Mechanism And Clinical Value Of Resistin-like Molecule β In Early Type2Diabetic Nephropathy

Part IScreening biomarkers of early diabetic nephropathy with antibody microarray technologyObjective:To find one or several biomarkers for incipient diabetic nephropathy (DN) through screening a number of proteins with antibody microarray technology.Methods:Using RayBiotech high-throughput antibody microarray,507proteins were screened in early type2DN patients, type2diabetic patients without nephropathy and the normal controls, in order to find the serological markers may be associated with the early stage DN.Result:Several proteins were differential expressed in early DN patients, diabetic patients without nephropathy and the normal controls. There are some differential expressed protein models. One group of34proteins, including resistin-like molecule p (RELMp), CXCR3, D6, Fas/TNFRSF6, FGF-12, can be detected only in DN patients. One group of6proteins (RANK, AgRP, BCMA, TIMP-3, CCR4and Smad-4) in early DN patients are higher than diabetic patients without nephropathy and the normal controls, while expression in diabetic patients without nephropathy are higher than the normal controls. One group of10proteins, including TGF-P, IL-18BPA, HCC-4/CCL16, TIMP-1and TREM-1, in early DN patients are higher than diabetic patients without nephropathy and the normal controls, while expression in diabetic patients without nephropathy are similar to the normal controls. Another group of18proteins, including M-CSF R, IL-1R9, and TNF-alpha can only be detected in DN patients and diabetic patients without nephropathy, and expression in DN patients are higher than the diabetic patients without nephropathy.Conclusions:Some proteins differentially expressed in early DN patients, diabetic patients without nephropathy and normal controls. The proteins expressed only in early DN patients, or expressed significantly higher in DN patients than diabetic patients without nephropathy and normal controls may be involved in the process of early onset of DN. Part IIRelationship between plasma RELMp and diabetic micro-albuminuria Objective:To verify the results from antibody microarray, measure the plasma levels of RELMβ in cross-sectional case-control study.Methods:1001subjects aged30-80years old were included in the study. There are654non-diabetic controls without albuminuria,123non-diabetic patients with albuminuria,152diabetes without albuminuria,59diabetes with micro-albuminuria and13diabetes with macro-albuminuria. Measure the plasma levels of RELMβ by ELISA assay. Correlation coefficients between RELMβ and clinical parameters were calculated by speraman rank correlation analysis. Risk factors and risk assessment were analysed by multiple linear regression and stepwise Logistic regression analysis.Result:The levels of RELMβ in diabteic micro-albuminuria patients (201.68±27.77pg/ml) were higher than diabetes without albuminuria (153.56±27.77pg/ml), non-diabetic controls without albuminuria (142.90±4.12pg/ml) and non-diabetic patients with albuminuria (141.41±8.85pg/ml)(P<0.05). There was no significant difference between fasting plasma RELMβ levels (150.92±1.49pg/ml) and OGTT2h RELMβ levels (151.89±1.50pg/ml)(P>0.05), and there was a strong positive correlation between them (r=0.95, P<0.05). In different case-control groups, no significant difference was found between different gender and age groups (P>0.05). There were high correlation between plasma RELMβ levels and waist circumference, waist-hip ratio, waist-stature ratio, OGTT2h glucose, glycated hemoglobin, serum creatinine, urine creatinine, glomerular filtration rate, urinary albumin creatinine ration, uric acid, interleukin-1receptor antagonist (P<0.05). Glycated hemoglobin, hip circumference, body fat, glomerular filtration rate, total cholesterol and body mass index were independent prognostic factors of plasma RELMβ. Participants in the highest quartile of RELMp had an odds ratio of2.155(95%CI1.214-3.825) for developing diabetic micro-albuminuria compared with participants in the lowest quartile. The association remained significant after further adjustment for age, gender, BMI, blood pressure, glucose and total cholesterol. As a candidate biomarker for diagnosing diabetic micro-albuminuria, the AUC for RELMβ was0.661, with the sensitivity of55.2%and specificity of65.1%. In the elderly female population, the diagnostic efficiency was0.719, with the sensitivity of70.6%and specificity of68.8%.Conclusions:Plasma RELMβ levels were higher in diabetic micro-albuminuria patients. In different case-control groups, no significant difference was found between different gender and age groups. Participants with high plasma RELMβ levels had an increased risk for developing diabetic micro-albuminuria. RELMβ may be a candidate biomarker for diagnosing early DN. Part ⅢThe localization and biological function of RELMβ protein in kidneyObjective:To find the localization of RELMβ expression in kidney tissue and cells. Explore the RELMβ expression under high glucose and the biological function of RELMβ protein on mesangial cells.Methods:To establish the rat model of DM and DN with intraperitoneally STZ injection. Explore the localization of RELMβ expression in kidney tissue and cells using immunohistochenistry and immunofluorescence. Treat the mesangial cells with high glucose and high lysophosphatidylcholine, detect the RELMβ expression by western blotting and ELISA assay. Treat the mesangial cells with different concentrations exogenous RELMβ protein, using the MTT assay to detect the proliferation of mesangial cells.Result:The urinary albumin excretion rate and kidney weight of DN SD rats increased significantly compared to the DM and normal SD rats. The blood glucose of DN and DM SD rats increased significantly compared to the normal SD rats, while body weight decreased significantly (P<0.05). The mesangial area of DN SD rats were significantly thicker than the DM and normal SD rats in PAS staining. The RELMβ protein was mainly expressed in glomerular mesangial area of the DN SD rats. The expression of RELMβ protein was higher in DN rats (grade3) than DM and normal rats (grade0). With immunofluorescence, RELMβ was mainly distributed in the cytoplasma of mesangial cells. The RELMβ protein secretion from mesangial cells increased under high glucose or high lysophosphatidylcholine condition in a time-dependent manner (P<0.05). High concentration of RELMβ protein (210ng/ml) can promote the proliferation of mesangial cells (P<0.05), while70ng/ml and140ng/ml RELMβ protein did not work.Conclusions:RELMβ protein was mainly expressed in mesangial cells in the kidney. RELMβ protein expressed in DN rats was obviously higher than in diabetic rats and normal rats. High glucose or high lysophosphatidylcholine can stimulate the mesangial cells to secrete RELMβ protein. RELMβ protein can promote the proliferation of mesangial cells, paticipate in the pathological process of early DN. Part IVThe effects of overexpression and interference RELMβ gene on theproliferation/apoptosis and signaling pathway of mesangial cells Objective:To explore the impact of RELMβ gene on the cell cycle, cell proliferation, cell apoptosis and MAPKs pathway of mesangial cells. Screen the possible cellular signal transduction pathway of RELMβ gene on mesangial cells.Methods:To construct a lentiviral vector of RELMβ gene overexpression or RNA interference and establish stable mesangial cell lines. Detect the cell proliferation by CCK-8assay, and detect the cell cycle and apoptosis by flow cytometry. Western blotting was to detect the change of MAPKs pathway. Search the differentially expressed gene in RELMβ-shRNA mesangial cell lines by cellular signal transduction pathway microarray. Real-time fluorescence quantitative PCR was to verify the PCR array results.Result:Overexpress RELMβ can stimulate the mesangial cells proliferation, and knockdown RELMβ gene can inhibit the mesangial cells proliferation (P<0.05). High glucose can reverse the inhibition under RELMβ gene knockdown. Overexpress RELMβ gene can decrease the cells number in cell G0-G1phase from74.36%to68.80%and increase the cells number in cell S phase from18.09%to23.20%(P<0.05). Knockdown RELMβ gene can increase the cells number in cell G0-G1phase from70.69%to87.14%and decrease the cells number in cell S phase from21.63%to11.59%(P<0.05), while high glucose and exogenous RELMβ protein can partially reversed the phenomenon. Overexpress RELMβ gene can upregulate the phosphorylation activity of p38MAPK and JNK, while RELMβ gene knockdown cells exert opposite effects, and high glucose and exogenous RELMβ protein can partially reversed it. P38MAPK inhibitor (SB202190) can significantly inhibit the mesangial cell proliferation caused by RELMβ overexpression. RELMβ gene has no effect on apoptosis of mesangial cells. Both the human signal transduction pathway finder PCR Array and RT-PCR test suggest that gene WISP1, IGFBP3, HK2, VEGFA, BCL2L1and so on are downregulated and gene CXCL9, CYP19A1, NOS2, PTGS2, TNF and so on are upregulated.Conclusions:RELMβ can promote human masangial cell proliferation and regulate the cell cycle of human masangial cell. RELMβ can paticipate in the pathological process of early DN by regulating the MAPKs pathway. WISP1, IGFBP3, CXCL9and so on may be important genes involved in the signal transduction pathway of diabetic nephropathy.