6 nephrectomized rats

European Journal of Pharmacology 709 (2013) 85–92

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Pulmonary, gastroinlistinal and urogenital pharmacologgy

Losartan ameliorates renal injury, hypertension, and adipocytokine imbalance in 5/6 nephrectomized rats Deng-Yuan Jian a,f, Yu-Wen Chao b,d, Ching-Heng Ting a, Seng-Wong Huang c,e, Chao-Fu Chang d, Chi-Chang Juan a,e,g,n, Jinn-Yang Chen b,h,nn a

Department of Physiology and Taipei Veterans General Hospital, Taipei, Taiwan Faculty of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan c School of Medicine, National Yang-Ming University, Taipei, Taiwan d Section of Nephrology, Department of Internal Medicine, Heping Branch, Taipei Veterans General Hospital, Taipei, Taiwan e Department of Education and Research, Taipei City Hospital, Taipei, Taiwan. f Division of Nephrology, Wen-Lin Hemodialysis Unit, Taipei, Taiwan. g Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan h Division of Nephrology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan. b

art ic l e i nf o

a b s t r a c t

Article history: Received 20 December 2012 Received in revised form 4 March 2013 Accepted 8 March 2013 Available online 23 March 2013

The mechanisms underlying insulin sensitivity and fat tissue distribution in chronic renal insufficiency remain unclear. Previous studies have shown the benefits of angiotensin II receptor blockers on moderately nourished to well-nourished patients with the metabolic syndrome. The current study explored the effect of losartan, the first selective angiotensin II receptor blocker, on insulin sensitivity and visceral fat tissue distribution in a 5/6 nephrectomized (N) rat model and investigated the expression of adipose tissue adipocytokines. Male Sprague-Dawley rats (200 g to 250 g) were subjected to 5/6 nephrectomy, and the adipocytes isolated from the visceral fat tissues were then studied. Results showed that desmin expression was significantly suppressed and systolic blood pressure was successfully normalized in the losartan-administered (NA) group. The weight of the visceral fat pad remarkably decreased in the N and NA groups (100 mg/500 ml drinking water) compared with the control group. The weight did not decrease further in the NA group compared with the N group. Insulin resistance was more remarkable in the N group compared with the control and NA groups. Moreover, the adipose tissue expression of adiponectin and leptin was downregulated whereas that of resistin was upregulated in the N group compared with the control group. However, the adiponectin, leptin, and resistin adipose tissue expression returned to their basal values in the NA group. These findings indicated that losartan administration ameliorated renal injury, systolic blood pressure, and adipocytokine imbalance of the adipose tissue in chronic renal insufficiency. Insulin sensitivity was not improved. & 2013 Elsevier B.V. All rights reserved.

Keywords: Angiotensin II receptor blocker Visceral fat Adipocytokine Insulin sensitivity Chronic kidney disease

1. Introduction Patients with chronic kidney disease present carbohydrate metabolism disorders and insulin resistance (Basturk and Unsal, 2012; Chen et al., 2003, 2004). Several studies have reported the association of metabolic syndrome and insulin resistance with a high risk for diabetes (Lorenzo et al., 2003) and kidney disease (Zhang et al., 2005). Diabetes and cardiovascular diseases are the main causes of end-stage renal disease, which is the dominant

n Corresponding author at: Yang-Ming University, Department of Physiology and Faculty of Medicine, No. 155, Section 2, Li-Nong St. Taipei 11221, Taiwan. Tel.: +886 2 28267085; fax: +886 2 28264049. nn Corresponding author at: Faculty of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan. Tel.: +886 2 28712121 2570; fax: +886 2 28757065. E-mail addresses: [email protected] (C.-C. Juan), [email protected] (J.-Y. Chen).

0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.03.024

outcome in patients with type-2 diabetic nephropathy. End-stage renal disease is characterized by decreased kidney function and substantial proteinuria (Lewis et al., 2001; Packham et al., 2012; Shahinfar et al., 2002). Non-diabetic patients with end-stage renal disease also present mild fasting hyperglycemia and abnormal glucose tolerance, whereas others maintain normoglycemia but at the price of hyperinsulinemia (DeFronzo et al., 1981; Mak and DeFronzo, 1992; Mak et al., 1983). Malnutrition, inflammation, and atherosclerosis syndrome are commonly found in dialysis patients with diabetes (Abe et al., 2011; Tonbul et al., 2006), and these conditions provide insight into patients' susceptibility to cardiovascular disease (Kalaitzidis and Bakris, 2009). Impaired insulin sensitivity is frequently recognized in uremic patients. However, whether insulin resistance is a cause or a consequence of chronic kidney disease or end-stage renal disease remains unclear. Hypertension is a key independent risk factor for kidney diseases and faster renal function loss. The pharmacological

