Role of Renal Perfusion Pressure Versus Angiotensin II on Renal Oxidative Stress in Angiotensin II–Induced Hypertensive Rats
Renal oxidative stress is thought to contribute to both the etiology and the associated renal injury in angiotensin (Ang) II–dependent hypertension. The contribution of Ang II versus elevated renal perfusion pressure (RPP) on albuminuria and renal oxidative stress in this model of hypertension was explored in the present study by chronically servocontrolling RPP to the left kidney and comparing responses with the right uncontrolled kidney and the left kidney of sham rats. Hypertension was produced in Sprague-Dawley rats fed a 4% NaCl diet by chronic IV infusion of Ang II (25 ng/kg per minute). The RPP to the left kidney was servocontrolled to mean daily pressures averaging ≈120 mm Hg, whereas the uncontrolled kidneys averaged ≈170 mm Hg over 14 days of Ang II infusion. Ang II infusion resulted in a 2.4-fold increase in albuminuria, which was RPP dependent. Kidneys exposed to both elevated RPP and Ang II (uncontrolled kidneys) displayed a 3.5-fold increase in malondialdehyde excretion and a 37% and 27% increase in renal cortical and outer medullary superoxide production, respectively. Elevated RPP significantly contributed to global renal oxidative stress (70% increase in malondialdehyde excretion) and outer medullary superoxide production. Elevated circulating levels of Ang II, per se, were responsible for a 1.5-fold and 2.0-fold increase in renal cortical and outer medullary NADPH oxidase activity, respectively. In summary, this study demonstrates that elevated RPP is directly responsible for the excess albuminuria in Ang II–infused rats, whereas both elevated RPP and Ang II directly contribute to the observed renal oxidative stress.
Excess levels of superoxide (O2·−) are found within the kidney of angiotensin II (Ang II)–dependent hypertensive rats1,2 and have been associated with both the etiology and injury in this form of hypertension.2–6 Both elevated Ang II2,4,5 and renal perfusion pressure (RPP)7 have been reported to independently increase renal O2·− production; however, the precise contribution of each on O2·− production within different regions of the kidney of Ang II–induced hypertensive rats remains incomplete. We investigated the relative contributions of RPP and Ang II on albuminuria, the in vivo production of renal reactive oxygen species, and the activities of NADPH oxidase and superoxide dismutase (SOD) in renal cortical and outer medullary tissue homogenates. We focused on these 2 enzyme systems because they have been reported previously to contribute importantly to the excess O2·− production in kidneys of Ang II–induced hypertensive rats.2 As we have described previously,6,8 a custom-built computerized servocontrol system of our own design was used to maintain RPP to the left kidney at baseline levels, whereas the contralateral right kidney was exposed to elevated RPP over 14 days of Ang II administration. This system enabled the precise determination of the role of Ang II versus elevated RPP in contributing to renal oxidative stress and pathways of O2·− production and scavenging within the renal cortex and outer medulla during hypertension.
Materials and Methods
All of the studies were performed on 12-week–old male Sprague-Dawley rats (Harlan) that were provided water ad libitum and given a 0.4% NaCl AIN-76 rodent diet (Dyets). All of the protocols were approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee.
Surgical Preparation and Chronic Servocontrol of RPP
As described previously,6,8 all of the rats were implanted with indwelling catheters in the right carotid and left femoral arteries and veins, and an inflatable silastic vascular occluder (1.5-mm lumen diameter, 2.5-mm OD; Kent Scientific Corp) was positioned around the aorta between the left and right renal arteries to allow for chronic servocontrol of the left RPP. Sham rats underwent identical surgical and implantation procedures; however, the vascular occluder cuff was never inflated. Arterial pressures above (carotid artery) and below (femoral artery) the vascular occluder cuff were monitored 24 hours per day throughout the study. Saline or drug was continuously administered intravenously at a rate of 6.9 μL/min throughout the study.
One group of rats was anesthetized with ketamine (30 mg/kg IM) and inactin (40 mg/kg IP) for bilateral ureteral urine collections after the 2-week administration of Ang II or saline. Rats were placed on a 37°C warming board, and Ang II infusion and servocontrol of left RPP were continued uninterrupted throughout the acute protocol. A tracheal tube was inserted to facilitate spontaneous respiration, and a catheter was implanted in the right femoral vein for the infusion (1 mL/h per 100 g of body weight) of 2% BSA to replace fluid loss during surgery. Through a midsagittal incision, the right and left ureters were catheterized with PE-10 tubing for the collection of urine. After a 1-hour equilibration period, urine was collected over two 30-minute intervals and immediately snap frozen and stored at −80°C.
