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. 2015 Sep 15;309(6):F540-50.
doi: 10.1152/ajprenal.00170.2015. Epub 2015 Jul 15.

Simultaneous deletion of Bax and Bak is required to prevent apoptosis and interstitial fibrosis in obstructive nephropathy

Hee-Seong Jang  1 , Babu J Padanilam  2
Affiliations

Simultaneous deletion of Bax and Bak is required to prevent apoptosis and interstitial fibrosis in obstructive nephropathy

Hee-Seong Jang et al. Am J Physiol Renal Physiol. .

Abstract

Proximal tubular injury and apoptosis are key mediators of the development of kidney fibrosis, a hallmark of chronic kidney disease. However, the molecular mechanism by which tubular apoptotic cell death leads to kidney fibrosis is poorly understood. In the present study, we tested the roles of Bcl-2-associated X (Bax) and Bcl-2 antagonist/killer (Bak), two crucial proteins involved in intrinsic apoptotic cell death, in the progression of kidney fibrosis. Mice with proximal tubule-specific Bax deletion, systemic deletion of Bak, and dual deletion of Bax and Bak were subjected to unilateral ureteral obstruction (UUO). Dual deficiency of Bax and Bak inhibited tubular apoptosis and atrophy. Consistent with decreased tubular injury, dual ablation of Bax and Bak suppressed UUO-induced inflammation and kidney fibrosis with decreased tubular cell cycle arrest, expression of fibrogenic and inflammatory cytokines, and oxidative stress in the kidney. Bax or Bak deficiency was insufficient to prevent apoptosis and all other aforementioned malevolent effects, suggesting compensatory mediation by each other in the respective signaling pathways. These data suggest that dual ablation of Bax and Bak in the kidney is required to prevent UUO-induced tubular apoptosis and the consequent kidney inflammation and fibrosis.

Keywords: Bcl-2 antagonist/killer; Bcl-2-associated X; apoptosis; chronic kidney disease; fibrosis; inflammation; oxidative stress.

