Injury Mechanism Of IRE1/JNK Signaling Pathway On Renal Tubular Epithelial Cells And Mesangial Cells During AKI To CKD Transition

Background:Acute kidney injury(AKI)is one of the most common critical diseases in clinic,with an incidence of intensive care units more than 50%.The etiology of AKI is diverse,the progression is complex and changeable as well as the prognosis is different.The occurrence and development of AKI is a complicated process of coexistence of injury and repair,among which,maladaptive repair is the key mechanism for the progression of AKI to chronic kidney disease(CKD)and even end-stage renal disease(ESRD).Endoplasmic reticulum stress(ER stress)and the secretion of inflammatory factors play an important role in maladaptive repair of renal tubular epithelial cells.Renal-tubular epithelial cells,as the main damaged cells in AKI,are the main source of inflammatory mediators of renal tissue.In addition,studies have found that ER stress is inextricably linked to inflammatory responses.Among them,IRE1 is most closely associated with the activation of inflammatory signals.Activated IRE1 can promote the phosphorylation of JNK and activate NF-κB,which encode the gene expression of the inflammatory mediators.Studies have found that the IRE1/JNK signaling pathway plays a role in a variety of tissues and cells,but there are few reports about the role of the IRE1/JNK signaling pathway in regulating inflammatory factors in AKI in the domestic and overseas reports.Grgic et al.found long-term repeated tubular damage can lead to glomerular segmental sclerosis in a mouse model of AKI with specific tubular damage,which suggests that renal tubular lesions may cause the glomeruli damage and sclerosis,but the mechanism is unclear.The accumulation of extracellular matrix(ECM)in the glomerulus is an important feature of glomerulosclerosis and the main ECM cell-secretors in glomerulus are mesangial cells.Studies have found that IL-6 can activate the signal transducer and activator of transcription 3(STAT3)pathway of mesangial cells,leading to the production of fibronectin,and the IL-6/STAT3 pathway is expressed in the renal interstitium and a variety of tissues,which can promote fibrosis.Therefore,the IL-6/STAT3 pathway may mediate the injury of mesangial cell and the increase of extracellular matrix(ECM)caused by inflammatory factors in AKI.In summary,the pathological changes of the renal tubules in AKI may partially damage the mesangial cells of the glomeruli,which may lead to the occurrence of fibrosis.This study aimed to explore how the mesangial cells were damaged and the ECM secretion was increased after the injury of renal tubular epithelial cells and the activation of the IRE1/JNK signaling pathway in AKI,and try to explore the potential mechanism of AKI to CKD transition.Method:In this study,human kidney tissue,human renal tubular epithelial cells(HK2),and human renal mesangial cells(HRMC)were selected as the research objects.1.Collection of human kidney tissue specimens:5 renal biopsy specimens from acute renal injury caused by insufficient renal perfusion and pathologically diagnosed as acute tubular injury were selected.5 cases of normal renal specimens excised from tumor margin more than 2 cm were selected as control.The specimens were taken from the First Affiliated Hospital of Zhengzhou University.2.Immunohistochemistry was used to detect the expression of ER stress markers and inflammatory factors in renal tubular epithelia-l cells of human kidney tissue.3.To induce hypoxia-reoxygenation injury model,HK-2 cells with serum free DMEM/F12 were exposed to 4 h of hypoxia(5%CO2,1%O2,94%N2 at 37℃),followed by 6,12 or 24 h of reoxygenation(5%CO2,95%air at 37℃)with normal medium.Controls were incubated in a humidified incubator with 5%CO2 at 37℃.4.Western Blot was used to detect the expression of ER stress markers in the hypoxia-reoxygenation HK2 model.The enzyme-linked immunosorbent assay(ELISA)was used to detect the secretion of inflammatory factors.And the morphology of endoplasmic reticulum was observed by transmission electron microscope.5.The effect of knocking down IRE1α or JNK1 expression of HK2 on the expression of ER stress markers in hypoxia-reoxygenation model was detected by Western Blot,and the effect ofthis interference on the secretion of inflammatory factors in hypoxia-reoxygenation model was detected by-ELISA.6.Western Blot was used to detect the activation of STAT3 pathway and the secretion of ECM in HRMC stimulated by IL-6.7.Western Blot was used to detect the effect of knocking down STAT3 expression of HRMC on the secretion ofECM in HRMC stimulated by IL-6.8.Co-culture of control HK2 and HK2 knocked down IRE1α or JNK1 expression which is normal or hypoxic with HRMC for 24 h respectively were performed by transwell co-culture technology.Western blot was used to detect the activity of STAT3 pathway and the secretion ofECM in HRMC after co-cultivation.Result:1.The expression of endoplasmic reticulum stress markers in renal tubular epithelial cells of patients with AKI increased compared with the normal control group,and the secretion of inflammatory factors also increased compared with the normal group.2.In the model of hypoxia-reoxygenation HK2,the expression of ER stress markers began to increase at 6 h,and reached apeak at 12 h,and decreased at 24 h.The secretion of inflammatory factors increased gradually with the prolongation of reoxygenation time,with a statistical difference(P<0.05).Compared with the control group,the endoplasmic reticulum of the hypoxia-reoxygenation groups was wider.3.Knocking down of IRE1α or JNK1 respectively,could lead to a significant decrease in the expression of the downstream ER stress markers and secretion of inflammatory factors in the HK2 hypoxia-reoxygenation model with a statistical difference(P<0.05).4.IL-6 stimulation of HRMC can lead to the activation of STAT3 pathway,reaching a peak at 24 hours,and the secretion of ECM increased significantly with the prolongation of stimulation time.5.Knocking down the expression of STAT3 in HRMC can lead to a significant decrease in the secretion of ECM in HRMC stimulated by IL-6.6.Co-cultivation with HK2 after hypoxia can activate the STAT3 pathway in HRMC as well as lead to the additional secretion of ECM.And knocking down the expression of IRE1α or JNK1 in HK2 can attenuate the activation of STAT3 pathway and the additional secretion of ECM.Conclusion:1.Renal tubular epithelial cell injury after AKI can cause endoplasmic reticulum stress,which in turn activates the IRE1/JNK signaling pathway and regulates the secretion of inflammatory factors.2.The stimulation of IL-6 can increase the secretion of ECM by activating the STAT3 pathway in HRMC.3.After hypoxia and reoxygenation,HK2 can activate the STAT3 pathway of HRMC by secreting IL-6,leading to an increase in the extracellular matrix secreted by HRMC,which may be one of the mechanisms for the progression of AKI to CKD.However,the depletion of IRE1α or JNK1 in HK2 can attenuate this injury.