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NDT Advance Access published online on October 14, 2008
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfn573
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Amelioration of diabetic tubulointerstitial damage in liver-type fatty acid binding protein transgenic mice

Atsuko Kamijo-Ikemori1,2, Takeshi Sugaya1,3, Ayako Sekizuka1, Kazuaki Hirata2 and Kenjiro Kimura1

1 Division of Nephrology and Hypertension, Internal Medicine 2 Department of Anatomy, St Marianna University School of Medicine, Kawasaki 3 CMIC CO. Ltd, Tokyo, Japan

Correspondence and offprint requests to: Kenjiro Kimura, Division of Medicine, Nephrology and Hypertension, St Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-Ku, Kawasaki 216-8511, Japan. Tel: +81-44-977-8111; Fax: +81-44-977-7873; E-mail: kimura{at}marianna-u.ac.jp



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 Conflict of interest statement
 References
 
Background. Renoprotection of liver-type fatty acid binding protein (L-FABP) was demonstrated in a streptozotocin (STZ)-induced diabetic mouse model.

Methods. Established human L-FABP (hL-FABP) transgenic (Tg) mice and wild-type (WT) mice were divided into two groups: diabetic mice were uninephrectomized and injected with STZ; control mice were uninephrectomized and injected with a citrate buffer alone. Although mouse L-FABP was not expressed in WT mice, hL-FABP was expressed in the proximal tubules of the diabetic Tg mice and in the control Tg mice at 8 and 14 weeks after these injections.

Results. The expression of renal hL-FABP increased significantly in diabetic Tg mice compared to control Tg mice. A number of macrophages (F4/80) infiltrating the interstitium, the gene expressions of MCP-1, MCP-3, TGF-β, Fas, Bax and RAGE were significantly lower in diabetic Tg kidneys compared with diabetic WT kidneys. In the diabetic Tg kidneys, the degree of the tubulointerstitial injury and the deposition of type IV collagen were significantly lower than that of diabetic WT kidneys. The expressions of catalase and glutathione peroxidase-1 were significantly lower in diabetic Tg kidneys compared with diabetic WT kidneys.

Conclusions. Renal L-FABP ameliorated the tubulointerstitial damage of type 1 diabetic mice.

Keywords: diabetic nephropathy; liver-type fatty acid binding protein; proximal tubule; tubulointerstitial damage



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary data
 Conflict of interest statement
 References
 
In spite of recent advances in the treatment of diabetes, diabetic nephropathy remains as the main cause of end-stage renal disease and the number of patients with diabetic nephropathy has steadily increased over the years. Because the progression of diabetic nephropathy is associated directly with tubulointerstitial damage [1,2], inhibition of tubulointerstitial damage may have merit as a new strategy in the treatment of progressive diabetic nephropathy.

Liver-type fatty acid binding protein (L-FABP) is a 14 kDa protein found in the cytoplasm of human proximal tubules [3]. Fatty acids are bound with L-FABP and transported to the mitochondria or peroxisomes, where fatty acids are beta-oxidized, and this may play a role in fatty acid homeostasis [4–6]. Moreover, L-FABP has high affinity and capacity to bind long-chain fatty acid oxidation products, and may be an effective endogenous antioxidant [7–9].

Renal L-FABP is rarely expressed in the kidneys of rodents [10]. In order to elucidate the pathophysiological role of renal L-FABP in kidney disease, human L-FABP (hL-FABP) chromosomal transgenic (Tg) mice were generated and the pathological significance of hL-FABP in experimental models of protein overload [11] and unilateral ureteral obstruction was determined [12], in which oxidative stress might contribute to the progression of tubulointerstitial injury. Under experimentally induced pathological conditions, the expression of renal hL-FABP was upregulated and the tubulointerstitial inflammation and fibrosis of Tg mice were attenuated compared to that of wild-type (WT) mice [11,12]. However, the pathophysiological significance of hL-FABP in an experimental model of diabetic nephropathy remains to be determined.

In this study, streptozotocin (STZ)-induced diabetic mice were used to assess the renoprotection of renal hL-FABP. Our observations demonstrated that, in the model of type 1 diabetic nephropathy, the expression of renal hL-FABP was not only upregulated in the diabetic Tg mice but was also protective against tubulointerstitial damage of diabetic nephropathy.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary data
 Conflict of interest statement
 References
 
Animals
Tg mice bearing hL-FABP gene were generated (patent no. WO0073791) [11]. In brief, the genomic DNA encoding the hL-FABP gene including its promoter region (13 kb) was microinjected into fertilized eggs obtained from Balb/c mice mated with CBA mice. ICR mice were used as the recipients for the transfected eggs. The resulting Tg mice were backcrossed for more than six generations onto C57/Bl6 to obtain homozygous mutant mice on an inbred background. Only male mice were used. Integration of the hL-FABP gene into the mice genome was confirmed by the polymerase chain reaction (PCR) using the genomic DNA, and the expression of hL-FABP in the proximal tubules of the Tg mice was confirmed by northern blot analysis, western blot analysis and immunohistochemistry [11]. The Tg mice did not show any obvious abnormalities in appearance and behaviour. The distribution of the hL-FABP expression was confirmed in the kidney, liver and the intestine of the Tg mice using an ELISA procedure described below [11]. Mice were housed in the animal facilities of St Marianna University School of Medicine with free access to food and water.

