Dietary polyunsaturated fatty acids slow the progression of diabetic nephropathy in streptozotocin-induced diabetic rats

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Nutrition Research 30 (2010) 217 – 225 www.nrjournal.com

Dietary polyunsaturated fatty acids slow the progression of diabetic nephropathy in streptozotocin-induced diabetic rats Meiko Yokoyama a , Kanae Tanigawa a , Tomoko Murata a , Yukiko Kobayashi a , Eriko Tada a , Isao Suzuki b , Yukihiro Nakabou c , Masashi Kuwahata a , Yasuhiro Kido a,⁎ b

a Graduate School of Human Environment Science, Kyoto Prefectural University, Kyoto 606-8522, Japan Graduate School of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto, Kumamoto 862-8502, Japan c Graduate School of Health and Welfare, Kawasaki University of Medical Welfare, Okayama 701-0193, Japan Received 18 January 2010; revised 21 February 2010; accepted 11 March 2010

Abstract Diabetic nephropathy is associated with lipid deposits in the kidney. We hypothesized that a diet containing polyunsaturated fatty acids (PUFAs) could ameliorate pathogenesis of diabetic kidney diseases associated with lipid depositions in the kidneys. We examined if the pathogenesis and progression of diabetic nephropathy are affected by the type of dietary fat using streptozotocin (45 mg/kg body weight, intravenous)-induced diabetic rats (5-week-old male Sprague-Dawley rats). Streptozotocin-induced diabetic rats were fed a lard diet containing saturated fatty acids or a rapeseed oil diet containing PUFAs (DML and DMR, respectively) for 11 days. Similarly, streptozotocinnontreated rats were fed a lard diet or a rapeseed oil diet (NL and NR, respectively) for 11 days. Hyperglycemia was induced in DML and DMR, compared with NL and NR groups. The levels of plasma ketone, total cholesterol, and triglyceride (TG) were significantly increased in the DML group. Moreover, albuminuria and renal TG content were enhanced in the DML group. The renal TG content correlated positively with urinary albumin excretion (P b .001). Oil-Red O staining of kidney sections indicated a marked accumulation of neutral lipids in both glomerular and tubular cells in the DML group. In addition, a renal sterol regulatory element-binding protein-1 mature protein increment was induced in the DML group. Conversely, sterol regulatory element-binding protein-1 expression in the kidney was maintained at normal levels in the DMR group. These results suggest that dietary PUFAs may slow the progression of diabetic nephropathy associated with lipid depositions in the kidney. © 2010 Elsevier Inc. All rights reserved. Keywords: Abbreviations:

Dietary fat; Diabetic nephropathy; Renal lipid accumulation; SREBP-1; Streptozotocin-induced diabetic rat Alb, albumin; DML, diabetic rats fed a lard diet containing saturated fatty acids; DMR, diabetic rats fed 1a rapeseed oil diet containing polyunsaturated fatty acid; FA, fatty acid; FFA, free fatty acid; Glc, glucose; NL, nontreated rats fed a lard diet; NR, nontreated rats fed a rapeseed oil diet; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid; SREBP, sterol regulatory element-binding protein; STZ, streptozotocin; TC, total cholesterol; TG, triglyceride.

1. Introduction The incidence of diabetes is increasing throughout the world [1,2]. The prevalence of diagnosed and undiagnosed diabetes in the United States has increased 1.5%, from 7.8% ⁎ Corresponding author. Tel.: +81 75 703 5402; fax: +81 75 703 5402. E-mail address: [email protected] (Y. Kido). 0271-5317/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2010.03.002

in 1988 to 1998 [3] to 9.3% in 1999 to 2002 [4]. As a result, the prevalence of complications from diabetes has also increased. In Asia, the prevalence of microalbuminuria and macroalbuminuria in type 2 diabetic patients was 59% [5]. Dyslipidemia is often observed in subjects with endstage renal failure [6]. Recent studies suggest that treatment with hydroxymethylglutaryl-CoA reductase inhibitors (statin) not only improves dyslipidemia but also reduces the

