Study On The Effect And Mechanism Of Notch Signaling In ADPKD

Background and objectiveAutosomal dominant polycystic kidney disease (ADPKD) is one of the most common human hereditary kidney diseases with a prevalence of 1/400-1/1000, estimated to affect more than 1.5 million people and account for about 5% of end stage renal disease patients requiring renal replacement therapy in China. The disease is characterized by the progressive formation of multiple renal cysts affecting all segments of the renal tubules and about 50% patients develop renal insufficiency in the fifth and sixth decades of life. Renal involvement is often accompanied by extra-renal manifestations, such as hepatic and pancreatic cysts, cardiac valvular defects, colonic diverticulosis and intracranial aneurysms. Transmitted in a dominant manner, ADPKD often affect 50% children of patients. So it is also a fatal systematic disease.The pathophysiology process of cysts genesis and progression in ADPKD is disorder of proliferation and apoptosis, remodeling of extracellular matrix, interstitial fibrosis and increase of fluid excretion. The pathogenesis of ADPKD is still unclear. Recent studies suggest that it may relate to several transduction pathways, such as cAMP-PKA pathway, EGF pathway, Wnt pathway, mTOR pathway and so on. The effects of one or more pathway result in excess-proliferation of cyst epithelium cell and ultra-excretion of hydatid fluid, and promote the growth of cysts.Notch is an ancient and conservative cell signaling system which exists in many species including Invertebrates and Vertebrates. The notch signaling includes receptors, ligands and DNA binding protein. Notch activity is dependent on ligand binding followed by a series of proteolytic cleavage and subsequent nuclear translocation of its cytoplasmic domain, Notch intracellular domain (NICD). NICD then complexes with the DNA-binding protein RBP-J (also known as CSL or CBF1) and Mastermind-like 1 (MAML 1) to exert a transcriptional effect on target gene expression that regulates cell fate specification, stem cell maintenance and initiation of differentiation in embryonic and postnatal tissues.One of the proteolytic enzymes involved in cleaving the membrane-bound Notch isγ-secretase. Inhibition ofγ-secretase can block activation of the Notch pathway.Four Notch receptors Notch-1, Notch-2, Notch-3, and Notch-4, five ligands, Jagged-1 and Jagged-2 belonging to the Serrate family, and Delta-1, Delta-3, and Delta-4 belonging to the Delta family and two target genes Hes and Hey family have been identified in mammals. It has revealed that Notch pathway is related to the differentiation of kidney, nerve stem cell, lung, pancreas, cartilage and prostate.As a tumor like disease, does Notch signaling exist in ADPKD? Or does Notch pathway play a role in the pathogenesis of progression of ADPKD? It still not reported. The objective of this study is to interpret the role of Notch signaling in ADPKD, to study the effect and mechanism of Notch signaling on the cyst lining epithelium cells in ADPKD and to reveal the cross talk between Notch signaling and mTOR pathway, so that to provide a new target for ADPKD therapy.Methodes1,We used real time PCR method to study the variance of Notch1, Notch2, Notch3, Notch4, Jagged1 and Hes1 between kidney tissue of ADPKD patients and normal human control;2,We used western blotting method to study the variance of Notch1, Jagged1 and Hes1 between kidney tissue of ADPKD patients and normal human control;3,We used immunohistochemistry to study the variance and cell location of Notch1, Jagged1 and Hes1 between kidney tissues of ADPKD patients and normal human control;4,Cystic epithelium cell line WT9-12 and normal renal tubular cell line RCTEC were treated withγ-secretase DBZ at various concentration(0,1,2,5,10nmol/L). Then we assessed cell viability by using the MTT assay;5,We interfered Notch1 by RNA interfere to downregulate the Notch signal. Then we observed the cell cycle by flow cytometry and established the result by western blotting;6,We treated the WT9-12 cell by various concentration(0,5,10nmol/L) of DBZ, then established the down regulation of Notch signal by detect the density of Notch1 and Hes1 by western blotting;7,We detected the mTOR and p-mTOR by western blotting after DBZ treating; 8,We detected the 4E-Bp1 and p-4E-Bp1 by western blotting after DBZ treating;9,We detected the p70S6K and p- p70S6K by western blotting after DBZ treatingResults1,We analyses the distribution of Notch1, Jagged1 and Hes1 by immunohistochemistry. We found Notch1, Jagged1 and Hes1 were expressed in the cytoplasm and nucleus in cyst lining epithelial cells with positive stain, however in normal kidney tissues they were slightly expressed in cytoplasm.2,The mRNA level of Notch1, Notch2, Notch3, Notch4, Jagged1 and Hes1 are elevated in kidney tissue of ADPKD patients.3,Notch1, Jagged1 and Hes1 are upregulated in in kidney tissue of ADPKD patients.4,γ-secretase inhibitor DBZ induced potent of growth inhibition in WT9-12 cells, nevertheless,there was no growth inhibiton in RCTEC cells. The growth inhibition of DBZ was dose dependent, but we did not observe time-dependent growth inhibition. The growth inhibition rate was almost the same between 24, 48 and 72h.5,After downregulating Notch1 by RNAi,we tested the cell cycle of WT9-12 by flow cytometry. We found cell cycle arrest in S phase. It was coincidence with the results established by western blotting.6,We treated the WT9-12 cells with DBZ. It showed that once Notch1/Hes1 pathway was downregulated, the level of mTOR,p-mTOR,p-4E-Bp1,p-p70S6K were decent as well.ConclusionWe firstly established the disorder of Notch pathway in ADPKD. It may play a key role in the pathogenesis of ADPKD. Down regulation of Notch signal can inhibit the growth rate of cyst line epithelium cells by arresting the cell cycle in S phase. Notch signaling can also affect the mTOR pathway. It suggested that Notch signaling may be a new effective target in ADPKD therapy.