Disease model
Twelve to 16-week-old male Tg mice (n = 23; body weight, 25.7 ± 0.3 g; mean ± SE) and WT littermates on a C57/Bl6 background (WT; n = 23; body weight, 25.1 ± 0.3 g) were used for this study. The presence of the transgene was ascertained by visualizing the mice under UV light. Because the transgene was fused with the green fluorescent protein gene, mice expressing the transgene were readily identified by green fluorescence [11,12].

All mice were subjected to left nephrectomy under intraperitoneal anaesthesia with pentobarbital, 75 mg/kg body weight. At 1 week after uninephrectomy, both Tg and WT mice were divided into two groups of control and diabetic mice. Diabetic mice were treated with STZ (Sigma Chemical Co., St. Louis, MO, USA) and control mice were treated with a citrate buffer adjusted to pH 4.5 (Mitsubishi Kagaku Iatron, Inc., Tokyo, Japan). STZ was dissolved in a sterile citrate buffer and injected intraperitoneally into mice (120 mg/kg, up to 0.4 ml) within 10 min of preparation. STZ or a citrate buffer was administered at three time points occurring at 48-h intervals during the first week of this study [13].

In order to confirm the hyperglycaemic status of STZ-injected mice, the glycaemic levels in blood collected from the tail vein were determined at the initial time and 2 weeks after injections using Free Style FreedomeTM (KISSEI Pharmacentical Co., Ltd, Tokyo, Japan). Before injection of STZ, the mean glycaemic level was 124 mg/dl in the Tg mice and 123 mg/dl in the WT mice. After injection of STZ, the glycaemic values were >200 mg/dl in all of the STZ-injected mice.

Urinary glucose level was monitored semiquantitatively with urinary dipsticks (WAKO Pure Chemical Industries, Ltd, Osaka, Japan) every week. Grading for urinary glucose was as follows: 0 (negative), 1+ (>100 mg/dl), 2+ (>250 mg/dl), 3+ (>500 mg/dl) or 4+ (>2000 mg/dl). To render animals hyperglycaemic without becoming ketoacidotic, when the urinary glucose level of diabetic mice was >4+, one unit of long-acting insulin (Humalin N, human insulin, Eli Lilly Japan KK, Kobe, Japan) was administered subcutaneously twice every week. Urinary glucose levels after insulin treatment of the mice remained at 4+ during the experimental period.

Mice were sacrificed at 8 and 14 weeks after injections of STZ. Five-hour urine samples were collected from each mouse 1 day before sacrifice using metabolic cages. Blood samples were obtained via the abdominal great vein at sacrifice.

The experimental protocol was approved by the Ethics Committees for Animal Experimentation of St Marianna University School of Medicine.

Serum biochemistry
Serum creatinine was measured by Jaffe's method (WAKO). Serum glycaemia and total cholesterol were measured by an enzymatic method, and serum lipid peroxidation was measured by the haemoglobin–methylene blue procedure [14,15] at the BCL Co. Research Service, Tokyo, Japan.

Urinary biochemistry
Urine samples were evaluated for proteinuria using the albumin/creatinine ratio. Albuminuria was determined using the Albuwell (Exocell, Inc., Philadelphia, PA, USA) mouse albumin ELISA, and creatinine was measured using The Creatinine Companion (Exocell) according to the protocols of the manufacturer.

The urinary measurement of oxidative stress was measured using an N{varepsilon}-(hexanoyl)lysine (HEL) ELISA kit (JaICA, Shizuoka, Japan) and was expressed as a ratio of urinary HEL to urinary creatinine.

Renal histological and morphometric analysis
The kidneys for light microscopy analysis were sliced axially into 3-mm-thick sections, fixed in methyl Carnoy's solution and embedded in paraffin. Paraffin sections (4 µm thick) were stained with periodic acid–Schiff (PAS) stain or Azan–Mallory stain. Tubulointerstitial injury was categorized as proximal tubule dilation with epithelial atrophy and extracellular matrix accumulation. At x100 magnification, 10 consecutive fields were randomly selected in the renal cortex [16,17], and the areas with tubulointerstitial damage and the whole cortical area were measured by using an image analyser (Leica Image Analyzer, Wetzlar, Germany). The degree of interstitial injury was defined as the ratio of the area of interstitial damage to the entire cortical area [12].

