Effecte Of HNaDC3 On The Cellular Energy Metabolism And Apoptosis In The Human Renal Proximal Tubular Epithelial Cells And The Role Of AngⅡ Regulation

Objective The Na+/dicarboxylate cotransporter3 (NaDC3) of the renal proximal tubular cells active transports Krebs cycle intermediates, such as succinate and maybe plays an important role in the development of kidney stone for regulation of urinary citrate concertration and in the senescence of living. Although NaDC is one of the highlight in the memrane transporters, we know little the effect of NaDC3 on the renal proximal tubular cells Except for this aspect, initiation of apoptosis by many agents is preceded by mitochondrial dysfunction and by disturbance of cell energy metabolism .Angiotensin II(AngII) is a multifunction for cells, it may triggers both proliferation and apotosis of renal proximal tubular cells . But the role of NaDC3 and Angllto renal proximal tubular cells during acute disturbance of cell energy metabolism is unknown. Moreover, the effect of AngII on the expression of NaDC3 in proximal tubular cells is to elucidate. In the present study, we evaluated the effect of human NaDC3(hNaDC3) on the cell energy metabolism and the role of hNaDC3 and Angllto the apoptosis of renal proximal tubular cells(HKC) duing acute ATP depletion., and reseached the role of AngII to hNaDC3 expression in HKC cells.Methods First, the different hNaDC3 expressed huamn renal proximal tubular cells (HKC) line were stained with specific new type fluorescent probe to cytoplastic membrane, DIBA4(3) and stained with specific new type fluorescent probe to mitochondrial membrane potential delta( m), JC-1. The cell fluorescent intensity were analysised by flow cytometry. and laser co-focus microscope,respectively. The cell production of ATP and H202were examined by the reverse-high pressure liquid chromatographer(HPLC) and by luminol (3-aminophthalhydrazide ) plus horseradish peroxidase-derived chemiluminescence with BPCL Ultra-weak luminescence analyzer . Active Na+ transport (Ouabain-sensitive Q02) and Oxygen consumption of the intact HKC cells were monitored with a clark electrode and the remainder of succinate in intaking -buffer were analyzed by capillary electrophoresis with indirect UV detection. Also Na+-K+-ATPase was determined in cellular lysates, by measuring the difference between total ATPase activity and ouabaine-insensitive ATPase and F0F1-ATPase activity of HKC cells were determined in freshly isolated HKC mitochondria by measuring the release of Pi from ATP . Then compare these results among the hNaDC3 hyper-expressed , normal- expressed and hype-expressed HKC cells. Second, the cultured HKC cells apoptosis model involving acute energy dysfunction (acute ATP depletion) was induced by incubation HKC cells with low dose of mitochondrial inhibitor CCCP (20uM) in glucose-free Krebs-Ringer bicarbonate buffer (RC way in shorts)containing succinate, . then the cells were incubated in Krebs-Ringer bicarbonate buffer or in DMEM containing variable concentration of AngII and succinate for another 1- 4 hours for further evaluation of the proapoptotic effects of hNaDC3 and ANG II. Third, 90%confluent HKC monolayers were incubated for 24h in serum-free DMEM, then were incubated for another 24h in serum-free DMEM containing Ang II (10~12 - 10-6M)and/or valsartan or CGP42112A(10-6M), the hNaDC3 mRNA and protein in HKC cells were analyzed by Northeren blot, Western blot and immunocytochemistry.Results: Of hNaDC3 hyper-expressed cells , the absorption of succinate was faster;the cell plasma membrane fluorescent intensity was lower;the ATP content , H202 production , Basal QO2, Oligomycine-sensitive QO, Uncoupled QO2 ,ouabain-sensitive QO2 and Mitochondrial membrane red fluorescent intensity were higher; the activity of Na+/k+-ATPase and the F0F1-ATPase activity also higher (p<0. 01) than these of hNaDCS normal-expressed cells. But there were not obvious defference between the hNaDCS normal- expressed and hypo-expressed cells (p>0.05) . In this apoptotic model induced byacute ATP depletion, the apoptotic rate and apoptotic index were dramatically higher in the HKC cells with higher level of hNaDCS expression than that...