The Effect of Salt on Renal Damage in eNOS Deficient Mice

Abstract

Hypertension is associated with significant end-organ damage such as stroke, myocardial infarctions, and renal disease. Although endothelial nitric oxide deficient mice (eNOS^−/−) are a transgenic model of hypertension, this model has not been studied extensively for hypertension-associated renal damage. Therefore, the purpose of the present study was to determine whether 1) there is hypertension-associated renal injury in older eNOS^−/− mice, and 2) dietary salt modulates the hypertension-associated renal injury. Six month old eNOS^−/− mice were placed on low (0.12% NaCl), control (0.45% NaCl), or high (8%) salt diet for eight weeks and blood pressure measured weekly. At the end of the eight-week period kidneys were collected and examined for tissue injury. Mice deficient of eNOS were hypertensive at baseline compared with wild type mice in all three groups (128 ± 3 vs.112 ± 3, p< 0.05 on 0.12%; 129± 5 vs. 111± 5 on 0.45%, p<0.05; 120 ± 2 vs. 109 ± 2 mmHg on 8%, p< 0.05). Blood pressure was significantly elevated from baseline in eNOS^−/− on 0.45% and 8% salt diets compared to baseline (p<0.02). While hypertensive eNOS^−/− mice had significant renal injury as compared to wild type mice, high salt intake exacerbated the injury (p < 0.001 vs. normal salt diet). These data show that hypertensive eNOS^−/− mice develop renal damage, and that a high salt diet potentiates the renal injury associated with hypertension. This study confirms previous pharmacologic models of eNOS deficiency and high salt effects on organ damage.

INTRODUCTION

Hypertension is a major risk factor for end-organ damage, including stroke, myocardial infarction, and renal disease. Although cardiovascular disease and other complications associated with hypertension have steadily decreased over the last several decades, the rate of decrease for end-stage renal disease (ESRD) has been much less ¹. Indeed, the rate of ESRD has been slowly rising over the past ten years ¹; the mechanism(s) involved in the development of ESRD is poorly understood.

One population that is disproportionately affected by ESRD is the African American ethnic group ². African Americans have a significantly higher incidence of hypertension as well as attendant end-organ injury ^{3, 4}. Furthermore, African Americans experience a 3–17-fold greater likelihood of developing ESRD ^5–7 as compared to other populations. Of interest, approximately seventy-five percent of hypertensive African Americans are characterized as salt-sensitive ^5–7, suggesting that salt may contribute to the pathophysiology of tissue organ damage associated with hypertension. Several studies in both humans and animals have shown that increased salt may be “toxic,” contributing to cardiac and renal tissue injury under certain conditions ^{8, 9}. These conditions may include states of nitric oxide deficiency ⁹. Recent evidence reveals that salt inhibits brain nNOS expression and increases sympathetic output in male rats (uninephrectomized + 1% saline) ¹⁰. Thus, available evidence indicates that nitric oxide deficiency plays both a permissive as well as an initiator role in the injurious effects of salt. Therefore, in this study we used endothelial knockout mice (eNOS−/−) to determine if 1) salt causes renal injury in this mouse model of hypertension, and 2) endothelial nitric oxide deficiency acts as a permissive or initiating agent in the effect of salt on renal injury associated with hypertension.

MATERIALS AND METHODS

Animals

Breeding pair stocks for eNOS knockout mice were obtained from Jackson Laboratory (Bar Harbor, ME; stock # 002684) and Dr. Paul Huang (Massachusetts General Hospital; Boston, MA). Wild type control (C57BL/6) breeding stock was obtained from Jackson Laboratory. All animals were maintained on regular rodent chow. Mice were entered into the study at six months of age. Systolic blood pressure was measured using the tail-cuff method (Visitech 2000, Apex, NC).

Protocol

Wild type and eNOS knockout mice were divided into three treatment groups. Group I mice were placed on a low salt diet (0.12% NaCl; Harlan Teklad, Madison WI; cat. no.T7034), Group II mice were maintained on a control regular salt diet (0.4% NaCl; LabDiet®, Richmond, IN; cat. no. 5001), and Group III mice were given a high salt diet (8%NaC; Harlan Teklad, cat. no.TD92012), all for eight weeks. Low and high salt diets were initiated in Groups I and III, respectively, once basal tail-cuff systolic blood pressures were obtained. Mice on control diets remained on this diet throughout the basal and experimental periods. Weekly blood pressures were taken throughout the eight-week study period. Protocols were approved by the Animal Care Committee at Boston University School of Medicine.