In regard to glomerulosclerosis quantification, the grade of sclerosis in each glomerulus stained with PAS was defined as follows: 0 = no sclerosis; 1 = 1–25% glomerular area affected by sclerosis; 2 = 26–50% glomerular area affected; 3 = 51–75% glomerular area affected and 4 = 76–100% glomerular area affected. At x200 magnification, the glomerulosclerosis score for each animal was calculated as [(1x the number of grade 1 glomeruli, %) + (2x the number of grade 2 glomeruli, %) + (3x the number of grade 3 glomeruli, %) + (4x the number of grade 4 glomeruli, %)] [13]. Twenty to forty glomeruli were examined for each animal. In addition, the glomerular size was measured at x200 magnification by using an image analyser (Leica Image Analyzer). The degree of glomerular size was defined as the average area of glomerulus.

Immunohistological analysis
Tissues were fixed in methyl Carnoy's solution and embedded in paraffin. An indirect immunoperoxidase method was used to identify the antigen. Macrophages were identified with a rat monoclonal antibody F4/80 (Medical and biological Laboratories Co., Ltd, Nagoya, Japan) [11,12], and type IV collagen was identified with a rabbit polyclonal antibody (Cedarlane Laboratories Ltd, Ontario, Canada). The degree of macrophage infiltration in the cortical interstitium was measured as the average number of F4/80-positive cells per field at x200 magnification by using the image analyser (Leica Image Analyzer) [12]. The positive area of type IV collagen in the interstitium was evaluated as the ratio of the positive area of type IV collagen to the entire cortical area at x100 magnification by using the image analyser (Leica Image Analyzer).

To perform immunohistochemistry with a monoclonal antibody against human L-FABP, tissues were fixed in 10% buffered formalin and embedded in paraffin. hL-FABP immunostaining in the kidneys of mice was performed with mouse monoclonal antibodies generated previously against human L-FABP and FABP-2, which reacted with endogenous mouse L-FABP expressed in the liver [11,12].

Taqman real-time PCR assay
Total RNA of the kidney was extracted using an RNAeasy mini kit (Qiagen Inc., CA, USA) according to the manufacturer's instructions [12]. Total RNA (0.5 µg) was reverse transcribed using an ExScriptTM RT reagent kit (Takara Shuzo, Kyoto, Japan) [12]. The TaqMan real-time PCR reactions were performed on a TaqMan ABI 7000 sequence detection system (Applied Biosystems, CA, USA) using TaqMan Universal PCR Master Mix (Applied Biosystems) [12]. hL-FABP, monocyte chemoattractant protein (MCP)-1, MCP-3, transforming growth factor-β (TGF-β), {alpha}1(I) procollagen ({alpha}1COL I), Fas, bcl-2 associated X protein (Bax), receptor for advanced glycation end products (RAGE), catalase and glutathione peroxidase-1 (Gpx1) and 18s rRNA were detected using TaqMan real-time PCR. Unlabelled specific primers and the TaqMan MGB probes (6-FAM dye-labelled) were purchased from Applied Biosystems. TaqMan conditions were as follows: after an initial hold of 2 min at 50°C and 10 min at 95°C, the samples were cycled 40 times at 95°C for 15 s and 60°C for 1 min. Expressions of hL-FABP, MCP-1, MCP-3, TGF-β, {alpha}1COL I, Fas, Bax, RAGE, catalase and Gpx1 mRNAs in each sample were evaluated after normalization with eukaryote 18s rRNA expression.

Measurement of renal and urinary hL-FABP by ELISA
In order to clarify the dynamics of renal hL-FABP in diabetic nephropathy, renal hL-FABP protein and urinary hL-FABP were measured with ELISA kits for hL-FABP (CMIC Co., Ltd, Tokyo, Japan) [11,18–20]. Renal protein was extracted by the previously described method [11,12]. The concentration of renal protein was measured using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA, USA). The concentration of renal hL-FABP was corrected for the total amount of protein and that of urinary hL-FABP was expressed as a ratio of urinary L-FABP to urinary creatinine.

Measurement of MCP-1 by ELISA
To determine the quality of MCP-1 proteins in the kidney, the protein extracted by the method described above was measured with an ELSIA kit for MCP-1 (R&D System, Minneapolis, MN, USA). The concentration of MCP-1 was corrected for the total amount of protein.

Western blot analysis of catalase
To determine protein expression of catalase, an antioxidant enzyme, a protein sample (40 µg), extracted as described above, was subjected to SDS–PAGE, and the separated protein bands were analysed by an enhanced chemiluminescence (ECL system, Amersham Int., Buckinghamshire, UK). The membrane was first blotted with a goat polyclonal antibody against catalase (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and then re-blotted with an antibody against a tubuline monoclonal antibody.

Statistical analysis
All values are expressed as mean ± standard error (SE). To compare the parameters from the two groups, the Mann–Whitney U-test for unpaired data and the Wilcoxon rank-sum test for paired data were used. Statistical significance was set at P < 0.05.



