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. Author manuscript; available in PMC: 2010 Dec 1.
Published in final edited form as: Hypertension. 2009 Oct 26;54(6):1269–1277. doi: 10.1161/HYPERTENSIONAHA.109.139287

Pressure-Induced Renal Injury in Angiotensin II Versus Norepinephrine-Induced Hypertensive Rats

Aaron J Polichnowski 1, Allen W Cowley Jr 1
PMCID: PMC2812436  NIHMSID: NIHMS161217  PMID: 19858406

Abstract

The susceptibility to renal perfusion pressure (RPP)-induced renal injury was investigated in angiotensin II (AngII) versus norepinephrine (NE)-infused hypertensive rats. To determine the magnitude of RPP-induced injury, Sprague-Dawley rats fed a 4% salt diet were instrumented with a servocontrolled aortic balloon occluder positioned between the renal arteries to maintain RPP to the left kidney at baseline levels while the right kidney was exposed to elevated RPP during a 2 week infusion of: 1) AngII i.v. (25 ng/kg/min), 2) NE i.v. (0.5, 1, and 2 ug/kg/min on Days 1, 2, and 3-14, respectively), or saline i.v. (sham rats). Over the 14 days of AngII infusion, RPP averaged 161.5 ± 8 mmHg to uncontrolled kidneys and 121.9 ± 2 mmHg to servocontrolled kidneys. In NE-infused rats, RPP averaged 156.3 ± 3 mmHg to uncontrolled kidneys and 116.9 ± 2 mmHg to servocontrolled kidneys. RPP averaged 111.1 ± 1 mmHg to kidneys of sham rats. Interlobular arterial injury and juxtamedullary glomerulosclerosis were largely RPP-dependent in both models of hypertension. Superficial cortical glomerulosclerosis was greater and RPP-dependent in NE versus AngII-infused rats, which was primarily independent of RPP. Outer medullary tubular necrosis and interstitial fibrosis was also primarily RPP-dependent in both models of hypertension; however, the magnitude of injury was exacerbated in AngII-infused rats. We conclude that elevated RPP is the dominant cause of renal injury in both NE and AngII-induced hypertensive rats and that underlying neurohumoral factors in these models of hypertension alter the pattern and magnitude of RPP-induced renal injury.

Keywords: angiotensin II, blood pressure, kidney, norepinephrine, renal injury

Introduction

Hypertension results in various pathological changes within the kidney, including vascular, glomerular, and tubulointerstitial injuries (1). The pattern and magnitude of renal injury varies according to the etiology of hypertension and/or the presence of underlying renal disease (1,2) and remains an important question with respect to the diagnostic accuracy of the progression of renal disease (3). Although hypertension is the second leading cause of end-stage renal disease (ESRD) (4), the direct effects of elevated renal perfusion pressure (RPP) in hypertension-induced renal injury remains ambiguous since the susceptibility to renal injury is known to vary greatly across human populations (2,5,6) as well as in experimental (2) and genetic (7,8) models of hypertension. The mechanisms responsible for the differences in the susceptibility to RPP-induced renal injury are uncertain, but are likely due to complex interactions between elevated RPP, altered paracrine and endocrine factors, genetic factors, and/or the presence of underlying renal disease.

We have begun to address these issues in a systematic way by first determining the role of underlying neurohumoral factors in altering the susceptibility to RPP-induced renal injury by comparing the pattern and magnitude of RPP-induced renal injury in norepinephrine (NE) versus angiotensin II (AngII)-induced hypertensive Sprague-Dawley rats. Various forms of hypertension are associated with elevated sympathetic activity (9) and/or inappropriate activation of the renin-angiotensin system (RAS) (10), in which both pressure-dependent and independent patterns of end-organ damage have been described (9,11-14).

The long-standing limitation of drawing conclusions about the contribution of elevated RPP versus the circulating endocrine factors in previous studies has been the inability to distinguish the relative contribution of each since the kidneys are simultaneously exposed to both potentially injurious factors. Using chronic arterial pressure servocontrol techniques adapted to rats, we have previously determined that elevated RPP, per se, accounts for the majority (∼ 80%) of juxtamedullary glomerular and outer medullary tubulointerstitial injury in AngII-infused rats fed a 4% NaCl diet for 14 days (14). In the current study, we utilized a chronic servocontrol of RPP technique to enable a more precise analysis and comparison of the magnitude of RPP dependent and independent patterns of renal injury in two different models of hypertension, one induced by chronic NE-infusion and the other by chronic AngII-infusion.

