The Expression And Significance Of FABP4 In Tissues Of Normal Rat And In Kindey Of Type 1 Diabetic Rats

Objective: Diabetic nephropathy (DN) is one of the most common complications of diabetes and has become the major cause of end-stage renal failure. Currently, there is a lack of diagnostic biomarker of early DN. Adipocyte fatty binding protein 4 (A-FABP or FABP4) is a member of the FABPs family and was first detected in adipose tissue. FABP4 mRNA is transcriptionally controlled by intracellular fatty acids, promotes the transportation of free fatty acid (FFA) and thus modulates systemic insulin sensitivity and energy metabolism. Long-chain fatty acid, insulin and agonist of peroxisome proliferator-activated receptor-γ(PPAR-γ) can induce the expression of FABP4. Circulating FABP4 has been shown to be closely associated with diseases of lipid metabolism disorder such as metabolic syndrome (MS) and atherosclerosis, and played an important role in the development of these diseases. Higher FABP4 levels can predict the development of MS and type 2 diabetic mellitus. Circulating levels of FABP4 are inversely associated with endothelial function in type 2 diabetic mellitus, and are an effector of vascular damage in type 2 diabetic mellitus.In our syudy, distribution, localization and content of FABP4 were detected Firstly by immunohistochemistry and western blot in normal Wistar rats;Then, time course of the fasting serun FABP4, FFA, and serum lipid were observed in STZ-induced diabetic rats model and finally expressive pattern and content of FABP4 in diabetic renal tissue were anaylized by immunohistochemistry and western blot at the 2^th ,4^th, 8^th, and 12^th weeks after injecting STZ.Materials and Methods1 Distribution, localization and content of FABP4 in various tissues16 Wistar rats (males and females were each 8) were used in the expriment, 10 of them (males were 5, females were 5) were perfusion fixed with 4% paraformaldehyde. The slices of the brain, eyes, cardiac muscle, lung, liver, kidney, fat, skeletal muscle, small intestine, pancreas, bladder, ovary and testis were fixed in 4% paraformaldehyde for HE stain and FABP4 immunohistochemistry. Cardiac muscle, lung, liver, kidney, fat, skeletal muscle, small intestine, pancreas, bladder, ovary and testis of other 6 rats were obtained and abstracted total protein for FABP4 western blot. 2 Induction of 1 type diabetic model by injecting STZ and detecting fasting FFA, lipid and other biochemical indicatorsA week after hemi-nephrectomize, 14 male Wistar rats were randomly divided into two groups: 9 of them were induced type 1 diabetic mellitus (DM group) by giving a single intraperitoneal injection of 10mg/ml STZ at a dose of 60mg/kg. 5 of them only received an injection of the same volume of 0.1mol/L sodium citrate as control group (C group). After 72 hours of the injection, the blood glucose was determined by bleed caudal vein. The model of diabetes was considered to be successful when the blood glucose was≥16.7mmol/L. 24h urine samples of 14 rats were collected in metabolic cages for the measurement of 24 h urine albumin by Elisa at the 3rd day, 1^th, 2^th, 4^th, 8^th, 12^th weeks. Fasting serum samples were obtained by separated peripheral venous blood and storaged at -20°C for detecting FABP4, FFA, FBG, lipid (TG, T-CHO) and other biochemical indicators at the 1^th , 2^th, 4^th, 8^th, 12^th weeks.3 Detecting the change of FABP4 expressio of renal tissue in DM model40 male Wistar rats were randomly divided into two groups: control group (C group,16)and STZ-induced diabetic mice (DM group, 29), modeling liked part 2. Groups of 10 rats, each group containing 6 DM rats (DM group) and 4 control rats(C group) were sacrificed at the 2^th, 4^th, 8^th and 12^th weeks after STZ injection. Serum samples were obtained by separated peripheral venous blood and storaged at -20°C for detecting GLU. 3 rats of DM group and 1 rats of control group were perfusion fixed liked part 1. Partial renal tissues were fixed in 4% paraformaldehyde, embedded in paraffin and stained the sections for HE, PAS and immunohistochemistry of osteopontin (OPN) and ED1. Other rats were weighed the body weight (BW), kidney weight (KW). Partial renal tissures were fixed in PLP stationary liquid, dehydrated by sucrose solution, embedded in OCT, compound and stained the frozen sections for immunohistochemistry of FABP4. Partial renal cortices were obtained to abstract total protein for FABP4 western blot.Results:1 Localization of FABP4 in normal tissues of Wistar rat1.1 Immunohistochemistry: (1) FABP4 was immunolocalized to cytoplasm and nucelus of all Adipocytes. (2) FABP4 was immunolocalized to cytoplasm and nucelus of endotheliocytes (Ecs) of capillaries and small veins in most tissuses. Different tissuses exist remarkably different on the expression of FABP4: In the fat, cardiac muscle, skeletal muscle, small intestine and pancreas, almost all Ecs had positive expression; In lung, kedney, liver , bladder, testis and ovary, FABP4 was immunolocalized to Ecs of partial capillaries and small veins; Eye and brain had no expression. (3) Macrophages: FABP4 was expressed in macrophages only in lung and Kupffer cell. (4) FABP4 was