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary data
 Conflict of interest statement
 References
 
Body weight
Body weights of the control Tg and WT mice increased during the entire experimental period, and were significantly higher than the body weights of the diabetic Tg and WT mice. In contrast, body weights decreased in the diabetic Tg and WT mice at 8 weeks but, thereafter, increased at 14 weeks. There was no significant difference between the body weights of control Tg and WT mice.

Expression of hL-FABP
TaqMan real-time PCR assay showed that the hL-FABP gene was expressed in the kidneys of Tg mice (Figure 1A) but not in WT mice (data not shown). In the kidneys of the diabetic Tg mice, gene expression of hL-FABP was significantly higher (relative expression in arbitrary units) at 8 weeks (0.45 ± 0.13; P < 0.05) compared with the kidneys of the control mice, and decreased at 14 weeks to 0.12 ± 0.02 (arbitrary unit), which was still significantly higher than in the control Tg mice (0.05± 0.01; P < 0.05) (Figure 1A).


Figure 1
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  		Fig. 1 Diabetes induced expression of hL-FABP in Tg mice. (A) The expression of hL-FABP transcripts was determined by TaqMan real-time PCR and was normalized to 18s rRNA. Values are represented as mean ± SE. (B) The expression of hL-FABP protein was determined by ELISA and was corrected for the total amount of protein. *P < 0.05 compared to the kidney of the control Tg mice. Figure 1P < 0.05 compared to the kidney of diabetic Tg mice at 8 weeks. (B–F) Photomicrographs of renal hL-FABP immunostaining at 8 and 14 weeks. Representative photomicrographs showing immunostaining of hL-FABP in the kidneys of (C) control Tg mice, (D) Diabetic Tg mice at 8 weeks and (E) Diabetic Tg mice at 14 weeks and (F) diabetic WT mice at 14 weeks. Original magnification: x100.

 
hL-FABP protein expressions were increased significantly at 8 weeks (5.6 ± 0.4; P < 0.05) and at 14 weeks (5.6 ± 0.6; P < 0.05) in the kidneys of the diabetic Tg mice compared with the kidneys of the control Tg mice (3.3 ± 0.4; P < 0.05) (Figure 1B). The levels of the renal hL-FABP expression in diabetic Tg mice were similar at 8 and at 14 weeks.

Immunohistochemical analysis showed that hL-FABP staining in the Tg mice was spread diffusely through the cytoplasm of the proximal tubules in the sham control Tg mice (Figure 1C) and in the diabetic Tg kidneys at 8 (Figure 1D) and 14 weeks (Figure 1E). Mouse L-FABP expression was not observed in the control WT mice (data not shown) and the diabetic WT kidneys (Figure 1F).

Serum biochemistry
Serum glycaemia, serum creatinine and total cholesterol in the control Tg mice were similar to that in the control WT mice (Table 1). At 8 and 14 weeks, in both diabetic Tg and WT mice, concentrations increased significantly compared to their corresponding controls. There was no difference between the diabetic WT and the diabetic Tg mice.


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  		Table 1 Variables measured during the study period

 
Serum lipid peroxidation in the control Tg mice was similar to that in the control WT mice (Table 1). The mean values in both diabetic Tg and WT mice at 8 and 14 weeks did not increase more than that in the controls of Tg or WT mice. There was no difference between the diabetic Tg and the diabetic WT mice (Table 1).

Urinary albumin and urinary hL-FABP
The levels of urinary albumin in the controls of Tg and WT mice were similar (Table 1). At 8 and 14 weeks, in both diabetic Tg and WT mice, the concentrations showed significant increases compared to the control Tg or WT mice, and to the initial values of the same group. There was no difference in the patterns of change between the diabetic WT and the diabetic Tg.

In regard to urinary hL-FABP, levels in the diabetic Tg mice at 8 and 14 weeks were significantly higher compared to the control Tg mice (Table 1). Urinary hL-FABP was barely detectable in the controls and diabetic WT mice.

Expression of MCP-1
By TaqMan real-time PCR, the renal expression of MCP-1 was similar in the control Tg and WT mice (Figure 2A). Gene expressions of MCP-1 in the diabetic Tg and WT kidneys at 8 weeks were significantly increased over their corresponding controls. The level of gene expression of MCP-1 in the diabetic Tg kidneys was significantly lower than that in the diabetic WT kidneys at 8 weeks [4.6 ± 1.3 (arbitrary unit) and 85.6 ± 19.6 (arbitrary unit), respectively; P < 0.05]. Thereafter, the expression in both of the diabetic Tg and WT kidneys decreased at 14 weeks, and there was no significant difference between the diabetic Tg and WT mice.


Figure 2
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  		Fig. 2 Expressions of MCP-1 in mice with diabetes. (A) Expression of MCP-1 mRNA transcripts was determined by TaqMan real-time PCR and was normalized to that of 18s rRNA transcripts in the same sample. (B) The expression of MCP-1 protein was determined by ELISA and corrected for the total amount of protein. Values are represented as mean ± SE. #P < 0.05 compared to the diabetic WT kidneys. *P < 0.05 compared to the kidney of the control Tg or WT mice.

