Volume 59, Issue 5, Pages 1779-1788 (May 2001) Bcl-XL translocation in renal tubular epithelial cells in vitro protects distal cells from oxidative stress  Leila Cuttle, Xiao-Ju Zhang, Zoltan H. Endre, Clay Winterford, Glenda C. Gobé  Kidney International  Volume 59, Issue 5, Pages 1779-1788 (May 2001) DOI: 10.1046/j.1523-1755.2001.0590051779.x Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 1 Percentage cell death (apoptosis) in control () or 1mmol/L hydrogen peroxide (▪; H2O2)-treated human kidney-2 (HK-2; proximal) or Madin-Darby canine kidney (MDCK; distal) renal epithelial cells. Cell death was minimal in control cultures. Both HK-2 and MDCK cultures had a significant increase in apoptosis after treatment (HK-2, P < 0.001, ▴); MDCK, P < 0.05, ▵). The percentage of cell death was significantly higher in the treated proximal compared with treated distal cultures (P < 0.001, •). Kidney International 2001 59, 1779-1788DOI: (10.1046/j.1523-1755.2001.0590051779.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 2 Morphological evidence of apoptosis in HE-stained, H2O2-treated MDCK cells (A) and a resin-embedded, toluidine-blue-stained section of HK-2 cells (B). Arrows demonstrate some examples of apoptosis. (C) High-power magnification of a blebbing apoptotic HK-2 cell. A, B = ×640; C = ×860. Kidney International 2001 59, 1779-1788DOI: (10.1046/j.1523-1755.2001.0590051779.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 3 Apoptotic DNA fragmentation is shown by gel electrophoresis. Lane 1 contains a marker of λ DNA cut with EcoRI and Hind III (21.23, 5.15, 4.98, 3.53, 2.03, 1.90, 1.58, 1.38, 0.95, 0.83, 0.56, and 0.12 kb). Lanes 2 and 3 contain 30 μg of DNA from control MDCK cells (lane 2) or H2O2-treated MDCK cells (lane 3; typical apoptosis banding patterns corresponding to multiples of 180 to 200 bp). Kidney International 2001 59, 1779-1788DOI: (10.1046/j.1523-1755.2001.0590051779.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 4 Western immunoblot and densitometry for control and treated MDCK cells. Both Bax (A) and Bcl-XL (B) show a nonsignificant increase (P> 0.05) when treated with 1mmol/L H2O2. Kidney International 2001 59, 1779-1788DOI: (10.1046/j.1523-1755.2001.0590051779.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 5 (A-C) Western immunoblot and densitometry for control and treated HK-2 cells. Bax and Bcl-2 show a nonsignificant increase (P> 0.05). Bcl-XL has a significant decrease in expression levels (*P < 0.05) in H2O2-treated cells. Kidney International 2001 59, 1779-1788DOI: (10.1046/j.1523-1755.2001.0590051779.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 6 Immunolocalization of the Bcl-2 proteins in control or H2O2-treated MDCK cells (A-D) or HK-2 cells (E and F) is indicated by black granular labeling. In control distal MDCK cells, Bax (A) was diffusely localized to the cytosol and some mitochondrial expression was found (arrows). This localization did not change for the H2O2-treated, surviving, cells (B). Bcl-XL (C) was spread throughout the cytosol, but showed little or no expression in mitochondria (arrows) of controls. However, in H2O2-treated cells, Bcl-XL (D) was found in mitochondria (arrows), as well as in the cytosol, indicating Bcl-XL was able to translocate to the organelles when the cells were stressed. In control HK-2 cells, Bcl-XL, Bcl-2, and Bax were diffusely spread throughout the cytosol, and this pattern did not alter in surviving cells after treatment with 1mmol/L H2O2. Examples are given for Bcl-XL under control (E) or treated (F) conditions (mitochondria are arrowed). Magnification for A, C, and E = ×39,000; B, D, and F = ×54,000. Kidney International 2001 59, 1779-1788DOI: (10.1046/j.1523-1755.2001.0590051779.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 6 Immunolocalization of the Bcl-2 proteins in control or H2O2-treated MDCK cells (A-D) or HK-2 cells (E and F) is indicated by black granular labeling. In control distal MDCK cells, Bax (A) was diffusely localized to the cytosol and some mitochondrial expression was found (arrows). This localization did not change for the H2O2-treated, surviving, cells (B). Bcl-XL (C) was spread throughout the cytosol, but showed little or no expression in mitochondria (arrows) of controls. However, in H2O2-treated cells, Bcl-XL (D) was found in mitochondria (arrows), as well as in the cytosol, indicating Bcl-XL was able to translocate to the organelles when the cells were stressed. In control HK-2 cells, Bcl-XL, Bcl-2, and Bax were diffusely spread throughout the cytosol, and this pattern did not alter in surviving cells after treatment with 1mmol/L H2O2. Examples are given for Bcl-XL under control (E) or treated (F) conditions (mitochondria are arrowed). Magnification for A, C, and E = ×39,000; B, D, and F = ×54,000. Kidney International 2001 59, 1779-1788DOI: (10.1046/j.1523-1755.2001.0590051779.x) Copyright © 2001 International Society of Nephrology Terms and Conditions

Figure 7 Western immunoblot and densitometry for Bcl-XL protein expression in soluble (cytosol) or membrane fractions from control or H2O2-treated MDCK or HK-2 cells are demonstrated. Only the MDCK membrane fraction had a significant increase in Bcl-XL protein expression (***P < 0.01) after treatment, with the HK-2 cells having nonsignificant decreases in Bcl-XL in both cytosolic and membrane fractions after treatment. Kidney International 2001 59, 1779-1788DOI: (10.1046/j.1523-1755.2001.0590051779.x) Copyright © 2001 International Society of Nephrology Terms and Conditions