Combination of Exercise and Enalapril Enhances Renoprotective and Peripheral Effects in Rats With Renal Ablation

AJH

2006; 19:80 – 86

Kidney

Combination of Exercise and Enalapril Enhances Renoprotective and Peripheral Effects in Rats With Renal Ablation Masayuki Kanazawa, Takayuki Kawamura, Lan Li, Yuko Sasaki, Kayomi Matsumoto, Hitomi Kataoka, Osamu Ito, Naoyoshi Minami, Toshinobu Sato, Tetsuya Ootaka, and Masahiro Kohzuki Background: It is suggested that appropriate chronic exercise (EX) may produce improvements of the physical strength in patients with chronic renal failure (CRF). Because acute exercise causes proteinuria and decreases the renal blood flow and glomerular filtration rate, it is necessary to consider the influence of EX on renal function. Therefore, we assessed the renal and peripheral effects of moderate to intense EX as well as the effects of the combination of EX and enalapril (ENA) in a rat model of CRF. Methods: Male 5/6-nephrectomized Wistar-Kyoto rats were divided into six groups according to the following treatment: 1) no exercise (C); 2) ENA (2 mg/kg/day, subcutaneously); 3) moderate exercise with treadmill running (20 m/min for 60 min/day, 5 days/week) (EXm); 4) intense exercise with treadmill running (28 m/min for 60 min/day, 5 days/week) (EXi); 5) EXm⫹ENA; and 6) sham operation (S). The rats were then treated for 12 weeks.

Results: Both EX and ENA blocked the development of hypertension, blunted increases in proteinuria, reduced serum creatinine and blood urea nitrogen, and improved the index of glomerular sclerosis (IGS) and the relative interstitial volume of the renal cortex (RIV). Moreover, IGS and RIV in the EXm⫹ENA group were the lowest among all other nephrectomized groups. Furthermore, EXm⫹ENA enhanced capillarization as well as the proportion of type-I fiber in the soleus muscle. Conclusions: These results suggest that EX and ENA have renoprotective effects. The findings also suggest that EXm⫹ENA provided greater renoprotective effects than those of ENA alone, and that EXm⫹ENA had some additional peripheral effects without any complications in this rat model. Am J Hypertens 2006;19:80 – 86 © 2006 American Journal of Hypertension, Ltd. Key Words: Chronic exercise, enalapril, rat, chronic renal failure, combination therapy.

he exercise capacity of patients with renal dysfunction decreases, and this phenomenon becomes more distinct as renal function deteriorates.1 On the other hand, it has been suggested that appropriate exercise may produce an improvement in the physical strength and the quality of life in patients with chronic renal failure (CRF).2– 4 However, it is necessary to consider the influence of exercise on renal function, because acute exercise causes proteinuria and decreases the renal blood flow (RBF) and glomerular filtration rate (GFR).5,6 At present, there are few reports about the optimal

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intensity and duration of exercise for patients with CRF. In addition, because appropriate animal models are few, there is no definite conclusion on whether chronic exercise (EX) has any renal protective effect or not. Previously, we reported that 4 weeks of treadmill exercise significantly attenuated the increase in proteinuria and the serum total cholesterol levels in spontaneously hypertensive rats (SHR) with 5/6-nephrectomy (NX).7 However, 4 weeks of moderate exercise was not long enough to decrease the serum creatinine (Scr) in SHR with CRF. Moreover, whether the renal protective effects of EX in SHR with

Received May 9, 2005. First decision July 12, 2005. Accepted July 16, 2005. From the Department of Internal Medicine and Rehabilitation Science (MK, LL, YS, KM, HK, OI, NM, MK), Center for Preventive Medicine and Well Being, Tohoku University Graduate School of Medicine; Tohoku Fukushi University (TK), Department of Blood Purification (TS), Tohoku University Hospital, School of Health Science (TO), Tohoku University, Sendai, Japan.