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inhibitor of the renin–angiotensin–aldosterone system more effectively retards the progression of advanced proteinuric chronic kidney disease than other antihypertensive agents (Akdag et al., 2008). Previous studies have shown the impact of angiotensin II receptor blockers on insulin sensitivity in hypertensive patients (Moan et al., 1994; Ran et al., 2006). Angiotensin II receptor blockers reportedly act on fat distribution in patients with metabolic syndrome and hypertension (Chujo et al., 2007; Shimabukuro et al., 2007). Losartan, the first selective angiotensin II receptor blocker discovered, controls hypertension (Bakris et al., 2003; Parrinello et al., 2009), treats proteinuria (de Zeeuw et al., 2004), and alleviates hypercholesterolemia (Petnehazy et al., 2006) during the course of chronic kidney disease toward endstage renal disease. Losartan also influences the proinflammatory mediator in atherogenesis (Han et al., 2007). Abdominal obesity is an evident risk factor for cardiovascular complications and chronic inflammation in chronic kidney disease and dialysis patients (Behn and Ur, 2006; Ishimura et al., 2011). The waist circumference is associated with chronic kidney disease and mortality incidents (Elsayed et al., 2008; Kalaitzidis and Siamopoulos, 2011). Three major adipose tissue cytokines (leptin, adiponectin, and resistin) have been separately investigated in chronic kidney disease and dialysis patients (Baldasseroni et al., 2013; Zoccali and Mallamaci, 2011; Zoccali et al., 2011). Reduced kidney function manifests as low adiponectin, high leptin, and high resistin levels in the plasma (Baldasseroni et al., 2013; Zoccali and Mallamaci, 2011). Reduced kidney function is also positively correlated with the metabolic syndrome/inflammatory response in hemodialysis patients (Lee et al., 2011; Tsai et al., 2011). Meanwhile, leptin reverses metabolic alterations (Zoccali and Mallamaci, 2011), and resistin circulation levels are reportedly relation to the inflammatory biomarkers of chronic kidney disease (Axelsson et al., 2006). The role of angiotensin II type 1 (AT-1) receptor activation in vascular inflammation regulation is also associated with the upregulation of pro-inflammatory and profibrotic pathways in obesity (Vaziri et al., 2005). Based on these findings, abdominal obesity and hypertension are considered to be inflammatory responses to the development of chronic kidney disease, and AT-1 reportedly regulates the inflammatory process. However, previous studies have been performed on moderately nourished to well-nourished subjects. Accordingly, the present study investigated the effects of angiotensin II receptor blocker on insulin sensitivity, kidney pathology, metabolic factors, and adipocytokines in a 5/6 nephrectomized (N) rat model, which is a model of impaired renal function that mimics chronic kidney disease in a poorly nourished environment (Shobeiri et al., 2010). The anti-inflammatory effects of losartan on obesity, especially in the adipose tissue expression of adipocytokines, were also explored.

2. Materials and methods 2.1. Animals Male Sprague-Dawley rats weighing 200 g to 250 g (6 weeks old) were purchased from a local breeder. The rats were housed four to a cage at 20 1C to 22 1C in a light-controlled room on an alternating 12 h light/dark cycle. The rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.) after one week of acclimatization, and ventral laparotomy was performed under aseptic conditions. The 5/6 nephrectomy was performed through a surgical resection of the upper and lower thirds of the left kidney followed by right nephrectomy one week later. The 5/6-nephrectomized rat model is the common model of impaired renal function to mimic advanced chronic kidney disease and a poorly