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rate of renal function decline in patients with chronic kidney disease [7-9]. In patients with diabetes, plasma triglyceride (TG) levels are increased and high-density lipoprotein cholesterol levels decreased compared with those of nondiabetic patients [10]. Dyslipidemia in diabetic patients may be associated with the pathogenesis of diabetic nephropathy. It is very important to balance the intake of fat and carbohydrate in the nutritional approach for diabetes. In humans with type 2 diabetes, decreasing the dietary fat and increasing carbohydrate intake can potentially worsen the dyslipidemia by lowering high-density lipoprotein cholesterol levels and increasing very lowdensity lipoprotein cholesterol and TG levels. The dyslipidemia can be ameliorated by body weight loss [11]. Briefly, diabetic patients should have a sufficient intake of dietary fat. We propose that the types of dietary fatty acids (FAs) may improve the prognosis of diabetic kidney diseases associated with lipid depositions in the kidneys, even with a high-fat diet. Dietary FA can be classified into saturated FAs (SFA), monounsaturated FAs, and polyunsaturated FAs (PUFA), based on their structure. Dietary PUFA have been shown to have beneficial effects on renal function in both patients with diabetes [12,13] and experimental animals with diabetes [14,15]. One of the mechanisms involved is the attenuation of renal structural damage associated with diabetes, including glomerulosclerosis and tubulointerstitial fibrosis [14]. Several studies have shown that lipid accumulation was observed in the kidneys of diabetic humans [16,17] and experimental animals [18]. In diabetic rats, studies have shown increased renal accumulation of lipids mediated by increased expression of sterol regulatory element-binding protein-1 (SREBP-1) [19,20]. Sterol regulatory elementbinding proteins have been shown to be master regulators of both FA and cholesterol metabolism [21,22]. A previous study indicated that PUFA could downregulate hepatic SREBP-1 [23]. Consequently, dietary PUFA could inhibit the alteration of lipid metabolism. However, the possibility of an increase in renal lipid accumulation originating from dietary fats has not been entertained. Furthermore, it has not been determined whether or not dietary PUFA can mediate diabetic kidney disease. Therefore, in the present study, we hypothesized that a diet containing PUFA under hyperglycemia could ameliorate the pathogenesis of diabetic kidney diseases associated with lipid depositions in the kidneys. We compared the effects of dietary PUFA with that of dietary SFA, which showed a higher intake in diabetic patients. We proposed that the type of dietary FAs may play an important role in the pathogenesis of diabetic kidney diseases associated with lipid depositions in the kidneys. A clear understanding of the role of the type of dietary fats, and their relationship to the progression of diabetic kidney disease may contribute greatly a better understanding of nutritional management for diabetic patients.

2. Methods and materials 2.1. Animal experiments This study was performed in accordance with the guidelines for animal experimentation at Kyoto Prefectural University. Five-week-old male Sprague-Dawley rats were purchased from Japan SLC, Inc (Hamamatsu, Japan). The rats were housed individually in metabolic cages with lights on from 0800 to 2000, in a controlled temperature (23 ± 2°C) and humidity (60% ± 10%) environment. After acclimatization for 3 days, the animals were randomly assigned to 2 groups. The first group was the normal group (N group), which was injected with a 50 mmol/L sodium citrate solution (pH 4.5), and the second group was injected with a 50 mmol/L sodium citrate solution (pH 4.5) of streptozotocin (STZ; 45 mg/kg body weight; Wako Pure Chemical Industries, Ltd, Osaka, Japan), in the jugular vein. Seven days after the injections, the diabetic rats were identified by measuring their tail vein blood glucose (Glc)

Table 1 Ingredient composition of the experimental diets fed to rats a Ingredients

Cb

Lc

Rd

Egg-white protein α-Starch Sucrose Control oil e, f Lard g Rapeseed oil h Minerals i Vitamins j Cellulose Biotin

