The Effect Of Mitochondria Injury On Structure And Function Of Primary Cilia On The Cystic Lining Epithelial Cells

Objective:This study aims to explore the presence of mitochondria injury in autosomal dominant polycystic kidney disease and discuss the possible mechanism of how mitochondria injury affect the structure and function of primary cilia, to learn mor about cilia pathogenic hypothesis, and provide new targets for intervention of polycystic kidney disease.Methods:1.Biological validation of mitochondria injury in polycystic kidney disease.(1)Expression of heat shock protein 75, translocases of the inner membrane 44, translocases of the outer membrane 20, cytochrome C is detected in pkdl-/- and pkdl+/+mice,human cystic lining epithelial cells of polycystic kidney (WT9-12) and human normal renal tubular epithelial cells (RCTEC) by Western-blot and real-time PCR(2)Mitochondrial structure is observed in WT9-12 and RCTEC by transmission electron microscope.2.Biological validation of abnormal primary ciliary structure and function in polycystic kidney disease.(1)Expression of kinesin 2 and IFT88 in pkdl-/- and pkdl+/+ mice, WT9-12 and RCTEC is detected by Western-blot.(2)Expression of kinesin 2 in WT9-12 and RCTEC is detected by real-time PCR.(3)Structure of primary cilia is observed in WT9-12 and RCTEC by transmission electron microscope and immunofluorescence.3.Regulation of HSP75 expression in human cystic lining epithelial cells and human normal renal tubular epithelial cells and the impact to structure and function of primary cilia.(1)Expression of TIM44, TOM20, kinesin 2, IFT88, cytochrome C in WT9-12 is detected after transfected plasmid which can overexpress HSP75 by Western-blot.(2)Expression of TIM44, TOM20, kinesin 2, IFT88, cytochrome C in RCTEC is detected after siRNA interference expression of HSP75 by Western-blot.(3)Intracellular ATP is detected in WT9-12 after transfected plasmid which can overexpress HSP75 and RCTEC after siRNA interference expression of HSP75 by chemiluminescence method.(4)Cell proliferation activity is detected in WT9-12 after transfected plasmid which can overexpress HSP75 and RCTEC after siRNA interference expression of HSP75 by MTT method.(5)Structure of primary cilia is observed in WT9-12 after transfeced plasmid which can overexpress HSP75 and RCTEC after siRNA interference expression of HSP75 by laser confocal microscope.(6)Intracellular calcium is detected in WT9-12 after transfected plasmid which can overexpress HSP75 and RCTEC after siRNA interference expression of HSP75 by flow cytometry method.Result:1.HSP75, TIM44, TOM20 expression is decreased in pkdl-/- mice kidney compared with pkd1+/+mice, while cytochrome C expression is increased. The same result is observed in WT9-12.2.Compared with RCTEC, mitochondria presents markedly swollen, mitochondrial cristae disappears in WT9-12.3.Compared with RCTEC, primary cilia presents less quantity, shorter length or even disappeared.4.After overexpression of HSP75 in WT9-12, TIM44, TOM20 expression is up-regulated, cytochrome C down-regulated, kinesin 2 up-regulated.5.After siRNA interference of HSP75 in RCTEC, TIM44, TOM20 expression is down-regulated, cytochrome C up-regulated, kinesin 2 down-regulated.6. The amount of intracellular ATP is decreased after overexpression of HSP75 in WT9-12 while the amount of ATP is increased after siRNA interference of HSP75 in RCTEC.7.Cell proliferation activity is enhanced after overexpression of HSP75 in WT9-12 while cell proliferation activity is abate after siRNA interference of HSP75 in RCTEC.8.After overexpression of HSP75 in WT9-12, the primary cilia length becomes longer. After siRNA interference of HSP75 in RCTEC, the primary ciliary becomes shorter.9. After overexpression of HSP75 in WT9-12, intracellular calcium concentration has no obvious difference. After siRNA interference of HSP75 in RCTEC, intracellular calcium concentration is significantly reduced after 72, hours.Conclusion:Our study indicates that mitochondria injury and abnormal structure and function of primary cilia exist in PKD renal cyst organizations and human cystic lining epithelial cells. Mitochondria injury may affect protein transport function of primary cilia through energy metabolism and leads to its structural abnormalities. Overexpression of HSP75 can improve mitochondria injury, promote proliferation of human cystic lining epithelial cell and make primary cilia length longer. Reduction of HSP75 leads to mitochondria injury and inhibit proliferation of human normal renal tubular epithelial cells and make primary cilia length shorter with dysfunction of regulating the flow of calcium.