Ischemia/Reperfusion-Induced CHOP Expression Promotes Apoptosis and Impairs Renal Function Recovery: The Role of Acidosis and GPR4

<div><p>Endoplasmic reticulum (ER) stress-induced apoptosis is implicated in a wide range of diseases, including ischemia/reperfusion injury (IRI). As a common feature of ER stress, the role of CCAT/enhancer-binding protein homologous protein (CHOP) in renal IRI has not been thoroughly investigated. We found that IR led to renal CHOP expression, accompanied by apoptosis induction. Renal IRI was markedly alleviated in CHOP^−/− mice. Observations from bone marrow chimeras showed that this was based on CHOP inactivation in renal parenchymal cells rather than inflammatory cells. In vivo and in vitro studies demonstrated that IRI induced CHOP expression in both endothelial and epithelial cells, which was responsible for apoptosis induction. These results were reinforced by the observation that CHOP knockout led to improvement of the postischemic microcirculatory recovery. In vitro studies revealed hypoxia-induced acidosis to be a major inducer of CHOP in endothelial cells, and neutralizing acidosis not only diminished CHOP protein, but also reduced apoptosis. Finally, knockdown of a proton-sensing G protein-coupled receptor GPR4 markedly reduced CHOP expression and endothelial cell apoptosis after hypoxia exposure. These results highlight the importance of hypoxia-acidosis in ER stress signaling regulation in ischemic kidneys and suggest that GPR4 inhibitors or agents targeting CHOP expression may be promising in the treatment of renal IRI.</p></div

Renal IRI after bone marrow transplantation (BMT).

<p>BMT (WT→WT, CHOP^−/−→WT, WT→CHOP^−/− and CHOP^−/−→CHOP^−/−) was performed 30 days before renal IR procedures. Blood and kidneys were harvested at 24 h after the initiation of reperfusion. Concentrations of serum creatinine (A) and BUN (B) were measured. Data were expressed as mean ± SD from 6 animals per group. *P<0.05 vs. BMT (WT→WT)/IR group and BMT (CHOP^−/−→WT)/IR group. (C–D) Survival of BMT mice after renal IR (n = 10 per group). Compared with BMT (WT→WT) group and BMT (CHOP^−/−→WT) group, BMT (WT→CHOP^−/−) group and BMT (CHOP^−/−→CHOP^−/−) group had a significant survival advantage by Kaplan-Meier analysis (log-rank test, P<0.05). (E) Representative pathological sections and corresponding histological scores of post-ischemic kidneys harvested at 24 h after reperfusion were shown. *P<0.05 vs. BMT (WT→WT)/IR group and BMT (CHOP^−/−→WT)/IR group.</p

The effect of CHOP inactivation on renal IRI. CHOP^−/− and wild-type mice were subjected to right nephrectomy, followed by IR (left kidney) or sham-operation.

<p>Serum creatinine (A) and BUN (B) concentrations at 24 h after the initiation of reperfusion were shown (n = 8 per group). (C) Survival of WT and CHOP^−/− mice after renal IR operations (n = 20 per group). CHOP knockout led to a significant survival advantage by Kaplan-Meier analysis (log-rank test, P<0.05). (D) Representative PAS-stained sections from post-ischemic kidneys harvested at 24 h (original magnification, ×200). (E) Representative renal MPO staining (original magnification, ×200). (F) TUNEL assay (original magnification, ×400). (G) Representative immunoblotting results of the non-IR right kidneys and the operated left kidneys (6 hours after reperfusion). (H–I) Quantitative analyses of the relative levels of protein expression. Cleaved caspase-3 and CHOP protein bands were quantified and normalized to β-actin. The mean value obtained from non-IR WT kidneys was arbitrarily defined as 1. There were 6 mice in each group and data were expressed as mean ± SD. *P<0.05 vs. WT/IR group.</p

8-bromo-cAMP induced CHOP expression in HUVECs.

<p>(A) 8-bromo-cAMP (500 µM) was added into the medium of HUVECs. 3 h, 6 h and 12 h later, cells were harvested and CHOP expression was assayed. (B) Quantitative analyses of the relative levels of CHOP expression. The mean value obtained from the control was arbitrarily defined as 1. Data were expressed as mean ± SD from four separate experiments. *P<0.05 vs. the control group.</p

The expressions of cleaved caspase-3 and CHOP in HK-2 cells (A) and HUVECs (B) after HR.

<p>The cells were harvested at the indicated time points after exposure to HR and total protein was subjected to immunoblotting analyses. Representative images of 6 separate experiments were shown.</p

Hydrochloric acid induced CHOP expression in HUVECs.

<p>(A) HUVECs were treated with acidic media (pH 6.4), and 3 h, 6 h and 12 h later cells were harvested and CHOP expression was assayed. (B) Quantitative analyses of the relative levels of CHOP expression. The mean value obtained from the control was arbitrarily defined as 1. Data were expressed as mean ± SD from four separate experiments. *P<0.05 vs. the control group.</p

Knockdown of CHOP reduced apoptosis in HR-confronted HUVECs.

<p>HUVECs were transfected with a scramble (control) or CHOP-specific siRNA at a final concentration of 80 nmol/l. 36 h later, cells were exposed to 4 h of hypoxia, followed by 6 h of reoxygenation. Then cells were collected and subjected to western blot analysis. Representative images of 6 separate experiments were shown (A). Quantitative analyses of the protein levels. Cleaved caspase-3 (B) and CHOP (C) protein bands were quantified and normalized to β-actin. The mean value obtained from the control was arbitrarily defined as 1. (D) The percentage of LDH release in the supernatant. Data were expressed as mean ± SD from 4–6 separate experiments. *P<0.05 vs. the control siRNA-transfected cells with HR treatment.</p

CHOP is prominently present in endothelial cells rather than epithelial cells in the early period after ischemic insult.

<p>Representative renal sections from wild-type mice harvested at 3 h and 24 h after ischemic insult (original magnification, ×400). Sections were stained with the indicated antibodies, followed by confocal microscopic analyses.</p

Cleaved caspase-3 and CHOP expression in the post-ischemic kidneys.

<p>(A) Representative immunoblotting results from post-ischemic renal samples. (B–C) Quantitative analyses of the relative levels of protein expression. Cleaved caspase-3 and CHOP protein bands were quantified and normalized to β-actin. The mean value obtained from sham-operated mice was arbitrarily defined as 1. There were 6 mice in each group and data were expressed as mean ± SD. *P<0.05 vs. sham-operated controls.</p

Neutralization of acidosis reduced CHOP expression and apoptosis in HR-treated HUVECs.

<p>The pH of culture media was adjusted to 7.4 with NaOH (1N) immediately after hypoxia exposure, followed by 6 h of reoxygenation. Then both cells and supernatants were harvested. (A) The expressions of cleaved caspase-3 and CHOP were determined by immunoblotting assay. (B–C) Quantitative analyses of the relative levels of protein expression. Cleaved caspase-3 and CHOP protein bands were quantified and normalized to β-actin. The mean value obtained from the control was arbitrarily defined as 1. (D) The percentage of LDH release in the supernatant. Data were expressed as mean ± SD from 4–6 separate experiments. *P<0.05 vs. HR-treated group without pH adjustment.</p