After 4 days of recovery from surgery, the diet of all of the rats was switched to a 4.0% NaCl AIN-76 rodent diet (Dyets) for the remainder of the study. Rats were recovered for 10 days after surgery, during which 3 days of baseline blood pressures were recorded. The intravenous infusion solution was then switched to either Ang II (25 ng/kg per minute) for servocontrol rats or saline for sham-operated rats. The chronic servocontrol of RPP began immediately after the infusion of Ang II and left RPP (femoral arterial pressure) was maintained within ±10 mm Hg of baseline pressures whereas the right kidney was exposed to elevated RPP throughout the 2 weeks of Ang II infusion. Two separate groups of rats were studied. Rats of group 1 were acutely anesthetized for bilateral ureteral urine collections after either 14 days of Ang II (n=6) or saline (n=7) infusion, as described above. Albumin excretion was measured as a physiological index of renal damage, and malondialdehyde excretion was measured as an in vivo index of oxidative stress, as described below. After 14 days of Ang II (n=9) or saline (n=10) infusion, rats of group 2 were euthanized by excess sodium pentobarbital (100 mg/kg), and the cortex and outer medulla were quickly separated and immediately snap frozen in liquid nitrogen. Tissues from 3 Ang II–infused rats and 4 sham rats from group 1 were included in the tissue homogenate analysis in group 2. The frozen tissue was then homogenized and centrifuged at 1000 g for 5 minutes, and the protein concentration of the supernatant was determined using a Coomassie blue protein assay (Pierce) with BSA used as a standard.9
O2·− Production by 2-Hydroxyethidium Measurements
Renal cortical and outer medullary tissue homogenates (20 μg of protein) were incubated with dihydroethidium (10 μmol/L), salmon testes DNA (0.5 mg/mL), and PBS (300 mmol/L) in a 96-well microtiter plate to determine O2·− production. NADPH oxidase–dependent O2·− production was assessed by adding 100 μmol/L of diphenylene iodonium (DPI) to tissue homogenates (20 μg protein) in separate wells to determine the decrease in O2·− production as compared with wells not incubated with DPI. This dose of DPI maximally inhibits NADPH oxidase, and we have previously used this approach to identify differences in NADPH oxidase activity in Dahl salt-sensitive versus SS.BN13 rats.9 After a 35-minute incubation, the increase in 2-hydroxyethidium fluorescence was measured at an excitation of 485 nm and an emission of 570 nm and used as an index of O2·− production, as described previously.9
Total SOD Activity
Total SOD activity was determined in renal cortical and outer medullary tissue homogenates by the disappearance of superoxide detected by a tetrazolium salt, as described previously.9
Measurement of Creatinine, Albumin, and Malondialdehyde in Urine
Urine samples were centrifuged at 5000 g for 10 minutes to remove debris, after which urine volumes were measured gravimetrically. Urine samples were stored at −80°C until the determination of creatinine, albumin, and malondialdehyde levels. Urinary creatinine was quantified using an assay based on the Jaffe reaction by autoanalyzer (ACE, Alfa Wasserman). Urinary albumin was quantified with Albumin Blue 580 dye (Molecular Probes) using a fluorescent plate reader (FL600, Bio-Tek). Malondialdehyde, a product of lipid peroxidation and an index of oxidative stress, was measured in urine samples using a Thiobarbituric Acid Reactive Substances kit obtained from Cayman.
Data are presented as mean±SE. A 2-way repeated-measures ANOVA, followed by a Tukey post hoc test, was used to determine daily differences in RPP across sham, servocontrolled, and uncontrolled kidneys. A paired t test was used to assess differences between servocontrolled and uncontrolled kidneys, whereas an unpaired t test was used to assess differences between uncontrolled kidneys and kidneys from sham rats. Pearson correlation and linear regression analyses were performed for sham, servocontrolled, and uncontrolled kidneys to assess the relationship between the average RPP over the 14-day infusion protocol and both albuminuria and malondialdehyde excretion. A P<0.05 was considered significant.