This work was supported by grants-in-aid for Scientific Research from the Japan Society for the Promotion of Science (16300179). Address correspondence and reprint requests to Dr. Masayuki Kanazawa, Department of Internal Medicine and Rehabilitation Science, Tohoku University Graduate School of Medicine, 1-1 Seiryo-cho, Aoba-ku, Sendai 980-8574, Japan; e-mail: makanaza@ sm.rim.or.jp

0895-7061/06/$30.00 doi:10.1016/j.amjhyper.2005.07.009

© 2006 by the American Journal of Hypertension, Ltd. Published by Elsevier Inc.

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CRF would appear in other rat models of CRF remains unknown. Therefore, in the present study, we assessed the renal and peripheral effects of chronic moderate to intense exercise in a rat model of CRF. We also assessed the effects of a combination of EX and an angiotensin converting enzyme (ACE) inhibitor, enalapril, on the muscle morphology and capillarity as well as on the renal function.

Methods Animals Eight-week-old male Wistar-Kyoto rats (WKY) (Charles River Japan Inc., Yokohama, Japan) were subjected to 5/6-NX by removal of the left and two-thirds infarction of the right kidney under ether anesthesia. The right kidney was exposed via a flank incision; the two poles were encircled with loops of ligatures and, after tightening the loops, the incision was closed. One week later, the left kidney was exposed via a flank incision, removed in total, and the flank incision closed. Sham operation was performed in age-matched male WKY. Rats were housed in a metabolic cage (model ST, Sugiyamagen, Tokyo, Japan) designed to prevent feces– urine contact, and the animals were kept in a humidity- and temperature-controlled (55 ⫾ 10% and 22 ⫾ 2°C) room with a 12-h light/dark cycle. The rats were fed a regular diet (0.18 wt% sodium, 1.13 wt% potassium, 18.3 wt% protein; Nosan Corp., Yokohama, Japan) and had free access to tap water. One week after the last operation, when the rats were 10 weeks of age, baseline measurements of body weight (BW), systolic blood pressure (SBP), urine volume (UV), and urinary excretion of protein (UP) were made. Treadmill test was performed, and the oxygen consumption (v˙O2) when rats were running at a speed of 20 m/min, 0 grade incline, and the peak v˙O2 were measured using an O2/CO2 metabolism measuring system8 (model MK-5000, MK-680AT/02R, Muromachikikai, Tokyo, Japan). The rats were then randomly assigned to six groups, according to the following treatment: 1) NX without exercise (C, n ⫽ 8); 2) NX without exercise and receiving enalapril (ENA, n ⫽ 8); 3) NX with treadmill running (KN-73, Natsume Industries Co., Tokyo, Japan) at a speed of 20 m/min, 0 grade incline for 60 min/day, 5 days/week (EXm, n ⫽ 9); 4) NX with treadmill running at a speed of 28 m/min, 0 grade incline for 60 min/day, 5 days/week (EXi, n ⫽ 9); 5) EXm⫹ENA (n ⫽ 8); 6) sham operation without exercise (S, n ⫽ 10). The rats were then treated for 12 weeks. Enalapril (2 mg/kg/day) was administered subcutaneously with osmotic minipumps (model 2002, Alza Corp., Palo Alto, CA). The SBP was monitored in conscious rats by the tailcuff method9 (UR5000, Elquest Corp., Ciba, Japan) without anesthesia. The UV was measured gravimetrically, and urine was collected and stored at ⫺80°C. Twelve weeks after the initiation of treatment the rats were killed by decapitation, and trunk blood was collected

