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Prevention and reversal of renal injury by leptin in a new mouse model of diabetic nephropathy

Takayoshi Suganami^*,1, Masashi Mukoyama^*,2, Kiyoshi Mori^*, Hideki Yokoi^*, Masao Koshikawa^*, Kazutomo Sawai^*, Shuji Hidaka^*, Ken Ebihara^*, Tomohiro Tanaka^*, Akira Sugawara^*, Hiroshi Kawachi, Charles Vinson, Yoshihiro Ogawa^*,1 and Kazuwa Nakao^*

^* Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, Japan;
Department of Cell Biology, Institute of Nephrology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan; and
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

²Correspondence: Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: muko{at}kuhp.kyoto-u.ac.jp

SPECIFIC AIMS

There has been a long debate about the reversibility of diabetic nephropathy, and the problem has been difficult to approach because of the lack of suitable animal diabetic nephropathy models closely resembling human diabetic glomerulosclerosis. We investigated and evaluated whether renal injury in a mouse model of lipoatrophic diabetes (A-ZIP/F-1 mice; A-ZIPTg/+) is consistent with clinical diabetic nephropathy. We examined whether leptin is effective in the prevention or treatment of renal injury in A-ZIPTg/+.

PRINCIPAL FINDINGS

1. Characteristics of renal injury in A-ZIP/F-1 mice
Lipoatrophic diabetes is a disorder characterized by paucity of adipose tissue, severe insulin resistance, and elevated triglycerides. A-ZIPTg/+, which express a protein that inactivates basic-zipper transcription factors in adipose tissue under the control of aP2 promoter, have been reported to exhibit severe lipoatrophic diabetes. Histological analysis revealed marked glomerular hypertrophy and mesangial expansion in A-ZIPTg/+ compared with wild-type mice (+/+) (Fig. 1 A–C). These changes were diffuse and global, and compatible with clinical diabetic nephropathy. Quantitative analysis revealed that the glomerular cross-sectional area in A-ZIPTg/+ was 1.6-fold higher than that in +/+ at 4 months and 1.9-fold higher at 10 months of age (Fig. 1D) . By immunohistochemistry and Northern blot analysis, transforming growth factor-ß and extracellular matrix proteins were markedly up-regulated in glomeruli of A-ZIPTg/+ compared with +/+. No apparent macrolipid deposits were observed in the kidney of A-ZIPTg/+ by Oil Red O staining (Fig. 1E ), although A-ZIPTg/+ showed remarkably high levels of serum triglyceride and cholesterol. Electron microscopic analysis revealed diffuse thickening of glomerular basement membrane, foot process effacement of podocytes, and marked expansion of mesangial matrix in A-ZIPTg/+ (Fig. 1F ) compared with +/+ (Fig. 1G ). Since recent studies have indicated that podocyte loss contributes to the progression of diabetic nephropathy in type 2 diabetic patients, we examined various podocyte markers by immunohistochemistry in order to elucidate podocyte injury in this diabetic model. Immunostaining of several podocyte markers including nephrin and WT-1 was apparently reduced in glomeruli of A-ZIPTg/+ compared with +/+. Urinary albumin excretion in A-ZIPTg/+ was significantly increased at 4 months (14-fold of +/+) and was further increased at 10 months (81-fold of +/+). These findings are compatible with functional and histological features of clinical diabetic nephropathy.

View larger version (111K): [in this window] [in a new window]   Figure 1. Representative renal histology of 10-month-old +/+ (A) 4-month-old A-ZIPTg/+ (B), and 10-month-old A-ZIPTg/+ (C). Periodic acid-Schiff stain, 400x. D) Quantitative analysis of glomerular hypertrophy. *P < 0.01, n = 6–7. E) Oil Red O staining of the kidney in A-ZIPTg/+. Inset: a positive control in the adipose tissue. Electron microscopy of A-ZIPTg/+ (F) and +/+ (G). Arrows: diffuse thickening of glomerular basement membrane. Arrowheads: foot process effacement of podocytes. #: marked expansion of mesangial matrix. Mes: mesangial cell. Pod: podocyte. Bow: Bowman’s capsule. Scale bar indicates 5 µm.

2. Prevention of renal injury in A-ZIP/F-1 mice by crossing leptin transgenic mice
We next genetically crossed A-ZIPTg/+ and leptin transgenic mice (LepTg/+) to produce double transgenic mice (A-ZIPTg/+:LepTg/+) in order to investigate the long-term effects of leptin on the development of renal injury (we previously reported that administration of leptin almost completely normalized metabolic disorders in A-ZIPTg/+). LepTg/+ are expressing mouse leptin cDNA in the liver under the control of serum amyloid P component promoter. Mean serum leptin concentrations were: +/+, 7.7 ng/mL; A-ZIPTg/+, 1.8 ng/ml; LepTg/+, 31 ng/mL; A-ZIPTg/+:LepTg/+, 52 ng/mL. LepTg/+ showed normal renal function and apparently normal histology at 10 months of age (Fig. 2B ), comparable to +/+ (Fig. 2A ). The double transgenic LepTg/+:A-ZIPTg/+ exhibited not only marked inhibition of albuminuria but also complete prevention of glomerular hypertrophy and mesangial expansion compared with A-ZIPTg/+ (Fig. 2C, D ). Loss of podocyte number and up-regulation of extracellular matrix proteins and TGF-ß1 gene expression were completely inhibited (Fig. 2 E-I ). LepTg/+ showed significant up-regulation of collagen type IV and fibronectin gene expression compared with +/+ (Fig. 2 F-I ), but the extent was not remarkable. These findings indicate that chronic overexpression of leptin effectively prevented the development of renal injury in A-ZIPTg/+.

