The Role And Mechanism Of Endoplasmic Reticulum-derived Transcription Factor CHOP In Renal Ischemia Reperfusion Injury

Acute kidney injury (AKI) is a common clinical syndrome caused by various diseases; itis rapidly progressive impairment of renal function by lead to kidney damage of structural orfunctional change. It is considered that renal ischemia reperfusion injury is one of the maincauses of AKI, with high morbidity and mortality, accompanied by a series of cellular eventsincluding necrosis, apoptosis, and infiltration of inflammatory cells and the release of theactive medium, which leads to tissue damage.Endoplasmic reticulum (ER) plays an important role in the organelles of eukaryotic cells,which is closely related with the synthesis and transport of proteins, glycosylationmodification, as well as the storage and distribution of the calcium ions. Under the stimulus ofa variety of physical and chemical factors (lack of oxygen, hunger, imbalance of calcium ion,chemicals, etc.), misfolded or unfolded proteins will aggregate within endoplasmic reticulum,which is known as endoplasmic reticulum stress (ERS).Renal ischemia reperfusion injury is a common stress disease caused by blood disorder.In the1970s, Glaumann reported that the ischemic injury could be aggravated after restoringthe blood supply of the ischemic kidney. It is confirmed that energy metabolism disorder isthe pathophysiology basis in the development of IR injury, and the free radicals and overloadof calcium as the key factor of the disease. Research has shown that kidney ischemiareperfusion injury is closely related to ERS. In the process of IR, ischemic, hypoxia, energydeprivation, ATP depletion, accumulation of free radicals and imbalance of calcium ion willinitiate ERS, and excessive ERS further aggravates the IR injury. but the specific mechanismhas not been elaborated. Recent studies have shown that in animal models of renal ischemiareperfusion injury, the expression of endoplasmic reticulum-derived transcription factorCHOP, known as growth arrest and DNA damage inducible153(GADDl53), increasedsignificantly, accompanied by the increased expression of caspase-11. Therefore, we hypothesized that CHOP plays an important role in renal ischemia reperfusion injury throughmediating apoptosis.We established an in vivo model of IR injury in wild type mice and CHOP knockoutmice to investigate the effect of CHOP in the development of ischemia reperfusion injury. Wefurther established an in vitro model of hypoxia reoxygenation (HR) in human renal tubularepithelial cells (HK-2) and human umbilical vein endothelial cells (HUVECs) to verify theimportant role of CHOP and its molecular mechanism in HR injury. It will help to clarify themechanism of ischemic renal injury and provide a scientific basis for searching newtherapeutic targets.1. Methods1.1AnimalsChop deficiency mice were purchased from Jackson Laboratory of USA. Adult malewild type and CHOP knockout mice, about22~26g, were used in this study. The animalswere taken care with a standard12:12h light-dark cycle and had ad libitum access to waterand standard chow.1.2Renal ischemia reperfusion injury modelAnimals were anesthetized with sodium pentobarbital (60mg/kg body weight i.p.),prepped for aseptic surgery and placed on homeothermic surgical tables to maintain bodytemperature of36°C through rectal probe. Following a midline abdominal incision and rightnephrectomy was performed; the left renal artery and vein were clamped with an atraumaticmicro-vascular clamp for25min. A sham operation was performed using the same surgicalprocedure without clamping the renal artery and vein. Animals were killed24h afterreperfusion by exsanguination, to obtain blood and renal samples for further analyses.1.3Bone Marrow Transplantation (BMT)Male recipient and donor mice aged8~10wk were used in this study. Recipient micewere lethally irradiated with a total dose of10.5Gy by Cobalt-60, with a4-hour interval.Bone marrow cells were harvested from the donor mice. Two hours after irradiation, therecipient mice received bone marrow cells (1×107) by tail-vein injection. Renal IR procedureswere performed30days after BMT. 1.4Cell culture and hypoxia reoxygenation (HR)Human proximal tubular cell line (HK-2) and HUVECs (Human umbilical veinendothelial cells), routinely grown in DMEM medium with10%FBS (fetal bovine serum) at37°C and5%CO2, were distributed onto6-well flat-bottomed plates at a concentration of5×105cells/well. According to the experiment scheme, cells were exposed to normoxiccondition (5%CO2,21%O2, and74%N2) or to hypoxia (5%CO2,1%O2, and94%N2).1.5siRNA interferenceThe CHOP sequence for siRNA targeting was synthesized, purified, and annealed byInvitrogen (Life Technologies Corporation, NY, USA). siRNA was synthesized using thefollowing sequences: GCUAGCUGAAGAGAAUGAATT; UUCAUUCUCUUCAGCUAGCTT. HK-2cells and HUVECs were transfected at a final siRNA duplex concentrationof80nM in Opti-Mem by using Lipofectamin2000.2. Results2.1CHOP led to apoptosis in renal IR injury2.1.1The protein expression of CHOP, cleaved caspase-3in renal IRCompared with the control group, the protein expression of CHOP and cleaved caspase-3began to increase after IR3h, reached to peak at6h, and remained high at24h, but stillsignificantly higher than the control group (p<0.05)。