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in polyethylene tubes for the determination of Scr and blood urea nitrogen (BUN). The UP, Scr, and BUN were measured by a standard autoanalysis technique (SynchronCX-3, Beckman Coulter Inc., Fullerton, USA). Soleus muscle was frozen in liquid isopentane, cooled in dry ice, and stored at ⫺80°C until sectioning. Renal Histology Portions of the remnant kidneys (removed when the rats were killed) were fixed in 10% neutral buffered formalin. Paraffin sections (3 ␮m in thickness) were cut, stained with periodic acid-Schiff’s reagent and Masson’s trichrome, and analyzed by an investigator with no prior knowledge of the groups to which the rats belonged. For calculating focal glomerular sclerosis, 150 to 200 glomeruli from each stained section were examined. The degree of sclerosis in each glomerulus was subjectively graded on a scale of 0 to 4: Grade 0, no change; Grade 1, sclerotic area ⱕ1/4 of glomerulus or distinct adhesions present between capillary tuft and Bowman’s capsule; Grade 2, sclerosis of 1/4 to 1/2 total glomerular area; Grade 3, sclerosis of ⬎1/2 the glomerulus but not global; Grade 4, global sclerosis. The index of glomerular sclerosis (IGS) was then calculated using the following formula:7,10 IGS ⫽ 共1 ⫻ N1 ⫹ 2 ⫻ N2 ⫹ 3 ⫻ N3 ⫹ 4 ⫻ N4 兲 ⁄ 共N0 ⫹ N1 ⫹ N2 ⫹ N3 ⫹ N4 兲 , where N is the number of glomeruli at each grade of sclerosis. We measured the relative interstitial volume of the renal cortex (RIV) to evaluate the degree of interstitial fibrosis. To estimate the RIV, single stained sections from each rat were examined by a color image analysis system (Olympus Optical Co. Ltd., Tokyo, Japan) that was composed of a main processor SP500F with a standard program software, an optical microscope BX40 with CCD color camera CS530MD, a high-resolution color display, and a personal computer for system control. Images were captured by an optical microscope connected with a CCD color camera at a magnification of ⫻200. The percentage of interstitium of the renal cortex except glomerulus, blood vessels, tubules per unit area were calculated; the values of RIV from 20 areas in each rat were measured and the mean values calculated.11 Muscle Morphology and Capillarity Serial transverse cross sections (10 ␮m thick) near the mid-belly portion of the soleus muscle were cut in a microtome cryostat at ⫺22°C, mounted on glass slides, and air dried. Each muscle fiber type (MFT) was determined by a myofibrillar adenosine triphosphatase (mATPase) staining method,12 after preincubation in alkaline (pH 10.7 to 10.9) and acid (pH 4.1 to 4.5) solutions. For capillarization analysis, another section from each

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using the Kruskal-Wallis test and Bonferroni/Dunn test. For C/F, CD, and MFT, comparisons between the different groups of rats were performed using one-way ANOVA and Tukey’s test. Values of P ⬍ .05 were considered statistically significant. The statistical analysis in the present study was performed using the statistical software Statview version 5.0 (Abacus Concepts Inc., Berkeley, USA) and SPSS version 10.0 (SPSS Inc., Chicago, IL). This study conformed to the principles for the use of live animals outlined in the Declaration of Helsinki and those of the ethics committee of Tohoku University Graduate School of Medicine.

Results FIG. 1. Sequential systolic blood pressure (SBP) values in the following groups (classified according to treatment): no exercise (C); sham operation (S); moderate exercise with treadmill running (20 m/min for 60 min/day, 5 days/week) (EXm); intense exercise with treadmill running (28 m/min for 60 min/day, 5 days/week) (EXi); enalapril (2 mg/kg/day, subcutaneously) (ENA); and EXm⫹ENA groups, during the 12-week experimental period. Values are expressed as the means ⫾ SEM. ●, C; 䡩, S; , EXm; □, EXi;
, ENA; , EXm⫹ENA. *P ⬍ .05, **P ⬍ .01, ‡P ⬍ .001, §P ⬍ .0001 v C group. ‘ a P ⬍ .05, bP ⬍ .01, dP ⬍ .001 v EXm and EXi groups. The SBP in the EXm group was not significantly different from that in the EXi group. The SBP in the ENA group was not significantly different from that in the EXm⫹ENA group.

sample was fixed at 4°C, then incubated at 37°C for 1 h in a lead (Pb)-ATPase staining medium to simultaneously stain for MFT and the surrounding capillaries in the same section.13 Capillary-to-fiber ratio (C/F), capillary density (CD), and fiber density (FD) were estimated using a 100point square grid test eyepiece at a magnification of ⫻100 (for fiber) or 200 (for capillaries) on a light microscope. Images were captured by an optical microscope connected with a CCD video camera (BX51, CS900, Olympus Optical Co., Tokyo, Japan) under the same microscope objective (⫻10) and projected on a monitor. For each muscle, at least 200 fibers and their associated capillaries were measured using the maximal number of non-overlapping fields.