159 444 222 110 – – 35 10 20 0.0025

(g/kg) 159 444 222 – 110 – 35 10 20 0.0025

159 444 222 – – 110 35 10 20 0.0025

a

Containing 15% protein, 25% fat, and 60% carbohydrate on a total energy basis. b C: Control oil diet. c L: Lard diet. d R: Rapeseed oil diet. e Rapeseed oil/soybean oil = 7/3. f Fatty acid composition of control oil (g/100 g oil): palmitic acid, 5.8; stearic acid, 2.5; palmitoleic acid, 0.2; oleic acid, 47.2; linoleic acid, 28.3; arachidonic acid, 0.0; α-linolenic acid, 7.1. g Fatty acid composition of lard (g/100 g fat): palmitic acid, 23.0; stearic acid, 13.0; palmitoleic acid, 2.3; oleic acid, 40.0; linoleic acid, 8.9; arachidonic acid, 0.1; α-linolenic acid, 0.5. h Fatty acid composition of rapeseed oil (g/100 g oil): palmitic acid, 4.0; stearic acid, 1.9; palmitoleic acid, 0.2; oleic acid, 58.0; linoleic acid, 19.0; arachidonic acid, 0.0; α-linolenic acid, 7.5. i AIN-76 mineral mixture (g/kg mixture): calcium phosphate dibasic, 500.0; sodium chloride, 74.0; potassium citrate, 220.0; potassium sulfate, 52.0; magnesium oxide, 24.0; manganese carbonate, 3.5; ferric citrate, 6.0; zinc carbonate, 1.6; cupric carbonate, 0.3; potassium iodate, 0.01; sodium selenite, 0.0066; chromium potassium sulfate, 0.55; sucrose, 118.03. j AIN-76 vitamin mixture (per g mixture): vitamin A, 400 IU; vitamin D3, 100 IU; vitamin E, 5 mg; vitamin K3, 0.005 mg; vitamin B1, 0.6 mg; vitamin B2, 0.6 mg; vitamin B6, 0.7 mg; vitamin B12, 0.001 mg; D-biotin, 0.02 mg; folic acid, 0.2 mg; calcium pantothenate, 1.6 mg; nicotinic acid, 3 mg; choline chloride, 200 mg; sucrose, 0.968 g.

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levels. The rats with a blood Glc level greater than 16.7 mmol/L were used in the experiment (DM group). During these pretreatment periods, all rats were fed the control oil diet (C) (Table 1). Both the N group and the DM group were further divided into 2 subgroups, which received the diets containing lard (L) or rapeseed oil (R) as a fat source (Table 1) for 11 days (NL, NR, DML, and DMR groups, respectively). All animals were allowed ad libitum access to food and water. Body weight and food intake were recorded at the same time every day. 2.2. Blood and tissue samples After 11 days of dietary treatment, the rats were killed during the early phase of the light cycle in a nonfasting state. The rats were euthanized by cervical dislocation under ether anesthesia, and blood samples drawn from the inferior vena cava were collected in tubes with heparin. After centrifugation (1500 × g for 5 minutes at 4°C), plasma was measured immediately for physiologic parameters. The liver, kidney, retroperitoneal fat, and epididymal fat were rapidly removed and weighed. The kidney was divided into 4 pieces. The first part of each kidney was lyophilized for renal protein and lipid contents. The second part was snap-frozen in liquid nitrogen and stored at −80°C for immunoblotting analysis. The third part was used immediately for total RNA extraction. The forth part was fixed in 10% neutral-buffered formalin for Oil-Red O staining. 2.3. Urinary samples All of the rats were maintained in individual metabolic cages during the experiments. Urine samples were collected for 24 hours from each rat at the end of the dietary treatment period, and the urine volume was measured. These specimens were centrifuged at 1000 × g for 5 minutes at 4°C. The supernatant samples were stored at −20°C.