2.1.2The effects of CHOP knockout on the protein expression of cleaved caspase-3,survival rates, renal function and pathological changesThe protein expression of cleaved caspase-3: the protein expression of cleaved caspase-3was significantly lower in CHOP knockout mice compared with WT mice after IR6h(p<0.05).The comparision of survival rates: the survival rate of CHOP knockout mice was80%,much higher than that of WT mice (0%)(p<0.05).The comparision of renal function: the levels of serum Cr and BUN in CHOP knockoutmice were significantly lower than WT mice (p<0.05).The comparision of pathological changes: the pathological changes of CHOP knockoutmice were much slighter than that of WT mice (p<0.05). 2.1.3CHOP knockout reduced IR injury by influencing renal microcirculationCompared with WT mice, CHOP knockout improved early renal microcirculation afterischemia and reduced the kidney IR injury.2.2BMT confirmed that CHOP in renal parenchymal cells but not inflammatory cellsled to attenuated renal IR injury2.2.1The comparision of survival rates in BMT miceBMT mice were observed for7days after IR and survival rates were evaluated. Theresults showed no significant difference between BMT (WT→WT)/IR mice and BMT(CHOP-/-→WT)/IR mice, and also between BMT (WT→CHOP-/-)/IR mice and BMT(CHOP-/-→CHOP-/-)/IR mice.2.2.2The comparision of renal function in BMT miceThere was no significant difference in levels of serum Cr and BUN between BMT(WT→WT)/IR mice and BMT (CHOP-/-→WT)/IR mice, and also between BMT(WT→CHOP-/-)/IR mice and BMT (CHOP-/-→CHOP-/-)/IR mice.2.2.3The comparision of pathological changes in BMT miceThere was no significant difference in pathological changes between BMT(WT→WT)/IR mice and BMT (CHOP-/-→WT)/IR mice, and also between BMT (WT→CHOP-/-)/IR mice and BMT (CHOP-/-→CHOP-/-)/IR mice.2.3CHOP in renal tubular epithelial cells and endothelial cells contributed to cellapoptosis2.3.1HR induced inflammatory injury in renal tubular epithelial cellsLDH expression: compared with the control group, the LDH leakage in HK-2cellsincreased6h after HR, and reached to peak at24h after HR (p<0.05).CHOP protein expression: compared with the control group, the expression of CHOPbegan to increase after HR6h, and reached to peak at24h (p<0.05).cleaved caspase-3protein expression: compared with the control group, the expression ofcleaved caspase-3began to increase after HR6h, and reached to peak at24h (p<0.05).2.3.2HR induced inflammatory injury in endothelial cellsLDH expression: compared with the control group, the LDH leakage in HUVECs increased6h after HR, and remained high until24h after HR (p<0.05).CHOP protein expression: compared with the control group, the expression of CHOPbegan to increase after HR6h, and remained high until24h after HR (p<0.05).cleaved caspase-3protein expression: compared with the control group, the expression ofcleaved caspase-3began to increase after HR6h, and remained high until24h after HR(p<0.05).2.3.3CHOP gene silencing significantly alleviated inflammatory injury induced by HRin renal tubular epithelial cellsCHOP siRNA significantly alleviated HR induced cell injury, the LDH leakage markedlydecreased (p<0.05).CHOP siRNA significantly inhibited CHOP protein expression in HK-2cells (p<0.05).CHOP siRNA significantly inhibited cleaved caspase-3protein expression at24h afterHR in HK-2cells (p<0.05).2.3.4CHOP gene silencing significantly alleviated inflammatory injury induced by HRin endothelial cellsCHOP siRNA significantly alleviated HR induced cell injury, the LDH leakage markedlydecreased at6h after HR (p<0.05).CHOP siRNA significantly inhibited CHOP protein expression in HUVECs (p<0.05).CHOP siRNA significantly inhibited cleaved caspase-3protein expression at6h afterHR in HUVECs (p<0.05).3. ConclusionsCHOP in renal parenchymal cells plays an important role in mediating cell apoptosis andrenal damage in IR injury, and CHOP knockout leads to reduced apoptosis and improvedrenal function.