Polypeptide AcSDKP Reduces The Established Renal Tubulointerstitial Fibrosis Induced By Aristolochic Acid In Mice

Objective At present,the progression of chronic kidney disease(CKD)to the end-stage renal disease(ESRD)cannot be reversed artificially.Renal tubulointerstitial fibrosis is one of the key factors in this progression.This study was designed to study whether and how the polypeptide N-acetyl-seryl-aspartyl-lysyl-proline(AcSDKP)could reduce the established renal tubulointerstitial fibrosis induced by aristolochic acid.Methods Part ?: renal tubulointerstitial fibrosis was induced by aristolochic acid in mice.The C57BL/6 mice were intraperitoneally injected with 5mg/kg/day aristolochic acid(AA)for 5 days after the single nephrectomy.Genome-wide expression profiling of micro RNA(mi RNA)and m RNA was detected by deep sequencing in kidney samples of the aristolochic acid(AA)group and the self-control group.Several significantly different expressed mi RNA(DE mi RNA)and m RNA(DE m RNA)were validated by real-time RT-PCR.All DE m RNA enriched function and pathway were analyzed using Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)software.Part ?: the acute aristolochic acid nephropathy was induced in C57BL/6 mice by intraperitoneal injection with 10mg/kg/day aristolochic acid for 5 days in the AA group and the AA+AcSDKP group.An additional intraperitoneal injection with 1.0 mg/kg/day AcSDKP for 15 days was given to the mice in the AA+AcSDKP group after a week break.Meantime,the mice in the AA group and the control group were injected with 0.9%saline intraperitoneally.HE staining and Masson staining were used to detect changes in renal histopathology and fibrosis.The serum creatinine,blood urea nitrogen(BUN)and microalbuminuria of the mice were tested by a biochemical analyzer.The m RNA expression levels of Serpine1(plasminogen activator inhibitor-1(PAI-1)gene),the extracellular matrix degradation inhibitory factor-1(TIMP-1),the extracellular matrix degradation inhibitory factor-3(TIMP-3),interleukin-11(IL-11),and the transforming growth factor beta 1(TGF?1)were detected by real-time RT-PCR.Western blot was used to detect PAI-1 protein levels in mouse kidney,mouse renal primary fibroblasts cultured in vitro and the cell culture supernatant.Immunohistochemistry was used to detect the protein levels and locations of Alpha-smooth muscle actin(?-SMA),the inducible nitric oxide synthase(i NOS),the leukocyte differentiation antigen 4(CD4),and the leukocyte differentiation antigen 8(CD8).The co-expression of?-SMA and apoptosis-related protein Fas,Bax or bcl-2 in mouse kidney was detected by immunofluorescence.Part ?: the chronic aristolochic acid nephropathy was induced in C57BL/6 mice by intraperitoneal injection with 5 mg/kg/day aristolochic acid for 4 weeks in the AA group and the AA + AcSDKP group.An additional intraperitoneal injection with 1.0 mg/kg/day AcSDKP for 4weeks was given to the mice in the AA+AcSDKP group after 16 weeks break.Meantime,the mice in the AA group and the control group were injected with 0.9% saline intraperitoneally.HE staining and Masson staining were used to detect changes in renal histopathology and fibrosis.The serum creatinine,BUN and microalbuminuria of the mice were tested by a biochemical analyzer.The m RNA expression levels of Serpine1 (PAI-1 gene),TIMP-1,TIMP-3,IL-11,and TGF?1 were detected by real-time RT-PCR.Western blot was used to detect PAI-1 protein levels in mouse kidney,mouse renal primary fibroblasts cultured in vitro and the cell culture supernatant.Immunohistochemistry was used to detect the protein levels and locations of ?-SMA,i NOS,CD4,and CD8.The co-expression of ?