats.2 4 In addition, the intrarenal Ang II levels in the remaining kidney were increased to levels similar to those found in the nonclipped kidney of 2K1C rats.2 However, the mechanisms of intrarenal Ang II augmentation remain undetermined. Several studies have indicated that although intrarenal Ang II is augmented in the nonclipped kidney of 2K1C rats and in the remaining kidney of Ang II–infused rats, there is marked depletion of renal renin contents and reduction of renal renin mRNA levels.2 5 6 7 8 9 These findings support the hypothesis that increases in circulating Ang II lead to augmentation of intrarenal Ang II via a renin-independent pathway. Our recent observation that losartan prevented the Ang II–induced increases in intrarenal Ang II levels suggests that the augmentation of intrarenal Ang II is due to involvement of the AT1 receptor, which may then be responsible for stimulation of endogenous intrarenal Ang II production and/or specific uptake of Ang II.10
For the present study, we sought to determine the relative contributions of infused Ang II and of endogenous Ang II to the observed enhancement of intrarenal Ang II. This was accomplished by infusing rats with [Val5]Ang II, which is not endogenously produced in the rat but has the same biological and immunoreactive properties as endogenous [Ile5]Ang II. Because [Val5]Ang II can be separated from [Ile5]Ang II by HPLC and quantified by radioimmunoassay, we were able to determine how much of the augmented Ang II in the kidney is due to accumulation of [Val5]Ang II and how much of it is endogenously formed Ang II.
Male Sprague-Dawley rats (Charles River Labs) were housed in wire cages and maintained in a temperature- and light-controlled room. Throughout the experiments, animals had free access to tap water and standard rat chow (Ralston Purina). All experiments were approved by the Tulane University Animal Care and Use Committee. Rats (180 to 200 g body weight) were anesthetized with pentobarbital anesthesia (50 mg/kg IP), and the right kidney was removed. Uninephrectomized rats were divided into three experimental groups: group 1 (control; n=6), rats infused with 0.9% NaCl vehicle; group 2 ([Ile5]Ang II; n=6), rats infused with [Ile5]Ang II; and group 3 ([Val5]Ang II; n=8), rats infused with [Val5]Ang II. Ang II (Novabiochem) was delivered continuously at a rate of 40 ng/min via an osmotic minipump (model 2002, Alza Corp) that was implanted subcutaneously at the dorsum of the neck.2
SBP was measured in conscious rats by tail-cuff plethysmography (Harvard Apparatus) to monitor the progression of hypertension. For measurement of PRA, plasma and renal Ang I, and [Ile5]Ang II and [Val5]Ang II levels, the conscious rats were decapitated on day 13. Trunk blood was collected, and the kidneys were immediately removed, quickly weighed, and homogenized in methanol. The time delay between decapitation and homogenization of the kidneys did not exceed 60 seconds.
Measurement of [Val5]Ang II and [Ile5]Ang II Levels in Plasma and Kidney
Collection and Extraction of Blood and Kidney
Trunk blood was collected in chilled tubes containing a mixed inhibitor solution (final concentrations: 5 mmol/L EDTA, 10 μmol/L pepstatin, 20 μmol/L enalaprilat, and 1.25 mmol/L 1,10-phenanthroline). After centrifugation at 4°C for 10 minutes at 1000g, plasma was separated and immediately applied to a phenyl-bonded solid-phase extraction column (Bond-Elut, Varian) that had been prewashed with methanol followed by water. After sample application, each solid-phase extraction column was washed sequentially with water, hexane, and chloroform. The water removed salts and other polar substances from the column. The hexane and chloroform eluted contaminating lipid and hydrophobic material from the column but did not affect Ang recovery.11 Ang peptides were eluted from the solid-phase extraction column with 100% methanol. The eluants were collected, evaporated to dryness under vacuum, and then subjected to HPLC to separate [Val5]Ang II from [Ile5]Ang II. One half of each kidney was immersed in cold methanol (100%) and homogenized with a glass homogenizer immediately on harvest. The supernatants from the kidney homogenates were dried overnight in a vacuum centrifuge. The dried residue was reconstituted in 4 mL assay diluent (50 mmol/L sodium phosphate buffer, pH 7.4, containing 0.1 mg/mL human serum albumin). These samples were extracted and evaporated as described above for plasma and subjected to HPLC.
Separation of [Val5]Ang II From [Ile5]Ang II by HPLC
The HPLC and radioimmunoassay methodologies for the measurement of Ang peptides have been reported.11 Briefly, the extract residue from each plasma and kidney sample was redissolved in 150-μL HPLC column equilibration solvent (35% methanol, 65% water, 0.1% H3PO4) and chromatographed at 1 mL/min on a 25×0.46-cm, 5-μm Vydac C18 reverse-phase HPLC column (Separations Group). To shorten the duration of the chromatography run, a combination of isocratic and step-gradient elution modes was used. As shown in Fig 1⇓, [Val5]Ang II eluted at 6 minutes (fractions 11 to 14), and [Ile5]Ang II had an elution peak of 9.5 minutes (fractions 18 to 22). Fractions were collected every 30 seconds, evaporated to dryness, reconstituted in assay diluent, and measured directly by radioimmunoassay.