Tail-cuff Blood Pressure Measurements

Mice tail-cuff blood pressures were measured using the BP2000 instrument [Visitech Systems (Apex, NC)]. Mice were trained one to two weeks prior to beginning blood pressure measurements. The mice were kept in restrainers during measurements, and these are magnetically held in place on a heated (37°C) platform. The cuff was gradually inflated, and systolic pressure was determined as that pressure when flow ceases as detected by a sensor.

Tissue Perfusion

At the end of the diet regimen, the animals were anesthetized with pentobarbital or isofluorane for blood collection and tissue perfusion. Once blood was collected, 5 mls of phosphate-buffered saline (PBS, pH 7.4) was perfused through the heart to remove the remaining blood within the tissues. Finally, 5 mls of 10% buffered formalin (Fisher) was perfused through the heart for fixation of tissues. The tissues were harvested for weighing, histological slide preparation, and staining (Histotechniques, Powell, OH). PAS was used for staining renal tissue, and Masson Trichrome staining was used for heart tissue.

Pathology Scoring

Glomerular injury included the presence of fibrosis, cellularity, sclerosis, and thrombosis; interstitial injury included the presence of inflammatory cells; tubular damage was defined as the presence of protein casts; and perivascular inflammatory injury included the presence of inflammatory cells, medial hypertrophy, and intimal proliferation. The presence of each of these was graded on a scale of 0 to 4 where 0 represented no injury and 4 represented maximal injury. The degree of injury was defined as the total of the scores for each of the types of injury. The extent of tissue injury score ranged from 1–3 where 1 represented focal areas of injury distribution, 2 represented multiple focal areas of injury, and 3 represented diffuse or global tissue damage. The final pathology score was calculated as the product of the degree and extent scores of tissue injury.

Statistical Analysis

Blood pressure time course data were analyzed by two-way analysis of variance (2-ANOVA), while blood pressure single point multiple group analysis was performed by one-analysis of variance (1-ANOVA) followed by Duncan’s multiple comparison. T-test or rank sum (for non-parametric data) analysis was used for basal and end-point comparisons. An among-group pathology comparison was made using 1-ANOVA followed by Student’s Newman Keuls (parametric analysis) or Dunn’s (non-parametric analysis) multiple comparison test.

RESULTS

Blood Pressure

All animals remained relatively healthy as indicated by body weight increases over the duration of the study (Table 1). As shown in Figure 1, all three groups of eNOS ^−/− mice had significantly higher baseline systolic blood pressures as compared to wild type control mice (WT). This pressure difference persisted throughout the eight-week study period. Interestingly, systolic blood pressure in eNOS ^−/− mice on control and high salt diets was significantly higher than the starting baseline blood pressure at week eight of the diets (0.45%:129 ± 4 vs. 145 ± 4, p = 0.004; 8%: 120 ± 2 vs. 135 ± 5, p = 0.017) (Table 1); however, blood pressure was unchanged in eNOS ^−/− on a low salt diet. Blood pressure in WT was unchanged from baseline over the eight-week period in all three diet groups.

Table 1.

Effect of Salt Diets on Systolic Blood Pressure and Body Weights

Strain	Salt Diet		Systolic Blood Pressure (mmHg)	Weight (grams)
WT	0.12% (n = 12)	Baseline	112 ± 4	28.5 ± 2
		Post-diet	111 ± 3	28.5 ± 1
	0.45% (n = 8)	Baseline	111 ± 5	27.6 ± 2
		Post-diet	117 ± 2	28.1 ± 2
	8% (n = 12)	Baseline	109 ± 2	27.5 ± 1
		Post-diet	107 ± 3	28.1 ± 1
ENOS−/−	0.12% (n = 17)	Baseline	128 ± 2*	26.5 ± 1
		Post-diet	127 ± 6*	27.9 ± 1
	0.45% (n = 21)	Baseline	129 ± 4*	27.7 ± 1
		Post-diet	145 ±4*,†	27.3± 1
	8% (n = 16)	Baseline	120 ± 2*	24.7 ± 1
		Post-diet	135 ± 5*,†	26.7 ± 1

Value = Mean ± SEM;

^*= p<0.01 vs. WT;

^†= p<0.02 vs. baseline using signed-rank test.

Figure 1.

Cardiac and Renal Hypertrophy

Because cardiac hypertrophy is frequently associated with hypertension, we also examined the eight-month old eNOS ^−/− transgenic mice for cardiac hypertrophy. Indeed, we observed cardiac hypertrophy in eNOS ^−/− relative to wild type control mice in all three diet groups (Figure 2, Panel A). Although salt had no effect on the hypertrophy associated with hypertension in the eNOS ^−/− mice, there was a dose-dependent effect of salt on cardiac hypertrophy in wild type mice with high salt causing a significant increase in cardiac size relative to the low salt group (p<0.01 vs. low salt treatment; 1-ANOVA and Newman-Keuls). Dietary salt had no effect on kidney size in either eNOS^−/− or WT (Figure 2, Panel B)

Figure 2.