Materials and Methods

An expanded Materials and Methods section is available in the online supplement at http:hyper.ahajournals.org.

Experimental Animals

All studies were performed on 12 week old male Sprague-Dawley rats (Harlan) that were provided water ad libitum and provided a 0.4% NaCl AIN-76 rodent diet (Dyets, Bethlehem, PA). All protocols were approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee.

Experimental Design and Chronic Servocontrol of Renal Perfusion Pressure

To determine the role of neurohumoral factors in altering the susceptibility to RPP-induced renal injury, all rats were surgically instrumented with an inflatable vascular occluder positioned on the aorta between the left and right renal arteries through a midsagittal abdominal incision (14,15). Three groups of rats were studied: 1) 14-day NE-infused rats (n = 7) 2) 14-day AngII-infused rats (n = 7) and 3) 14-day saline-infused sham rats (n = 7). Four days following surgery, the diet was switched to a 4.0% NaCl AIN-76 rodent diet (Dyets, Bethlehem, PA) for all rats for the remainder of the study. Rats were allowed to recover for 10 days during which 3 stable days of baseline blood pressure were recorded. After baseline blood pressure was obtained, the intravenous solution was switched to either NE or AngII while saline was continuously infused for sham operated rats. During NE or AngII infusion, the vascular occluder was chronically servocontrolled to maintain RPP at baseline levels to the left kidney while the right kidney was exposed to elevated RPP for 14 days. The vascular occluder cuff was never inflated for saline-infused sham rats. For the AngII group, 25 ng/kg/min was infused (i.v.) continuously for 14 days as we have reported previously (14). In a separate group of rats, the dose of NE was increased over the first 3 days which produced a sustained elevation of MAP over 14 days that was comparable to the pattern of MAP elevation in AngII-infused rats. Specifically, on day 1, a dose of 0.5 μg/kg/min NE was administered intravenously for 24 hours, increased to 1.0 μg/kg/min on day 2, and increased again on day 3 to 2.0 μg/kg/min for the remainder of the study. The chronic servocontrol of left RPP began at the start of drug administration and continued for the entire duration of the experimental protocol in NE and AngII groups.

Histological Analysis

Histological and immunohistochemical analyses were performed to quantify the magnitude of RPP dependent and independent superficial cortical and juxtamedullary glomerular injury, interlobular arterial remodeling and injury, and outer medullary tubular necrosis and interstitial fibrosis, as we have described previously (14,15) and is provided in detail in the online supplement at http:hyper.ahajournals.org.

Statistical Methods

Data are presented as mean ± SE. A two-way repeated measures ANOVA followed by a Tukey post hoc test was used to determine daily differences in blood pressure and heart rate across groups over the 17 day experimental protocol. A paired t-test was used to assess differences in indices of renal injury between servocontrolled and uncontrolled kidneys while an unpaired t-test was used to compare servocontrolled and sham kidneys within a group and for the comparison of servocontrolled or uncontrolled kidneys between groups. Pearson correlation and linear regression analysis were performed for sham, servocontrolled, and uncontrolled kidneys within each model to assess the relationship between MAP and indices of renal injury. A p < 0.05 was considered significant.

Results

MAP to Left and Right Kidneys

The average daily MAP for sham, NE, and AngII rats are summarized in Figure 1. MAP was significantly elevated over the 14 days of NE and AngII infusion as compared to the 3 baseline days (p < 0.05). In both NE and AngII groups, MAP to the uncontrolled kidney remained significantly elevated above the servocontrolled MAP and the MAP of sham rats for days 1-14 (p < 0.05). There were no significant differences in MAP between the servocontrolled kidneys and kidneys from sham rats across all time points. The MAP to the uncontrolled kidneys in NE and AngII groups were similar across the 14 days of infusion, except for day 3 of infusion, in which MAP was significantly (p < 0.05) higher in AngII versus NE-infused rats.

Figure 1.