View larger version (76K): [in this window] [in a new window]   Figure 2. Representative renal histology of 10-month-old +/+ (A), LepTg/+ (B), A-ZIPTg/+ (C), and LepTg/+:A-ZIPTg/+ (D). Periodic acid-Schiff stain, x400. E) Podocyte number. F–I) Renal gene expression of COL4A1, fibronectin, and TGF-ß1. *P < 0.05, **P < 0.01 vs. +/+, #P < 0.05, ##P < 0.01 vs. A-ZIPTg/+, n = 5–6.

3. Reversal of renal injury in A-ZIP/F-1 mice by continuous leptin administration
To further investigate whether advanced diabetic nephropathy is reversible, we examined the effects of continuous leptin administration on renal injury in 10-month-old A-ZIPTg/+. Mean serum leptin concentrations after the 2 wk treatment of leptin or saline were: saline-treated group, 1.3 ng/mL; leptin-treated group, 40 ng/mL. Metabolic disorders were markedly ameliorated in the leptin-treated group compared with the saline-treated group. Glomerular hypertrophy as well as TGF-ß up-regulation was significantly ameliorated by leptin treatment. Moreover, the leptin-treated group exhibited 66% reduction of albuminuria compared with the saline-treated group. These findings indicate that leptin treatment can reverse renal injury of A-ZIPTg/+ functionally and histologically, at least partly, even at the advanced stage of diabetic nephropathy.

CONCLUSIONS AND SIGNIFICANCE

In the present study, A-ZIP/F-1 mice exhibited massive proteinuria as well as diffuse and global glomerular lesions characterized by marked glomerular hypertrophy, mesangial expansion, foot process effacement, and thickening of glomerular basement membrane. TGF-ß as well as extracellular matrix gene expression and protein were markedly increased in the kidney of A-ZIP/F-1 mice. These findings in A-ZIP/F-1 mice are consistent with functional and histological features of clinical diabetic nephropathy and seem to be more pronounced than those in other diabetic models. We propose that A-ZIP/F-1 mice can be a new mouse model of human diabetic nephropathy, although lipoatrophic diabetes is not common with regard to the pathogenesis. Similar to most other animal models of diabetic nephropathy, there was no typical nodular lesion developed until 10 months of age in this model.

In the present study, leptin completely prevented the development of renal injury in A-ZIP/F-1 mice throughout the observation period. Continuous administration of leptin significantly ameliorated not only metabolic disorders but also established renal injuries (i.e., glomerular hypertrophy with marked mesangial expansion, up-regulation of TGF-ß and extracellular matrix, and proteinuria). These findings suggest the potential of leptin to prevent or to treat renal injury in clinical lipoatrophic diabetes. Several previous studies showed the reversibility of established diabetic nephropathy by pancreas transplantation or with anti-TGF-ß antibody. Our data reveal significant reduction of TGF-ß expression in the glomeruli after the treatment of leptin, suggesting the causative role of TGF-ß in the progression of renal injury in A-ZIP/F-1 mice. Further study is needed to explore whether renal injury returns to normal by the long-term treatment of leptin.

It has been reported that high-dose leptin administration to rats up-regulates TGF-ß1 and collagen IV expression in the glomeruli and causes mild proteinuria, suggesting a possible pathogenic role of leptin in obesity-related glomerulopathy. It has been reported that leptin exerts profibrotic action in the liver. Consistent with these reports, LepTg/+ showed statistically significant up-regulation of collagen type IV and fibronectin gene expression at 10 months of age, although no apparent histological and functional alterations were observed in LepTg/+ compared with the wild-type. Serum leptin concentrations in LepTg/+ and in mice with continuous leptin administration are within the levels almost comparable to those of the patients with morbid obesity. Collectively, leptin seems to be primarily profibrotic on the glomerulus but has potential to prevent or reverse renal damage by normalizing metabolic disorders including hyperglycemia and hyperlipidemia.

Regarding the clinical application of leptin treatment on diabetic nephropathy, it is important to clarify the effects of leptin on diabetes. We have reported that leptin treatment alone is effective in KKA^y mice, a model of type 2 diabetes at younger ages when they are of normal weight. In contrast, hyperleptinemia does not prevent the progression of diabetes in KKA^y mice at older ages when they develop obesity at least partly because of obesity-induced resistance to leptin. In calorically restricted KKA^y mice, however, leptin can accelerate recovery from diabetes. It is conceivable that leptin treatment with diet therapy might be effective in renal injury of obese type 2 diabetic patients. We have recently reported that a combination therapy of leptin and insulin is effective for the treatment of hyperglycemia in streptozotocin-induced diabetic mice, although administration of leptin alone has no effects on blood glucose concentrations. Taken together, these data suggest that leptin may be therapeutically useful as an antidiabetic agent for various types of diabetes.

Our data summarized in the schematic diagram (Fig. 3 ) clearly show that the effects of leptin on renal injury in A-ZIP/F-1 mice, a new mouse model of diabetic nephropathy. Leptin can prevent and reverse glomerular injuries in A-ZIP/F-1 mice, suggesting the potential therapeutic usefulness of leptin for treating clinical nephropathy in lipoatrophic diabetes. This mouse model will provide a new tool to analyze the molecular mechanism underlying the progression and reversal of diabetic nephropathy.

View larger version (34K): [in this window] [in a new window]   Figure 3. Prevention and reversal of renal injury by leptin in A-ZIP/F-1 mice.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2183fje;

¹ Present address; Department of Molecular Medicine and Metabolism, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.

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