 
The level of renal MCP-1 protein expression (measured by ELISA) of the control Tg mice was similar to that in the kidneys of the control WT mice (Figure 2B). In the diabetic Tg and WT kidneys, MCP-1 protein expressions at 14 weeks increased significantly (P < 0.05) compared with their corresponding controls. At 14 weeks, MCP-1 protein expression in the diabetic Tg kidneys was significantly lower than that in the diabetic WT kidneys (19.2 ± 4.5 pg/mg protein and 40.9 ± 4.4 pg/mg protein; P < 0.05).

Expression of MCP-3
TaqMan real-time PCR showed that renal expressions of MCP-3 mRNA in the controls of Tg and WT mice were nearly undetectable (Figure 3). In the diabetic Tg and WT kidneys at 8 weeks, gene expressions of MCP-3 were significantly increased over their corresponding controls. The level of gene expression of MCP-3 in the diabetic Tg kidneys was significantly lower than that in the diabetic WT kidneys at 8 weeks [0.59 ± 0.21 (arbitrary unit) and 94.5 ± 41.1 (arbitrary unit), respectively; P < 0.05] (Figure 3). In both groups of mice, MCP-3 expression decreased from 8 to 14 weeks. However, the level of MCP-3 remained higher in the diabetic WT than in the diabetic Tg mice at 14 weeks [51.4 ± 42.7 (arbitrary unit) and 0.43 ± 0.23 (arbitrary unit), respectively; P < 0.05].


Figure 3
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  		Fig. 3 Expression of MCP-3 in mice with diabetes. Expression of MCP-3 mRNA transcripts was determined by TaqMan real-time PCR and was normalized to that of 18s rRNA transcripts in the same sample. Values are represented as mean ± SE. #P < 0.05 compared to the diabetic WT kidneys. *P < 0.05 compared to the kidney of the control Tg or WT mice.

 
Expressions of TGF-β and {alpha}1COL I
The expressions of TGF-β and {alpha}1COL I were examined in the kidneys. The levels of gene expressions of TGF-β (Figure 4A) and {alpha}1COL I (Figure 4B) measured by TaqMan real-time PCR in the control Tg mice were similar to those in the kidneys of the control WT mice. In the diabetic Tg and WT mice, gene expressions of TGF-β and {alpha}1COL I were significantly increased over their corresponding controls. At 8 weeks, the levels of gene expressions of TGF-β and {alpha}1COL I in the diabetic Tg kidneys [1.67 ± 0.36 and 0.17 ± 0.06 (arbitrary units), respectively] were significantly lower than those in the diabetic WT kidneys [25.0 ± 6.8 and 9.3 ± 2.4 (arbitrary units), respectively] (P < 0.05). Thereafter, their expressions in both diabetic Tg and WT kidneys decreased at 14 weeks, and there were no significant differences between the diabetic Tg and the diabetic WT.


Figure 4
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  		Fig. 4 Expressions of TGF-β and {alpha}1COL I in mice with diabetes. (A, B) Expressions of TGF-β and {alpha}1COL I mRNA transcripts were determined by TaqMan real-time PCR and were normalized to that of 18s rRNA transcripts in the same samples. Values are represented as mean ± SE. #P < 0.05 compared to the diabetic WT kidneys. *P < 0.05 compared to the kidney of the control Tg or WT mice.

 
Histological and immunohistochemical evaluation of kidneys
In the control kidneys of Tg (Figure 5A) and WT mice (Figure 5B), tubulointerstitial damage was not observed. In the diabetic Tg kidneys or the diabetic WT kidneys at 8 (data not shown) and 14 weeks (Figure 5C and D), the areas of tubulointerstitial damage were significantly greater than those in the kidneys of their corresponding controls. The areas damaged in the diabetic Tg kidneys were similar to that in the diabetic WT kidneys at 8 weeks (0.05 ± 0.01 and 0.07 ± 0.03, respectively; NS) but thereafter were significantly lower than in the diabetic WT kidneys at 14 weeks (0.29 ± 0.04 and 0.59 ± 0.10, respectively; P < 0.05) (Figure 5E).


Figure 5
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  		Fig. 5 Histological evaluation of tubulointerstitial change in the Tg and WT mice. At 14 weeks, representative photomicrographs of Azan–Mallory stained sections from (A) the control Tg mice, (B) the diabetic Tg kidneys, (C) the control WT mice and (D) the diabetic WT kidneys. Tubulointerstitial injury was assessed semiquantitatively as described in the Materials and Methods section; values are represented as mean ± SE. #P < 0.05 compared to the diabetic WT, *P < 0.05, {ddagger}P < 0.01, §P < 0.001 and {dagger}P < 0.005 compared to the kidney of the control Tg or WT mice; original magnifications: x100 (A–D).