Figure 1

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Daily 24 hour averages of MAP to servocontrolled (□, n=7) and uncontrolled (■, n=7) kidneys from AngII (25 ng/kg/min) rats, servocontrolled (○, n=7) and uncontrolled (●, n=7) kidneys from NE (day 1: 0.5 μg/kg/min, day 2: 1.0 μg/kg/min, days 3-14: 2.0 μg/kg/min) rats, and kidneys from saline-infused sham (Δ, n=7) rats. Values are mean ± SE. † p < 0.05 vs. servocontrolled and sham groups over days 1-14. * p < 0.05 vs. NE uncontrolled at day 3.

HR, SBP, DBP, and Pulse Pressure in AngII versus NE-infused Rats

The average daily heart rate (HR) over the 14 days of drug or saline infusion was not significantly different among sham (416 ± 1 bpm), NE (401 ± 5 bpm), and AngII (402 ± 5 bpm) infused rats; however, HR was significantly (p < 0.05) lower on days 1 and 2 of drug infusion in both NE and AngII-infused rats as compared to sham rats, but subsequently approached the level of sham rats on days 3-13. No significant differences in HR were observed between NE and AngII infused rats.

The average daily systolic blood pressure (SBP) and diastolic blood pressure (DBP) in uncontrolled kidneys of both NE and AngII-infused rats were significantly (p < 0.05) elevated as compared to servocontrolled kidneys and kidneys from sham rats over days 1-14. While there were no significant differences in DBP between uncontrolled kidneys of NE and AngII-infused rats, SBP was significantly higher in NE-infused rats on days 12-14 of drug infusion as compared to AngII-infused rats. Furthermore the standard deviation of the average daily SBP, used as an index of BP variability, over the 14 days of drug infusion averaged 19 ± 0.9 mmHg in uncontrolled kidneys of NE-infused rats and 13 ± 0.6 mmHg in AngII-infused rats, which were both significantly higher than the variability observed in servocontrolled kidneys and kidneys from sham rats (6.7 ± 0.1 mmHg). The average daily SBP variability was significantly (p < 0.05) higher in uncontrolled kidneys of NE versus AngII-infused rats on days 5, 8, 10, 11, and 14 of drug infusion, suggesting a greater lability of blood pressure in the NE model of hypertension.

The average daily pulse pressure (PP) over the 14 days of drug infusion was significantly lower in servocontrolled kidneys of NE (15 ± 0.5 mmHg) and AngII (12 ± 0.7 mmHg) infused rats as compared to kidneys from sham rats (26 ± 0.4 mmHg). Pulse pressure in uncontrolled kidneys from NE (31 ± 2 mmHg) and AngII (23 ± 0.9 mmHg) infused rats was significantly higher as compared their respective servocontrolled kidneys over the 14 days of drug infusion. The average daily PP in uncontrolled kidneys in NE-infused rats was significantly higher as compared to AngII-infused rats and kidneys from sham rats on days 11-14 of drug infusion.

Interlobular arterial remodeling and injury

The vascular smooth muscle wall/lumen cross-sectional area (CSA) ratio of interlobular arteries is presented in Figure 2A. Elevated RPP was responsible for ∼ 90% of the increase in the wall/lumen ratio of interlobular arteries in both NE and AngII infused rats (p < 0.05). The direct effect of NE or AngII did not result in a significant increase in the interlobular wall/lumen ratio. The wall/lumen ratio was significantly (p < 0.05) correlated with the average MAP across 14-days in the both AngII (r = 0.81) and NE (r = 0.84) groups.

Figure 2.

Figure 2

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Analysis of vascular smooth muscle wall/lumen cross sectional area (CSA) ratio (A) and injury (B) in 20 randomly selected interlobular arteries in kidneys from 14-day sham, servocontrolled (SC), and uncontrolled (UC) kidneys. A significant elevation in the wall/lumen ratio and injury was observed in interlobular arteries exposed to elevated RPP in both the AngII and NE-induced models of hypertension. Values are mean ± SE. * p < 0.05 vs. respective servocontrolled kidney.

