Study On The Effect And Mechanism Of Hippo Pathway In ADPKD

Background and objectiveAutosomal dominant polycystic kidney disease (ADPKD) is one of the most commonhuman hereditary kidney diseases which characterized by the progressive formation ofmultiple renal cysts affecting all segments of the renal tubules. Renal involvement is oftenaccompanied by extra-renal manifestations, such as hepatic cysts, pancreatic ductdilatation, colonic diverticulitis, aneurysm,and heart valve deformed. ADPKD is asystemic disease, which seriously hazard to human health. Patients that developed toend-stage renal failure have to rely on dialysis or kidney transplantation to sustain life.Thatplaces a significant burden to society and families. Currently known the disease causinggenes for ADPKD is pkd1and pkd2, but the fundamental pathogenesis of the disease is notyet clear. There are mainly two theories:"second hit" and "cilia pathogenic". Cystepithelial cells over-proliferation is one of the pathogenesis of ADPKD,so it is thougt tobe a tumor like disease. Existing studies have demonstrated that, the pathophysiologicalprocesses of ADPKD are: kidney cyst epithelial cell tumor-like continued proliferation andapoptosis abnormality; cell polarity change and poorly differentiated; cysts liquid withinthe cysta large concentration of accumulation to squeeze the surrounding renalparenchyma. The above process makes the normal renal tissues constantly produce newcysts, the existing cyst gradually increasing, then compress the normal kidney tissue,finally developed to ESRD.As a tumor like disease, ADPKD is closely relate to signaling pathways that regulatorgan size. MTOR signaling pathway is associated with abnormal cell proliferation andtumor occurrence. In recent years, scholars have discovered except mTOR pathway otherthan a specific involvement of organ size control signaling pathways, namely the Hippopathway, the latter mainly through a comprehensive regulation of cell proliferation andapoptosis plays a role in the control of organ size. Because of the important function ofHippo pathway, it plays an important role in human tumor and other disease mechanism.The Hippo protein is a serine/threonine kinase, named after Hippo kinase in2007using genetic mosaic scans discovered in Drosophila. Hippo pathways are highlyconserved among mammals. There are a lot of studies have shown that the main signalingmolecule of this pathway has an important role in regulating the size of the organ growth. Existing evidence has proved that the Hippo pathway has an important impact ontumorigenesis.Human renal carcinoma cell line ACHN and human melanoma tissue wereobserved to have gene mutations with SAV1and Mob1. The expression of Mob1was lowerin colorectal carcinoma than normal colon tissue. The downregulation of Lats1/2exists ina variety of tumors (such as human ovarian carcinoma, invasive breast cancer, astrocytoma,retinoblastoma and acute lymphoblastic leukemia etc.). Human soft tissue sarcoma andcarcinoma of the colon was also observed decreased expression of Mst1/2.Conditionalknockout of Mst1and Mst2genes in liver cause a huge hepatocellular carcinoma in mice,but in these mice again, the YAP gene knock-out can reverses cancer cell phenotypes. Asthe downstream effector molecules of Hippo pathway, an increase of YAP expression andnuclear translocation were also observed in a variety of human tumors such as liver cancer,colon cancer, ovarian cancer, lung cancer and prostate cancer.As a signaling pathway found in recent years, so far, the Hippo pathway in ADPKDresearch is limited to the expression change of pathway target protein YAP in cystepithelial cells and expanded tubular epithelial cells in ADPKD animal model,the Pkd-1gene knockout mice. But other molecular changes in the Hippo pathway and changes inthe human kidney and other animal models has not been reported. In addition, whetherHippo pathway is involved in cell proliferation related signal pathway, cause epithelial cellgrowth and apoptosis abnormality also haven't reported. Therefore, this study wasdesigned to observe the Hippo pathway molecules and target molecules YAP and TAZexpression in ADPKD changes and explore the impaction of Hippo pathway for theexcessive proliferation of cyst epithelial cells and its mechanism,so as to foundexperimental and theoretical basis for effective treatment of ADPKD.Methodes1, We used immunofluorescence