Quantitation of [Val5]Ang II and [Ile5]Ang II by Radioimmunoassay
The reconstituted plasma and kidney fractions were incubated with rabbit anti–Ang II antisera (Arnel) and 125I-radiolabeled Ang II (Sigma Chemical Co) for 48 hours at 4°C. Bound and free Ang peptides were separated by dextran-coated charcoal, and the supernatants were counted by a computer-linked gamma counter for 3 minutes. Immunoreactivities of the antibody for [Val5]Ang II and [Ile5]Ang II were virtually identical. Results are reported in femtomoles per gram of kidney weight or femtomoles per milliliter of plasma. The sensitivity of the Ang II assay was 1.05±0.15 fmol. For the Ang II assays, the specific binding was 44.17±2.36%, with a nonspecific binding of 1.38±0.14%.
PRA and Ang I Assays
For renin determination, trunk blood was collected in chilled tubes containing EDTA (5 mmol/L). Plasma was separated and stored at −20°C until assayed with a commercially available Ang I radioimmunoassay kit (Incstar) as described previously.2 For Ang I measurement, blood and kidney samples were collected, extracted, and quantified by radioimmunoassay as reported previously.2
All data are presented as mean±SEM. The statistical analyses for plasma and kidney levels were performed using ANOVA and Fisher’s least significant difference post hoc test. 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.
Influence of [Val5]Ang II on Arterial Pressure
SBP in both [Ile5]Ang II– and [Val5]Ang II–infused rats exhibited progressive increases over a 12-day period and averaged 173±5 and 197±7 mm Hg, respectively. The SBP in the control rats remained stable and normotensive throughout the duration of the experiment (125±1 to 127±2 mm Hg) (Fig 2⇓).
Effects of Ang II Infusions on PRA
Fig 3⇓ shows that the control PRA averaged 5.0±1.2 ng Ang I/mL per hour, and that the [Val5]Ang II– and [Ile5]Ang II–infused rats had almost complete suppression of PRA, with values of 0.2±0.1 and 0.5±0.2 ng Ang I/mL per hour, respectively.
Effects of Ang II Infusions on Plasma and Kidney Ang I
As predicted from the PRA data, plasma Ang I levels in the [Ile5]Ang II– and [Val5]Ang II–infused rats were reduced by 95% and 98%, respectively. Similar trends were observed in the intrarenal Ang I contents. [Val5]Ang II–infused rats had an 80% decrease in intrarenal Ang I content, whereas the [Ile5]Ang II–infused group exhibited a 40% reduction in intrarenal Ang I levels (Fig 4⇓). The greater effect of [Val5]Ang II to suppress intrarenal Ang I levels could be due to the slightly but significantly greater levels of systolic arterial pressure reached in these rats.
Effects of Ang II Infusions on Plasma and Kidney Ang II
As shown in Fig 5⇓, total plasma Ang II levels were elevated in both [Ile5]Ang II– and [Val5]Ang II–infused groups compared with controls. Only [Ile5]Ang II was detected in the plasma of rats infused with [Ile5]Ang II. In the [Val5]Ang II–infused rats, about half of the plasma Ang II was in the form of [Val5]Ang II, whereas [Ile5]Ang II levels were maintained at concentrations similar to those found in control rats despite marked renin depletion and reduction in Ang I concentration.
The intrarenal Ang II contents, however, showed a distinctly different pattern (Fig 6⇓). In agreement with previous findings from our laboratory, the [Ile5]Ang II–infused rats exhibited a marked increase in total intrarenal Ang II content (358±54 versus 116±11 fmol/g in controls), which, as expected, was detected only in the form of [Ile5]Ang II. Total intrarenal Ang II contents in the [Val5]Ang II–infused rats were elevated to almost the same extent (371±57 fmol/g) as in the [Ile5]Ang II–infused rats. A substantial portion of total intrarenal Ang II in the [Val5]Ang II–infused rats was in the form of [Val5]Ang II. Renal [Val5]Ang II content in the kidney was 256±44 fmol/g and greatly exceeded plasma concentrations of 57±17 fmol/mL. Intrarenal [Ile5]Ang II contents were maintained in the [Val5]Ang II–infused rats compared with controls even though PRA and plasma and renal Ang I levels were markedly suppressed. Thus, this dissociation between the plasma and intrarenal Ang II levels suggests that intrarenal Ang II levels were regulated independently of the circulating Ang II levels.