Vascular injury, as assessed by the presence of either interlobular arterial hyalinosis or fibrinoid necrosis, was similarly increased in uncontrolled kidneys exposed to elevated RPP in both AngII and NE-infused rats (Figure 2B). Elevated RPP significantly (p < 0.05) contributed to vascular injury in both groups as the percentage of vessels injured in uncontrolled versus servocontrolled kidneys averaged 30.7 ± 9% vs. 0.7 ± 0.7%, respectively, in NE treated rats and 25.7 ± 10% vs. 1.4 ± 0.9%, respectively, in AngII treated rats. A significant (p < 0.05) correlation was observed between vascular injury and the average MAP across 14-days in both NE (r = 0.78) and AngII (r = 0.85) infused rats. Vascular injury was minimal in both servocontrolled kidneys of AngII and NE-infused rats as compared to kidneys from sham rats (0.7 ± 0.7%). These data indicate that elevated RPP is the dominant source of interlobular arterial remodeling and injury in both AngII and NE-induced hypertensive models.

Glomerular Injury

In both NE and AngII-infused rats, glomerulosclerosis exhibited a focal pattern of injury. The percent of sclerosed superficial cortical and juxtamedullary glomeruli for all groups is compared in Figures 3A and 3B, respectively. Photomicrographs, obtained with a 40× objective lens, representative of this pathology from sham, NE, and AngII rats are shown in Figures 3A and 3B. The extent of glomerulosclerosis differed considerably between superficial and juxtamedullary glomeruli.

Figure 3.

Figure 3

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Summary of the % of superficial cortical and juxtamedullary glomerulosclerosis in kidneys from 14-day sham and servocontrolled (SC) and uncontrolled (UC) kidneys. Photomicrographs (40× objective lens, H&E) representative of glomerulosclerosis are shown on the right in 3A and 3B. 30 superficial cortical and 20 juxtamedullary glomeruli were examined for sclerosis twice in a blinded fashion. Superficial cortical glomerulosclerosis exhibited a RPP-dependent injury only in the NE-infused rats, whereas elevated AngII was the dominant contributor to glomerular injury in AngII-infused rats. A higher magnitude of RPP-dependent glomerulosclerosis was observed in juxtamedullary glomeruli in both models of hypertension. Values are mean ± SE. * p < 0.05 versus respective servocontrolled kidney. † p < 0.05 vs. sham. calibration bar = 100 μm

Superficial cortical glomerulosclerosis

Superficial cortical glomerulosclerosis in NE-infused rats, elevated RPP contributed to 92% of superficial cortical glomerulosclerosis with the uncontrolled kidneys exhibiting 24.1 ± 2.6 % of 30 glomeruli with sclerosis as compared to the servocontrolled kidneys, in which only 5.5 ± 1.5 % of 30 glomeruli were sclerosed (p < 0.05). The direct effects of NE (8% contribution) did not result in significant superficial cortical glomerulosclerosis as determined by comparison to the kidneys from sham rats.

In AngII-infused rats, superficial glomerulosclerosis was moderately, but significantly (p < 0.05), elevated in both servocontrolled (9.8 ± 2.5 % of 30 glomeruli) and uncontrolled (13.8 ± 2.7 % of 30 glomeruli) kidneys as compared to kidneys from sham rats. There was no difference in superficial glomerulosclerosis between the servocontrolled and uncontrolled kidneys of AngII-infused rats. Elevated AngII was responsible for ∼60% of superficial cortical glomerulosclerosis while elevated RPP only contributed ∼40%, which indicates that elevated AngII, per se, was the dominant cause of superficial glomerular injury, which is similar to our previous study (14). These data indicate that the magnitude of superficial cortical glomerular injury is significantly higher (p < 0.05) in NE versus AngII-infused rats, with the susceptibility to RPP-induced superficial cortical glomeruloslcerosis being greater in the presence of elevated levels of circulating NE as compared to AngII.

Juxtamedullary glomerulosclerosis

Juxtamedullary glomerulosclerosis elevated RPP was responsible for 87% of juxtamedullary glomerulosclerosis in NE-infused rats. Sclerosis was significantly (p < 0.05) higher in uncontrolled (37.1 ± 4.7% of 20 glomeruli) versus servocontrolled (9.8 ± 2.2% of 20 glomeruli) kidneys. The direct effects of elevated circulating levels of NE on juxtamedullary glomerulosclerosis was minimal and did not reach statistical significance as compared to kidneys from sham rats.