nourished environment (Shobeiri et al., 2010). Control rats were subjected to a mock operation. All procedures were carried out in accordance with the guidelines of the Taiwan Government Guide for the Care and Use of Laboratory Animals. The study protocol was approved by the animal welfare committees. The rats were bred for one month and grouped into three: 5/6 nephrectomized rats (N group; n ¼8), 5/6 nephrectomized rats administered with losartan (NA group; n ¼8), and mock control rats (control group; n¼ 8). Losartan was administered to the rats by dissolving it in their drinking water (100 mg/500 ml) as described by Yang et al. (2002). Blood pressure was measured by the non-invasive tail-cuff method. Renal function was determined by measuring the plasma creatinine and blood urea nitrogen levels. Insulin sensitivity was measured by the oral glucose tolerance test (OGTT). Whole body fat distribution was evaluated by computed tomography (CT) scanning. Renal impairment was evaluated by histological examination. Isolated adipocytes were used to evaluate the mean and distribution of fat cell size. Adipocytokine expression in adipocytes (adiponectin, leptin, and resistin) was measured by reverse transcription (RT)−polymerase chain reaction (PCR). 2.2. Blood pressure measurement Blood pressure was measured using a programmable sphygmomanometer (BP-98A; Softron, Japan) by the tail-cuff method. The small animal study unit of the system has a rat-holder base with a built-in warming element to raise the ambient temperature to 37 1C and maintain adequate circulation in the tail for indirect blood pressure measurements. The animal was positioned in Lucite housing with its tail firmly held outside. The occluding metal tubular cuff (9 mm to 12 mm internal diameter) and the pneumatic pulse sensor-transducer were then placed on the tail and connected to the sphygmomanometer. The occluding cuff pressure was controlled at a pre-adjusted inflation−deflation rate until the first pulse was recorded. All measurements were carried out in a quiet room starting at 10:00 AM because the normal blood pressure shows an intrinsic diurnal variation and may be disturbed by environmental conditions. The order of testing for the different groups was varied on subsequent testing days. An experienced technician took three to five measurements per rat from 20 min to 30 min. 2.3. Plasma measurement Blood sampling was carried out in a quiet room starting at 10:00 AM. The order of testing for the different groups was varied on subsequent testing days. Blood samples for the glucose and insulin measurements (approximately 500 μl) were collected by tail bleeding into a 1.5 ml heparin-coated polyethylene microfuge tube on ice. Trunk blood was collected from each rat after decapitation. Plasma was separated by centrifugation and stored at –20 1C until analysis. Plasma glucose was measured on a glucose analyzer (Model 23A; Yellow Springs Instrument Company, Yellow Springs, OH, USA). Plasma insulin, triglyceride, nonesterified fatty acids, creatinine, and urea were measured with commercial kits. 2.4. Measurement of visceral fat tissues A CT scan was performed on the rats in three different regions at an ultra-high resolution setting. Three slices were examined at the level of the sacroiliac joint, the upper part of the iliac crest, and 1.5 cm above the second slice. A density range between –150 and – 40 Hounsfield units was used to define the fat area, and a range between –40 and 250 Hounsfield units was used to define the muscle area (water density ¼0). All CT scans were performed with the anesthetized rats in a prone position. The total adipose and

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muscle tissues in these intervals were calculated using a computer. The intra-abdominal cavity was manually outlined with a cursor, including the retroperitoneal space. The adipose tissue in this area was calculated by subtracting the intra-abdominal area from the total abdominal area to yield the subcutaneous area. The average ratio of visceral fat area to subcutaneous fat area of the three slices was used for statistical analysis. 2.5. OGTT Rats were subjected to an OGTT after overnight fasting. A zero minute blood sample was collected from each rat. The rats were then given a glucose solution (concentration: 0.2 g/0.1 ml, 0.1 ml/ 100 g body weight) by gavages without delay, and blood samples were collected at 30 min, 60 min, 90 min, and 120 min. Plasma insulin and glucose concentrations were determined.

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2.9. Western blot analysis Whole cell lysates were prepared by sonication in lysis buffer (1% Triton X-100, 50 mM KCl, 25 mM Hepes, pH 7.8, 10 μg/ml leupeptin, 20 μg/ml aprotinin, 125 μM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride). Some 50 μl samples (100 μg of total protein) of the Laemmli sample buffer were boiled for 10 min and resolved on 15% mini-sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The gel contents were then transferred onto a polyvinylidene difluoride membrane. The membrane was preblotted in 5% skimmed milk in phosphatebuffered saline for 30 min at room temperature and immunoblotted with a primary antibody for 24 h at 4 1C, followed by horseradish peroxidase-conjugated secondary antibody for 60 min at room temperature. The membrane finally subjected to chemiluminescence reagent (Amersham Biosciences, Buckinghamshire, England) analysis.