 
Glomerulosclerosis and glomerular hypertrophy were observed in both diabetic Tg and WT kidneys at 8 and 14 weeks. For glomerulosclerosis, in the TG mice, the 8- and 14-week values were 0.81 ± 0.09 and 0.57 ± 0.11, while in the WT mice, the values were 0.75 ± 0.21 and 0.78 ± 0.09. For glomerular hypertrophy, in the TG mice, the 8- and 14-week values were 88.6 ± 3.9 and 94.8 ± 4.0. In the WT mice, the values were 95.7 ± 4.3 and 107.9 ± 4.7 (values are given in arbitrary units). The degrees of glomerulosclerosis and glomerular hypertrophy were not significantly different between the diabetic Tg kidneys and the diabetic WT kidneys at 8 and 14 weeks.

Macrophage infiltrates were found in the interstitium of both diabetic Tg and WT kidneys at 8 (data not shown) and 14 weeks (Figure 6A–D). The extent of macrophage infiltration was significantly greater than in the kidneys of their corresponding control mice. There was significantly less infiltrate in the diabetic Tg kidneys than in the diabetic WT kidneys at both 8 and 14 weeks (Figure 6E).


Figure 6
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  		Fig. 6 Immunostaining of the tubulointerstitium with F4/80 in the diabetes Tg and WT mice. At 14 weeks, F4/80 immunostaining in the kidney of (A) control Tg mice, (B) diabetic Tg mice, (C) control WT mice and (D) diabetic WT mice. (E) The number of F4/80 positive cells that had infiltrated the interstitium was assessed semiquantitatively as described in Materials and Methods; values are represented as mean ± SE. £P < 0.01 and #P < 0.05 compared to the diabetic WT kidneys, {dagger}P < 0.005 and $P < 0.0001 compared to the kidney of the control Tg or WT mice; original magnifications: x100 (A–D).

 
At 8 (data not shown) and 14 weeks, the deposition of interstitial type IV collagen in the diabetic Tg or WT kidneys was significantly greater than in the kidneys of their corresponding control mice (Figure 7A–D). At 8 weeks, the extent of deposition in the diabetic Tg kidneys was similar to that in diabetic WT kidneys (0.04 ± 0.01 and 0.04 ± 0.01, respectively). At 14 weeks, however, the extent of deposition in the diabetic Tg kidneys was significantly less than in the diabetic WT kidneys (0.06 ± 0.01 and 0.11 ± 0.02, respectively; P < 0.05) (Figure 7E).


Figure 7
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  		Fig. 7 Type IV collagen immunostaining in the diabetic Tg and WT mice. At 14 weeks, type IV collagen immunostaining in the kidney of (A) control Tg mice, (B) diabetic Tg, (C) control WT mice, and (D) diabetic WT. (E) The positive area of type IV collagen was assessed semiquantitatively as described in the Materials and Methods section; values are represented as mean ± SE. #P < 0.05 compared to the diabetic WT kidneys, *P < 0.05 compared to the kidney of the control Tg or WT mice; original magnifications: x100 (A–D).

 
Expression of FAS and Bax
The expressions of the proapoptotic genes FAS and Bax [21,22] were examined in the kidneys. The gene expressions of FAS (Figure 8A) and Bax (Figure 8B) measured by TaqMan real-time PCR in the control Tg and WT mice were similar. In the diabetic Tg and WT mice, gene expressions of FAS and Bax were significantly increased over their corresponding controls. At 14 weeks, the levels of FAS and Bax in the diabetic Tg kidneys [0.50 ± 0.06 (arbitrary unit) and 1.00 ± 0.12 (arbitrary unit), respectively] were significantly lower than that in the diabetic WT kidneys [0.75 ± 0.07 (arbitrary unit) and 1.58 ± 0.17 (arbitrary unit), respectively] (P < 0.05).


Figure 8
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  		Fig. 8 Expressions of FAS and Bax in mice with diabetes. (A, B) Expressions of FAS and Bax mRNA transcripts were determined by TaqMan real-time PCR and were normalized to that of 18s rRNA transcripts in the same sample. Values are represented as mean ± SE. #P < 0.05 compared to the diabetic WT kidneys. *P < 0.05 compared to the kidney of the control Tg or WT mice.

 
Expression of RAGE
RAGE is the best-characterized cell surface molecule to which AGE binds [23] and enables AGE to accelerate the transdifferentiation of epithelial cells to form myofibroblasts and aggravate the tubulointerstitial damage [24]. The levels of gene expression of RAGE (Figure 9) measured by TaqMan real-time PCR in the control Tg mice were similar to those in the kidneys of the control WT mice. In the diabetic Tg and WT mice, gene expressions of RAGE were significantly increased over their corresponding controls at 8 and 14 weeks (P < 0.05). At 14 weeks, gene expression of RAGE in the diabetic Tg kidneys [0.90 ± 0.12 (arbitrary unit)] was significantly lower than in the diabetic WT kidneys [2.47 ± 0.73 (arbitrary unit)] (P < 0.05).