RPP of Sham, Servocontrolled, and Uncontrolled Kidneys
Figure 1 summarizes the average RPP to servocontrolled and uncontrolled kidneys of Ang II–infused rats (n=12), as well as kidneys of saline-infused rats (n=13). The right uncontrolled kidneys of these rats were exposed to an average RPP of ≈170 mm Hg over the 14 days of Ang II infusion, whereas RPP to servocontrolled kidneys was maintained within ±10 mm Hg of the baseline RPP. There were no significant differences in RPP between the sham rats and the servocontrolled kidneys of the Ang II–infused rats across all of the time points.
Urinary Albumin and Malondialdehyde Excretion
As determined on day 14 of the study in the anesthetized rats (Figure 2), elevated RPP resulted in a greater (P<0.05) urinary albumin:creatinine ratio in uncontrolled kidneys (3.54±0.70 mg/mg) as compared with both servocontrolled kidneys (1.39±0.40 mg/mg) and kidneys from sham rats (1.45±0.20 mg/mg). A significant (P<0.05) correlation existed between the albumin:creatinine ratio and the average RPP over the 14 days of Ang II infusion (r=0.68) among all of the kidneys. No significant differences in the albumin: creatinine ratio were observed between servocontrolled kidneys and kidneys from sham rats. Using the albumin:creatinine ratio as an index of renal injury, these data indicate that the renal injury in Ang II–infused hypertensive rats was primarily attributable to elevated RPP.
There was a 3.5-fold increase in malondialdehyde excretion, an in vivo index of oxidative stress (Figure 3), in uncontrolled kidneys versus kidneys from sham rats. Elevated RPP directly led to a 1.7-fold (P<0.05) increase in renal oxidative stress, as demonstrated by the higher malondialdehyde excretion in uncontrolled (0.47±0.07 nmol/min) compared with servocontrolled kidneys (0.28±0.05 nmol/min). Furthermore, there was a significant (P<0.05) correlation between malondialdehyde excretion and the average RPP across 14 days of infusion (r=0.8) among all of the kidneys. The source of the greater levels of malondialdehyde excreted from the uncontrolled kidneys could have been either from the renal cortex or medulla but not from nonrenal sources, because such changes would have been reflected by excretion from both kidneys. The mean arterial pressure over the two 30-minute urine collection periods averaged 149±13 mm Hg in uncontrolled kidneys and 124±11 mm Hg in servocontrolled kidneys. Because these pressures were well within the autoregulatory range of renal blood flow10 and glomerular filtration rate,7 differences in malondialdehyde excretion between servocontrolled and uncontrolled kidneys cannot be explained in this way. These data provide evidence that elevated RPP significantly contributes to renal oxidative stress in Ang II–induced hypertensive rats.
Malondialdehyde excretion was 2-fold (P<0.05) higher in servocontrolled kidneys compared with kidneys from sham rats (0.13±0.01 nmol/min). This difference in malondialdehyde excretion between servocontrolled kidneys and kidneys from sham rats could have resulted from sources other than the kidney and, thus, does not provide direct evidence of Ang II–induced stimulation of renal oxidative stress. The pressure-independent effects of Ang II on renal oxidative stress are demonstrated by the tissue assay of O2·− production described below.
In Vitro Renal O2·− Production, NADPH Oxidase, and SOD Activity
Figure 4 summarizes O2·− production, reported in raw fluorescent units (RFU), in renal cortical and outer medullary tissue homogenates in servocontrolled and uncontrolled kidneys from Ang II–infused rats and kidneys from saline-infused sham rats. In the renal cortex, there was a 32% and 37% increase in O2·− production in servocontrolled and uncontrolled kidneys, respectively, from Ang II–infused rats as compared with kidneys from saline-infused sham rats (P<0.05). These data indicate that Ang II, independent of RPP, was the primary contributor to O2·− production in the renal cortex. In the outer medulla, there was a 17% (P<0.05) and 27% (P<0.05) increase in O2·− production in servocontrolled and uncontrolled kidneys, respectively, from Ang II–infused rats as compared with kidneys from saline-infused sham rats. In contrast to the renal cortex, elevated RPP significantly increased O2·− production in the outer medulla, as indicated by the significantly higher RFUs in uncontrolled versus servocontrolled kidneys (P<0.05). Furthermore, O2·− production was higher (P<0.05) in the outer medulla versus the cortex among all of the kidneys.
