Functional Identification And Regulation Mechanism Of Podocyte Receptor Tyrosine Kinase?TYRO₃?

Part 1.Identification of TYRO3 function based on zebrafish modelObjecive:Identification of TYRO3 expression in kidney and functional identification of TYRO3 in zebrafish.Methodolgy:(1)The localization of TYRO3 in human glomeruli was determined by immunofluorescence staining and laser confocal microscopy.The change of TYRO3 in diabetic nephrology(DN)and db/db mice was detected by immunohistochemistry.(2)tyro3 morpholino and CRISPR/Cas9 gene editing techniques were applied to achieve tyro3 knockdown and deletion in zebrafish.The effects of tyro3 knockdown and and deletion on zebrafish's filtration membrane and podocytes were studied by edema rate,proteinuria test and electron microscopy.Result:(1)Colocalization studies detected TYRO3 protein along with the podocyte marker SYNAPTOPODIN in glomeruli.No colocalization was observed between TYRO3 and endothelial marker(CD31)or mesangial cells marker(GATA3).(2)IHC demonstrated TYRO3 and phosphorylated TYRO3 were downregulated in the glomeruli from DN patients.(3)The similarity of human TYRO3 protein sequence to zebrafish TYRO3 protein sequence was 47%,The results of RT-PCR of isolation glomeruli from zebrafish showed that tyro3 was expressed on zebrafish glomeruli.(4)Knockdown of tyro3 by morpholino(MO)in zebrafish larvae exhibited pericardial,yolk sac and pericardial edema.Glomerular filtration rate of fluorescent labeled 70KD dextran increased significantly,indicating impaired glomerular filtration membrane.Electron microscope results showed foot-process effacement.Rescue experiment showed that zebrafish co-injected with synthetic zebrafish tyro3 mRNA and MO were morphologically normal in terms of edema and foot process.(5)CRISPR/Cas9 gene editing technology has effectively knocked out the base of tyro3,tyro3 gene knockout zebrafish showed edema and foot process.Conclusion:TYRO3 special localized at podocyte in glomerulus.TYRO3 is downregulated in DN patients at protein level.Inhibition of tyro3 contributes to disruption of filtration membrane in zebrafish.tyro3 is a novel protein that might play a crucial role in zebrafish podocyte homeostasis.Part 2.Study on the regulation mechanism of TYRO3 in podocyteObjecive:To observate the changes of TYRO3 expression after injury and the effect of changing TYRO3 expression on the function of human podocyte,and analysis the underlying molecular mechanism of TYRO3 in the maintenance the normal function of podocyte.Methodolgy:(1)Detection of the expression of TYRO3 ligand GAS6 and PROS1 in human glomeruli by immmunohistochemistry.(2)The expression of TYRO3 in human podocytes was detected by qRT-PCR and Western Blot after high glucose and TGF beta treatment.(3)Knockdown TYRO3 in podocyte with siRNA,Flow cytometry analysis of apoptosis,Phalloidin staining of skeleton.(4)Inhibition and overexpression of the expression of TYRO3 in cultured podocytes in vitro to detect the change of phosphorylation level in the downstream signal pathway of TYRO3 using Western blot.(5)Using Western blot to detect the expression of P53 in podocyte under TYRO3 expression changes and injury stimulation.(6)The effect of TYRO3 on the activity of p53 promoter was detected by the luciferase reporter gene test.Result:(1)Immunohistochemical results showed that the TYRO3 ligand GAS6 and PROS1 were expressed on the glomeruli.(2)After the treatment of high glucose and TGF beta,the expression of TYRO3 was significantly decreased by qRT-PCR and Western Blot.(3)Inhibition of TYRO3 expression induced increase of apoptosis in podocyte,cytoskeleton damaged.(4)The results of Western blot showed that the phosphorylation of JNK were significantly decreased after knockdown of TYRO3,while the phosphorylation of p38 increased.Consistently,the phosphorylation of JNK was increased in podocytes with TYRO3 overexpression,while the phosphorylation of p38 decreased.(5)TYRO3 inhibits the expression of p53 and the expression of p53 increased in podocyt after injury stimulation.(6)The luciferase reporter gene experiment showed that TYRO3 inhibited the activity of p53 promoter and the block recoverd after JNK inhibition.Conclusion:High glucose and TGF beta can reduce the expression of TYRO3 in the podocyte,Reduction of TYRO3 expression induce podocyte apoptosis and skeleton injury.Changes of TYRO3 expression lead to changes of phosphorylation of p38 and JNK.The suppression of p53 by TYRO3 is mediated by the JNK signaling pathway.