Physical Training Increases Renal Injury in Rats with Chronic NOS Inhibition

Abstract

Nitric oxide (NO) is involved in regulation of vascular tone and renal hemodynamics. Inhibition of NO synthase (NOS) by N^ω-nitro-l-arginine methyl ester (L-NAME) promotes systemic hypertension and glomerular damage. Exercise is effective in reducing elevated blood pressure in hypertensive individuals and rats treated with L-NAME. We investigated the effects of regular aerobic exercise on renal injury in hypertensive rats with NOS inhibition. Adult Wistar rats were divided into four groups: sedentary or exercising, nonhypertensive (two groups) and hypertensive, sedentary or exercising (two groups). Treadmill running exercise was prolonged for 4 weeks (60 min.day^{− 1}, 5 days/week, 20 m.min^{− 1}, no incline), and hypertension was induced by L-NAME given orally to rats for 4 weeks (25 mg.kg^{− 1}.day^{− 1} in drinking water). Blood pressure was monitored at baseline and then once a week throughout L-NAME administration. Kidney sections were examined for renal histopathology. Hypertensive animals exhibited elevated blood pressure, and exercise partly prevented this elevation. Renal injury observed as arteriolar wall thickening, focal tubular atrophy, and interstitial inflammatory infiltration was apparent in hypertensive animals, and exercise induced further renal damage in hypertensive animals. The present training protocol exacerbates renal insufficiency in NOS-blockage hypertension in rats.

• nitric oxide
• nitric oxide synthase
• exercise training
• renal injury

Introduction

Nitric oxide (NO) is an important physiological regulator of vascular tone and renal hemodynamics.[1&2] NO is enzymatically synthesized from l-arginine by a process that could be competitively inhibited by l-arginine analogues like N^ω-nitro-l-arginine methyl ester (L-NAME).[3] Acute and chronic NO synthase (NOS) enzyme inhibition increases vascular resistance and, in turn, blood pressure.[4] Chronic administration of L-NAME induces a dose- and time-dependent sustained elevation in blood pressure, and renal glomerular damage accompanies hypertension.[5] Increased renal vascular resistance and proteinuria were also observed in NOS inhibition, coincident with the appearance of renal histopathology as glomerular sclerotic injury and arteriolar narrowing as reported elsewhere.[4-6]

Along with drug therapy, regular physical activity is an approach to the treatment of hypertension. Exercise-related beneficial effects on blood pressure have been well documented in hypertensive individuals[7&8] and animals.[9-11] In most studies of hypertensive rats, it has been shown that when performed regularly, low- to moderate-intensity aerobic exercise reduces elevated arterial blood pressure.[9&10] We already found that exercise reduces the arterial pressure in rats with chronic NOS inhibition[11] and a similar observation was also reported, though the decrease in blood pressure was not statistically significant.[12] We also revealed that exercise reduces elevated blood pressure in L-NAME-treated hypertensive rats via increasing NOS activity, and changes in vessel structure with exercise may also be involved in this effect.[11]

There are several conclusions related to the effects of exercise training on renal function in different types of chronic renal insufficiency.[13-16] Although exercise effectively reduces blood pressure in L-NAME-treated rats, the influence of physical training on renal injury has not yet been clearly revealed in hypertension induced by NOS inhibition. Thus, the aim of the present study was to evaluate the effect of exercise on renal injury in rats with chronic L-NAME treatment.

Methods

Thirty-six 8-week-old, male Wistar rats had free access to standard rat chow and tap water and were housed in a temperature controlled room (22–25°C) under a 12:12 dark–light cycle. All procedures were approved by the Akdeniz University Animal Care and Usage Committee and followed the guidelines for using the animals in experimental research. Rats were randomly divided into four groups: sedentary, nonhypertensive (Sed, n = 9); exercising, nonhypertensive (E, n = 9); sedentary, hypertensive (H, n = 9); and exercising, hypertensive (HE, n = 9) groups.

Hypertension was induced by systemic NOS blockage with L-NAME (Sigma Chemical Co.), orally administered by dissolving in drinking water. A dose of 25 mg.kg^{− 1}.day^{− 1} L-NAME was used during 4 weeks in H and HE groups. L-NAME was concomitantly given during the training protocol.

