Hypothermia Alleviates Reductive Stress, a Root Cause of Ischemia Reperfusion Injury

Abstract

Ischemia reperfusion injury is common in transplantation. Previous studies have shown that cooling can protect against hypoxic injury. To date, the protective effects of hypothermia have been largely associated with metabolic suppression. Since kidney transplantation is one of the most common organ transplant surgeries, we used human-derived renal proximal tubular cells (HKC8 cell line) as a model of normal renal cells. We performed a temperature titration curve from 37 °C to 22 °C and evaluated cellular respiration and molecular mechanisms that can counteract the build-up of reducing equivalents in hypoxic conditions. We show that the protective effects of hypothermia are likely to stem both from metabolic suppression (inhibitory component) and augmentation of stress tolerance (activating component), with the highest overlap between activating and suppressing mechanisms emerging in the window of mild hypothermia (32 °C). Hypothermia decreased hypoxia-induced rise in the extracellular lactate:pyruvate ratio, increased ATP/ADP ratio and mitochondrial content, normalized lipid content, and improved the recovery of respiration after anoxia. Importantly, it was observed that in contrast to mild hypothermia, moderate and deep hypothermia interfere with HIF1 (hypoxia inducible factor 1)-dependent HRE (hypoxia response element) induction in hypoxia. This work also demonstrates that hypothermia alleviates reductive stress, a conceptually novel and largely overlooked phenomenon at the root of ischemia reperfusion injury.

Keywords: cellular respiration; hypothermia; hypoxia; ischemia reperfusion injury; organ transplant; reductive stress.

Figure 1

Cirbp (A) and Rbm3 (B) gene expressions at various temperatures and oxygen concentrations. Asterisks refer to a statistically significant difference with respect to 37 °C control unless indicated otherwise. Numbers in bars indicate sample size. Values are expressed as mean ± SEM. Statistical analysis was performed with One-way ANOVA with post-hoc Tukey HSD Test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 2

Ratio of ATP to ADP (A), ratio of extracellular lactate to pyruvate (B), extracellular lactate levels (C), and gene expression of LDH-A (D,E) and PDK1 (F,G) at various temperatures and oxygen concentrations. Treatments were performed for 4 or 24 h. Results are normalized relative to 37 °C group (A–C). Asterisks refer to a statistically significant difference with respect to 37 °C control unless indicated otherwise. Numbers in bars indicate sample size. Values are expressed as mean ± SEM. Statistical analysis was performed with One-way ANOVA with post-hoc Tukey HSD Test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 3

Ratio of mitochondrial DNA to nuclear DNA at various temperatures and oxygen concentrations at 24 h time point (A). EtBr (50 ng/mL, 48 h) was used as a positive control to reduce mitochondrial DNA (B). BNIP3 gene expression at various temperatures and oxygen concentrations (C,D). Treatments were performed for 4 or 24 h. Effect of acute hypothermia on respiration in normoxia (E). Effect of acute hypothermia on CO₂ flux in anoxia (F). Effect of acute hypothermia on the recovery of respiration after anoxia (G). Experiment design (H). Asterisks refer to a statistically significant difference with respect to 37 °C control unless indicated otherwise. Numbers in bars indicate sample size. Values are expressed as mean ± SEM. Statistical analysis was performed with One-way ANOVA with post-hoc Tukey HSD Test or unpaired t test with Welch correction (B). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 4

Transcriptional activity of HRE luciferase reporter at various temperatures and oxygen concentrations (A,B). Treatments were performed for 4 and 24 h. Results are normalized relative to 37 °C group. Asterisks refer to a statistically significant difference with respect to 37 °C control unless indicated otherwise. Numbers in bars indicate sample size. Values are expressed as mean ± SEM. Statistical analysis was performed with One-way ANOVA with post-hoc Tukey HSD Test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 5

Lipid content (A) and PPARG gene expression (B) under mild hypothermia (32 °C) and 1% O₂. Treatments were performed for 4 or 24 h. Scale bar = 100 μm. Asterisks refer to a statistically significant difference with respect to corresponding genotype at 37°C unless indicated otherwise. Numbers in bars indicate sample size. Values are expressed as mean ± SEM. Statistical analysis was performed with unpaired t test with Welch correction (A) or One-way ANOVA with post-hoc Tukey HSD Test (B). *, p < 0.05; **, p < 0.01.