Renal Pathology

Figure 3 shows the renal histology of WT and eNOS^−/− mice on a high salt diet. As can be seen in Panel B, the hypertensive eNOS ^−/− mice have contracted and thrombosed glomeruli that are not observed within the WT. Additionally, there was perivascular and interstitial inflammation dispersed throughout the kidney of eNOS^−/− mice (Panels B, D and E). Protein casts within the tubules (Panel F), were particularly prominent within kidneys from eNOS^−/− on a high salt diet. Although there was a tendency for glomerular damage to increase as dietary salt increased, there was no statistical difference among the treatment groups (Table 2). However, we did find that the high salt diet caused a significant increase in tubular injury as indicated by the larger pathology scoring for protein casts (Table 2; 8% diet: 2.46 ± 0.65 vs. normal 0.45% diet 0.29 ± 0.16; 1-ANOVA and Dunn’s test, p<0.004). There was no significant effect of salt on perivascular and interstitial inflammation.

Figure 3.

Table 2.

Effect of Dietary Salt on Renal Injury

Strain	Salt Diet	Glomerular Damage	Tubular Damage	Interstitial Inflammation	Vascular Inflammation
WT	0.12% (n = 11)	0.0 ± 0.0	1.1 ± 0.4	2.2 ± 0.8	0.6± 0.6
	0.45% (n = 8)	0.0 ± 0.0	1.1 ± 0.4	0.8 ± 0.5	0.1 ± 0.1
	8% (n = 11)	0.2 ± 0.2	0.9 ± 0.4	1.5 ± 0.8	0.9 ± 0.6
ENOS−/−	0.12% (n = 14)	6.5 ± 2.4*	0.9 ± 0.4	4.5 ± 1.0*	7.1 ± 1.6*
	0.45% (n = 21)	8.6 ± 1.7*	0.3 ± 0.2	3.7 ± 0.7*	3.8 ± 0.7*
	8% (n = 16)	12.5 ± 2.3*	2.5 ± 0.7*,§	6.5 ± 1.7*	10.3 ± 3.3*

Value = mean ± SEM;

^*= p<0.05 vs. WT on same diet by t-test comparison;

^§= p<0.05 vs. 0.45% NaCl diets by 1-ANOVA.

This degree of renal injury was not present within the WT animals (Table 2). Additionally, increasing dietary salt did not significantly alter the histological evidence of renal injury in WT.

DISCUSSION

This study demonstrates that significant renal damage occurs in older (8 months old) hypertensive eNOS-deficient mice as compared to wild type mice and that this damage can be modulated by dietary salt. Specifically, high dietary salt leads to greater tubular damage in eNOS^−/− mice but not in wild type mice. Interestingly, there was a trend for a dose-dependent effect of salt on glomerular injury, while both low salt and high salt diets tended to increase perivascular inflammation in eNOS^−/− mice. Additionally, there was cardiac hypertrophy associated with the hypertension in this model of hypertension, but the hypertrophy was not modulated by dietary salt in eNOS^−/− mice. Unlike what was observed in eNOS ^−/− mice, there was a dose-dependent effect of salt on cardiac hypertrophy in WT.

In the present study we found that systolic blood pressure remained elevated throughout the two-month low dietary salt intervention in eNOS^−/− mice (Figure 1). Following one week of high salt, blood pressure increased from 120 ± 2 to 148 ± 5 mmHg, a 28 mmHg increase (Figure 1, Panel C; p<0.05). We observed a similar blood pressure response to a normal salt diet in this strain (Figure 1, Panel B). Although blood pressure decreased the subsequent week, it remained above baseline for the remainder of the study period in both the normal and high salt diet groups. It is not clear why blood pressure increased during the first week, althoughit is unlikely due to acclimation to the tail-cuff blood pressure measurement since all mice were exposed to a one-to-two week training period. The rise may have been due to some undetected environmental stressor. Leonard and associates have also reported similar time course effects of high dietary salt on blood pressure in eNOS^−/− mice ¹¹. They show an early peak at two weeks followed by a sustained increase throughout the remainder of the six-week time period. The results from the normal salt group suggest that a normal salt diet may be an inappropriately high salt diet in the absence of eNOS.