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resuspended in isopropanol for TC and TG analysis using the autoanalyzer. 2.5. Preparation of nuclear extracts Nuclear extracts were prepared from frozen kidneys using the method described by van der Veen et al [26]. Approximately 100 mg of renal tissue was homogenized in buffer A containing 20 mmol/L Tris-Cl (pH 7.4), 2 mmol/L MgCl2, 0.25 mol/L sucrose, 10 mmol/L sodium EDTA, 10 mmol/L sodium ethylene glycol bis-(β-aminoethylether)-N, N,N′,N′-tetraacetic acid, and protease inhibitor cocktail (Complete Mini; Roche Diagnostics K.K., Mannheim, Germany). The homogenate was filtered through sterile gauze and centrifuged at 1000 × g for 5 minutes at 4°C. The pellet was washed twice with buffer A. The nuclear pellet was resuspended in buffer B containing 20 mmol/L HEPES (pH 7.6), 2.5% (vol/vol) glycerol, 0.42 mol/L NaCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L bis-(βaminoethylether)-N,N,N′,N′-tetraacetic acid, and protease inhibitor cocktail at 4°C for 1 hour. After centrifugation at 100 000 × g for 30 minutes at 4°C, the supernatant was collected and stored at −80 °C. Protein concentration was determined using the Bradford method [27]. 2.6. Immunoblotting Equal amounts of renal nuclear extracts (15 μg) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10% wt/vol), as described by Laemmli [28], and transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc, Benicia, CA). The membranes were blocked in 5% dried milk powder in 1 × TBST containing 20 mmol/L Tris-Cl (pH 7.6), 150 mmol/L NaCl, 0.1% (vol/vol) Tween-20, for 60 minutes at room temperature and incubated overnight at 4°C with antibodies for SREBP-1 (Abcam plc., Cambridge, Mass; 1:5000). The SREBP-1 antibody recognizes both SREBP-1a and SREBP1c. The membranes were washed in 1 × TBST and incubated for 60 minutes at room temperature in a secondary antibody

2.4. Blood, urine, and kidney analysis Plasma concentrations of Glc, albumin (Alb), creatinine (Cr), ketone, total cholesterol (TC), TG, and free FA (FFA) were measured using the following kits: GLU-HL, BCG-N, CRE-L, ketone-T, TCHO-L, TG-L (Serotec Co. Ltd, Sapporo, Japan) and NEFA-HA (Wako Pure Chemical Industries), respectively. Samples were analyzed using an autoanalyzer (CL-8000, Shimadzu Co, Kyoto, Japan). Urinary Cr level was also measured using the CRE kit, and urinary Alb concentration was measured using the Nephrat enzyme-linked immunosorbent assay kit (Exocell Inc, Philadelphia, PA). Kidney samples were homogenized in saline, and renal protein content was determined using the Lowry method [24]. Renal lipid content was extracted from lyophilized kidney tissue using the Folch method [25]. Extracts were evaporated under a stream of nitrogen gas and

Table 2 Body weight, food intake, and food efficiency of normal and diabetic rats fed lard or rapeseed oil diets NL

NR

DML

DMR

Body weight Initial (g) 193 ± 4 192 ± 6 173 ± 5 172 ± 7 266 ± 8a 199 ± 13b 208 ± 12b Final (g) 264 ± 6a Food intake (g/d) 17 ± 1a 16 ± 1a 33 ± 2b 27 ± 2b Food efficiency 0.42 ± 0.02a 0.46 ± 0.01a 0.07 ± 0.03b 0.14 ± 0.03b Normal rats fed L diet (NL: n = 6) or R diet (NR: n = 6) and diabetic rats fed L diet (DML: n = 11) or R diet (DMR: n = 10) for 11 days. Data are expressed as means ± SEM. Values in the same row that do not share the same superscript letter are significantly different at P b .05 for analysis of variance with post hoc Bonferroni (initial body weight, food intake, and food efficiency) or Kruskal-Wallis test with post hoc Mann-Whitney U tests with the Bonferroni inequality (final body weight).

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Table 3 Organ weights of normal and diabetic rats fed lard or rapeseed oil diets

Liver (g) Kidney (g) Retroperitoneal fat (g) Epididymal fat (g)

NL

NR

DML

DMR

11.7 ± 0.7 1.57 ± 0.04a 3.51 ± 0.60

11.5 ± 0.4 1.67 ± 0.05a 3.73 ± 0.51

9.4 ± 0.8 2.68 ± 0.11b 0.01 ± 0.01

10.7 ± 0.8 2.49 ± 0.12b 0.32 ± 0.16

3.50 ± 0.28a

3.19 ± 0.28a

0.52 ± 0.09b

0.90 ± 0.20b

Polymerase chain reaction results were normalized to β-actin mRNA levels. Primers for SREBP-1 (Applied Biosystems, Assay ID; Rn01495763_g1) and β-actin (Applied Biosystems, Assay ID; Rn00667869_m1) were purchased from Applied Biosystems (Foster City, Calif). 2.8. Oil-Red O staining Kidneys fixed in 10% neutral-buffered formalin were embedded in Tissue-Tek O.C.T. compound (Sakura Finetek Japan Co, Ltd, Tokyo, Japan) and were snap-frozen in liquid nitrogen. To determine the renal accumulation of neutral fats, frozen sections (4 μm) were stained for 30 minutes with OilRed O solution in propylene glycol and counterstained for 3 minutes in hematoxylin. Twenty glomerular profiles from each renal section were scanned with a microscope. The sections were imaged with a microscope (Olympus, Co, Tokyo, Japan).

