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. 2006 Feb 21;103(8):2809-14.
doi: 10.1073/pnas.0506854103. Epub 2006 Feb 14.

Cytoprotective gene bi-1 is required for intrinsic protection from endoplasmic reticulum stress and ischemia-reperfusion injury

Béatrice Bailly-Maitre  1 , Constantino Fondevila, Fady Kaldas, Nathalie Droin, Fréderic Luciano, Jean-Ehrland Ricci, Rhonda Croxton, Maryla Krajewska, Juan M Zapata, Jerzy W Kupiec-Weglinski, Douglas Farmer, John C Reed
Affiliations

Cytoprotective gene bi-1 is required for intrinsic protection from endoplasmic reticulum stress and ischemia-reperfusion injury

Béatrice Bailly-Maitre et al. Proc Natl Acad Sci U S A. .

Abstract

Ischemia-reperfusion (IR) injury induces endoplasmic reticulum (ER) stress and cell death. Bax Inhibitor-1 (BI-1) is an evolutionarily conserved ER protein that suppresses cell death and that is abundantly expressed in both liver and kidney. We explored the role of BI-1 in protection from ER stress and IR injury by using bi-1 knockout mice, employing models of transient hepatic or renal artery occlusion. Compared to wild-type bi-1 mice, bi-1 knockout mice subjected to hepatic IR injury exhibited these characteristics: (i) increased histological injury; (ii) increased serum transaminases, indicative of more hepatocyte death; (iii) increased percentages of TUNEL-positive hepatocytes; (iv) greater elevations in caspase activity; and (v) more activation of ER stress proteins inositol-requiring enzyme 1 and activating transcription factor 6 and greater increases in expression of ER stress proteins C/EBP homologous protein and spliced XBP-1 protein. Moreover, hepatic IR injury induced elevations in bi-1 mRNA in wild-type liver, suggesting a need for bi-1 gene induction to limit tissue injury. Similar sensitization of kidney to ER stress and IR injury was observed in bi-1(-/-) mice. We conclude that bi-1 provides endogenous protection of liver and kidney from ER stress and IR injury. Analysis of components of the bi-1-dependent pathway for protection from IR injury may therefore reveal new strategies for organ preservation.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
BI-1 deficiency sensitizes to cell death induced by OGD. Hepatocytes were isolated from adult wild-type (+/+) and knockout (−/−) mice. (A and B) Hepatocytes were cultured for 60 min in normal medium (C) or in medium containing 20 mM deoxyglucose (DG), 2.5, 5, or 10 mM KCN, or the combination (OGD), then washed and cultured 1 day in normal medium with or without 50 μM benzoyl-Val-Ala-Asp-fluoromethyl-ketone (z). As positive controls, hepatocytes were also treated with 10 μM staurosporine (STS) or 500 ng/ml anti-Fas (Fas) for 24 h. After treatments, the percentages of viable cells (A) (relative to control) (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay) and TUNEL-positive cells (B) were quantified (mean ± SD; n = 5). (C and D) Hepatocytes were cultured for 1–3 h in medium lacking glucose in 1% O2, then washed and switched to normal medium and atmosphere, with or without 50 μM z. The percentages of viable (C) and TUNEL-positive (D) cells were quantified (mean ± SD; n = 4).
Fig. 2.
Fig. 2.
Increased apoptosis and caspase activity in post-IR livers of bi-1 knockout mice. (A) TUNEL Assay. Apoptotic mouse hepatocytes were visualized by TUNEL assay in tissue sections from livers of bi-1−/− (KO) and bi-1+/+ (WT) mice (mean ± SD; n = 5–8). (B–D) Caspase activities. Liver homogenates from ischemic (I) and nonischemic (NI) lobes were prepared from bi-1−/− (KO) and bi-1+/+ (WT) mice after IR injury, normalized for total protein content, and caspase protease activities were measured by using fluorigenic tetrapeptide substrates: Ac-DEVD-AFC (B); Ac-LEHD-AFC (C) and Ac-IETD-AFC (D). Data represent Vmax (mean ± SD; n = 5–8). Statistical significance (∗) was determined by the Mann–Whitney U test (P < 0.05, KO versus WT).
Fig. 3.
Fig. 3.
Analysis of molecular markers of ER stress. Mice were subjected to 90 min of hepatic ischemia, followed by 6 h of reperfusion. The ischemic (I) and normal liver lobes (NI) were then collected, and total proteins were extracted. The expression of ER stress and apoptotic proteins was compared in bi-1+/+ and bi-1−/− livers by immunoblot analysis. Examples of representative blots are shown at the top. Quantification of the most relevant data were performed by scanning densitometry and is shown at the bottom (mean ± SEM; n = 3–7). Statistical significance was determined by t test and is denoted by asterisks (P ≤ 0.05).
Fig. 4.
Fig. 4.
Analysis of apoptosis markers in kidney from bi-1−/− and bi-1+/+ mice. (Upper Left) TUNEL-positive nuclei were quantified in tissue sections from kidney of bi-1−/− (KO) and bi-1+/+ (WT) mice and expressed as a percentage of total nuclei counted (mean ± SD; n = 4). The histograms summarize the expression levels of active caspase-3 and cleaved DFF40 as determined by immunoscore (Upper Center and Upper Right) and immunohistochemical densitometry (Lower).
Fig. 5.
Fig. 5.
BI-1 deficiency results in expression of ER stress proteins in kidney. Total proteins were extracted from ischemic (I) and nonischemic (NI) kidneys of bi-1+/+ and bi-1−/− mice. The expression of ER and apoptotic proteins was compared by immunoblotting. Examples of representative blots are shown at top. Data were quantified by scanning densitometry at bottom (mean ± SEM; n = 3–4). Statistical significance was determined by t test (∗, P ≤ 0.05).
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