Effects Of ERK Signal Pathway On Extracellular Matrix Synthesis Of Human Proximal Tubular Epithelial Cells Treated With Albumin

Background and Objective Tubulointerstitial fibrosis(TIF) is the final common pathway by which all chronic kidney diseases (CKD) leads to end stage renal disease(ESRD), the main pathological features of which are proliferation of renal interstitial fibroblasts and excessive accumulation of extracellular matrix such asⅠ,Ⅱ,Ⅲ,Ⅳcollagen, fibronectin and laminin. Long studies have shown that proteinuria is not only a sign of kidney damage, but also as an independent risk factors involved in renal disease progression. albumin, the dominant protein in proteinuria,has been shown to be involved in tubulointerstitial fibrosis by inducing the excessive accumulation of extracellular matrix. Many studies have shown mitogen-activated protein kinase (MAPK) involved in normal renal physiology and various forms of renal injury, including renal fibrosis. Extracellular signal-regulated kinase(ERK) signaling pathway is one of the key members of MAPKs'family, its specific inhibitor PD98059 can block the activation of ERK signaling pathway. To further clarify the impact of proteinuria on extracellular matrix of renal tubular epithelial cells, we have used human proximal tubular epithelial cell strain—human kidney epithelial cells(HK-2 cells) treated with human serum albumin in vitro to observe the synthesis of extracellular matrix, and through observation the effect of PD98059 on extracellular matrix synthesis in human serum albumin-treated HK-2 cells to explore the mechanisms of proteinuria-induced tubulointerstitial fibrosis.Methods Cultured HK-2 cells in vitro were randomly divided into 4 groups:normal control group (no irritants,group A);human serum albumin treated group (5mg/ml human serum albumin, group B);ERK signaling pathway inhibitor PD98059 treated group (10μmol/L PD98059,group C);human serum albumin and PD98059 co-treated group (treated with 5mg/ml human serum albumin 45 min after 10μmol/L PD98059 pretreatment,group D).The expression of matrix metalloproteinase-9(MMP-9),tissue inhibitor of metalloproteinase-1(TIMP-1) and type IV collagen(col-Ⅳ) mRNAs were detected by RT-PCR at four time points (0,12,24,48 h).Results①Compared with group A:MMP-9 mRNA expression in group B increased first and then decreased(all p<0.01),and it reached a peak at 12 h;TIMP-1 mRNA expression in group B increased in a time-dependent way(all p< 0.01),and it reached a peak at 24 h;the ratio of TIMP-1 to MMP-9 in group B decreased first and then increased(all p<0.01),and it was higher than group A from 24 h;col-ⅣmRNA expression in group B also increased in a time-dependent way(all p< 0.01),and it reached a peak at 24 h.②Compared with group B,MMP-9 mRNA expression in group D decreased markedly (p<0.01);at 12 h and 24 h,it's expression was lower than group B but higher than group A(all p<0.01);at 48 h,it's expression was lower than group B and group A(all p<0.01).TIMP-1 mRNA expression in group D decreased markedly when compared with group B(p< 0.01);at 48 h,the expression of TIMP-1 mRNA had no difference between group D and A(p>0.05).Compared with group B,the ratio of TIMP-1 to MMP-9 in group D increased first and then decreased(p<0.05 or p<0.01);at 12 h,it's ratio was higher than group B but lower than group A(p<0.01);at 24 h,it's ratio was lower than group B,it's ratio had no statistical difference compared with group A(p>0.05);at 48 h,it's ratio was lower than group B but higher than group A(all p<0.01).col-ⅣmRNA expression in group D decreased markedly when compared with group B(p<0.01);at 48 h,the expression of col-ⅣmRNA had no difference between group D and A(p> 0.05).③The expression of MMP-9,col-Ⅳ,TIMP-1 mRNA and the ratio of TIMP-1 to MMP-9 had no difference between group C and A at each time point(all P>0.05).Conclusion 1,After treated with human serum albumin, the expression of MMP-9 mRNA in HK-2 cells first increased and then decreased;TIMP-1 mRNA expression was upregulated in a time-dependent way in HK-2 cells;the ratio of TIMP-1 to MMP-9 decreased first and then increased;the expression of col-ⅣmRNA was also upregulated in a time-dependent way in HK-2 cells.2,In human serum albumin-treated HK-2 cells,ERK pathway inhibitor PD98059 can reduce the expression of MMP-9, TIMP-1 and col-ⅣmRNA,and can partially correct the imbalance of TIMP-1/MMP-9,indicating that human serum albumin may promote ECM synthesis and inhibit ECM degradation through the ERK pathway,induce ECM accumulation and thus participate in tubulointerstitial fibrosis.