Normal rats fed L diet (NL: n = 6) or R diet (NR: n = 6) and diabetic rats fed L diet (DML: n = 11) or R diet (DMR: n = 10) for 11 days. Data are expressed as means ± SEM. Values in the same row that do not share the same superscript letter are significantly different at P b .05 for analysis of variance with post hoc Bonferroni (liver, kidney, and epididymal fat) or Kruskal-Wallis test with post hoc Mann-Whitney U tests with the Bonferroni inequality (retroperitoneal fat).

conjugated to horseradish peroxidase (Jackson Immunoresearch Laboratories, Inc, West Grove, Pa; 1:20 000). After additional washes, chemiluminescence detection was carried out using an Immobilon Western kit (Millipore Co, Bedford, Mass) and hyperfilms (Hyperfilm ECL; GE Healthcare Ltd., UK). The membranes were stripped and reprobed with an antibody recognizing α-tubulin (SigmaAldrich Co, Louis, MO).

2.9. Statistical analyses Before assessing the different variables, we carried out a Kolmogorov-Smirnov test to check the normal distribution of the variables. Data that fit the normal distribution were compared by 1-way analysis of variance. The Levene test for homogeneity was used to test for equal variance between samples. When equal variance could be assumed, the Bonferroni post hoc test was used to identify significant differences between multiple test groups. When equal variance could not be assumed, the Games-Howell post hoc test was applied. A nonparametric Kruskal-Wallis test with post hoc Mann-Whitney U tests with the Bonferroni inequality was performed on the final body weight, kidney, retroperitoneal fat, plasma TG level, urinary Cr excretion, and renal SREBP-1 mRNA data, because it was in contrast with the normality hypothesis. The correlation coefficient was calculated using Spearman rank correlation coefficient. Data (NL and NR group, n = 6; DML group, n = 11; DMR group, n = 10) were presented as means ± SEM. The level of significance was set at P b .05. Analyses were performed using SPSS 11.0 for Microsoft Windows (SPSS, Chicago, Ill). Sample sizes were determined based on power analysis using freeware (http://www.stat.uiowa.edu/~rlenth/Power).

2.7. Preparation of total RNA and quantitative real-time polymerase chain reaction Total RNA was extracted using the Agilent total RNA isolation mini kit (Agilent Technologies, Inc, Santa Clara, Calif) according to the manufacturer's protocol. cDNA was prepared by reverse transcription of 1 μg total RNA, and the reverse transcription reaction mixture was amplified with primers specific for SREBP-1 in a total volume of 20 μL. Samples were amplified at 95°C for 7 seconds, 60°C for 30 seconds, 72°C for 20 seconds, and cycles of 54 times. Real-time polymerase chain reaction (PCR) was performed with the DNA Opticon system and Opticon Monitor software (MJ Research, Inc, Watertown, Mass) using TaqMan for detection. All of the tissue samples were tested in duplicate in a single 96-well reaction plate, and data were analyzed according to the ΔΔCT method. Table 4 Biochemical parameters of normal and diabetic rats fed lard or rapeseed oil diets NL Plasma Glc (mmol/L) Plasma ketone (μmol/L) Plasma FFA (mmol/L) Plasma TC (mmol/L) Plasma TG (mmol/L) Plasma Cr (mmol/L) Urinary Cr (mmol/d)

NR a

8.6 ± 0.6 123 ± 15a 1.01 ± 0.07a 1.9 ± 0.2a 4.4 ± 0.3a 0.6 ± 0.1a 0.48 ± 0.04

DML a

8.9 ± 0.4 105 ± 17a 0.84 ± 0.07a 1.7 ± 0.2a 4.5 ± 0.2a 0.8 ± 0.1a 0.48 ± 0.03

51.4 ± 5150 ± 1.94 ± 15.2 ± 23.6 ± 17.2 ± 0.62 ±

DMR b

5.2 890b 0.13b 1.5b 2.4b 4.6b 0.08

38.7 ± 2.8b 2172 ± 734a 1.68 ± 0.10b 4.2 ± 0.6a 10.6 ± 2.0a 2.7 ± 0.3c 0.45 ± 0.05