2.6. Measurement of the remnant kidney 2.10. Statistical analysis Rat kidneys were removed, weighed, and processed for light microscopy. The specimens were fixed in 10% neutral formaldehyde and then embedded in paraffin. Serial cross-sections (4 μm) were cut perpendicular to the longest axis of the solid organs and stained with hematoxylin−eosin and periodic-acid Schiff. The stained slides were examined under a microscope with the slide reviewers blinded to the treatment group. Representative images were automatically captured using a digital spot camera. 2.7. Preparation of adipocytes At the end of the experiment, the rats were sacrificed by decapitation after overnight fasting. The epididymal fat pads from each group of rats were pooled to isolate the adipocytes through the Rodbell method with minor modifications. In a typical procedure, fat tissue was minced and incubated for 1 h at 37 1C in a Krebs−Ringer bicarbonate buffer containing 1% bovine serum albumin (KRBB) and 0.1% collagenase in an oxygen-rich shaking chamber (5:95 CO2:O2, 75 strokes/min). The suspension was then filtered through a nylon mesh and centrifuged at 100  g for 1 min. After collecting the supernatant containing the adipocytes, the cells were washed twice and resuspended in KRBB. The number of cells in the adipocyte suspension was determined after fixing with 2% osmium tetraoxide. The lipocrit and cell size were measured before, during, and after each experiment to check cell viability. 2.8. RT−PCR analysis One microgram of total RNA was reverse transcribed using poly (dT) 12–18 primers and SUPER RT reverse transcriptase in a 50 μl reaction volume at 42 1C for 1 h. Five microliters of these reactions was used in PCR in a 50 μl reaction volume mixture under the following conditions: 1 cycle of 95 1C  5 min, 35 cycles of 95 1C  30 s, 55 1C  30 s, 72 1C  30 s, and a final 7 min extension period. The primers used were as follows: adiponectin sense primer, 5′-TGGAG AGAAG GGAGA GAAGG-3′; adiponectin antisense primer, 5′-CCATA CACTT GGAGC CAGAC-3′; leptin sense primer, 5′-CCTAT GTTCA AGCTG TGCC-3′; leptin antisense primer, 5′-TGTTG ATAGA CTGCC AGGG-3′; resistin sense primer, 5′-CTGCC ACGTA CTTAA CAGGA TGAAG-3′; resistin antisense primer, 5′-TCAGG AACCA ACCCG CAGGG TACA-3′; β-actin sense primer, 5′-GAGAA GATTT GGCAC CACAC-3′; and β-actin antisense primer, 5′-CATCA CAATG CCAGT GGTAC-3′. The PCR products were electrophoresed on 2% agarose gel. After staining the gel with ethidium bromide, the DNA band densities were measured for densitometry. Gene expression was normalized to the β-actin expression.

The experiments were repeated at least four times. The results are expressed as the mean 7S.E.M. Statistical significance was assessed by one-way analysis of variance or Student's t-test. Po 0.05 was considered statistically significant.

3. Results 3.1. Blood pressure, renal function, and histopathological examination of the remnant kidney Plasma blood urea nitrogen (BUN) and creatinine levels significantly increased in the N and NA groups compared with the control group (Fig. 1A and B), and no significant difference was found between the N and NA groups. The histopathological examination results showed no significant difference among the three groups. The expression of desmin, a marker of podocyte damage, was also examined to evaluate podocyte injury in the three groups. Immunofluorescent staining detected a low expression of desmin in the control group (Fig. 1C). By contrast, a strong positive signal was observed in the glomeruli of the N group, and desmin expression was significantly suppressed in the NA group. These observations were confirmed by a semi-quantitative measurement of desmin immunofluorescent staining (Fig. 1D). Hypertension was significantly induced in the N group and successfully ameliorated by angiotensin II receptor blocker administration (Table 1). 3.2. Effect of losartan on visceral fat distribution Table 1 shows the body weight, visceral fat pad weight, fasting glucose, fasting insulin, and plasma triglyceride concentrations after the four-week experimental period. The body and the visceral fat pad weights significantly decreased in the N and NA groups (P o0.05) compared with the control group. The plasma fasting glucose concentration slightly decreased in the N group compared with the control and NA groups. By contrast, the fasting insulin concentration had no significant difference among the three groups. The plasma triglyceride level significantly increased in the N group, and the angiotensin II receptor blocker administration did not suppress this parameter in the NA group. Table 2 shows the CT scanning results of the fat compositions in the three groups. The average ratio of visceral fat area to subcutaneous fat area of the three slices (kidney, iliac, and inguinal levels) significantly decreased in the N and NA groups (Po0.05). The weight of total adipose tissue in relation to body weight decreased in the N

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and NA groups compared with that in the control group, consistent with the CT scanning result. The mean adipocyte size and size distribution were determined in the epididymal adipose tissue. The size distribution curve of adipocytes shifted to the left in the N and NA groups compared with that in the control group (Fig. 2).