Figure 9
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  		Fig. 9 Expression of RAGE in mice with diabetes. The expression of RAGE mRNA transcripts were determined by TaqMan real-time PCR and was normalized to that of 18s rRNA in the same sample. #P < 0.05 compared to the diabetic WT kidneys, *P < 0.05 compared to the kidney of the control Tg or WT mice.

 
Evaluation of oxidative stress
The level of urinary HEL, a lipid hydroperoxide-derived protein modification generated at the early phase by oxidative stress [25,26], in the control Tg mice was similar to that in the control WT mice (Table 1). At 8 weeks, in the diabetic WT mice, the concentration showed significant increases compared to the control WT mice, and to the values of the same group at 8 weeks, and was decreased at 14 weeks. In the diabetic Tg, urinary HEL level increased at 8 and 14 weeks, but this increase was not statistically significant. Urinary HEL level was significantly higher at 8 weeks in the diabetic WT compared to the diabetic Tg (P < 0.05). There was no difference in urinary HEL between the diabetic WT and the diabetic Tg at 14 weeks.

Expression of catalase
The gene expression of catalase was examined in the kidneys by TaqMan real-time PCR. The levels of gene expression of catalase (Figure 10A) in the control Tg and WT mice were similar. The diabetic Tg and WT mice showed significant increases of catalase gene expressions compared with their corresponding control. At 8 weeks, the level of gene expression of catalase in the diabetic Tg kidneys [0.58 ± 0.16 (arbitrary unit)] was significantly lower than in the diabetic WT kidneys [18.0 ± 5.4 (arbitrary unit)] (P < 0.05). Thereafter, the expressions of catalase in both the diabetic Tg and WT kidneys decreased at 14 weeks, and there was no significant difference between the diabetic Tg and the diabetic WT mice.


Figure 10
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  		Fig. 10 Expressions of catalase in mice with diabetes. (A) The expression of catalase mRNA transcripts was determined by TaqMan real-time PCR and was normalized to that of 18s rRNA in the same sample. #P < 0.05 compared to the diabetic WT kidneys, *P < 0.05 compared to the kidney of the control Tg or WT mice. (B) Western blot analysis of catalase expression (antibody dilution 1:100).

 
The protein expression of catalase was not observed in western blot analysis of the control Tg and WT mice (Figure 10B). Although the catalase protein was not detected during these experiments in the diabetic Tg mice, it was weakly expressed at 8 and 14 weeks in the diabetic WT mice.

Expression of GPX-1
The gene expression of Gpx1 was examined in the kidneys by TaqMan real-time PCR. The levels of gene expression of Gpx1 (Figure 11) in the control Tg and WT mice were similar. In the diabetic Tg and WT mice, gene expressions of Gpx1 showed significant increases compared with their corresponding control mice. At 8 weeks, the level of gene expression of Gpx1 in the diabetic Tg kidneys [16.7 ± 4.4 (arbitrary unit)] was significantly lower than in the diabetic WT kidneys [1987.0 ± 462.5 (arbitrary unit)] (P < 0.05). Thereafter, the expressions of Gpx1 in both the diabetic Tg and WT kidneys decreased at 14 weeks, and there was no significant difference between the diabetic Tg and the diabetic WT mice.


Figure 11
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  		Fig. 11 Expression of Gpx-1 in mice with diabetes. The expression of Gpx-1 mRNA transcripts was determined by TaqMan real-time PCR and was normalized to that of 18s rRNA in the same sample. #P < 0.05 compared to the diabetic WT kidneys, *P < 0.05 compared to the kidney of the control Tg or WT mice.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary data
 Conflict of interest statement
 References
 
In the experimental model of diabetic nephropathy used in this study, we have demonstrated that the expression of renal hL-FABP was upregulated in the diabetic Tg, and the production of MCP-1, MCP-3, TGF-β, {alpha}1COL I, FAS, Bax, RAGE, catalase and Gpx1 was suppressed in the diabetic Tg mice. HEL, a urinary marker of oxidative stress, was significantly higher in the diabetic WT than in the Tg mice at 8 weeks. The number of infiltrating macrophages at 8 and 14 weeks and the deposition of type IV collagen at 14 weeks were significantly lower in the diabetic Tg kidneys than in the diabetic WT kidneys. Tubulointerstitial damage was significantly attenuated in the diabetic Tg kidneys compared with the diabetic WT kidneys at 14 weeks. These results suggested the possibility that renal hL-FABP inhibited fibrosis and production of inflammatory cytokines and attenuated the tubulointerstitial damage of the diabetic model via reduction of oxidative stress.