Peak v˙O2 in the NX group (6.1 ⫾ 0.2 mL/min/100 g BW) was significantly (P ⬍ .01) decreased compared with that in the S group (7.4 ⫾ 0.1 mL/min/100 g BW). The v˙O2 when NX rats were running at a speed of 20 m/min and 28 m/min corresponded to about 80% and to about 90% of the peak v˙O2, respectively. The SBP in the C group progressively increased to 198 ⫾ 7 mmHg during the 12-week experimental period and was significantly higher (P ⬍ .0001) than that in the S group (144 ⫾ 2 mmHg) at 12 weeks (Fig. 1). The SBP in the EXm, EXi, ENA, and EXm⫹ENA groups (169 ⫾ 6, 164 ⫾ 3, 129 ⫾ 4 and 142 ⫾ 6, respectively) was significantly decreased (P ⬍ .01, P ⬍ .001, P ⬍ .001 and P ⬍ .01, respectively) compared with that in the C group at 12 weeks (Fig. 1). The SBP in the ENA and EXm⫹ENA groups was significantly decreased (P ⬍ .05) compared with that in the EXm and EXi groups. The UP in the C group progressively increased and was significantly higher (P ⬍ .0001) than that in the S group (Fig. 2). The UP was significantly decreased in the EXi,

Statistical Analysis Values are expressed as the means ⫾ SEM. With respect to peak v˙O2, a comparison between NX group and S group was performed using the unpaired t-test. For Scr, BUN, and RIV, comparisons between the different groups of rats were performed using one-way analysis of variance (ANOVA) and the Bonferroni/Dunn test. For SBP, UP, and BW, comparisons between the different groups of rats were performed by ANOVA with repeated measures over the duration of the study. Statistically significant differences on each day were assessed between groups by the Bonferroni/Dunn test. With respect to the IGS, comparisons between the different groups of rats were performed

FIG. 2. Urinary excretion of protein (UP) during 12-week experimental period. Values are expressed as the means ⫾ SEM. ●, C; 䡩, S; , EXm; □, EXi;
, ENA; ‘, EXm⫹ENA. *P ⬍ .05, **P ⬍ .01, ‡P ⬍ .001, § P ⬍ .0001 v C group. aP ⬍ .05, bP ⬍ .01, cP ⬍ .001, dP ⬍ .0001 v EXm and EXi groups. The UP in the ENA group was not significantly different from that in the EXm⫹ENA group. Abbreviations as in Fig. 1.

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Table 1. Values of serum creatinine (Scr), blood urea nitrogen (BUN), body weight (BW), index of glomerular sclerosis (IGS), and relative interstitial volume of the renal cortex (RIV) at 12 weeks Group S C ENA EXm EXi EXm⫹ENA

Scr (mg/dL) 0.29 1.69 0.89 0.83 0.99 0.70

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.03* 0.11† 0.05*† 0.04*† 0.07*† 0.10*†‡

BUN (mg/dL) 17.2 63.7 46.5 43.8 44.5 42.2

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.3* 2.8† 1.0*† 3.1*† 2.6*† 5.3*†

BW (g) 374 307 310 317 308 316

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

4 9† 3† 6† 4† 7†

IGS 0.05 2.53 2.05 2.10 2.26 1.90

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.01* 0.09† 0.03*† 0.06*† 0.05†‡ 0.04*†§储

RIV (%) 1.2 17.8 8.8 8.9 8.6 6.0

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.1* 0.9† 0.8*† 0.5*† 0.5*† 0.7*†‡

C ⫽ no exercise; ENA ⫽ enalapril; EXi ⫽ intense exercise with treadmill running; EXm ⫽ moderate exercise with treadmill running; S ⫽ sham operation. Values are expressed as the means ⫾ SEM. [Scr, BUN, BW]: * P ⬍ .0001 v C group; ‡ P ⬍ .01 v EXi group; †P ⬍ .0001 v S group. The BW was not significantly different among the C. EXm, EXi, ENA, and EXm⫹ENA groups. IGS: * P ⬍ .0001, ‡ P ⬍ .001 v C group: § P ⬍ .05 v EXm and ENA groups; 储 P ⬍ .0001 v EXi group; † P ⬍ .0001 v S group. RIV: * P ⬍ .0001 v C group; ‡ P ⬍ .01 v EXm, EXi, and ENA groups; † P ⬍ .0001 v S group.