Cardiac hypertrophy has not been previously reported in eNOS^−/− mice ^12–15; however, we report significant cardiac hypertrophy in our eNOS^−/− mice. We used two eNOS^−/− stocks, obtained from Jackson Laboratory and Dr. Paul Huang (Huang, 1995 #583; Boston, MA), and did not detect any difference in blood pressure or hypertrophy response between the two breeding stocks. The reason for this apparent discrepancy in cardiac hypertrophy reporting is not known but the use of younger aged animals in the earlier studies may explain the failure to observe cardiac hypertrophy. In this study we used six month old mice; consequently, the mice were eight months old at the time of measurement, an age comparable to middle age in humans. Babikat and associates also studied 6–8 month old eNOS^−/− with comparable systolic blood pressures as in our study, and found modest but insignificant hypertrophy at this age. This discrepancy cannot be explained by differences in control dietary salt since Babikat and colleagues used chow containing 0.6% NaCl and we used chow containing 0.45% NaCl. However, it is important to note that Yang and colleagues have reported left ventricular hypertrophy in 12-week old eNOS^−/− mice ¹⁶, a finding consistent with our study. It is not clear why the presence of cardiac hypertrophy is so variable in this model; however, despite this variability, these studies, taken together, suggest that sustained hypertension in eNOS^−/− can produce cardiac hypertrophy.

As with cardiac hypertrophy, previous examination of renal histology of 14 week-old eNOS^−/− mice revealed no obvious histological differences from that of WT ¹⁴. The effect of sustained hypertension on renal injury in eNOS^−/− mice, however, has not been investigated. In this study we show that there is significant renal injury at eight months of age in mice lacking the eNOS gene. eNOS deficient mice on normal rat chow have enlarged and collapsed glomeruli, tubular casts, interstitial and perivascular inflammation, and arterial wall thickening. Similar renal injury has been reported in chronic nitric oxide inhibition models of hypertension with the major features of renal injury being enlarged and collapsed glomeruli ^{17, 18}. Other histological findings in NO inhibition models of hypertension include capillary thrombi and sclerosis, arteriolar fibrosis, and proliferation providing the onion skin appearance to the arterioles ^{19, 20}. Thus, this mouse model of hypertension appears to be comparable to the pharmacologic NO inhibition models.

We also examined the effect of low and high salt diets on cardiac hypertrophy and renal injury. We found a dose-dependent effect of salt on cardiac hypertrophy in the WT; however, we did not find such an effect in the eNOS^−/− mice. The ‘toxic’ effect of salt on organ damage independent of blood pressure has been previously reported ^21–23. This effect of salt on cardiac mass is seen in animals and humans ^{15, 21, 24–27}. Thus, our cardiac hypertrophy response to salt in the WT is consistent with previous findings in models of normal blood pressure.

Interestingly, African Americans have significant hypertension-associated renal injury ^{28, 29} and tend to be salt-sensitive ⁴ with diminished NO bioavailability ^30–32. It is for these reasons that we examined the effect of salt on renal injury in eNOS deficient mice. Renal pathology has been reported for eight-week old eNOS^−/− made diabetic with streptozotocin ³³. These authors do not report any renal injury in mice not treated with streptozotocin. To our knowledge, this report is the first to demonstrate renal injury in eNOS^−/− mice. This may be due to an age difference as compared with prior studies; we used mice that were much older (eight months of age) than what others have reported (often 12 weeks of age). Dietary salt significantly modulated the extent of tubular casts seen in this model of hypertension. Although statistically insignificant, glomerular injury tended to increase as dietary salt increased. The inflammation seen in this model was not altered by dietary salt; however, it is interesting to note that perivascular inflammation tended to be higher under both low and high salt conditions. Moreover, we also show that increased dietary salt aggravates the tubular injury seen in this model. The renal pathological scores were unaffected by dietary interventions in the wild type, normotensive control mice. These findings suggest that prolonged exposure to hypertension in this mouse model is associated with significant renal injury. In addition, these data suggest that in the setting of impaired NO bioavailability, a low salt diet may protect against hypertension and tissue injury associated with hypertension.

In summary, prolonged hypertension in eNOS deficient mice is associated with cardiac hypertrophy and renal injury. While increased dietary salt does cause cardiac e a hypertrophy in wild type controls, high salt does not further aggravate the effect of hypertension on heart mass in eNOS^−/− mice. There is a dose-dependent effect of salt on renal tubular pathology in eNOS^−/− that is not seen in wild type control mice. This renal pathology response to dietary salt appears to be associated with an increase in systolic blood pressure. Therefore, this mouse model of hypertension has similar target organ damage as the hallmark characteristics seen in human and other animal models of hypertension and may a useful model for hypertension observed in African Americans.