This finding demonstrated that the mean adipocyte size was smaller in the N and NA groups than in the control group. 3.3. Effect of losartan on the insulin sensitivity and adipose tissue expression of adipocytokines The OGTT results obtained for the three groups are shown in Fig. 3 and Table 3. The baseline plasma glucose and insulin levels did not significantly differ among the three groups (Fig. 3). All groups showed similar plasma glucose levels in response to oral glucose challenge. Plasma insulin levels in the N and NA groups decreased compared with that in the control group after oral glucose loading for 30 min, but the difference was not significant. Likewise, no significant difference was observed in the AUCglucose, AUCinsulin, ΔAUCglucose, and ΔAUCinsulin among the three groups Table 2 The average of ratio of visceral-to-subcutaneous fat area was measured, from three slices of CT scan (kidney, iliac, and inguinal levels). C Kidney level Iliac level Inguinal level

N

10.14 74.98 19.7078.75 5.7071.10

NA a

8.66 73.09 10.27 71.54a 3.85 70.26a

8.71 71.94a 10.3574.27a 4.3371.09a

Unit: % of total cross-section area. C: mock control rats; N: 5/6-nephrectomized rats; NA: 5/6-nephrectomized rats, administered with losartan. Data ae expressed as the mean 7 S.E.M. (n ¼8). a

Fig. 1. (A) Plasma BUN and (B) creatinine levels in three groups (C, mock control rats; N, 5/6 nephrectomized rats; NA, 5/6 nephrectomized rats administered with losartan). (C) Remnant kidney histopathological examination and desmin immunofluorescent staining in the control and experiment groups. (D) Semi-quantitative evaluation of the immunofluorescent staining of desmin. Data are expressed as the mean 7S.E.M. (n ¼8). n versus control group, P o 0.05; # versus 5/6 nephrectomized group, Po 0.05.

versus control group, Po 0.05.

Fig. 2. Mean adipocyte size in the control and experimental groups. The mean adipocyte size and size distribution were determined in epididymal adipose tissue. C, mock control rats (white bar; solid line with open circle); N, 5/6 nephrectomized rats (black bar; solid line with filled circle); NA, 5/6 nephrectomized rats administered with losartan (gray bar; dashed line with filled square). Data are for pooled eight mice in each group.

Table 1 Body weight, visceral fat weight, and metabolic parameters. C Body weight (g) Epidymal fat pad weight (g) Restroperitoneal fat pad weight (g) Fastin insilin (μU/ml) Fastin glucose (mg/dl) Fastin triglyceride (mg/dl) Systolic blood pressure (mmHg)

494.37 23.0 5.3 7 1.7 6.8 7 2.5 19.4 7 6.1 126.5 7 13.1 72.8 7 4.8 121.6 7 1.4

N

NA a

413.3 732.1 3.0 70.7a 2.3 70.8a 20.5 714.1 116.4 710.3a 107.3 78.9a 146.2 77.0a

415.7 739.1a 3.0 71.1a 2.8 71.0a 20.1 713.7 122.7 715.3 113.5 710.5a 109.2 72.3a,b

C: mock control rats; N: 5/6-nephrectomized rats; NA: 5/6-nephrectomized rats, administered with losartan. Data are expressed as the mean 7 S.E.M. (n ¼8). a b

versus control group, Po 0.05. versus 5/6-nephrectomized group, Po 0.05.

D.-Y. Jian et al. / European Journal of Pharmacology 709 (2013) 85–92

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Table 3 Calculation of AUCglucose and AUCinsulin among control and experimental groups.

AUCglucose ΔAUCglucose AUCinsulin ΔAUCinsulin ΔAUCglucose/ΔAUCinsulin

C

N

NA

355.8 7 18.1 97.4 7 16.8 65.9 7 7.7 29.2 7 3.9 3.6 7 0.7

360.17 16.2 134.2 7 19.7 59.0 7 12.5 28.5 7 7.8 5.6 7 0.7a

364.37 8.4 119.6 7 8.4 51.8 7 4.8 23.6 7 3.9 5.7 7 0.8a

Units: AUCglucose, mg/dl h, AUCinsulin, μU/ml h. AUC: area under the curve during the OGTT. ΔAUC: change in the area under the curve during OGTT. C: mock control rats; N: 5/6-nephrectomized rats; NA: 5/6-nephrectomized rats administered with losartan (NA). Data are expressed as the mean7 S.E.M. (n¼ 8). Fig. 3. Measurement of plasma glucose and insulin levels by oral glucose tolerance test. No significant difference was observed in glucose and insulin values at different time points. C, mock control rats (open circle); N, 5/6 nephrectomized rats (filled circle); NA, 5/6 nephrectomized rats administered with losartan (filled square). Data are expressed as the mean 7 S.E.M. (n ¼8).

a

versus control group, Po 0.05.

3.4. Malnutrition and adipocytokine imbalance in chronic renal insufficiency Data from the three groups were pooled and correlated as fat tissue adipocytokines, systolic blood pressure, body weight, blood test (BUN, creatinine, and fasting glucose), and fat pad weight (Table 4). We identified the correlation factors in the three groups that mimicked different stages of chronic renal insufficiency. The results showed that the blood BUN and creatinine levels were negatively correlated with the body and fat pad weights. Body weight was positively correlated with fat pad weight. Body weight loss was similar to fat pad weight loss in chronic renal insufficiency. The decreased visceral fat distribution reflected the key point of malnutrition. Moreover, the blood fasting glucose was positively correlated with the body and fat pad weights but negatively correlated with the blood BUN and creatinine levels. Chronic renal insufficiency presented decreased body and fat pad weights associated with malnutrition, thereby leading to low fasting blood glucose. Resistin was negatively correlated with adiponectin and leptin, but positively correlated with the systolic blood pressure in the fat tissue adipocytokines. Furthermore, adiponectin negatively correlated with the blood BUN level. Adiponectin and leptin were downregulated in the visceral fat tissue of the N group in contrast to resistin. This observation indicated that an adipocytokine imbalance existed in chronic renal insufficiency, and the elevated resistin level was related to high systolic blood pressure.

4. Discussion

Fig. 4. Measurement of adiponectin, leptin, and resistin expression in adipocytes isolated from the three groups by RT–PCR. All three adipocytokines were corrected in 5/6 nephrectomized rats administered with losartan (NA). C, mock control rats; N, 5/6 nephrectomized rats; NA, 5/6 nephrectomized rats administered with losartan. Data are expressed as the mean7 S.E.M. (n¼ 8). n versus control group, Po 0.05; # versus 5/6 nephrectomized group, P o0.05.

(Table 3). However, the ratio of ΔAUCglucose to ΔAUCinsulin significantly increased in the N and NA groups, suggesting that these two groups were more insulin resistant than the control group. The adipose tissue expression of adiponectin, leptin, and resistin isolated from the three groups was also evaluated. Adiponectin and leptin expression was downregulated in the N group, whereas resistin was upregulated in the N group compared with the control (Fig. 4A–C). Angiotensin II receptor blocker administration ameliorated the abnormal adipocytokine expression in the NA group (P o0.05).

The renal protective effect of angiotensin II receptor blocker has been found in patients with nephropathy caused by type-2 diabetes mellitus (Lewis et al., 2001; Shahinfar et al., 2002). Thus, the health implications of insulin resistance and metabolic syndrome are gaining increased attention. The effect of insulin sensitivity and fat distribution induced by angiotensin II receptor blockers has been proven in patients with primary hypertension, metabolic syndrome, and excess energy (Chujo et al., 2007; Moan et al., 1994; Ran et al., 2006; Shimabukuro et al., 2007). The current study explored the effect of chronic renal insufficiency induced by 5/6 nephrectomy on insulin sensitivity and fat distribution in rats treated with angiotensin II receptor blockers, specifically, losartan. The N rat model is a well recognized animal model for chronic kidney disease study. After removing 5/6 of the kidney, the remaining 1/6 part took on the workload of the whole kidney. The remnant kidney underwent a dramatic hemodynamic change, including high perfusion pressure and high filtration rate. The nephrons in the remnant kidney compensated for the increased loading and hemodynamic disorder through hyperplasia and

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Table 4 Parameters of body weight, visceral fat pad weight, and metabolic factors, were pooled and correlated among control and experimental groups. Glucose Glucose Adiponectin Leptin Resistin BUN Creatinine SBP BW Fat

NS NS NS o0.05 o0.05 NS o0.02 o0.01

Adiponectin

Leptin

Resistin

BUN

Creatinine

SBP

BW

Fat

0.352

0.217 0.284

−0.397 −0.432 −0.639

−0.413 −0410 −0.205 0.140

−0469 −0278 −0.138 0.357 0.786

−0.085 −0318 −0.383 0.639 −0.108 0.176

0.507 0380 0.057 −0.039 −0.806 −0.690 −0.690

0.628 0279 0.231 −0.260 −0.759 −0.754 −0063 0.787

NS o 0.05 o 0.05 NS NS NS NS

o 0.01 NS NS NS NS NS

NS NS o 0.01 NS NS

o 0.01 NS o 0.01 o 0.01

NS o 0.01 o 0.01

NS NS

o 0.01

The left lower area indicates the P value, and the right upper area indicates positive, or negative correlation.