ENA and EXm⫹ENA groups (P ⬍ .0001) compared with that in the C group. The UP in the ENA and EXm⫹ENA groups was significantly decreased (P ⬍ .001) compared with that in the EXi group. The values of Scr and BUN in the C group were the highest (P ⬍ .0001) (Table 1). The Scr in the EXm⫹ENA group was significantly lower (P ⬍ .01) than that in the EXi group. The Scr and BUN in the EXm, EXi, ENA, EXm⫹ENA groups were significantly higher ([Scr]:P ⬍ .0001, P ⬍ .0001, P ⬍ .0001, P ⬍ .001, respectively; [BUN]: P ⬍ .0001) than those in the S group. The BW in the S group was significantly heavier (P ⬍ .0001) than that in the C group (Table 1).

.0001) (Table 1). The RIV in the EXm⫹ENA group was significantly lower (P ⬍ .01) than that in the EXm, EXi and ENA groups and was significantly higher (P ⬍ .0001) than that in the S group. Muscle Morphology and Capillarity Figure 4 shows the cross-sections of stained soleus muscle. The C/F in the C group was significantly lower (P ⬍ .05) than that in the EXm, Exi, and EXm⫹ENA groups

Renal Histology The C group demonstrated focal and segmental glomerular structural lesions and increased cortical interstitial volume. These lesions were milder in the EXm, EXi, ENA, and EXm⫹ENA groups than in the C group (Fig. 3). The value of IGS in the C group was the highest (Table 1). The IGS in the EXm⫹ENA group was significantly lower than that in the EXm, EXi and ENA groups (P ⬍ .05, P ⬍ .0001, P ⬍ .05, respectively) and was significantly higher (P ⬍ .0001) than that in the S group. The value of RIV in the C group was the highest (P ⬍

FIG. 3. Light micrographs of the kidneys from rats of the S, C, ENA, EXm, EXi, and EXm⫹ENA groups (periodic acid-Schiff, ⫻200). Abbreviations as in Fig. 1.

FIG. 4. Representative cross sections of soleus muscle from rats in the (a) C, (b) ENA, (c) EXm, (d) EXi, (e) EXm⫹ENA, and (f) S groups. Sections were assayed for myofibrillar adenosine triphosphatase activity after different preincubation pH treatments. The type-I fiber stained dark after acid treatment (pH 4.45), whereas the others (ie, type-II fibers) stained light. Scale bar ⫽ 200␮m. Abbreviations as in Fig. 1.

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Table 2. Values of capillary-to-fiber ratio (C/F), capillary density (CD), fiber density (FD), and proportion of type-I fiber at 12 weeks Group S C ENA EXm EXi EXm⫹ENA

C/F 2.37 2.08 2.30 2.97 2.96 3.64

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.10 0.04 0.18 0.24* 0.30* 0.08*†

CD (no./mm2) 725 668 736 940 792 1020

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

18 24 30 70* 53 55*†

FD (no./mm2) 305 321 334 321 274 272

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

8 10 40 29 22 14

Type-I fiber (%) 88.7 82.6 94.4 90.3 98.0 97.3

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

2.7 5.0 2.1 2.4 1.9* 1.8*

Abbreviations as in Table 1. Values are expressed as the means ⫾ SEM. C/F: * P ⬍ .05 v C group; † P ⬍ .05 v ENA and S groups. CD: * P ⬍ .05 v C group; † P ⬍ .05 v ENA, S, and EXi groups. Proportion of type-I fiber: * P ⬍ .05 v C group. The C/F in the ENA group was not significantly different from that in the C and S groups. The C/F was not significantly different among the EXm, EXi, and EXm⫹ENA groups. The CD in the ENA group was not significantly different from that in the C group. The CD in the EXm⫹ENA group was not significantly different from that in the EXm group. The FD was not significantly different among the EXm, EXi, and EXm⫹ENA groups. The proportion of type-I fiber in the EXm⫹ENA group was not significantly different from that in the EXi group.