As summarized in Figure 5, chronic Ang II infusion resulted in an increase in NADPH oxidase–dependent O2·− production. This was a consequence of elevated levels of circulating Ang II, per se, as shown by the 1.5-fold and 2.0-fold greater (P<0.05) inhibition of O2·− production by DPI in the renal cortex and outer medulla, respectively, in both servocontrolled and uncontrolled kidneys from Ang II–infused rats as compared with kidneys from sham rats.
As shown in Figure 6, renal cortical SOD activity was not significantly altered by elevated Ang II or RPP. However, outer medullary SOD activity in uncontrolled kidneys (2.0±0.2 U/mL per microgram of protein) exposed to both elevated Ang II and RPP was 28% (P<0.05) lower as compared with kidneys from sham rats (2.7±0.2 U/mL per microgram of protein). Ang II directly accounted for 12% of this reduction in SOD activity, whereas elevated RPP was responsible for the remaining 16% reduction, although neither of these values individually reached statistical significance. These data suggest that the significant decrease in outer medullary SOD activity in Ang II–infused hypertensive rats may be explained by the additive effects of elevated Ang II and RPP.
The major findings of the present study were that, after 2 weeks of Ang II–induced hypertension, elevated RPP, per se, is the major contributor to albuminuria; both elevated RPP and Ang II independently contribute to renal oxidative stress; and Ang II, independent of elevated RPP, increases NADPH oxidase activity in the renal cortex and outer medulla.
Ang II–induced hypertension results in significant renal injury,6,11,12 which can lead to the appearance of excess levels of albumin excretion.13 This is the first study that directly implicates chronically elevated RPP as the primary cause of the increased albuminuria in Ang II–induced hypertension in rodents. These observations are consistent with our previous histological analyses showing that elevated RPP is responsible for the majority of the juxtamedullary glomerulosclerosis and the interstitial fibrosis in the outer medulla found in Ang II–induced hypertension.6,12
Hypertension-induced albuminuria has been reported to result from podocyte injury with the subsequent loss of the glomerular filtration barrier.14–16 Mechanical forces related to RPP-induced increases in glomerular capillary pressure, such as shear stress and stretch, have been reported to alter the structure and function of podocytes, which can ultimately lead to an increase in the permeability of the glomerular filtration barrier to albumin.16–18 Our study demonstrates that it is mainly the lowering of arterial pressure that is the most important factor in normalizing albuminuria in Ang II–induced hypertensive rodents. As reviewed previously,19 this is in agreement with several clinical trials and emphasizes the importance of blood pressure control in the prevention of renal injury.
RPP-Induced Superoxide Production
The 1.7-fold greater urinary excretion of malondialdehyde in uncontrolled versus servocontrolled kidneys provides evidence that chronically elevated RPP, independent of Ang II, results in renal oxidative stress in the Ang II–induced model of hypertension. It is relevant that the RPP-dependent stimulation of renal oxidative stress was much more apparent in the intact kidney on the basis of malondialdehyde excretion compared with O2·− production in tissue homogenates, where only a small RPP-induced effect was observed in the outer medulla. This suggests that mechanisms within the context of the intact kidney are necessary to enable increases of RPP to fully stimulate oxidative stress. It is likely that the critical signals required for elevations of RPP to stimulate oxidative stress are lost when the kidney is removed and tissue homogenized and incubated for the various assay studies. Mechanical forces associated with increased levels of RPP, such as stretch and/or shear stress, can increase O2·− production in blood vessels and mesangial cells.20–24 In isolated perfused medullary thick ascending limbs, we and others have demonstrated that increases in tubular flow rate,25,26 stretching,27 and delivery of luminal NaCl25 increase O2·− production. Furthermore, we have found that acute increases of RPP within the kidney autoregulatory range stimulate the production of H2O2 within the renal outer medulla.7 These studies support the stimulation of renal oxidative stress by mechanical forces, which may have resulted in the greater amount of RPP-induced oxidative stress observed in the intact kidney versus tissue homogenates.