Blood pressure was measured by a noninvasive tail-cuff method, and data were obtained with the MAY-BPHR200 unit and MP 100A-CE data acquisition system (BIOPAC Systems, Inc., Santa Barbara, CA, USA) connected to a personal computer. Mean arterial pressure (MAP) values were determined at baseline and then once a week in all groups, and measurements were performed without anesthesia at room temperature in a silent room.

Exercise was performed on a motor driven treadmill (MAY-TME 9805, Commat Ltd., Ankara, Turkey) in the dark period. The training program consisted of running at 20 m.min^{− 1} for 60 min.day^{− 1}, 0% grade, five times per week, and prolonged for 4 weeks in exercising groups. Exercising animals were familiarized to the treadmill and ran for < 10 min.day^{− 1} during 3 days until the initiation of the training protocol. The speed of the treadmill was gradually increased until the animals were running at the designated speed. Mild electrical shock was used to prod the animals to run; however, most animals ran voluntarily during their initial run.

Animals were sacrificed under ether anesthesia 24 h after the last exercise session, and tissue samples were obtained. Kidneys were carefully dissected and isolated, and fixed in neutral formalin. Paraffin-embedded tissue samples were used to prepare sections with 5–6 µm thickness for histopathological examination. Tissue sections were stained with common hematoxylin-eosin dye and were examined on a blinded basis for the level of glomerular sclerosis by a semiquantitative method as described previously.[17] One hundred glomeruli were randomly selected, and the severity of lesion was graded from 0 to 4 + according to the percentage of glomerular involvement. The number of glomeruli for each degree of scoring was multiplied by the score level, and the results were summed to calculate the glomerular sclerosis index for the given specimen.

Results are expressed as mean ± SE. One-way ANOVA and Newman-Keuls post-test were used for comparison of results between four groups. The value p < 0.05 was accepted as statistically significant.

Results

Weight gain was not different between the groups (Table 1). Daily water intake and food consumption were also not different (data not presented). In addition, the heart:body weight ratio was not different between the Sed, E, and H groups, but it was higher in the HE group (p < 0.05) as compared to both Sed and H groups (Table 1).

Table 1 Weight gain (expressed in grams as percent of initial weight) and heart/body weight (expressed as mg.g^{− 1}) in experimental groups

	Sed	E	H	HE
Weight gain (g, % of initial)	29.21 ± 3.48	23.52 ± 3.68	24.31 ± 1.12	21.78 ± 2.54
Heart/body weight (mg.g^− 1)	2.78 ± 0.005	2.96 ± 0.008	2.83 ± 0.040	3.16 ± 0.050,

Sed: sedentary, nonhypertensive; E: exercising, nonhypertensive, H: sedentary, hypertensive; HE: exercising, hypertensive groups. Values are mean ± SE.

^*p < 0.05 Sed versus HE group.

^†p < 0.05 H versus HE group.

MAP values at the beginning of the experiment were similar in all groups. MAP levels of the H and HE groups were higher after the first week of L-NAME intake compared to the Sed group. The HE group exhibited a significant decrease in MAP compared to the H group after 3 weeks of exercise (initial and periodic data are not presented). The elevated blood pressure in both groups was maintained during the period of L-NAME intake. Final blood pressure values were significantly higher (p < 0.001) in both hypertensive groups compared to those in the Sed group. However, MAP levels in the HE group were significantly lower (p < 0.001) in comparison with the MAP values of animals in H group (Figure 1).

Figure 1 Final blood pressure levels in experimental groups. (MAP, mean arterial pressure; Sed: sedentary, nonhypertensive; E: exercising, nonhypertensive; H: sedentary, hypertensive; HE: exercising, hypertensive groups. ^*** p < 0.001 significantly different from Sed group, ††† p < 0.001 difference between H and HE groups.)

Stained sections from kidneys were examined for the typical lesions of hypertension. Interstitial inflammatory infiltration was observed in a few specimens from the E group, while there were no signs for arteriolar wall thickening and focal tubular atrophy in both Sed and E groups. Arteriolar wall thickening and arteriole near obliteration predominated in sections obtained from animals with elevated blood pressure (trained or not), and focal tubular atrophy was also detected, although not in all samples. Mesangial matrix proliferation was present in some glomeruli. Another pathology observed in these groups was the interstitial inflammatory infiltration. The mentioned microscopic changes were more prominent in the HE group than in the H group. Renal injury scores were significantly higher in both H and HE groups as compared to the Sed group (p < 0.001), and the HE group exhibited a higher score when compared to the H group p < 0.001 (Figure 2).

