The Role And Mechanism Of CHOP In Inflammatory Response After Kidney Acute Ischemic Injury

Acute kidney injury (AKI) is associated with a high degree of morbidity and mortality. In many surgical and medical settings , ischemic AKI plays a dominant role in the pathogenesis of AKI. AKI is also an inflammatory disease, but the mechanisms how inflammatory response is initiated after kidney ischemia-reperfusion have not been fully understood. Endoplasmic reticulum is the largest cellular organelle responsible for protein synthesis, assembly and modification, and plays an important role in cell survival and normal function. It has been proved that ischemia, hypoxia or energy deprivation could activate a series of partner proteins, transcription factors and protein kinases, and then initiate endoplasmic reticulum stress (ERS). Studies have shown that appropriate ERS could maintain cell homeostasis and preserve cell function; however, prolonged and/or excess ERS leads to cell injury and cell death.. Endoplasmic reticulum molecular chaperone glucose-regulated protein 78 (GRP78) and oxygen-regulated protein 150 (ORP150) are considered as markers of ERS. One recent study showed that strong endoplasmic reticulum stress and renal tubular epithelial cell apoptosis could be observed after the renal arteries clamped for 10 minutes. Another study found that excessive ERS could induce not only cell apoptosis but also inflammatory response. CHOP (also known as a growth arrest or DNA damage inducible gene 153, GADD153) is an important pro-apoptotic molecule distributed in the endoplasmic reticulum. Recent study found that activation of CHOP could cause inflammatory response, for example, CHOP knockout could decrease the concentrations of inflammatory cytokines and attenuate the tissue injury in mouse model of pneumonia and pancreatitis induced by LPS. In the present study, we observed the dynamic changes of CHOP and its relationship with inflammatory response in renal tissue after kidney ischemia-reperfusion injury.Methods1. ERS and the expression of inflammatory cytokines after kidney ischemia and reperfusion Injury The renal ischemia and reperfusion (I/R) model was performed by clamping the bilateral renal pedicles for 40 min and reperfusion for 1, 6, 12 and 24h. Blood SCr and BUN levels were detected by chemistry analyzer. Renal tissue injury was measured by PAS stain. The expression of GRP78 and CHOP in the ischemic renal tissue was detected by immunohistochemistry, fluorescence quantitative RT-PCR and Western blot, respectively. Renal tissue IL-1β, IL-6 and IL-8 levels were detected by ELISA.2. ERS and the expression of inflammatory cytokines induced by Hydrogen Peroxide (H₂O₂) in HKCCells were cultured in 10% FBS DMEM culture medium and 37℃, 5% CO₂ incubator. When the density was over 95%, serum-free DMED was used for synchronization. HKC was stimulated for 4h with 50, 100, 200, 500, 1000, 5000μmol H₂O₂, respectively. Then the HKC and the culture supernatant were collected. on the other hand, HKC was stimulated with 100umol H₂O₂ for 1, 2, 4, 8, 12 and 16h, respectively, then collected the HKC and culture supernatant. LDH was detected with LDH kit. Cell survival viability was detected by MTT. IL-1β, IL-6 and IL-8 in culture medium were detected by ELISA. The mRNA and protein expression of GRP78, CHOP, IL-1β, IL-6 and IL-8 were detected by Real-time PCR and Western blot, respectively.3. The effects of CHOP over expression and CHOP gene silencing on ERS and the expression of inflammatory cytokins induced by H₂O₂HKC was transfected for 24h with liposome Lipofectamine ? 2000 5μl, PEGFP-CHOP 4μg, PEGFP-N1 4μg, CHOP siRNA 8ul, control siRNA 8ul, respectively. The transfection efficiency was observed with fluorescence microscope. Then the expression of CHOP mRNA and protein was detected by Realtime-PCR and western blot.HKC was stimulated with 100μmol H₂O₂ for 12h. Cells were divided into HKC group, HKC + H₂O₂ group, CHOP siRNA + H₂O₂ group, control siRNA + H₂O₂ group, PEGFP-CHOP + H₂O₂ group, PEGFP-N1 + H₂O₂ group, respectively. LDH in culture supernatant, cell survival viability, IL-1β, IL-6, IL-8 levels, CHOP mRNA and protein levels were detected.Results1. At 1 h after kidney I/R, tubular damage score was 1.17, SCr levels and renal tissue IL-1β, IL-6 and IL-8 levels were all markedly increased compared with those in sham group, and reached to peak at 12 h after I/R. Compared to those in sham group, the protein and mRNA expressions of GRP78 and CHOP in kidney I/R groups increased distinctly in renal tissue at 1h after I/R. Correlation analysis showed that renal tubular injury score and inflammatory cytokines levels were positively correlated with the expression of CHOP.2. With the concentration of H₂O₂ increased or the stimulation prolonged, the LDH leakage in HKC increased; the survival rate of HKC declined; the expression of IL-1β, IL-6 and IL-8 increased; the expression of GRP78 and CHOP increased, too. the levels of inflammatory cytokines and the expression of CHOP had positive correlation.3. Green fluorescent signal in HKC was detected. CHOP expression was significantly higher in PEGFP-CHOP transfected HKC and was notably lower in CHOP siRNA transfected HKC than that in the control group respectively. Incubating PEGFP-CHOP transfected HKC with 100μmol H₂O₂ for 12h, the LDH levels increased; cell survival viability reduced; IL-1β, IL-6 and IL-8 levels were significantly higher than that in the HKC + H₂O₂ group and PEGFP-N1 + H₂O₂ group respectively.Incubating CHOP siRNA transfected HKC with H₂O₂ for 12h, the LDH levels reduced; cell survival viability increased; IL-1β, IL-6 and IL-8 levels were lower than that in the HKC + H₂O₂ group and control siRNA + H₂O₂ group respectively.ConclusionsI/R and H₂O₂ can increase the expressions of CHOP and inflammatory cytokines in renal tissues and HKC; Over expression of CHOP can increase the production of inflammatory cytokines and worsen the HKC injury induced by H₂O₂; Inhibit the expression of CHOP can reduce the production of inflammatory cytokines and attenuate the HKC injury induced by H₂O₂. These results suggest that the increased expression of CHOP induced by ERS plays an important role in the mechanisms of ischemic AKI.