The specific sources of RPP-induced oxidative stress remain to be determined. Increased NADPH oxidase activity and reduced SOD activity are 2 commonly reported sources of the excess O2·− levels observed in kidneys of Ang II–infused rats. On the basis of the activity of these enzymes in tissue homogenates, the present study suggests that only outer medullary SOD activity is influenced by chronically elevated RPP. It is apparent that NADPH oxidase activity is significantly elevated by Ang II, independent of RPP, in both the renal cortex and outer medulla. Although these results are direct evidence of an RPP-independent effect of Ang II to increase NADPH oxidase activity, they do not discount a role of RPP-induced stimulation of NADPH oxidase. For example, several of the mechanical forces that have been reported to stimulate renal O2·− production, as described in the previous paragraph, operate through NADPH oxidase.22,23,26 It is possible that the lack of RPP-induced stimulation of NADPH oxidase activity in tissue homogenates was the result of NADPH oxidase–induced O2·− production being assessed in vitro and, thus, removed from the various in vivo mechanical stimuli, as described above. In summary, our study demonstrates that elevated RPP significantly contributes to renal oxidative stress in Ang II–induced hypertensive rats and suggests that in vivo detection of renal oxidative stress may be necessary to detect the full extent of RPP-induced oxidative stress.
Ang II–Induced Superoxide Production
Independent of changes of RPP, O2·− production and NADPH oxidase activity were significantly enhanced by Ang II, per se, in both renal cortical and outer medullary tissues. As demonstrated in Figure 5, NADPH oxidase activity was increased to a similar extent in both the cortex and outer medulla by the direct effects of Ang II. This unequivocal demonstration of the direct actions of Ang II on NADPH oxidase activity is consistent with observations by others2,4,28 and appears to be mediated through Ang II type 1 receptor pathways. The use of the servocontrol system uniquely enabled the in vivo separation of the direct effects of Ang II on these enzymes from the physical forces on the kidney that inevitably occur with hypertension. Even chronic subpressor infusion of Ang II in rats appears capable of simulating oxidative stress, as shown by increased plasma concentrations of 8-isoprostanes29 and increased expression of genes related to oxidative stress and extracellular matrix formation in the renal outer medulla.30 Although much work remains regarding the acute and chronic cellular mechanisms by which Ang II stimulates NADPH oxidase in different renal structures, this is the first study to demonstrate that renal NADPH oxidase activity can be directly regulated by Ang II, per se, in Ang II–induced hypertensive rats. As reviewed recently,31 excess reactive oxygen species production within the kidney can stimulate sodium reabsorption and vasoconstriction, both of which can result in a rightward shift in the long-term pressure-natriuresis curve and lead to hypertension. Consistent with previous studies,2,28 our study supports a direct role of Ang II–induced stimulation of NADPH oxidase to contribute to the prohypertensive actions of Ang II.
The consequence of reduced renal function in the initiation of many experimental and human forms of hypertension has long been established.32,33 On the other hand, the extent to which the secondary effects of elevated arterial pressure can lead to further renal dysfunction and injury has remained a difficult question to answer. It is recognized that renal injury is minimal in some individuals with hypertension, whereas end-stage renal disease develops at an early age in others.34,35 The question of the how much RPP contributes directly to the renal injury and the participating mechanisms is important as it relates to how vigorously one should pursue the lowering of arterial pressure of hypertensive individuals and by what means. It is unlikely that the chronic impact of arterial pressure on the kidneys could ever be directly addressed in human subjects in a manner that enables the independent control of the critical variable, renal arterial perfusion pressure. However, an awareness of these issues is reflected by several clinical trials to ascertain the effectiveness of various antihypertensive drug combinations in reducing renal and cardiac injury.36,37 Several studies from our laboratory have now begun to unravel some aspects of this chicken and egg conundrum using experimental animal models that mimic various forms of the human condition.6,8,12 Much remains to be done, especially with regard to an understanding of how genetic background can predispose an individual to renal injury in the face of elevated arterial pressure and the pathways that could be therapeutically targeted to minimize such effects.
We thank David Eick, Mike Kloehn, and Greg McQuestion for design and maintenance of the servocontrol system and Lisa Henderson, Jenifer Goepfert, and Camille Torres in the Biochemical Core Laboratory for the measurement of albumin and malondialdehyde.
Sources of Funding
This work was supported by National Heart, Lung, and Blood Institute grants HL-081091 and HL-29587 and a predoctoral fellowship from the American Heart Association (AHA-0615590Z).
- Received February 3, 2010.
- Revision received February 19, 2010.
- Accepted March 18, 2010.
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