and laser scanning confocal to study the varia nceand cell location of LATS1,YAP and TAZ between kidney tissues of ADPKD animalmodel Han:SPRD rats cy/+and Han:SPRD rats+/+.2, We used western blotting method to study the variance of LATS1, P-LATS1,YAP,P-YAP and TAZ between kidney tissues of ADPKD animal model Han:SPRD ratscy/+and Han:SPRD rats+/+.3, We used real time PCR method to study the variance of MST1/2, LATS1/2, YAPand TAZ between kidney tissue of ADPKD patients and normal human control.4, We interfered YAP and TAZ in cystic lining epithelium cell line WT9-12by RNA interfere. Then we assessed cystic lining epithelium cell line WT9-12viability by using theMTT assay.5, After downregulating YAP and TAZ by RNAi, we observed the cell apoptosis byflow cytometry and established the result by western blotting.6, After downregulating YAP and TAZ by RNAi, we observed the cell cycle by flowcytometry and established the result by real-time PCR and western blotting.7, We interfered LATS1in cystic lining epithelium cell line WT9-12by RNAinterfere.Then we assessed YAP and P-YAP to verify wheter LATS1phosphorylate YAP inWT9-12cells.8, After downregulating LATS1by RNAi, we assessed cystic lining epithelium cellline WT9-12viability by using the MTT assay.9, After downregulating LATS1by RNAi, we observed the cell apoptosis by flowcytometry and established the result by western blotting.10,After downregulating LATS1by RNAi, we observed the cell cycle by flowcytometry and established the result by western blotting.Results1,We analyses the distribution of LATS1,YAP and TAZ by immunofluorescence andlaser scanning confocal. We found LATS1expressed less in cystic lining epithelium inHan:SPRD rats cy/+, however in Han:SPRD rats+/+it was positive stained. YAP actuallywas strongly positive expression in Han:SPRD rats cy/+, and are distributed in bothcytoplasm and nucleus, while the Han:SPRD rats+/+exists only in the cytoplasm. TAZexpression and distribution of two kinds of rat has no significant difference.2,LATS1, P-LATS1and P-YAP are downregulated while YAP is upregulated inkidney tissue of Han:SPRD rats cy/+.3,The mRNA of MST1/2, LATS1and TAZ are decreased in kidney tissue of ADPKDpatients.4,The growth inhibition rate in WT9-12were2.66%,31.41%and33.37%afterdownregulating YAP by RNAi for48,72and96h.But downregulating TAZ by RNAi onlyhas slightly growth inhibition.5, After downregulating YAP and TAZ by RNAi,we tested the cell apoptosis ofWT9-12by flow cytometry. We found downregulat YAP and TAZ expression does notincrease the apoptosis of WT9-12cells. Western blotting also showed PARP,the substrateof pro-apoptotic protein caspse-3, did not change significantly.It is illustrated that the down-regulation of YAP and TAZ can not induce apoptosis;6,After downregulating YAP by RNAi,we tested the cell cycle of WT9-12by flowcytometry. We found cell cycle arrest in G0/G1phase. It was coincidence with the resultsestablished by reat-time PCR and western blotting.7,P-YAP are upregulated after downregulating LATS1by RNAi inWT9-12bywestern blotting.8,The proliferation rate in WT9-12were113.22%,116.27%,111.80%and106.81%after downregulating LATS1by RNAi for24,48,72and96h.9,After downregulating LATS1by RNAi,we tested the cell apoptosis of WT9-12byflow cytometry. We found downregulat LATS1expression does not decrease the apoptosisof WT9-12cells. Western blotting also showed PARP,the substrate of pro-apoptoticprotein caspse-3, did not change significantly.It is illustrated that the down-regulation ofLATS1can not reduce apoptosis;10,After downregulating LATS1by RNAi,we tested the cell cycle of WT9-12byflow cytometry. We found downregulating LATS1could accelerate the cell cycle, so thatmore cells in S phase. It was coincidence with the results established by western blotting.ConclusionWe established the disorder of Hippo pathway in ADPKD. The inhibition of Hipposignaling pathway accompany with actived YAP is one of the leading cause of autosomaldominant polycystic kidney disease. In vitro experiments confirmed that reduce the activityof YAP can inhibit the cyst lining epithelial cell proliferation by arrestting cell cycle in G0/G1phase and inhibitting cell division. Decrease the expression of LATS1can promote thecyst lining epithelial cell proliferation through accelerating cell cycle and promoting celldivision. It suggested that Hippo pathway may be a new effective target in ADPKDtherapy.