The important role for intrarenal Ang II levels in the development of hypertension has received support from recent studies showing that chronic low-dose Ang II infusion increases intrarenal Ang II levels and mimics 2K1C hypertension.2 The observation that the increases in intrarenal Ang II levels in the Ang II–infused rats were prevented by AT1 receptor antagonist with losartan despite elevated plasma Ang II levels indicates that the enhanced intrarenal Ang II is not just a nonspecific consequence of the elevated levels but is an active receptor-mediated process.10 These findings2 10 and those of others4 12 suggest an important role for increased intrarenal Ang II levels in the hypertensinogenic process. The objective of this study was to investigate the possible mechanisms of intrarenal Ang II augmentation by determining how much of the infused Ang II accumulated in the kidney. By infusion with [Val5]Ang II, which is not produced in the rat but has similar biological activity, the development of hypertension and enhancement of intrarenal Ang II observed in the [Ile5]Ang II–infused model were reproduced. Since these two isoforms can be separated by HPLC, this substitution approach enabled determination of the relative contributions of uptake of exogenously infused [Val5]Ang II versus endogenously formed [Ile5]Ang II to the elevated intrarenal Ang II content.
In the present study, chronic subcutaneous [Val5]Ang II infusion resulted in a similar pattern and degree of hypertension as observed in the [Ile5]Ang II–infused rats, confirming that [Val5]Ang II has a similar biological effect on arterial pressure. As expected, PRA levels were markedly suppressed in both [Val5]Ang II– and [Ile5]Ang II–infused rats. These results confirm previous findings that Ang II, together with elevated arterial pressure, exerts a negative feedback effect on renin release.2 5 6 13 14 15 16 Plasma Ang I levels in both [Val5]Ang II– and [Ile5]Ang II–infused groups were also suppressed.
In agreement with previous results, the [Ile5]Ang II–infused rats exhibited a marked increase in intrarenal Ang II content. A similar increase in intrarenal Ang II levels occurred in the [Val5]Ang II–infused rats. However, an important finding of this study is that a substantial fraction of the intrarenal Ang II found in the kidneys of [Val5]Ang II–infused rats was in the form of [Val5]Ang II, which indicates that the kidney has the capability to take up or sequester circulating Ang II into intrarenal sites that protect against degradation and metabolism. Quantitatively, the tissue [Val5]Ang II contents expressed as femtomoles per gram are three to four times greater than the plasma concentrations expressed as femtomoles per milliliter, suggesting that the intrarenal Ang II contents cannot be explained simply by nonspecific trapping of circulating Ang II in plasma and extracellular fluid within the kidney. Our recent observation that losartan treatment prevents intrarenal Ang II augmentation during chronic low-dose Ang II infusion10 provides further evidence that the renal uptake of Ang II is an active process and does not represent nonspecific trapping of circulating Ang II within the kidney extracellular spaces.
One possible explanation for the present findings is that circulating Ang II is protected subsequent to binding with AT1 receptors, which are widely distributed throughout the kidney.17 18 This possibility is supported by a study from Anderson and Peach19 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. Likewise, Wang et al20 reported receptor-mediated internalization of Ang II in primary culture of bovine adrenal medullary chromaffin cells. Thus, our present findings could be explained by receptor-mediated internalization of circulating Ang II into an intracellular compartment devoid of proteolytic enzymes so that Ang II is protected from degradation and metabolism. Sequentially, the internalized Ang II could then serve as an intracellular and/or nuclear signaling function, which could participate directly in regulating its own gene expression or that of other systems.21 22 23 24 25
An interesting phenomenon also found in this study is that circulating and intrarenal [Ile5]Ang II contents in the [Val5]Ang II–infused rats were maintained despite markedly reduced PRA and plasma and kidney Ang I levels. These results support the notion that the elevated circulating Ang II levels stimulate endogenous Ang II formation. Previous studies have shown that intrarenal angiotensin-converting enzyme 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.1 2 Moreover, renal angiotensinogen mRNA and angiotensinogen contents were found to be maintained in the 2K1C and Ang II–infused rats, demonstrating the ability of the kidney to provide a sustained source of substrate for continued intrarenal Ang II production.5 6 Collectively, the data suggest that elevated circulating Ang II levels stimulate intrarenal production of Ang II as a consequence of maintained angiotensinogen levels coupled with an increased angiotensin-converting enzyme activity.
In summary, the results of the present study indicate that the effect of [Val5]Ang II is equivalent to that of [Ile5]Ang II in raising arterial pressure and intrarenal Ang II levels. More than half of the intrarenal Ang II found in the [Val5]Ang II–infused rats was in the form of [Val5]Ang II, suggesting that augmentation of intrarenal Ang II is due in large part to uptake of circulating Ang II into a protected environment. The finding that [Ile5]Ang II levels were maintained in the kidneys of [Val5]Ang II–infused rats in a setting of renin and Ang I reduction further supports the hypothesis that elevated circulating Ang II stimulates endogenous production of Ang II via a renin-independent pathway.
Selected Abbreviations and Acronyms
|2K1C||=||two-kidney, one clip|
|AT1||=||angiotensin II type 1|
|HPLC||=||high-performance liquid chromatography|
|PRA||=||plasma renin activity|
|SBP||=||systolic blood pressure|
This study was supported by National Heart, Lung, and Blood Institute grant HL-26371. Li-Xian Zou is a postdoctoral fellow supported by an International Society of Nephrology fellowship training award. The authors express their gratitude to Paul Diechmann for his technical assistance.
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