The Clinical And Basic Study Of Soluble Urokinase Receptor Levels And FSGS-like Lesions In Patients With IgA Nephropathy | Posted on:2017-03-05 | Degree:Doctor | Type:Dissertation | Country:China | Candidate:S M Guo | Full Text:PDF | GTID:1314330482994301 | Subject:Internal Medicine | Abstract/Summary: | PDF Full Text Request | Part Ⅰ Soluble urokinase receptor levels are correlated with focal segmental glomerulosclerosis lesions in IgA nephropathyBackground:Soluble urokinase receptor (suPAR) may be involved in the pathological mechanisms of focal segmental glomerulosclerosis (FSGS) changes. However, it remains unclear whether suPAR is correlated with the FSGS-like lesions in IgA nephropathy (IgAN).Methods:We measured the plasma suPAR levels in 138 patients with IgAN, and then their clinical and pathological relationships were analyzed.Results:We found that the plasma suPAR levels were significantly correlated with age and renal function by both univariate and multivariate analysis in our IgAN patient cohort. Female had higher plasma suPAR levels and no significant correlation was observed between plasma suPAR levels and 24-h urine protein and highly sensitive C-reaction protein with multivariate analysis. In our cohort, sixty of these IgAN patients could be diagnosed with a type of FSGS lesions. The plasma suPAR levels were higher in the IgAN patients with FSGS lesions than in the IgAN patients without FSGS lesions by univariate (P <0.0001) and multivariate (P<0.001) analysis adjusting for other predictor variables, which might be helpful to differentiate the pathological changes with and without FSGS lesions. And the optimal cutoff value was 1806 pg/ml in this study. The plasma suPAR concentrations were also associated with the degree of tubular atrophy/interstitial fibrosis in both univariate and multivariate analysis. In multivariate analysis, the plasma suPAR levels were correlated with the percentage of crescents, not global sclerosis and arterial lesions.Conclusions:Our study suggested that the plasma suPAR levels were associated with age, gender, renal function, the degree of tubular atrophy/interstitial fibrosis and the percentage of crescent formation. The plasma suPAR might be a potential predictor for the presence of FSGS pathological lesions in Chinese patients with IgAN.Part Ⅱ Urokinase receptor protien promotes mice primary podocyte movement abilityBackground:Although our previous study has shown that circulating suPAR levels were significantly correlated with FSGS lesions in patients with IgA nephropathy, whether uPAR protein promotes podocyte injury needs further research.Methods:We detected the purity of mice primary podocytes using immunofluorescence and flow cytometry technology. With would healing assay and transwell migration assay, we analyzed the effect of uPAR protein on mice primary podocyte movement ability. Moreover, we analyzed the expression differences of cell movement-related proteins between groups by Western blot technology.Results:Mice primary podocytes extracted had high expression levels of podocyte specific proteins including Nephrin, Synaptopodin and WT-1. The cell purity of primary podocytes was 98.4% by flow cytometry. We found that 10μg/ml uPAR recombinant protein significantly enhanced primary podocyte movement ability within 24 hours and 48 hours compared with normal control group by the would healing assay (P<0.05). The transwell migration assay also had similar results.5μg/ml (P<0.05) and 10μg/ml (P<0.01) uPAR recombinant protein both significantly promoted primary podocyte migration ability within 24 hours and 48 hours. 10μg/ml uPAR also could significantly enhance the expression levels of RhoA, Rac1 and Cdc42 proteins and reduce the production of Neprhin protein.Conclusion:uPAR protein could enhance mice primary podocyte movement ability thought the regulation of expression level of RhoA, Rac1 and Cdc42 proteins.Part Ⅲ Urokinase receptor affects mice mesangial cell proliferation depending on different culture conditionsBackground:Deregulated injury of mesangial cells is the common pathological changes of different kinds of kidney diseases. Urokinase receptor (uPAR) is a kind of multifunctional glycosylphosphatidylinositol-anchored protein. Its role in regulating pathological injuries of mesangial cells remains unclear.Methods:We detected the effect of uPAR recombinant protein on mice mesangial cell proliferation ability under the condition of normal, high glucose or serum-free using MTT and Brdu experimental techniques. Western blot method analyzed cell proliferation-related protein expression changes, including AKT, P-AKT, MEK, P-MEK, ERK and P-ERK protein. With the MTT method, we also assessed whether RGD peptide interfered the cell proliferation regulation process of uPAR recombinant protein.Results:5ug/ml and lOug/ml uPAR protein significantly reduced mice mesangial cell proliferation rate under normal medium within 24 hours and 48 hours. High glucose or serum-free treatment both significantly reduced mesangial cell proliferation ability within 24 hours or 48 hours compared with normal control group. uPAR enhanced the inhibition degree of cell proliferation initiated by high glucose. In contrast, uPAR promoted mesangial cell proliferation under serum-free medium. The role of 10μg/ml uPAR protein was most significant. Accordingly, compared with high glucose treatment group,10μg/ml uPAR protein significantly reduced mesangial cell protein expression of P-AKT, MEK, P-MEK and P-ERK under high glucose medium. On the contrary, 10μg/ml uPAR protein significantly increased expression levels of these proteins of mesangial cells under serum-free medium. The expression levels of the AKT and ERK proteins did not significantly change. RGD peptide could significantly inhibit the ability of promoting mesangial cell proliferation of uPAR protein under serum-free medium, while it could not significantly antagonize uPAR-induced inhibition process of mesangial cell proliferation under high glucose.Conclusion:uPAR could further inhibit mice mesangial cell proliferation under high glucose medium, and promote mice measangial cell proliferation under serum-free medium thought the regulation of expression level of P-AKT, MEK, P-MEK and P-ERK. Integrin protein is involved in the process of promoting mice mesangial cell proliferation of uPAR protein under serum-free medium.Part Ⅳ The association between urokinase receptor and complement pathway activationBackgroud:Our previous studies have shown that complement pathway activation and plasma suPAR level both were correlated with the FSGS-like lesions in patients with IgA nephrology. However, it remains unclear whether there is a relationship between complement pathway activation and the expression of suPAR.Methods:We detected the plasma suPAR levels of 210 patients with IgA nephrology, and analyzed their correlation with glomerular mesangial area complement C3 deposition. Then the expressions of C3aR and C5aR of mice mesangial cells and primary podocytes were detected using RT-PCR technology. With western blot technology, we analyzed the effect of different concentrations of uPAR protein on the expressions of C3aR and C5aR proteins of mice mesangial cells and primary podocytes. And we also detected the impact of different concentrations of complement C3a and C5a on the expression of uPAR protein of mice mesangial cells and primary podocytes.Results:IgAN patients with 3+-4+glomerular mesangial area C3 deposition had significantly higher plasma suPAR levels compared with the IgAN patients with 2+ C3 deposition (P<0.001) and 0~1+C3 deposition (P<0.0001). Mice mesangial cells and primary podocytes both expressed C3aR and C5aR mRNA. uPAR protein significantly enhanced the expressions of C3aR and C5aR proteins in mice mesangial cells and primary podocytes in a does-dependent manner compared with control group. In turn, complement C3a and C5a also could both significantly promote the expression of uPAR protein in mice mesangial cells and primary podocytes in a does-dependent manner.Conclusion:There may be an interaction relation between uPAR protein expression and complement pathway activation in kidney disease. | Keywords/Search Tags: | soluble urokinase receptor, focal segmental glomerulsclerosis, IgA nephropathy, urokinase receptor, primary podocyte, cell movement ability, mesangial cell, high glucose, serum-free, complement pathway, renal inherent cell | PDF Full Text Request | Related items |
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