hypertrophy; consequently, nephron sclerosis developed. The function of the remnant kidney worsened with its decreased toxin clearance rate and the progress of renal dysfunction (Kliem et al., 1996; Lu et al., 2011). The main findings of the present study included increased plasma BUN and creatinine levels as well as podocyte injury. Hypertension was also significantly induced in the N rats. The administration of angiotensin II receptor blockers successfully ameliorated 5/6 nephrectomy-induced podocyte injury and hypertension. The results showed that 5/6 nephrectomy induced chronic renal insufficiency, and that losartan was effective in ameliorating podocyte injury and renal hypertension. These results were consistent with the study of Aunapuu et al. (2003), who found that nephrectomized rats have higher proteinuria and systolic blood pressure than losartan-treated rats. Podocytes demonstrate hypertrophy and foot process effacement in a nephrectomized group compared with a losartan-treated group (Aunapuu et al., 2003). Kööbi et al. (2003) studied losartan–ameliorated renal hypertension in nephrectomized rats and provided a mechanical explanation, i.e., impaired endothelium-mediated relaxation in response to acetylcholine. The present study successfully created a losartantreated nephrectomized rat model. In previous studies, the model proved the antihypertensive and renoprotective effects of the drug. Our data also suggested that losartan treatment prevented continuous podocyte loss and renal function deterioration in the N animal model, but did not recover normal renal function. Therefore, decreasing the plasma BUN and creatinine levels to normal values was difficult in the NA group. Without losartan administration, podocyte loss continued and plasma BUN and creatinine levels further increased. Consequently, end-stage kidney disease occurred in the N group. These results were comparable to the findings of Kim et al. (2005) and Arozal et al. (2009). They both demonstrated that treatment with AT-1 receptor blockade significantly reduces proteinuria but not the BUN level in different chronic renal failure models. Insulin resistance was observed in the N and NA rats in the current experiment. The N and NA rats had decreased visceral fat mass and adipocyte size compared with the control group. Losartan administration did not further decrease the visceral fat mass and the adipocyte size in the N group, in contrast to the findings of Chujo et al. (2007) and Huang et al. (2011). They reported that telmisartan treatment decreases visceral fat accumulation in hypertensive and obese patients who are moderately nourished to well-nourished. Insulin sensitivity and vascular inflammation markers are also improved after 24 weeks of telmisartan therapy. Iwashita et al. (2012) explored the molecular mechanism underlying the insulin-sensitizing effect of angiotensin II receptor blockers and valsartan in a lipopolysaccharide-induced macrophage co-cultured adipocyte model. In the present study, low body weight and low visceral fat tissue indicated that poor

nutrition was the major condition in the nephrectomized rat model, and losartan did not decrease visceral fat accumulation. To our knowledge, malnutrition occurs in a chronic renal insufficiency condition and may significantly affect the effectiveness of losartan on visceral fat accumulation. Moreover, the ratio of ΔAUCglucose to ΔAUCinsulin significantly increased in the N and NA groups and was equal in both groups. This finding, which agreed with the report of Hung and Ikizler, 2011, indicated that insulin resistance in chronic renal insufficiency situation was complex and not completely normalized by angiotensin II receptor blocker therapy only. The primary reason may be the short duration of losartan therapy (one month). No significant difference was observed between the NA and control groups. Poor nutrition was another factor that affected the failure of losartan therapy to improve insulin resistance. Recent studies have shown that adiponectin and leptin are involved in the pathogenesis of renal damage in obesity (Zoccali and Mallamaci, 2011), and that the reduction of renal function has a key role in elevating adipocytokine levels (Baldasseroni et al., 2013). Several reports have also indicated that elevated systemic levels do not hinder the downregulation of leptin and adiponectin mRNA levels in the adipose tissue of mildly malnourished and obese subjects (Marchlewska et al., 2004; Nordfors et al., 1998). Furthermore, a high plasma level of resistin affects endothelial dysfunction and predicts myocardial infarction in overweight subjects (Baldasseroni et al., 2013; Weikert et al., 2008). The present study evaluated the expression of adiponectin, leptin, and resistin in adipocytes isolated from N, NA, and control groups (Fig. 4A–C). The adipose tissue expression of adiponectin and leptin in the N group was downregulated whereas resistin was upregulated compared with the control group. The low leptin expression of adipose tissue reflected poor nutrition in nephrectomized rats, whereas the high resistin expression of adipose tissue can be correlated with high systolic blood pressure. In addition, angiotensin II receptor blocker administration improved the abnormal expression of all three adipocytokines in adipocytes in the N group. Therefore, losartan exerted a protective effect by ameliorating adipocytokine imbalance in chronic renal insufficiency with poor nutrition. Abdominal visceral adiposity is closely associated with cardiovascular complications and chronic inflammation in patients with impaired renal function (Behn and Ur, 2006; Ishimura et al., 2011; Kato et al., 2011). Furthermore, lean hemodialysis patients are more prone to malnutrition and inflammation (Małgorzewicz et al., 2010). The data in the present study showed that visceral fat mass and body weight decreased in the N and NA groups, and that abnormal adipocytokine expression of adipocytes in the N group improved after angiotensin II receptor blocker administration. Malnutrition with decreased visceral fat mass was also found in chronic renal insufficiency, and the co-existing adipocytokine imbalance was ameliorated by an angiotensin II receptor blocker.

