Bcl-X-L translocation in renal tubular epithelial cells in vitro protects distal cells from oxidative stress

Background. The molecular pathogenesis of different sensitivities of the re nal proximal and distal tubular cell populations to ischemic injury, includ ing ischemia-reperfusion (IR)-induced oxidative stress, is not well-defined . An in vitro model of oxidative stress was used to compare. the survival o f distal [Madin-Darby canine kidney (MDCK)] and proximal [human kidney-2 (H K-2)] renal tubular epithelial cells, and to analyze for links between indu ced cell death and expression and localization of selected members of the B cl-2 gene family (anti-apoptotic Bcl-2 and Bcl-X-L, pro-apoptotic Bar and B ad), Methods. Cells were treated with 1 mmol/L hydrogen peroxide (H2O2) Or were grown in control medium for 24 hours. Cell death (apoptosis) was quantitate d using defined morphological criteria. DNA gel electrophoresis was used fo r biochemical identification. Protein expression levels and cellular locali zation of the selected Bcl-2 family proteins were analyzed (West ern immuno blots, densitometry, immunoelectron microscopy). Results. Apoptosis was minimal in control cultures and was greatest in trea ted proximal cell cultures (16.93 +/- 4.18% apoptosis) compared with treate d distal cell cultures (2.28 +/- 0.85% apoptosis, P < 0.001). Endogenous ex pression of Bcl-X-L and Bax, but not Bcl-2 or Bad, was identified in contro l distal cells, Bcl-X-L and Bar had nonsignificant increases (P > 0.05) in these cells. Bcl-2, Bar, and Bcl-X-L, but not Bad, were endogenously expres sed in control proximal cells. Bcl-X-L was significantly decreased in treat ed proximal cultures (P < 0.05), with Bas and Bcl-2 having nonsignificant i ncreases (P > 0.05). Immunoelectron microscopy localization indicated that control and treated hut surviving proximal cells had similar cytosolic and membrane localization of the Bcl-2 proteins. In comparison, surviving cells in the treated distal cultures showed translocation of Bcl-X-L from cytoso l to the mitochondria after treatment with H2O2, a result that was confirme d using cell fractionation and analysis of Bcl-XL expression levels of the membrane and cytosol proteins. Bar remained distributed evenly throughout t he surviving distal cells, without particular attachment to any cellular or ganelle. Conclusion. The results indicate that in this in vitro model, the increased survival of distal compared with proximal tubular cells after oxidative st ress is best explained by the decreased expression of anti-apoptotic Bcl-X- L in proximal cells, as well as translocation of Bcl-X-L protein to mitocho ndria within the surviving distal cells.