Normal rats fed L diet (NL: n = 6) or R diet (NR: n = 6) and diabetic rats fed L diet (DML: n = 11) or R diet (DMR: n = 10) for 11 days. Data are expressed as means ± SEM. Values in the same row that do not share the same superscript letter are significantly different at P b .05 for analysis of variance with post hoc Bonferroni (plasma FFA), Games-Howell posttest (plasma Glc, ketone, TC, and urinary Cr ) or Kruskal-Wallis test with post hoc Mann-Whitney U tests with the Bonferroni inequality (plasma TG and Cr).

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3. Results 3.1. Rat experiment We conducted the experiment using normal or STZinduced diabetic rats fed a lard or rapeseed oil diet to test the effects of different dietary fats. No rats died during the present study. As shown in Table 2, dietary fats had no affect on body weight, food intake, or food efficiency. However, food intake significantly increased in diabetic rats compared with normal rats; food efficiency was significantly reduced in diabetic rats compared with normal rats associated with reduced body weight gain. Although the liver weights were similar in each group, the kidney weights were significantly increased in the DML and DMR groups compared with the NL and NR groups. No differences in the retroperitoneal fat and epididymal fat weights were found between the NL and NR groups, but the degree of decrement of these fat weights was greater in the DML group than in the DMR group (Table 3). As shown in Table 4, plasma Glc levels of STZinduced diabetic rats (DML and DMR) were markedly higher than those of normal rats (NL and NR) and tended to decrease in the DMR group, compared with the DML group. The levels of plasma ketone, FFA, TC, and TG in diabetic rats (DML and DMR) were significantly increased compared with those in normal rats (NL and NR). Although dietary fats showed no effect on these plasma levels in normal rats (NL and NR), in diabetic rats, these levels tended to increase more in the DML group compared with the DMR group. Plasma Cr levels in the diabetic groups (DML and DMR) were significantly increased compared with the levels in the normal groups (NL and NR). The plasma Cr level in the DMR group was not increased compared with that in DML group (Fig. 3A-C). There were no significant differences in the urinary Cr excretion among the 4 groups. 3.2. Urinary albumin excretion and renal TG contents To study the effects of dietary fats on the progression of diabetic nephropathy, we measured urinary Alb excretion and renal TG contents. Dietary fats showed no effect on either urinary Alb excretion or renal TG contents in normal rats (Fig. 1A, B). However, urinary Alb excretion in DML was remarkably increased compared with that in the DMR group (Fig. 1A). Moreover, renal TG contents in DML were also increased compared with those in the DMR (Fig. 1B). In the present study, urinary Alb excretion was significantly related to renal TG contents (Fig. 1C). A positive linear relationship was found between urinary Alb excretion and renal TG contents (r = 0.754, P b .001; Fig. 1C). 3.3. Oil-Red O staining To determine whether short-term (11 days) dietary fats can cause an alteration in the lipid metabolism, we

Fig. 1. Urinary albumin excretion (A), renal TG content (B), and correlation between urinary albumin excretion and renal TG content (C) in normal rats fed L or R diet for 11 days (NL or NR, respectively) and diabetic rats fed L or R diet (DML or DMR, respectively) for 11 days. Urine samples were collected for 24 hours at the end of the dietary treatment period. Data are expressed as means ± SEM (A and B) (NL and NR, n = 6; DML, n = 11; DMR, n = 10). Values in the same row that do not share the same superscript letter are significantly different at P b .05 by analysis of variance with post hoc Games-Howell (A) and Bonferroni (B). The correlation analysis performed with Spearman coefficient test (C) of NL (open circle), NR (solid circle), DML (open square), and DMR (solid square) is shown for all of the groups.

performed Oil-Red O staining, which revealed the accumulation of neutral lipids in both glomerular and tubular cells of STZ-treated rats (Fig. 2). Oil-Red O staining of kidney sections indicated a marked accumulation of neutral lipids in both glomerular and tubular cells. There was an increased Oil-Red O staining in the