In AngII-infused rats, elevated RPP was responsible for 80% of juxtamedullary glomerulosclerosis as sclerosis was significantly (p < 0.05) higher in uncontrolled (35.7 ± 9.6% of 20 glomeruli) versus servocontrolled (13.2 ± 2.0% of 20 glomeruli) kidneys. The direct effects of elevated circulating levels of AngII (20%) on juxtamedullary glomerulosclerosis were modest, but did reach statistical significance as compared to kidneys from sham rats (p < 0.05). These data indicate that elevated RPP is the dominant cause of juxtamedullary glomerulosclerosis in both NE and AngII-induced hypertensive rats.

Outer medullary tubular injury and fibrosis

Tubular Injury

As shown in Figure 4A, elevated RPP increased outer medullary tubular injury in both NE (p < 0.05) and AngII (p < 0.05) infused rats. The magnitude of tubular injury present in the uncontrolled kidneys of AngII-infused rats was significantly (p < 0.05) greater as compared to the uncontrolled kidneys of NE-infused rats. Furthermore, the difference in outer medullary tubular injury between the uncontrolled and servocontrolled kidneys of rats receiving AngII (1.0 ± 0.3) was significantly greater (p < 0.05) when compared to rats receiving NE (.28 ± 0.1). Elevated AngII, per se, was directly responsible for 10% of outer medullary tubular injury (p < 0.05 as compared to sham) and resulted in more renal injury (p < 0.05) as compared to the direct effects of NE (0 % contribution to injury). A significant (p < 0.05) correlation between outer medullary tubular injury and the 14-day average MAP was found in both NE (r = 0.81) and AngII (r = 0.89) infused rats. Representative images, obtained with a 10× objective lens, depicting the pattern of outer medullary tubular injury are shown in Figure 4A. These results suggest that elevated RPP is responsible for the majority of outer medullary tubular injury in both models of hypertension; however, AngII greatly increases the susceptibility to RPP-induced outer medullary tubular injury as compared to NE.

Figure 4.

Figure 4

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Summary of outer medullary tubular injury (% tubular cast positive region – H&E, A) and interstitial fibrosis (% α-SMA positive region, B) in kidneys from sham rats and servocontrolled (SC) and uncontrolled (UC) kidneys. Photomicrographs (10× objective lens) representative of outer medullary tubular injury and fibrosis are shown on the right of 4A and 4B. Elevated AngII, per se, significantly contributed to both tubular injury and interstitial fibrosis, whereas this RPP-independent pattern of injury was not observed in NE-infused rats. Elevated RPP was the primary source of outer medullary injury in both groups; however the magnitude of RPP-induced injury was exacerbated in AngII-infused rats. Values are mean ± SE. * p < 0.05 NE servocontrolled. † p < 0.05 vs. sham and NE servocontrolled. ‡ p < 0.05 vs. NE uncontrolled. calibration bar = 200 μm

Interstitial Fibrosis

The percent positive α-SMA region within the outer medulla was used as an index of outer medullary fibrosis (see supplemental materials), as we have described previously (14,16). As shown in Figure 4B, elevated RPP was responsible for 78% of outer medullary fibrosis in NE (p = 0.09) and 65% in AngII (p < 0.05) infused rats. Outer medullary interstitial fibrosis was significantly (p < 0.05) greater in the uncontrolled kidneys of AngII as compared to the uncontrolled kidneys of NE-infused rats. Furthermore, the difference in outer medullary fibrosis between the uncontrolled and servocontrolled kidneys of rats receiving AngII (2.1 ± 0.8) was higher (p = 0.06) as compared to rats receiving NE (0.4 ± 0.2). AngII directly resulted in 35% of outer medullary fibrosis, which was significantly (p < 0.05) greater as compared to the direct effects of NE (22%). A significant correlation between outer medullary fibrosis and the 14-day average MAP was found in both NE (r = 0.43) and AngII (r = 0.70) infused rats. As shown in Figure 4B, the dark brown regions are positive for α-SMA and tended to be localized to only vasa recta capillaries in kidneys with minimal interstitial fibrosis. In kidneys with significant fibrosis, α-SMA staining was more diffuse with more intense staining in both vasa recta capillaries and tubules. Thus, elevated RPP was responsible for the majority of outer medullary interstitial fibrosis and AngII increased the susceptibility of the kidney to RPP-induced outer medullary fibrosis.