(Table 2). The C/F in the EXm⫹ENA group was significantly higher (P ⬍ .05) than that in the ENA and S groups. The CD in the C group was significantly lower (P ⬍ .05) than that in the EXm and EXm⫹ENA groups (Table 2). Proportion of high-oxidative type-I fiber was significantly higher (P ⬍ .05) in both EXi and EXm⫹ENA groups than in the C group (Table 2).

Discussion In the present study, we assessed the renal and peripheral effects of moderate to intense EX in a rat model of CRF. We also assessed the effects of the combination of EX and ENA on the muscle morphology and capillarity as well as renal function. We observed focal and segmental glomerular sclerosis and an increased cortical interstitial volume associated with a progressive increase in proteinuria in nephrectomized WKY; these findings are consistent with the development of hypertension. Both EX and ENA blocked the development of hypertension, blunted the increases in proteinuria, reduced Scr and BUN, and improved IGS and RIV. In particular, IGS and RIV in the EXm⫹ENA group were the lowest among all nephrectomized groups. Moreover, EX enhanced the capillarization as well as the proportion of type-I fiber in soleus muscle. These results suggest that the combination of EX and ENA has some additional effects without complications in this rat model. Human muscle fibers are commonly divided into three major types, designated types I, IIa, and IIb. These are analogous to animal muscle fibers that have been classified on the basis of their directly determined functional properties as slow twitch, fast twitch–fatigue resistant, and fast twitch–fatiguable fibers, respectively.14 Type-I fibers are red cells that contain relatively slow-acting myosin ATPases and hence contract at a slow rate. Type-I fibers have numerous mitochondria, mostly located close to the periphery of the fiber, near the blood capillaries, which provide a rich supply of oxygen and nutrients. These fibers

have a high capacity for oxidative metabolism; they are resistant to fatigue; and they are specialized for the performance of repeated strong actions over prolonged periods.14 Both cross-sectional15 and longitudinal studies16 have indicated that endurance training increases the CD of skeletal muscle. The capillary bed of muscle plays a crucial role in providing a surface for exchange between muscle and blood. Increasing the number of capillaries surrounding individual muscle fibers means that when a fiber is recruited it becomes more effectively exposed to the flow of blood delivered to the muscle during exercise. These adaptations of the vasculature could contribute to the increased oxygen extraction observed in trained muscle and the increase in whole-body maximal oxygen intake (v˙O2max) observed after a program of endurance training.17 The present results also indicate that the effects of EX must be distinguished from the acute effects of exercise. In other words, a decrease in RBF, contraction of renal efferent arterioles, contraction of mesangial cells, and a decrease in the GFR caused by accelerated sympathetic nerve activity are the acute effects of exercise. Simultaneously, the acceleration of renin secretion because of decreased inflow of sodium and chloride to the distal renal tubules, activation of the renin-angiotensin system and a rise in blood pressure are induced as the acute effects of exercise.5 Moreover, there is an increase in proteinuria immediately after acute exercise because of changes in renal hemodynamics during exercise, the loss of negative charges in glomerular barrier, exceeding the proximal tubular reabsorption capacity induced by activation of the sympathetic nervous system and renin-angiotensin system.18 The acute exercise-induced oxidant stress may also contribute to postexercise proteinuria.19 Furthermore, nitric oxide may have a role in the relevance of exerciseinduced proteinuria.18 It has not yet been elucidated whether such influences of acute exercise cause nephropathic development and