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One recent research has suggested that renin–angiotensin system inhibitors potentially benefit the plasma levels of high-molecularweight adiponectin in hemodialysis patients and can improve adipocytokine-related metabolic abnormalities (Nakagawa et al., 2011). A recent review article has summarized the use of angiotensin II receptor blockers by presenting reduced plasma adiponectin with an unknown effect through an unknown mechanism in chronic kidney disease patients, with adiponectin resistance implied as a possible causative factor (Slee, 2012). The current study proposed that persons with chronic renal insufficiency who have low body mass and low visceral fat mass may abnormally secrete adipocytokines through adipose tissues. Losartan, a rennin–angiotensin system inhibitor, may ameliorate adipocytokine-related metabolic abnormalities. The association among angiotensin II, adipocytokines, visceral fat, and metabolic syndrome/inflammatory responses in chronic renal insufficiency with coexisting malnutrition warrants further investigations. The present study successfully induced chronic renal insufficiency, insulin resistance, fat mass loss, and abnormal fat cell size distribution in N rats. Losartan administration was found to decrease renal injury and systolic blood pressure as well as improve abnormal adipocytokine expression. However, insulin sensitivity was not ameliorated by losartan in patients with poor nutritional status. Further studies are needed to prove the benefits of adipocytokine normalization even in chronic renal insufficiency with coexisting malnutrition.

5. Disclosures statement The authors declare no conflict of interest.

Author contributions J.Y.C. and C.C.J. conception and design of research. Y.W.C., C.H.T., and S.W.H. performed experiments. C.H.T. and C.C.J. analyzed data. D.Y.J., Y.W.C., C.F.C., J.Y.C., and C.C.J. interpreted results of experiments. D.Y.J., Y.W.C., and C.C.J. prepared figures. D.Y.J. drafted manuscript. J.Y.C. and C.C.J. edited and revised manuscript. C.C.J. approved final version of manuscript.

Acknowledgments We thank Miss Yi-Hsiu Lin for her assistance in animal care, RNA extraction, and RT-PCR assays. Grants: This study was supported by research grants from the Department of Health, Taipei City Government (99-18) and from the Ministry of Education, Aim for the Top University Plan, Taipei, Taiwan. References Abe, M., Okada, K., Maruyama, T., Maruyama, N., Matsumoto, K., Soma, M., 2011. Relationship between erythropoietin responsiveness, insulin resistance, and malnutrition–inflammation–atherosclerosis (MIA) syndrome in hemodialysis patients with diabetes. Int. J. Artif. Organs 34, 16–25. Akdag, I., Yilmaz, Y., Kahvecioglu, S., Bolca, N., Ercan, I., Ersoy, A., Gullulu, M., 2008. Clinical value of the malnutrition–inflammation–atherosclerosis syndrome for long-term prediction of cardiovascular mortality in patients with end-stage renal disease: a 5-year prospective study. Nephron Clin. Pract. 108, c99–c105. Arozal, W., Watanabe, K., Veeraveedu, P.T., Ma, M., Thandavarayan, R.A., Suzuki, K., Tachikawa, H., Kodama, M., Aizawa, Y., 2009. Effects of angiotensin receptor blocker on oxidative stress and cardio-renal function in streptozotocin-induced diabetic rats. Biol. Pharm. Bull. 32, 1411–1416. Aunapuu, M., Pechter, U., Arend, A., Suuroja, T., Ots, M., 2003. Ultrastructural changes in the remnant kidney (after 5/6 nephrectomy) glomerulus after losartan and atenolol treatment. Medicina (Kaunas) 39, 975–979.

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