The Relationship Between Zinc-alpha2-glycoprotein And Renal Interstitial Fibers In Primary Glomerular Disease And The Degree Of Albumin-induced EMT In NRK-52E Cells

Objective Renal tissue fibrosis plays a crucial role in the evolution and prognosisof primary glomerular disease. Currently, there is no effective diagnostic method besiderenal biopsy. Zinc alpha2-glycoprotein(ZAG) is a multifunctional glycoprotein，expresses in renal tubular epithelial cell, and the change of urine ZAG may representrenal tubular-interstitial damage. Our experiment is to study the relationship betweenZAG and the degree of renal interstitial fibrosis in primary glomerular disease, inorder to find the new biology mark to renal tissue fibrosis in diagnose primaryglomerular disease.Albumin overload can induce renal tubular epithelial transform into mesenchymal.Our study also discussed the relationship between ZAG and the degree of renal tubularepithelial transform into mesenchymal(EMT) in renal tubular epithelial cell(NRK-52E), provided the theory and experimental evidence for the diagnosis of renalinterstitial fibrosis in primary glomerular disease.Methods (1)16patients were selected, who were diagnosed as primary glomerulardisease with the method of Percutaneous renal biopsy.20healthy persons wereselected. We collected blood samples and urine samples in the morning and patents’general information(gender, age, Scr, BUN, Cys C, Alb, TRIG, CHOL and24-hoururine total protein). Urine and serum ZAG were detected by ELISA. Calculate theratio of urine ZAG and urine albumin concentration. Masson staining was used inhistopathology in order to evaluate the level of renal tissue fibrosis at low magnification. We analyzed the relationship between the blood and urine ZAGconcentration with the level of serum bio-indicates. Furthermore, we observed therelationship between urine ZAG and renal interstitial fibrosis in renal biopsy. Allpatients were informed consent.(2) NRE-52E cells were cultured in different concentrations of d-BS (0、0.5、1、5、10、30mg/ml) for48hour, and10mg/ml d-BSA for different times (0、2、6、12、24、48hour). The total protein was collected. The level of α-SMA protein and ZAGwere detected by western blotting method.Results (1) Compared with healthy control, the levels of blood ZAG, urinal ZAG,cholesterol were higher significantly (P<0.05), there was no different in gender, age,blood urea nitrogen, serum creatinine, triglyceride between the two groups.(2) There wereno relationships between the serum and urine ZAG levels with serum urea, Cys C，BUN, Scr, TRIG, CHOL, eGFR (p>0.05).(3) The levels of the interstitial fibrosiswere evaluated by the Banff classification. The outcomes showed:0grade (0case),1grade (9cases),2grade (7cases),3grade (o cases). The main pathology types were:IgA nephropathy (7cases), Mild mesangial proliferative glomerulonephritis(5cases),Ⅱphase membranous nephropathy(2case), Hyperplasiaof sclerosing glomerulonephrit–is(1case), anaphylactic purpura nephritis (mild mesangial proliferative glomerulonep-hritis)(1case).(4) the degrees of tissue fibrosis in16patients with primary glomerulardisease were positively correlated with urinal ZAG, serum cystatin C.(5) InducedNRK-52E cells by different d-BSA concentrations for48hours showed increasedZAG and α-SAM protein expressions. The expressions of ZAG and α-SAM increasedon concentration–dependent manner.(6) Induced NRK-52E cells by10mg/ml d-BSAfor different times(0、2、6、12、24、48hour) showed that the expressions of ZAG andα-SMA increased at24h and48h, and the expression of ZAG and α-SMA proteins at48h were higher significantly. The expressions of ZAG were time-dependent.(7) Withthe increasing expression of α-SMA in NRK-52E cells, the expression of ZAGincreased too.Conclusions (1) The levels of the serum and urine ZAG in patient with primaryglomerular disease were higher than health control.(2) The urine ZAG concentration is related with the level of renal tissue fibrosis, the urine ZAG may be a biomarker forthe diagnosis of renal tissue fibrosis.(3) The expression of ZAG increased onconcentration-and time-dependent manners in d-BSA induced-NRK-52E cell.(4)With the increasing expression of α-SMA in NRK-52E cells, the expression of ZAGincreased too. ZAG may be associated with EMT in d-BSA induced-NRK-52E cell.