Discussion

Results of the present study show that elevated RPP was the dominant cause of renal damage in both NE and AngII-induced hypertensive rats; however, the pattern and magnitude of RPP-induced injury varied between the models. Injury of the superficial cortical glomeruli was greater in NE-infused rats and largely RPP dependent while in comparison, the elevation of RPP did little damage to cortical glomeruli in AngII-infused rats. In contrast, outer medullary tubular injury and interstitial fibrosis was greatly exacerbated by elevations of RPP in AngII when compared to NE-induced hypertensive rats. This was true despite a similar degree of RPP-induced vascular and juxtamedullary glomerular injury observed in these two models of hypertension. The results of this study demonstrate that different neurohumoral factors alter the susceptibility to RPP-induced renal injury in different ways. It is evident that the mechanisms responsible for hypertension-induced renal injury in the superficial cortex differ from those of the outer medulla in these two forms of hypertension.

Vascular hypertrophy and injury

The vascular wall/lumen ratio in interlobular arteries was significantly elevated in uncontrolled kidneys exposed to elevated RPP in both the NE and AngII models of hypertension. Remodeling and injury in small renal resistance vessels is common in individuals with primary hypertension and in most experimental and genetic models of hypertension (1). Hypertrophy of resistance vessels in response to chronic elevations in RPP is a physiological adaptation to reduce the amount of vascular wall stress and contributes to maintaining a relatively constant RBF and GFR in the face of elevated systemic pressures. Over time, however, with excessive vascular hypertrophy autoregulation is impaired as the diffusion distance of oxygen across the smooth muscle wall increases, which can result in injury to both the vasculature and downstream structures (17). In both the NE and AngII models of hypertension, elevated RPP, per se, accounted for 90% of the total amount of vascular hypertrophy in superficial cortical vessels. This is consistent with our previous servocontrol study in AngII-infused rats (14), and indicates that elevated RPP, per se, is the primary stimulus for hypertrophy of interlobular arteries in both NE and AngII-induced models of hypertension.

Our data indicate that elevated RPP results in significant interlobular arterial injury in both NE and AngII-induced models of hypertension. Interestingly, elevation of circulating NE or AngII, per se, at the concentrations used in this study, did not directly stimulate significant vascular remodeling or injury. Although previous studies suggest that vascular injury is in part due to direct consequences of elevated AngII, many of these studies have been performed in cell culture models or with the use of various antihypertensive drugs administered to different groups of animals (10,18), making it difficult to control for the contributions of elevated RPP, as was done in the present study. Importantly, our data demonstrate that elevated RPP was the dominant cause of interlobular arterial remodeling and injury in both NE and AngII models of hypertension. That is, the normalization of arterial blood pressure prevented most of the vascular complications arising as a result of hypertension in these rats.

Glomerulosclerosis

Superficial cortical glomerulosclerosis

Superficial cortical glomerulosclerosis superficial cortical glomerular injury in hypertension is typically modest unless there are severe elevations in blood pressure, such as in malignant nephrosclerosis, or in models of hypertension associated with impaired renal blood flow autoregulation or underlying renal disease (2). In the present study, superficial glomeruli of AngII-infused rats were greatly protected from elevated RPP as we have previously observed (14). The increase in superficial cortical glomerulosclerosis, albeit relatively small, could be attributed to the direct effects of elevated circulating AngII, as predicted based on evidence that AngII directly stimulates free radical production, extracellular matrix production, inflammation, and TGF-β production (19-22).

NE-infused rats exhibited greater RPP-dependent levels of superficial glomerulosclerosis than that elicited by the direct effects of AngII. Although it has previously been reported that 8 weeks of chronic phenylephrine infusion in rats (23) resulted in only a relatively small degree of glomerular injury, it is difficult to compare the magnitude of glomerular injury between studies. Specifically, the present study produced higher levels of arterial pressure for a shorter duration and could be expected to exhibit greater levels of renal injury. The goal of our study was to produce the same levels of hypertension with both AngII and NE.