also immunolocalized in cytoplasm and nucelus of transitional epithelial cells in mucous membrane of renal pelvis and bladder, cytoplasm and nucelus of the lutein cells in ovary and cytoplasm of islet cells.1.2 Western blot: FABP4 protein was expressed in the cardiac muscle, pancreas, fat, lung, renal cortex, small intestine, testes, skeletal muscle, ovary, bladder. The most content of FABP4 is the fat, the second is the cardiac muscle, renal cortex is lower. Liver had no expression.2 Time course of fasting FFA and lipid in DM rats2.1 Compared with control group, fasting FABP4 levels in DM group were obviously higher, with statistical significance (p<0.05) at different times. There were no difference significant among four DM groups.2.2 Compared with control group, fasting FFA levels in DM group were obviously higher, with statistical significance (p<0.05) at different times. There were no difference significant among five DM groups. 2.3 The levels of FBG, TG, T-CHO and BUN at different time points in DM group were significantly higher than those in C group (p<0.05) except T-CHO at the 2^th week.2.4 24h urine albumin quantification in DM group were obviously higher at the 3rd day, significantly higher than those in C group and increased gradually at the 1^th, 2^th, 4^th, 8^th weeks.2.5 Serum ALB levels at different time points in DM group was higher than those in C group. There were difference significant only at the 8^th and 12^th week (p<0.05).2.6 The results of correlation analysis: The fasting FABP4 positively correlated FFA(r=0.512, p=0.000),FBG(r=0.434,p=0.003),TG(r=0.360, p=0.017),and 24h urine albumin (r=0.583,p=0.000) and has negative correlation with serum ALB (r=-0.485,p=0.001). But it has no correlation with T-CHO. The fasting FFA positively correlated with FBG (r=0.490, p=0.000), TG (r=0.542, p=0.000), T-CHO (r=0.37, p=0.002) and 24h urine albumin (r=0.634, p=0.000), but has negative correlation with serum ALB (r=-0.278, p=0.002).3 The expression and significant of FABP4 in renal tissues of DM rats3.1 Clinical parameters, GLU and glomerular diameter: The BW of DM group was obviously lower than C group (p<0.05). The KW, KW/BW, GLU, and glomerular diameter were significantly higher than those in C group (p<0.05).3.2 Morphology: Protein casts were formed in some tubules at the 2^th week. Vacuolar degeneration was observed in some tubular epithelium at the 2^th week. These lesions become more serious at the 12^th week. Mild hyperplasia mesangial matrix in glomerulus was observed in DM group at the 4^th week. Proliferation of mesangial cells was observed at the 8^th week.3.3 Count of ED1 positive cells both in glomeruli and renal interstitium: The numbers both in glomerulus and interstitium of renal cortex were significantly higher than those in C group (p<0.05).3.4 The expression of FABP4: Expression of FABP4 both in Ecs of capillaries and small veins become widely and extend to the surface of renal cortex at the 2^th week. Western blot of renal cortex showed that the expression of FABP4 protein in DM group were significant higher from the 2^th week (p<0.05). From 4^th week, positive cells was observed in epithelium of Bowman's capsule and in glomerulus. But as one marker of tubular damage, there were no significant difference expression of OPN between DM group and C group.Conclusion:1 Confirming there was abundant expression of FABP4 in adipocytes. macrophages of FABP4 expression were only observed in those of lung and Kupffer cells of liver. These findings suggest that the different results both in vivo and in vitro. FABP4 expression of macrophage at physiological conditions was different with that in pathological conditions.2 We observed systematically the distribution and the quantitation of FABP4 in Wistar rats normal tissues for the first time. In many tissues' capillaries and small veins, FABP4 was immunolocalized to cytoplasm and nucelus of Ecs, but in different organs, the expression of FABP4 in Ecs varied wildly. The different expression should be further studied. Although adipose tissue was considered as a source of circulating FABP4, our results support the hypothesis that endothelial cells of capillaries and venules is the important source of circulating FABP4.3 FABP4 was also immunolocalized in cytoplasm and nucelus of transitional epithelial cells in mucous membrane of renal pelvis and bladder, cytoplasm and nucelus of lutein cells and cytoplasm of pancreas. Distributive pattern of FABP4 may suggest its many functions.4 The levels of Fasting FABP4, FFA in DM group were significantly higher than those in C group, pointing out that increase fasting FABP4, FFA may be the early event in DM.5 In STZ Induced type 1 diabetic rats, the number of the positive staining of FABP4 in Ecs of capillaries and small veins in kidney has increased at the early period of nephrosis. Along with course of disease, and the change persisted to the 8^th week. But OPN, one marker of injured renal tubule, has no significant difference between DM group and C group. Further, we will detect the level of FABP4 both in serum and urine of these DM rats. Detecting FABP4 may be used at the same time to provide biomarker for early diagnosis of DN.