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progressing. Furthermore, there have been few reports regarding the influence of EX on the progression of renal disease and the results have been controversial.5,20 –22 Heifets et al. reported a 24% increase in GFR, a 59% decrease in UP, and an alleviation of glomerulosclerosis compared with values in sedentary rats brought about by swimming for 2-h/day for 2 months in Sprague-Dawley rats with 3/4-NX.20 Osato et al. reported a decrease in Scr, an increase in GFR, and an alleviation of glomerulosclerosis resulted from 2-h/day swimming for 20 weeks with relative restriction of food in adriamycin-treated Lewis rats, a model of sclerosing glomerulonephritis with nephrotic syndrome.21 In contrast, Cornacoff et al. reported that in an acute serum nephritis rabbit model, exercise by 45 to 60 min treadmill running at a speed of 7.2 m/min for 4 weeks increased BUN and UP.22 It was also reported that EX did not affect renal disease. Bergamaschi et al. reported that treadmill exercise (65% to 75% v˙O2max) for 30 min, 5 times/week for 60 days in Munich-Wistar rats with 5/6-NX did not show any significant change in GFR, UP, and the IGS.5 The reason for the discrepant effects of EX in the present study may be that the animal models used as well as the variety, intensity, and duration of exercise were different. Moreover, these reports suggested that EX may have an unfavorable influence on renal function and on nephric lesions depending on the nephropathic cause and stage. The mechanism by which EX protected the remnant kidney in the present study has not been fully elucidated. Renal ablation results in a transmission of the systemic hypertension to the glomerulus and an elevation of the intraglomerular capillary pressure. This hemodynamic alteration is associated with a sharp increase in UP and an acceleration of focal and segmental glomerular sclerosis.23 Although we did not assess the intraglomerular hypertension and hyperfiltration in the present experiment, if the glomerular capillary pressure is reduced by EX, renal protective effects may appear. Bergamaschi et al reported the dilation of renal efferent arterioles and an improvement of glomerular hypertension by EX in Munich-Wistar rats with CRF prepared by 5/6-NX.5 Provided that these findings are also true for WKY with CRF, it is conceivable that EX shows a protective effect on kidneys by improving the glomerular hypertension. Moreover, if the reduction of RBF by acute exercise is repeated by EX, it is thought that the glomerular capillary pressure would be reduced by it and that the kidneys may thereby be protected. In addition, EX may have shown a renal protective effect by inhibiting nephropathic risk factors such as hypertension or proteinuria through some mechanism. Further investigations will be needed to determine the mechanisms of the renal protective effects of EX. In the present study, we set two kinds of exercise intensity. The v˙O2 when NX rats were running at a speed of 20 m/min and 28 m/min corresponded to ⬃80% and to ⬃90% of the peak v˙O2. Although UP and the proportion of high-oxidative type-I fiber in soleus muscle in the EXi group were better than those in the EXm group, the effects

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of both intensities of exercise on the other evaluated items were similar. Moreover, UP in the ENA and EXm⫹ENA groups was significantly less than that in the EXi group, and the proportion of type-I fiber in the EXm⫹ENA group was not significantly different from that in the EXi group. Therefore, it is suggested that, to obtain more renal protective effects compared with those in the EXm group, ENA should be added instead of increasing exercise intensity. The ACE inhibitors reduce hyperfiltration damage in remnant kidney nephrons in CRF,24,25 and meta-analyses have shown that these agents have the beneficial effects of reducing UP and preserving renal function.26,27 However, the renal protective effects of ACE inhibitor monotherapy in the present experiment were less than perfect. Based on the present results, we may state that EX in combination with ENA is stronger than ENA monotherapy in terms of renal protection, muscle morphology, and capillarity. Moreover, the demonstration of renal protective effects of EX in a WKY remnant kidney model of CRF suggests that it is important to reconsider the relationship between CRF and exercise. The present results show that EX lowers blood pressure, ENA completely prevents the hypertension, and that there is no additional effect of combining ENA and EX with regard to blood pressure. There also was no additional effect of combining EX and ENA on proteinuria or BUN. On the other hand, there was an additional benefit of combining ENA and EX on Scr, IGS, and RIV. Therefore, when we perform a longer experiment than that in the present study, the combination of EX and ENA may result in a beneficial effect on blood pressure, proteinuria, or BUN. In conclusion, the renal protective effects of both moderate to intense EX and ENA were demonstrated, and simultaneous treatment of moderate EX and ENA enhanced the capillarization as well as the proportion of type-I fiber in soleus muscle. These results also suggest that the simultaneous treatment of moderate EX and ENA provided greater renoprotective effects than those of ENA alone, and that the combination of moderate EX and ENA has some additional peripheral effects without complications in this rat model.

Acknowledgment The authors are grateful to Banyu Pharmaceutical Co., Ltd. (Tokyo, Japan) for supplying enalapril.

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