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Figures

Fig. 1.
Fig. 1.
Inhibition of unilateral ureteral obstruction (UUO)-induced kidney tubular apoptosis by dual ablation of Bax and Bak. Four groups of mice {wild-type (WT), Bcl-2-associated X (Bax)fl/fl;Pepck-Cre [Bax proximal tubule (PT)-specific knockout (KO)], Bcl-2 antagonist/killer (Bak)-null (Bak KO), and Baxfl/fl;Pepck Cre; Bak-null [double KO (DKO)] mice} were subjected to UUO or sham operation for 7 days. A: tubular apoptosis was evaluated by TUNEL assay. B: TUNEL-positive cells were measured in five randomly chosen fields per kidney. C: expression of cleaved caspase-3 was examined by Western blot analysis. Anti-GAPDH antibody was used as a loading control. D: Western blot band densities were evaluated using ImageJ software. Green fluorescence indicates TUNEL-positive cells. Arrow, tubular TUNEL-positive cells. CLK, contralateral kidney. Data are expressed as means ± SE; n = 3. *P < 0.05 vs. respective sham-operated mice; #P < 0.05 vs. UUO in WT mice.
Fig. 2.
Fig. 2.
Dual ablation of Bax and Bak decreases kidney tubular atrophy during UUO. Four groups of mice (WT, Bax PT-KO, Bak KO, and DKO mice) were subjected to UUO or sham operation for 7 days. A: tubular necrosis was evaluated by Periodic acid-Schiff staining. *Atrophied tubules in Periodic acid-Schiff-stained kidney sections. B and C: tubular atrophy (B) and dilatation (C) were measured in five randomly chosen fields per kidney. Data are expressed as means ± SE; n = 3. *P < 0.05 vs. respective sham-operated mice; #P < 0.05 vs. UUO in WT mice.
Fig. 3.
Fig. 3.
Dual ablation of Bax and Bak reduces UUO-induced recruitment of neutrophils and macrophages into the kidney. Mice were subjected to UUO or sham operation for 7 days. Paraffin-embedded kidney sections were used for immunohistochemistry staining with anti-polymorphonuclear neutrophil (PMN; A) or F4/80 (C) antibodies. B and D: PMN-positive (B) and F4/80-positive (D) cells were quantified in five randomly chosen fields per kidney. Data are expressed as means ± SE; n = 3. *P < 0.05 vs. respective sham-operated mice; #P < 0.05 vs. UUO in WT mice.
Fig. 4.
Fig. 4.
Dual ablation of Bax and Bak alleviates inflammatory signals and oxidative stress during UUO. Mice were subjected to UUO or sham operation for 7 days. A: expression of ICAM-1, IL-6, TNF-α, and IL-1β was examined by Western blot analysis. Anti-GAPDH antibody was used as a loading control. B–E: Western blot band densities of ICAM-1 (B), IL-6 (C), TNF-α (D), and IL-1β (E) were evaluated using ImageJ software. F: kidney oxidative stress was examined by lipid peroxidation using a lipid hydroperoxide assay. Data are expressed as means ± SE; n = 3. *P < 0.05 vs. respective sham-operated mice; #P < 0.05 vs. UUO in WT mice.
Fig. 5.
Fig. 5.
Dual ablation of Bax and Bak blocks UUO-induced tubular cell cycle arrest. Mice were subjected to UUO or sham operation for 7 days. A: paraffin-embedded kidney sections were used for immunofluorescent staining with anti-phosporylated (p-)histone H3 antibody. B: tubular p-histone H3-positive cells were measured in five randomly chosen fields per kidney. C: expression of cyclin B1, cyclin D1, and p53 were examined by Western blot analysis. Anti-GAPDH antibody was used as a loading control. D and E: Western blot band densities of the cyclin B1-to-cyclin D1 ratio (D) and p53 (E) were evaluated using ImageJ software. Arrow, p-histone H3-positive cells. Data are expressed as means ± SE; n = 3. *P < 0.05 vs. respective sham-operated mice; #P < 0.05 vs. UUO in WT mice.
Fig. 6.
Fig. 6.
Ablations of both Bax and Bak prevent kidney fibrosis. Mice were subjected to UUO or sham operation for 7 days. A and C: collagen deposition (A) and α-smooth muscle actin (α-SMA)-positive areas (C) were evaluated by Sirius red and immunofluorescent staining with anti-α-SMA antibody in paraffin-embedded kidneys, respectively. B and D: Sirius red-positive (B) and α-SMA-positive (D) areas were measured in five randomly chosen fields per kidney using ImageJ software. E: fibronectin expression was examined by Western blot analysis. Anti-GAPDH antibody was used as a loading control. F: Western blot band densities were evaluated using ImageJ software. Data are expressed as means ± SE; n = 3. *P < 0.05 vs. respective sham-operated mice; #P < 0.05 vs. UUO in WT mice.
Fig. 7.
Fig. 7.
Ablations of both Bax and Bak inhibit the expression of fibrogenic signals in UUO-induced kidney fibrosis. Mice were subjected to UUO or sham operation for 7 days. A: expression of p-EGF receptor (EGFR), p-Smad3, p-JNK, transforming growth factor (TGF)-β, and connective tissue growth factor (CTGF) was examined by Western blot analysis. Anti-GAPDH antibody was used as a loading control. B–F: Western blot band densities of p-EGFR (B), p-Smad3 (C), p-JNK (D), TGF-β (E), and CTGF (F) were evaluated using ImageJ software. G: paraffin-embedded kidney sections were used for immunohistochemistry staining with anti-p-Smad3 antibody. Data are expressed as means ± SE; n = 3. *P < 0.05 vs. respective sham-operated mice; #P < 0.05 vs. UUO in WT mice.
Fig. 8.
Fig. 8.
Dual ablation of Bax and Bak reduces UUO-induced interstitial cell proliferation in the kidney. Mice were subjected to UUO or sham operation for 7 days. A: paraffin-embedded kidney sections were used for immunofluorescent staining with anti-Ki67 antibody. B and C: interstitial (B) and tubular (C) Ki67-positive cells were measured in five randomly chosen fields per kidney. Arrow, interstitial Ki67-positive cells. Data are expressed as means ± SE; n = 3. *P < 0.05 vs. respective sham-operated mice; #P < 0.05 vs. UUO in WT mice.
Fig. 9.
Fig. 9.
A scheme for the role of Bax/Bak in UUO-induced kidney fibrosis. PTC, proximal tubule cells; ECM, extracellular matrix.
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