Superficial cortical glomerular injury that could be attributed to the direct effects of circulating NE was minimal. These observations suggest that the greater degree of NE versus AngII related RPP-induced glomerulosclerosis was a consequence of relatively less preglomerular vasoconstriction in the NE model of hypertension. The data of previous studies is unclear on this issue with isolated arterioles from rats suggesting preferential postglomerular constriction with Ang II compared to NE (24) while others (25) suggest, using micropuncture techniques in acutely anesthetized rats, that AngII results in greater constrictor actions (independent of RPP) on afferent arterioles. Others have found that GFR does not decrease following chronic administration of NE, whereas a similar level of hypertension produced by AngII resulted in a decrease in GFR (26). Thus, the lack of RPP-induced superficial glomerulosclerosis in the AngII model of hypertension may be a result of increased preglomerular vasoconstriction, which could confer protection from RPP-induced glomerular injury. Furthermore, the small RPP-independent pattern of superficial cortical glomerulosclerosis in the AngII-induced model of hypertension may have been a consequence of ischemia due to increased preglomerular vasculature constriction (27,28).

Another possible explanation for the greater RPP-induced superficial cortical glomerulosclerosis in NE-infused rats could be related to the sensitivity of the tubuloglomerular feedback (TGF) mechanism. Previous studies have shown that AngII, but not NE, serves as an important modulator of TGF sensitivity (29,30), resulting in higher levels of afferent arteriolar constriction in the presence of elevated distal tubule flow rates or increased distal NaCl delivery. NE may fail to sensitize TGF-mediated afferent arteriolar constriction in response to a RPP-induced increase in distal NaCl delivery.

Additionally, the superficial cortical glomerulosclerosis in NE-infused rats could have occurred as a result of the differences observed in the arterial pulse waves and variability of pressure with NE-induced hypertension. It has been demonstrated that SBP (31), PP (32), and blood pressure variability (33) can influence the magnitude of hypertension-induced renal injury. Although the average MAP was similar between the uncontrolled kidneys of NE and AngII models of hypertension, the PP and both the magnitude and variability of SBP were significantly higher in NE-infused rats over the second week of drug infusion as compared to AngII-infused rats. The myogenic mechanism of renal blood flow autoregulation intrinsic to the preglomerular vasculature is thought to protect glomeruli from RPP-induced injury (34); however, the higher SBP as well as the greater magnitude of SBP fluctuations in NE-infused rats may have resulted in a greater fraction of systemic pressure being transmitted to the glomerular capillaries.

Juxtamedullary glomerulosclerosis

In the present study, juxtamedullary glomerulosclerosis was prominent in both NE and AngII-infused rats and was almost entirely a direct consequence of elevated RPP. Hypertension-induced juxtamedullary glomerulosclerosis is typically more severe compared to superficial cortical glomerulosclerosis and is often observed in the early phases of hypertension (35,36). The increased susceptibility to RPP-induced injury in juxtamedullary glomeruli is thought to be due to the larger diameters and shorter lengths of the preglomerular vessels thereby exposing these glomeruli to elevated capillary pressures and ultimately barotrauma related injury (37).

Our results stand in strong contrast to the commonly accepted belief that the direct effects of AngII are largely responsible for the glomerular and interstitial injury through the downstream activation of various pathways, such as the production of reactive oxygen species and TGF-β, in a RPP-independent manner (19-22). Clearly, these pathways do contribute to the injury as supported by observations that inhibitors of the RAS are found to be superior to other antihypertensive treatments due to their RPP-independent renal protection (38). The results of this study, however, suggest that the majority of glomerular, primarily juxtamedullary, damage observed in AngII-infused rats is a direct result of elevated RPP, an idea that is gaining strength (39).

Outer medullary tubulointerstitial injury

A major finding from this study is that the susceptibility to RPP-induced outer medullary tubulointerstitial injury and fibrosis is substantially greater in AngII-infused as compared to NE-infused hypertensive rats despite a similar magnitude of hypertension in both groups. We have previously demonstrated a RPP-dependent pattern of outer medullary injury in AngII-infused rats (14) and have proposed that this could be explained by the poor autoregulation of blood flow in the region of the outer medulla (40,41). The attenuation of RPP-induced outer medullary injury in NE-infused rats does not appear to be explained by greater preglomerular constriction because juxtamedullary glomerular RPP-induced injury was as great in NE as that observed in AngII-infused rats.