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Fig. 2. Oil-Red O staining in the kidneys of normal and diabetic rats in normal rats fed L or R diet for 11 days (NL or NR, respectively) and diabetic rats fed L or R diet (DML or DMR, respectively) for 11 days. Kidneys were fixed in 10 % neutral-buffered formalin were embedded in Tissue-Tek O.C.T. compound and frozen in liquid nitrogen. Cryosections were sliced into 4-μm sections, stained with Oil-Red O and counterstained with hematoxylin. Lipid droplets appear as red spots. Magnification, ×100.

glomerular and tubular cells of STZ-treated rats. Accumulations of neutral lipids in both glomerular and tubular cells were observed more in the DML group than in the DMR group (Fig. 2). 3.4. Expression of SREBP-1 in kidney and liver To determine whether there was a marked increase of SREBP-1 mRNA and SREBP-1 mature protein levels due to dietary fats in STZ-treated rats, the SREBP-1 mRNA and SREBP-1 mature protein levels were measured by real-time PCR and Western blotting, respectively (Fig. 3). Although there were no significant differences in the renal SREBP-1 mRNA level between normal (NL and NR) and diabetic (DML and DMR) rats (Fig. 3A), the renal SREBP1 mature protein level in the DML group was higher than that in the NL, NR and DMR groups (Fig. 3B). Conversely, hepatic SREBP-1 mRNA levels in the DML group were significantly decreased compared with the level in the NL group (Fig. 3C). The hepatic and renal SREBP-1 levels in the DMR group were both maintained at a normal level (Fig. 3A-C).

4. Discussion The major finding of this study is that normal levels of SREBP-1 mRNA and mature protein were maintained by feeding rats rapeseed oil, containing PUFA. The maintenance of SREBP-1 was responsible for a reduced accumulation of neutral lipid in the kidney in STZ-treated rats, resulting in the inhibition of the progression of diabetic renal failure. In the present study, plasma Glc and FFA levels were higher in both the DML and DMR groups, compared with the NL and NR groups. Recent reports indicate that hyperglycemia and elevated FFA negatively impact β-cell functions and glucotoxicity induced by prolonged hyperglycemia which causes β-cell dysfunction and altered β-cell mass [29,30]. Hence, it was likely that the partial failure of pancreatic β-cells with STZ treatment was due to further inflammation caused by the elevation of plasma Glc and FFA levels, inducing increasingly insulin deficiency. However, ketoacidosis and dyslipidemia were strongly enhanced in diabetic rats fed a lard diet, but not in diabetic rats fed a rapeseed oil diet. The results suggest that the

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progression of pancreatic β-cell dysfunction was more effectively suppressed by the rapeseed oil diet and that lipid metabolism was controlled by insulin. We also examined whether dietary fats had an effect on lipid metabolism, not only in the plasma but also in the kidney. Both FA synthesis and FA oxidation are factors controlling lipid metabolism. In the present study, lipogenesis was investigated by an analysis of the SREBP-1 expression. Jiang et al [31] demonstrated that a high-fat diet using lard induced renal FA synthesis via SREBP-1 but that it did not induce renal FA oxidation, compared with a

