ADAM10 Mediates Proximal Tubule Development And Renal Fibrosis By Activating Notch Pathway

Background Disturbed intrauterine development increases the risk of renal disease,chronic kidney disease(CKD)can begin in early life and driven by environmental insults in utero.Accumulating evidence indicates that kidney development can be aberrantly programmed in response to adverse conditions and result in permanent structural and functional changes.At present,environmental pollution has been regarded as a risk factor of kidney diseases,and pesticide pollution is a prominent problem in China due to its widespread use and loose regulation.The Notch signaling pathway has been confirmed to play a significant role in kidney development,it is believed that NOTCH2 of Notch pathway promotes the formation of proximal tubules.In addition to the vital role in kidney development,Notch was also found to be involved in kidney fibrosis.A disintegrin and metalloproteinase domain 10(ADAM10),an upstream protease of Notch pathway,is also reportedly involved in renal fibrosis.However,how ADAM10 interacts with Notch pathway and causes renal fibrosis is not fully understood.Objective This study was aimed at exploreing the role of the ADAM10-Notch signal axis in proximal tubule development and renal fibrosis,and to identify potential targets for early intervention to prevent chronic kidney disease.Methods 8-week old ICR mice were used to establish prenatal chlorpyrifos exposure model.CPF 5mg/kg/d was administrated by subcutaneous injection in CPF-treated pregnant mice from pregnant day 7.5-11.5 while the controls were injected with DMSO.Embryonic kidney samples at E 12.5,E 14.5,E 16.5 and E 18.5 were obatained of experimental groups and the control group;offspring mice were sacrificed at 4-week(4 w),8-week(8 w),6-month(6 m),9-month(9 m),both kidney and blood samples were collected.Embryonic kidney samples at different time points were subjected to RNA transcriptome sequencing(RNA-seq),gene set enrichment analysis(GSEA)was performed to explore the Notch pathway enrichment.Gene chip of Notch signaling pathway was performed on normal embryonic kidneys at 4 time points to clarify the relationship between the expression trend of Adam10 and Notch pathway molecules.m RNA and protein levels of Adam10,Notch pathway molecules and fibrosis related factors of embryonic kidneys and adult kidneys were further detected.We used the antisense oligonucleotide Morpholino to block the expression of Adam10(Adam10-MO)to observe the kidney development,proximal tubules differentiation and the activity of Notch pathway in vitro.Knock down and overexpressing experiments of ADAM10 in human 293 T cells were performed to observe the changes in Notch pathway activity and fibrosis related factors.81 patients with pathological diagnosis of Ig A nephropathy(Ig AN)were included for correlational analysis.Renal interstitial fibrosis was assessed by Masson’s trichrome staining,Immunohistochemistry staining was performed to evaluate the ADAM10 expression score and NOTCH2 expression score.The Chi-square tests,correlation analysis,and Cox regression analysis were used to analyze the relationship between ADAM10 expression,NOTCH2 expression,degree of fibrosis and renal function.Results Gene set enrichment analysis of RNA-seq data showed that Notch pathway was enriched in the embryonic kidneys of prenatal CPF exposure mice.We also found that the proximal tubule marker Aqp1 was enriched in the CPF group,while nephron progenitor-related markers were downregulated.Gene Chip of Notch pathway showed that Adam10 and Notch receptors Notch1,Notch2 had the same expression trend.RT-q PCR and western blotting confirmed that the m RNA levels and protein levels of Adam10,Notch1,Notch2 were increased in the CPF group,the nephron progenitor marker Six2 was decreased in the CPF group.Immunofluorescence and immunohistochemistry showed that the number of proximal tubules increased in the CPF group.After treated with Adam10-MO,the in vitro cultured embryonic kidneys showed hindered kidney development,reduced proximal tubules differentiation and decreased Notch pathway activity.Expressions of Adam10,Notch1,Notch2,and Aqp1 continued to increase in the offspring adult mice kidneys,and the number of proximal tubules also increased.The 6 m and 9 m mice showed spontaneous renal fibrosis kidneys in the CPF group,the positive Masson staining area increased,the expressions of interstitial markers increased.Transfected with si RNA-ADAM10 in human 293 T cells showed no significant reduce in Notch activity and fibrosis markers.Transfected both si RNA-ADAM10 and si RNA-ADAM17,Notch activity and fibrosis markers were down-regulated.Transfected with plasmid pc DNA-ADAM10-HA resulted in increased Notch activity and fibrosis markers levels.Chi-square test showed that ADAM10 expression was significantly correlated with NOTCH2 expression in Ig AN patients;ADAM10 and NOTCH2 expressions were significantly correlated with the degree of renal fibrosis;ADAM10 expression was significantly correlated with serum creatinine levels,Cystatin C levels,and e GFR levels.Correlation analysis showed the similar results.Univariate Cox regression analysis showed that ADAM10 expression levels,serum creatinine levels,blood urea nitrogen levels,e GFR levels,and Cystatin C levels were risk factors for renal fibrosis in Ig AN patients.Further multivariate Cox regression analysis showed that ADAM10 expression levels and e GFR levels were risk factors for renal fibrosis in Ig AN patients.Conclusions Embryonic kidneys of prenatal CPF exposed mice existed the ADAM10-Nocth signal axis over-activated,causing more nephron progenitor cells differentiate into proximal tubules.This abnormal phenotype persisted in adult mice kidneys.Continuous activation of the ADAM10-Nocth axis promoted spontaneous renal fibrosis in 6-month-old mice.Knock down and overexpressing experiments of ADAM10 in human 293 T cells confirmed the role of ADAM10 in fibrosis.In Ig AN patients,the expression of ADAM10 and NOTCH2 is related to the degree of renal fibrosis,and the increased expression of ADAM10 is related to renal function.These findings indicated that ADAM10 may be a potential target for early intervention to prevent renal fibrosis in chronic kidney disease.