The mechanisms responsible for the reduced susceptibility to RPP-induced outer medullary injury in NE-induced hypertension remains unclear, but recent studies provide some possible explanations. It is recognized that increased RPP results in decreased reabsorption of NaCl in the proximal tubule, which increases distal tubule flow rate and NaCl delivery (42). We have recently found that acute elevations of RPP increase the production of superoxide in the medullary thick ascending limb (mTAL) (43) and interstitial hydrogen peroxide levels in the outer medulla (44), responses that appear to be mediated through stimulation of superoxide production via NADPH-oxidase within mTAL (45). Since studies have shown that AngII, but not NE, increases the expression of NADPH oxidase (46), the increased NaCl delivery to mTAL associated with elevated RPP may stimulate greater amounts of superoxide and hydrogen peroxide in the AngII model of hypertension resulting in greater tissue injury and fibrosis of the outer medulla.

Alternately, or in parallel with the release of reactive oxygen species, AngII may have resulted in greater ischemia in the outer medulla as compared to NE. Schachinger et al. (28) found in humans that acute administration of AngII, but not NE, resulted in decreases in renal oxygen tension despite similar increases in blood pressure. Thus, the outer medullary injury caused by the direct actions of AngII, independent of RPP, observed in the present study could have resulted from ischemia due to increased preglomerular constriction, as discussed previously. Furthermore, it is also possible that AngII sensitizes the myogenic response of the afferent arteriole (47), which could produce excessive levels of vasoconstriction and ischemia in response to elevated RPP, and exacerbate the magnitude of outer medullary ischemia. It is recognized that hypoxia can be both a cause and consequence of renal injury, i.e. RPP-induced injury (48), and additional studies will be required to determine the specific role of ischemia in AngII-induced hypertensive renal injury.

In conclusion, it is recognized that the extent and pattern of renal injury differs widely in subjects with hypertension and in various animal models and the contribution of elevated RPP, per se, to the injury is poorly understood. The insight provided by this study is the demonstration that neurohumoral factors can influence the pattern and magnitude of RPP-induced injury. Having for the first time precisely controlled RPP to one kidney and exposing the contra lateral kidneys to the same levels of hypertension, we can with confidence conclude: 1) that elevated RPP significantly contributes to renal injury in both NE and AngII models of hypertension and 2) the magnitude of RPP-induced injury varies between models with the extent of outer medullary damage being considerably greater in AngII-infused rats, whereas superficial cortical glomerular injury is more severe in NE-infused rats.

Perspectives

The incidence of ESRD has risen enormously over the last two decades (4), with hypertension recognized as the second leading cause of ESRD. It is likely that the variation in the susceptibility to RPP-induced injury in different human populations is related to the genetic heterogeneity and the complex etiologies among subjects. Assuming that the rat data are relevant to humans, the results of the present study indicate that excess adrenergic activity associated with hypertension would influence the pattern and magnitude of RPP-induced injury differently than subjects with inappropriately elevated AngII. As similar studies are carried out in strains of various congenic and transgenic inbred rats with controlled genetic backgrounds, the genomic and functional pathways that determine susceptibility to RPP-induced injury may be elucidated. This would provide a better understanding of the RPP-dependent and independent mechanisms of injury and guide the implementation and optimization of antihypertensive therapies which could also minimize renal injury.

Supplementary Material

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Acknowledgments

The authors thank David Eick, Mike Kloehn, and Greg McQuestion for design and maintenance of the servocontrol system, Glenn Slocum for microscopy assistance, and Carol Bobrowitz, Barb Fleming, and Mary Kaldunski for assistance with histological and immunohistochemistry protocols. The authors also thank Dr. Carl Becker for his expert input regarding the evaluation of the renal histology.

Sources of Funding: This work was supported by National Heart, Lung, and Blood Institute grants HL-081091 and HL-29587 and predoctoral fellowship from American Heart Association AHA-0615590Z.

Footnotes

Online Supplement contains detailed methods.

Disclosures: None

References

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NIHMS161217-supplement-supp1.pdf (28.9KB, pdf)
supp2
NIHMS161217-supplement-supp2.pdf (259.9KB, pdf)
supp3
NIHMS161217-supplement-supp3.pdf (140KB, pdf)

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