-SMA and apoptosis-related protein Fas,Bax or bcl-2 in mouse kidney was detected by immunofluorescence.Results Part ?: Integrative micro RNA and m RNA expression profiling in acute aristolochic acid nephropathy in mice in this self-control study.The histopathology changes revealed the renal injury and tubulointerstitial fibrosis induced by aristolochic acid in mice.82 mi RNAs and 4605 m RNAs were differently-expressed between the AA treatment group and the self-control group.Part of them was validated by real-time RT-PCR.Profibrotic mi R-21 family,mi R-433 family,and mi R-132 family were significantly increased.Anti-fibrotic mi R-122-5p and let-7a-1-3p were significantly decreased.This study found 767 opposite direction differently-expressed pairs of mi RNAs and their m RNA targets.Among them,regulatory networks of mi RNAs and m RNAs were analyzed in KEGG enriched signaling pathways and the extracellular matrix(ECM)associated pathways.Part ?: Polypeptide AcSDKP reduces the established tubulointerstitial fibrosis in aristolochic acid acute nephropathy in mice.In the acute aristolochic acid nephropathy model,the polypeptide AcSDKP significantly reduced the AA-induced tubulointerstitial fibrosis,tubulopathy,and renal function injury.Compared with the control group,in the AA Group,there was significant increase in mRNA levels of PAI-1 and TIMP-1 and IL-11(pro-fibrosis factor),the protein level of PAI–1,and the number of myofibroblasts(?-SMA+),monocytes/macrophages(i NOS+),T Helper lymphocytes(CD4+)and cytotoxic T lymphocytes(CD8+)in mouse kidney.Aristolochic acid also induced a significantly increased resistance of apoptosis in renal myofibroblasts in the AA Group than the control group.Compared with the control group,the apoptotic bcl-2 was significantly up-regulated,and the pro-apoptotic Fas,Bax and the ratio of Bax/bcl-2 were significantly down-regulated in renal myofibroblasts(?-SMA+)in the AA group.The polypeptide AcSDKP treatment after AA treatment could significantly reverse the above AA-induced effects.There were significantly decreased m RNA levels of PAI-1,TIMP-1,TGF?1 and IL-11,and protein level of PAI-1 in AA+AcSDKP group than the AA group.In the AA+AcSDKP group,there were significantly decreased numbers of myofibroblasts(?-SMA+),monocytes/macrophages(i NOS+),T Helper lymphocytes(CD4+)and cytotoxic T lymphocytes(CD8+).In the AA +AcSDKP group,the protein levels of apoptotic protein Fas and Bax,and the ratio of Bax/bcl-2 in myofibroblasts(?-SMA+),were significantly increased,and the BCL-2 protein level was significantly decreased.The mouse kidney primary fibroblasts were cultured in vitro.The peptide AcSDKP inhibited the up-regulated PAI-1 protein expression induced by TGF?1 in the primary fibroblasts and the cell culture supernatants. Part ?: Polypeptide AcSDKP reduces the established tubulointerstitial fibrosis in aristolochic acid chronic nephropathy in mice.In the aristolochic acid chronic kidney disease model,the polypeptide AcSDKP also significantly reduced the established AA-induced tubulointerstitial fibrosis,tubulopathy,and renal function injury.In the AA+AcSDKP group,the polypeptide AcSDKP reversed the significantly increased m RNA expression of PAI 1,TIMP–1,TGF?1 and IL-11,the protein level of PAI-1,the protein level of Bcl-2 in myofibroblasts(?-SMA+),as well as the number of the myofibroblasts(?-SMA+),macrophages/monocytes(i NOS+),T Helper lymphocytes(CD4+)and cytotoxic T lymphocytes(CD8+)in the AA group.The significantly increased levels of Fas and Bax,as well as the increased ratio of Bax/bcl-2were reversed by AcSDKP in the AA group.Conclusion In this study,we found that AA-induced renal tubulointerstitial fibrosis could be reduced by the polypeptide AcSDKP in mice.Firstly,renal tubulointerstitial fibrosis could be induced by Aristolochic acid in mice,accompanied with the significantly changed genome-wide expression profile of mi RNA and m RNA.Secondly,both in aristolochic acid acute and chronic nephropathy model,AA-induced renal tubulointerstitial fibrosis could be reduced by AcSDKP in mice.On the one hand,it was contributed by reducing the synthesis of ECM components,as AcSDKP reduced AA-induced increase of the number of renal myofibroblasts and lymphocytes,the resistance of apoptosis in myofibroblasts,and pro-fibrosis factors TGF?1 and IL-11.On the other hand,it was contributed by the promoting of ECM degradation,as AcSDKP reduced AA-induced expression of PAI-1 and TIMP-1,leading to an increased activity of MMPs and plasmin on the extracellular matrix(ECM)degradation.