The pathological role of renal L-FABP has not been extensively studied. Previously, we reported that the expression of renal L-FABP inhibited tubulointerstitial damage in the experimental protein overload model [11] and unilateral ureteral obstruction [12]. In this study using the STZ-induced diabetic model, the expression of renal hL-FABP significantly suppressed the expressions of MCP-1 and MCP-3, which are principal cytokines involved in chemotaxis and activation of macrophages [27–30]. TGF-β and {alpha}1COL I, which are associated with fibrosis [31,32], were also suppressed. In addition, the expression of renal L-FABP significantly attenuated macrophage infiltration, deposition of type IV collagen, the progression of tubulointerstitial damage and the expressions of FAS, Bax and RAGE in the diabetic Tg kidneys compared to the diabetic WT kidneys. These results suggested the possibility that L-FABP had a renoprotective function in various renal diseases.

HEL adduct is the lipid-peroxidation product generated only in the early phase of diabetic nephropathy by oxidative stress [33], and it was reported that urinary HEL significantly increased in the diabetic and hyperlipidaemic rats compared to control [25]. In the STZ-induced diabetic model, urinary HEL in the diabetic WT increased sharply at 8 weeks. From this result, oxidative stress might be strongly generated at 8 weeks and promote the upregulation of the genes expression of hL-FABP in the diabetic Tg and MCP-1, MCP-3, TGF-β and {alpha}1COL I in the diabetic WT at 8 weeks.

Because there is a possibility that L-FABP prevents the peroxidation of intracellular fatty acids by promoting fatty acid metabolism and by regulating gene expression involved in lipid metabolism, L-FABP may have a cytoprotective function [34]. Recently, it was reported that a cell line expressing L-FABP had a reduced concentration of intracellular reactive oxygen species (ROS) and that L-FABP had a role against oxidative stress [8]. In the diabetic Tg mice, urinary HEL level and gene expression of RAGE induced by oxidative stress were significantly lower than in the diabetic WT mice. Moreover, gene expressions of catalase [35,36] and Gpx1 [37,38], which are the principal antioxidant enzymes in the kidney, were significantly lower in the diabetic Tg than in the diabetic WT mice. At the same time, hL-FABP expression in the proximal tubules of diabetic Tg kidneys was upregulated. These results suggested the possibility of an antioxidative potential for renal hL-FABP.

Because severe tubulointerstitial damage was rarely observed in the STZ-induced diabetic mouse model, mice were subjected to uninephrectomy before STZ injection. Uninephrectomy is known to accelerate the development of advanced diabetic nephropathy [39]. In a preliminary study of uninephrectomized mice, blood pressure, urinary albumin and urinary hL-FABP showed no increases and were similar to mice without uninephrectomy. Tubulointerstitial damage was not induced by uninephrectomy. Therefore, in this study, only uninephrectomized mice served as the control group, which was compared with the diabetic mice.

Renal hL-FABP was upregulated, and urinary excretion of hL-FABP increased in the STZ-induced diabetic Tg mice. Because the Tg mice used in this study were generated by microinjections of the genomic DNA of hL-FABP including its promoter region, it is possible for the transcription of the hL-FABP gene in the Tg mice to be regulated in the same mode as in humans. Conceivably, the dynamics of hL-FABP in the experimental diabetic model might reflect its dynamics under similar pathological conditions in humans. In some clinical studies, the increased levels of urinary hL-FABP paralleled the progression of type 2 diabetic nephropathy [40,41].

Serum creatinine and urinary albumin levels, which reflect the degree of glomerular damage, were not improved in the diabetic Tg mice. Because hL-FABP is expressed in the proximal tubules, tubulointerstitial damage but not glomerular damage was ameliorated in the diabetic Tg mice compared to the diabetic WT mice.

The underlying cause for the increase of urinary hL-FABP in the diabetic Tg mice that led to the amelioration of tubulointerstitial damage was not determined in this study. Because hL-FABP has a high affinity and capacity to bind long-chain fatty acid oxidation products [7], it is likely that renal hL-FABP binds the oxidation products and excretes them into urine to relieve intracellular oxidative stress. Further study will be needed to clarify this problem.

In conclusion, we found that hL-FABP expressed in the proximal tubules was up-regulated in the STZ-induced diabetic model and suppressed the development of tubulointerstitial damage. Renal L-FABP is likely to have an effective endogenous antioxidant function. Our study suggested that the agents that up-regulate the expression of renal L-FABP in the proximal tubules could serve as an important therapeutic target for the prevention of tubulointerstitial damage in diabetic nephropathy.



   Supplementary data
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
Conflict of interest statement
 References
 
Supplementary data is available online at http://ndt.oxfordjournals.org.



   Conflict of interest statement
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 Conflict of interest statement
References
 
None declared.



   Acknowledgments
 
We acknowledge a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science. We thank Seiko Hoshino, Aya Sakamaki, Kayoko Yamashita and Junko Igarashi-Migitaka for technical assistance.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 Conflict of interest statement
 References
 

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Received for publication: 21. 6.08
Accepted in revised form: 18. 9.08


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