Figure 2 Renal injury scores in experimental groups. (Sed: sedentary, nonhypertensive; E: exercising, nonhypertensive; H: sedentary, hypertensive; HE: exercising, hypertensive groups. ^*** p < 0.001 Sed versus H and HE groups; ††† p < 0.001 H versus HE group.)

Discussion

The beneficial effects of regular physical activity in reducing the elevated blood pressure were revealed in human studies[7&8] and animal hypertension models such as spontaneously hypertensive rats,[18] Dahl salt-sensitive and salt-resistant rats,[19] deoxycorticosterone acetate (DOCA)-induced hypertension[9] and hypertension due to manipulation of kidney arteries (Goldblatt model).[20] Exercise training is advised as a regular habit of life style for hypertensive individuals.[7&8] Different exercise protocols were applied for evaluation of training-related effects in experimental hypertension models. It was clearly concluded that regularly performed aerobic exercise reduces the high blood pressure[9&10] or delays the rise in blood pressure in rats with spontaneous hypertension.[18] Moderate intensity exercise training in the present study significantly reduced blood pressure in rats with chronic NOS inhibition, as in our previous study.[11]

Renal injury is a common state in accelerated hypertension, and the malignant phase of increased blood pressure might lead to renal vascular changes.[21] In contrast, benign essential hypertension is attributed to cause much less damage to the kidney than to other target organs such as the heart and brain.[21] Published data provide evidence for progression of renal disease induced by systemic hypertension.[22] More complex hypertension is also associated with specific renal vascular, glomerular, and interstitial lesions that are often accompanied by atherosclerosis.[23] Renal pathology also accompanies hypertension induced by NOS inhibition, and severe renal injury is mostly developed by long-term NOS blockade.[1] Beside the systemic hypertension, chronic inhibition of NO synthesis also induces glomerular capillary hypertension and glomerular damage.[5] Renal structural injury consisting of glomerular ischemia, glomerulosclerosis, and interstitial expansion were also promoted by chronic NOS inhibition.[4&5] The higher renal injury scores of hypertensive animals in our study are consistent with previously reported findings. Interstitial inflammatory infiltration and vascular damage were typical findings in hypertensive animals.

Beside the well-documented effects of exercise on renal function in normal man contradictory reports exist in the field of exercise action on impaired renal function and renal histopathology. The first detailed study that reported that moderate-intensity exercise lessens the extent of glomerulosclerosis in rats with renal insufficiency was done by Heifets et al.[13] Exercise was also attributed to induce an improvement in the glomerulosclerosis index in two different models of renal insufficiency used in separate studies by the same research group.[15], [24] The improving effects of regular exercise on renal function in individuals with moderate renal insufficiency were only reported in a study by Pechter et al.[25] Human studies revealed that physical activity usually does not affect renal function.[14], [26] However, renal function was not found to be positively or negatively affected by exercise in many experimental models.[27-30] Despite our findings for the reducing effect that regular physical activity has on blood pressure, we observed an increased glomerulosclerosis index in the exercising hypertensive group. Although our findings are not in accordance with available literature, Cornacoff et al. reported similar effects in rabbits.[31]

The present study does not include parameters concerning mechanisms for increased glomerulosclerosis index in hypertensive animals with exercise. Beside the increased renal sympathetic activity during exercise, the activation of the renin–angiotensin system and elevated endothelin-I levels might also lead to negative effects. Exercise-induced oxidative stress could also worsen renal function. Leaf et al. observed that patients on chronic maintenance dialysis have greater lipid peroxidation compared with control subjects at rest and during and after physical exercise.[32]

In conclusion, our study is one of the rare reports suggesting that training enhances renal insufficiency, although agreement about the relation between regular exercise and renal function does not exist. Detailed investigation of different pathways is required to reveal the training effects on impaired renal function.

Acknowledgments

This study was supported by Akdeniz University Research Projects Unit.