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low-fat diet. Moreover, another article presented information on the overexpression of SREBP-1 mature protein in the kidney, but not in the liver [19]. Our results also revealed downregulation of hepatic SREBP-1 mRNA and overexpression of renal SREBP-1 mature protein in the DML group compared with the NL group. However, SREBP-1 expression in both the liver and kidney was maintained at normal levels in the DMR group. The present study showed that dietary fats affected renal lipogenesis by alterating renal SREBP-1 expression in the STZ-treated rats. In fact, kidneys in the DML group showed an accumulation of renal TG, but renal TG deposits in the DMR group were similar to that in the NR group. Oil-Red O staining of the kidney sections also indicated a marked accumulation of neutral lipids in both glomerular and tubular cells in the DML group, whereas histopathologic findings in the DMR group revealed more intact tissue compared with the DML group. Interestingly, there was a positive correlation between renal TG contents and urinary Alb excretion. The results indicate that diabetic renal failure is associated with lipid deposits in the kidney via an increase in the SREBP-1 mature protein. The study using SREBP-1 knockout mice provide additional evidence for the progression of diabetic nephropathy. In mice fed a high-fat diet for 12 weeks, SREBP-1c knockout did not prevent renal TG accumulation and markedly attenuated the mRNA upregulation of plasminogen activator inhibitor-1, vascular endothelial growth factor, type 4 collagen, and fibronectin, although wild-type mice fed a high-fat diet showed increased renal TG contents and overexpression of these mRNA [31]. These reports suggest that overexpression of SREBP-1 should induce inflammatory glomerulosclerosis and tubulointerstitial fibrosis. Transcriptional activation of SREBP-1 could be upregulated by insulin [32] and glucose [33]. The alteration of renal SREBPFig. 3. Real-time PCR of SREBP-1 mRNA in kidney (A), Western blot of SREBP-1 in kidney nuclear extracts (B), and real-time PCR of SREBP-1 mRNA in liver (C) in normal rats fed L or R diet for 11 days (NL or NR, respectively) and diabetic rats fed L or R diet (DML or DMR, respectively) for 11 days. Total RNA was extracted from fresh kidney, and cDNA was prepared by reverse transcription of total RNA. When using the relative quantitative real-time PCR method, the cycle thresholds of the genes of interest are compared with the housekeeping genes to determine relative changes in expression. CTtarget − CTcontrol (CT) as the preferred method of detecting differences in the threshold cycles between the target and control genes. The relative expression levels of mRNA were calculated according to the formula 2-CT, where CT is the difference in threshold cycle (CT) values between the target and the internal control, and between the target and the calibrator. Snap-frozen kidney nuclear protein extracts (15 μg) from each group were subjected to immunoblot techniques using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and probed with mouse monoclonal anti–SREBP-1. The relative values of protein levels were determined by Western blot using anti–SREBP-1 and antitubulin antibodies. Data are expressed as means ± SEM (NL and NR, n = 6; DML, n = 11; DMR, n = 10). Values in the same row that do not share the same superscript letter are significantly different at P b .05 for Kruskal-Wallis test with post hoc Mann-Whitney U tests with the Bonferroni inequality (A) or analysis of variance with post hoc Bonferroni (B and C).

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1 mature protein might be due to this factor through insulin deficiency. Moreover, one of the triggers in renal SREBP-1 activation was likely the FA composition of plasma FFA. The plasma FA composition of obese nondiabetic subjects increased in the ratio of SFA/PUFA after intragastric infusion of palm oil compared with that of safflower oil [34]. The FA composition of phosphatidylcholine in red blood cells in type 1 diabetic patients showed either an increase of SFA or decreases of n-3 PUFA [35]. In addition, recent research has shown that PUFA using fish oil, eicosapentaenoic acid, or docosahexaenoic acid regulated SREBP-1 expression [36-40]. Rapeseed oil contains a large amount of α-linolenic acid (another n-3 or omega-3 PUFA). In the present study, we found that rapeseed oil containing αlinolenic acid was also effective in the upregulation of renal SREBP-1 mature protein, even with this short-term study using a high-fat diet. Many reports have shown that diabetic patients have higher saturated fat intake and poorly controlled diabetes [41,42]. Our study demonstrated that rapeseed oil had several beneficial effects, which include the prevention of renal SREBP-1 activation, reduced renal TG contents, and a decrease in urinary Alb excretion, compared with lard in STZ-induced diabetic rats. However, the normal groups showed no change in the SREBP-1 expression, and dietary fat in this study revealed no modulation of lipid metabolism. The present study suggests that rapeseed oil could be effective for preventing the progression of diabetic nephropathy under diabetes conditions, even with a high-fat diet. Our study demonstrated at least partly that dietary rapeseed oil containing PUFA had several beneficial effects, such as the prevention of diabetic nephropathy under diabetic conditions. Similar beneficial effects might be expected from other vegetable oils, for example, safflower oil. Moreover, the results are still preliminary and do not directly relate to humans. Rats received only one source as a dietary fat type, so the action of a dietary mixture of fats and the best dietary fat intake for humans remains unknown. Although the concept may be controversial, we believe that diabetic patients may need to reconsider the type of dietary fat included in their diet.

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