Protection Mechanism Of Death-associated Protein(DAP5) On Cisplatin-induced Renal Tubular Epithelial Cell Apoptosis

Background and objectives:Cisplatin is currently one of the most commonly used drugs in combination chemotherapy for cancer. Its nephrotoxicity limits its clinical application. Cisplatin-induced acute renal tubular epithelial cell apoptosis is one of the major mechanisms of its nephrotoxicity. As an important factor in regulation of protein translation, when cap-dependent protein translation pathway is inhibited in the classical stress or apoptosis state, death-associated protein (DAP5) can promote the translation of certain proteins via the pathway of the internal ribosome entry site (IRES) to keep normal cell survival. However, the mechanism of DAP5 in cisplatin-induced tubular cell apoptosis is still unclear. In this study we established cisplatin-induced renal tubular epithelial cell apoptosis models in vivo and in vitro. In vitro we observed the changes of DAP5, and its effect on apoptosis-related protein and apoptosis. Then we learned whether DAP5 was regulated by important signal pathway (PI3K/Akt/mTOR signaling pathway) to find new prevention methods for cisplatin-induced acute kidney injury.Methods:part I. Model establishment of cisplatin-indued acute renal tubular epithelial cells in vivo and in vitro:In vivo model, after intraperitoneal injection of cisplatin into Wistar rats for 72 hours, degrees of kidney injury were assessed based on renal fuction and renal histopatholgy. Tubular epithelial cell apoptosis was detected by TUNEL staining, western blot was used to examine the proteins the expression of caspase-3, caspase-8, caspase-9, Bcl-2 and Bax. In vitro model, human renal tubular epithelial cells (HKC) were stimulated for 24h by using different concentrations of cisplatin. Degrees of apoptosis were detected by Hochest staining and flow cytometry analysis. Expression of apoptosis-related proteins were verified by western blot.Part II. Regulatory mechanism of DAP5 on cisplatin-induced renal tubular epithelial cell apoptosis:After upregulation of DAP5 expression by plasmid transfection and downregulation of DAP5 expression by siRNA-DAP5 in HKC cells, apoptosis of the two HKC cells were induced by cisplatin. The degree of cell apoptosis was assessed by flow cytometry analysis. The expression of Bax and Bcl-2 proteins were detected by western blot and. The effect of DAP5 on cisplatin-induced cell apoptosis was investigated. In addition, change of PI3K/Akt/mTOR signaling pathway in cisplatin-induced HKC apoptosis cells was detected. After inhibitting PI3K/Akt/mTOR signaling pathway by the Akt inhibitor triciribine and mTOR inhibitor rapamycin, the expression of PI3K/Akt/mTOR signal pathway and DAP5 were detected by western blot analysis. Then the relationship between PI3K/Akt/mTOR signaling pathway and DAP5 were evaluated.Results:After the cisplatin application for 72h, significant deterioration of renal function was found with increased levels of serum creatinine and blood urea nitrogen. Histopathology analysis showed vacular degeneration and granular degeneration of tubular epithelial cells, proximal tubular dilatation, tubule brush border loss, cells derangement. Protein casts and TUNEL-positive tubular cells can be seen in some tubular-interstitial regions. We conducted further detections of caspase-3, caspase-8, caspase-9, Bcl-2 and Bax protein by western blot and then found that the expression of caspase-3, caspase-8, caspase-9, Bax in the model group were markedly increased, but Bcl-2 was decreased compared with the control group. In vitro we stimulated HKC cells for 24h by using different concentrations (1～100μM) of cisplatin, then found the degree of apoptosis was increased with the concentration of cisplatin by Hochest staining and flow cytometry analysis. The same change patterns of proteins related to apoptosis were found. The expressions of caspase-3, caspase-8, caspase-9 and Bax were increased gradually with higher concentration of cisplatin, while the expression of Bcl-2 was decreased.In the meantime, in the progress of cisplatin-induced HKC cells apoptosis, DAP5 was cracked into protein fragments 86KDa-DAP5/p86. Overexpression of DAP5/p97 and DAP5/p86 increased the translation of Bcl-2 and reduced the degree of cisplatin-indued apoptosis. Inhibitting DAP5 expression by siRNA-DAP5 decreased the translation of Bcl-2, and increased the degree of apoptosis. But these had no effect on the expression of Bax. In the cisplatin-induced apoptosis state we found the PI3K/Akt/mTOR signaling pathway was blocked. Triciribine and rapamycin decreased the expression of DAP5, as well as the expression of Bcl-2 and Bax. Therefore, DAP5 expression was positively regulated by the PI3K/Akt/mTOR signaling pathway.Conclusions:In this study, we successfully established the cisplatin-induced apoptosis of renal tubular epithelial cells models in vitro and in vivo. For the first time we studied the mechaism of DAP5 involved in cisplatin-induced apoptosis of renal tubular epithelial cells. During the apoptotic process, DAP5 can be cracked into DAP5/p86. DAP5/p97 and DAP5/p86 enhanced the translation of anti-apoptotic genes Bcl-2, and inhibited cispaltin-induced apoptosis. PI3K/Akt/mTOR signaling pathway positively regulated the expression of DAP5. Thus, overexpression of DAP5 enhanced the tolerance of tubular epithelial cells to cisplatin treatment and reduced the severity of apoptosis. This study would provide benefit for the prevention and treatment of cisplatin nephrotoxicity.