Mechanisms And Effects Of Omi/HtrA2 On The Apoptosis Of Rat Renal Tubular Epithelial Cells

Objective: To investigated the mechanisms and effects of Omi/HtrA2 through observing the variety of the apoptosis of rat renal tubular epithelial cells(NRK-52E), in which the protein expression of Omi/HtrA2 was inhibited using RNAi.Methods: The cells were divided into 5 groups: normal groups(n=5), cells were cultured under routine condition; model groups(n=5), hypoxia-reoxygenation injury was performed; HK groups(n=5), transfected with Pgenesil-1/HK; shRNA1 groups, transfected with Pgenesil-1/Omi/HtrA2 shRNA1; shRNA2 groups(n=5), transfected with Pgenesil-1/Omi/HtrA2 shRNA2. The cells were incubated in 6 well plates; after cells were 50%-60% confluence, the transfection was performed, and the medium was refreshed after the first 8 hours with complete medium. After 24 hours, the transfected monoclone cells were examined by the fluorescent microscopy, and the cells were incubated with complete medium containing G418(300μg/ml). After 2 weeks, and the transfected monoclone cells were incubated with medium containing G418(150μg/ml). Except the normal group, the other four groups were cultured with medium oxygen-free under the condition of N295%+O21%+CO25% for an hour, then cultured with complete medium for 12 hours. Collected the cells after the hypoxia–reoxygenation. The apoptosis of every groups was analysised by DNA ladder and tunnel, and protein expression of Omi/HtrA2 and caspase3 were examined by Western blot.Results: The cells with green fluorescent were visible in transfected cells. The protein expression of Omi/HtrA2 of the model group increased compared with the normal, and the protein expression of Omi/HtrA2 is significantly decreased in shRNA1 groups and in shRNA2 groups compared with the model (p<0.05) by Western blot. The distinctive ladder pattern was visible in the model group, and then the ladder was not obvious in the shRNA1 and shRNA2 groups by DNA ladder. After Omi/HtrA2 was inhibited, positive cells also lessened by tunnel.Conculsions: Omi/HtrA2 played an important role in apoptosis of the NRK-52E. The apoptosis of NRK-52E was mitigated by inhibited the expression of Omi/HtrA2 in hypoxia-reoxygenation.