The Study On The Molecular Mechanism Of TMEM67in Recessive Polycystic Kidney Disease

Polycystic Kidney Disease (PKD) is a cystic genetic disorder of the kidneys. There are two types of PKD:ADPKD and ARPKD. PKD is one of the most common disorders caused by mutations in a single gene. It affects about500,000people in the United States. The ADPKD is much more common than the ARPKD. ADPKD affects1in500-1,000people, while the ARPKD occurs in an estimated1in20,000-40,000people. B6C3Fe α/a-bpck is a mouse model of ARPKD. The lack of gene TMEM67has the homologue with human MKS3and the Wistar rat’s polycystic kidney model.Purpose:After making sure the possibility of the model as ARPKD, construct the vector with the target gene TMEM67. Test the expression of TMEM67protein to analyse the function of TMEM67gene in ARPKD pathogenesis. To understand the molecular and cellular mechanisms of pathogenesis of ARPKD, we performed a microarray gene expression profiling.Methods:1Design primes, prepare the mouse tail, then use PCR to identify the mouse genotyping. Collect different periods WT and MUT mouse kidneys, HE staining. Observe the renal cystic changes in different stages, then to make sure the possibility of mouse model as ARPKD.2use molecular biology experiment methods to construct the vector pFlag-CMV-TMEM67which include TMEM67full lenghth.3Culture HEK293cell, transfect the void vector and the vector with TMEM67full lenghth. After different controls, use Western blot to study the TMEM67function in signaling pathway.4Collect the WT and MUT mouse kidney tissues at postnatal day3. Use a microarray gene expression profiling, screen the differential gene. Then use Real time PCR to verity the validity of the result.results: 1Through obseving the mouse renal cystic structure in different stages, the result is that the cystic structure exist on E15.5day early. As the time going, the cystic structure begin to expand. The dying mouse was found on E18.5and was MUT mouse after genotyping. The mouse renal structure is instead of cystic structure at postnatal day18. It is thought that it is possible using B6C3Fe a/a-bpck as ARPKD mouse model.2The plasmid pFlag-CMV-TMEM67sequences are correct after restriction endo-nuclease digestion and gel electrophoresis. Design3pairs of primers, gene sequencing results show that the plasmid pFlag-CMV-TMEM67contains TMEM67.3Meckelin, gene product of TMEM67, was not a tyrosine-phospho-protein from Western blot analysis. After the experiment of the overexpresson of TMEM67in HEK293cell and the WT, MUT mice in the different stages, increased activation of JNK and ERK were tested. TMEM67function may be mediated through JNK/Erk signaling pathways not mTOR signaling pathway.4A microarray gene expression profiling was performed. Genes with over1.5-fold expression changes in mutant kidneys compared with age matched WT tissues were selected for analysis. About255genes were satisfied by the comparison filtering criteria.conc|usions:1It is feasible for B6C3Fe a/a-bpck to study ARPKD.2The vector pFlag-CMV-TMEM67is successfully constructed.3TMEM67function may be mediated through JNK/Erk signaling pathways not mTOR signaling pathway.4The genes that exhibited significantly altered expression in microarray profiling, were shown to be specific for renal development and/or linked to the PKD process, possessing a wide spectrum of biological functions. The pathways include EGF/TGF pathway, a/EGFR pathway, TGF-β pathway, apoptosis pathway, integrin-mediated pathway and NF-kB pathway.