The Role And Mechanism Of Irhom2 In Acute Kidney Injury

Acute kidney injury(AKI)is a clinical syndrome caused by rapid reduction of kidney function in a short period of time.AKI has high morbidity and mortality,and long-term outcomes of AKI can include the development of chronic kidney disease(CKD)and end-stage renal disease(ESRD).Renal ischemic reperfusion injury and toxic responses to medications such as cisplatin are frequent causes of AKI.The pathophysiological mechanism of AKI is relatively complex.At present,patients with AKI are mainly treated by renal dialysis.Therefore,it is of great significance to actively explore the pathogenesis of AKI and identify effective therapies to prevent or ameliorate AKI.iRhom2(inactive Rhomboid protein 2,encoded by the Rhbdf2 gene),a member of the iRhoms subfamily of the Rhomboid family,belongs to an inactive rhomboid serine protease.Studies have shown that iRhom2 plays an important role in inflammatory diseases such as arthritis and pathogen infection by participating in the regulation of the transport process of intracellular proteins(Tumor necrosis factor-α-converting enzyme,Stimulator of interferon genes,etc.)from endoplasmic reticulum to Golgi apparatus and perinuclear microsomes.Inflammation is now recognised as key mediator of tubular cell injury in AKI.At present,whether iRhom2 is involved in AKI pathophysiology has not been reported.Therefore,this study mainly focuses on the role and preliminary mechanism of iRhom2 in AKI.Research Objectives:1.To explore the expression pattern of iRhom2 in AKI by in vivo animal experiment,in vitro cell experiment and clinical specimen detection.2.To explore the role of iRhom2 in AKI by in vivo animal experiments and in vitro cell experiments.3.To clasrify the preliminarily mechanism by which iRhom2 promoting AKI.Research methods and resultsPart I:The expression of iRhom2 is significantly increased in AKI1.1 The expression of iRhom2 was significantly increased in ischemia-reperfusion induced AKIMale C57BL/6J mice aged 8 weeks were used in renal Ischemia reperfusion injury(IRI)model by ligation of bilateral renal arteries for 30 minutes and reperfusion for 6 h,12 h,24 h and 48 h,respectively.The concentration of serum creatinine and urea nitrogen showed that AKI model was successfully established.The second generation transcriptome gene sequencing analysis showed that the expression of iRhoms subfamily was increased after renal IRI,especially Rhbdf2.Meanwhile,real-time quantitative PCR(RT-qPCR),Western blot(WB)and Immunohistochemical staining(IHC)results showed that the expression of iRhom2 increased in a time-dependent manner and was relatively high at 12 h after ischemia-reperfusion.Immunofluoresce(IF)experiments showed that iRhom2 expression was significantly increased in distal convoluted renal tubules after renal IRI.1.2 iRhom2 expression was significantly increased in a variety of hypoxia models in vitroRat renal tubular epithelial cells(NRK-52E)were used in hypoxia model in vitro.The results of RT-qPCR and WB showed that the expression of iRhom2 was significantly increased under oxygen and glucose deprivation(OGD),anti-mycin A,2-hypoxic glucose(AA/2-DG)and cobalt chloride(CoCl2)stimulation,respectively.1.3 iRhom2 expression was significantly increased in the kidney tissue of cisplatin-induced AKIAfter intraperitoneal injection of cisplatin(25 mg/kg),samples were collected at 1 d,2 d and 3 d.The concentration of serum creatinine and urea nitrogen showed that the model was successfully established.Results of RT-qPCR,WB and IHC showed that the expression of iRhom2 increased at different time points after cisplatin injection,and the expression was relatively high at 2 d.1.4 The expression of iRhom2 was significantly increased in human kidneys from the patients with ATNIHC results showed that iRhom2 expression in renal tubules was significantly elevated in human kidneys from the patients with ATN compared with paracancarcinoma tissue sections.Part II:The role of iRhom2 in AKI2.1 Generation of Rhbdf2 knockout miceRhbdf2-/-mice were generated by using the Cre-loxp system.The homozygous female Rhbdf2 mice with loxp locus were mated with dppa3-cre+/-male mice,and finally Rhbdf2-/mice were obtained.Mouse tail DNA was extracted to identify the mouse genotypes,and the results showed that Rhbdf2 was successfully knockout.The knockout efficiency of iRhom2 in renal cortex detected by RT-qPCR and WB was 78%.2.2 Generated conditional knockout mice in which Rhbdf2 is specifically ablated in renal tubular epithelial by using Cre-loxp recombination system.The renal tubular epithelial cells knockout Rhbdf2 mice--Cdh16-cre+/-Rhbdf2fl/fl(marked as KSP-cre/Rhbdf2fl/fl in the results of this paper)was generated by using the Cre-loxp system,and the mouse tail DNA was extracted to identify the genotypes.The results showed that KSP-cre/Rhbdf2fl/fl was the mouse with specific knockout of Rhbdf2 in renal tubular epithelial cells,and the analysis of second-generation transcriptional sequencing results showed that the location of Rhbdf2 exon 4-9 was successfully knocked out.Further RT-qPCR and IF detection results showed that the expression of iRhom2 in renal tubular epithelial cells was significantly reduced,which proved that conditional knockout mice were successfully generated.2.3 Rhbdf2 deficiency protects against renal ischemia-reperfusion injuryThe IRI model was constructed by bilateral renal ischemia for 30 min and reperfusion for 24 h in 8-week-old male Rhbdf2fl/fl,Rhbdf2-/-and KSP-cre/Rhbdffl/fl mice.The concentration in serum creatinine and urea nitrogen induced by ischemia-reperfusion was significantly reduced after Rhbdf2 deficiency,suggesting that iRhom2 deficiency ameliorated renal ischemia-reperfusion injury.Hematoxylin-eosin staining(HE)and Periodic acid-schiff staining(PAS)results showed that Rhbdf2 deficiency could significantly improve the edema and swelling of renal tubules,brush edge shedding,and nuclear shedding caused by ischemia-reperfusion.IHC results showed that Kidney injury molecule-1(Kim-1)and Neutropil gelatinase-associated lipocalin(NGAL)were significantly reduced after Rhbdf2 deficiency.In situ terminal transferase labeling(TUNEL staining)further showed that TUNEL positivity was significantly increased after renal ischemia-reperfusion,while apoptosis of renal tubular epithelial cells was significantly reduced after Rhbdf2 deficiency.These results suggest that Rhbdf2 deficiency protects against renal ischemia-reperfusion injury.Moreover there is no significant difference between the IRI group of Rhbdf2-/-and KSP-cre/Rhbdf2fl/fl suggesting that iRhom2 expressed in renal tubules plays an important role in ischemia-reperfusion injury.2.4 Rhbdf2 deficiency protects against cisplatin-induced AKIThe 10-week-old male Rhbdf2fl/fl and Rhbdf2-/-mice were used in cisplatin-induced AKI.The concertration of serum creatinine and urea nitrogen,HE staining,PAS staining and IHC results of NGAL showed that Rhbdf2 deficiency protects against cisplatin-induced AKI.Part III:Preliminarily study on the mechanism of iRhom2 in ischemia-reperfusion induced AKI3.1 iRhom2 promots ischemia-reperfusion injury by aggravating inflammatory responseMacrophages were labeled with CD68 and neutrophils were labeled with Ly6b.IHC results showed that macrophages and neutrophils infiltration increased significantly in Rhbdf2fl/fl mice after renal IRI,and the infiltration of macrophages and neutrophils in Rhbdf2-/-and KSP-cre/Rhbdf2fl/fl mice significantly reduced.The mRNA expression levels of TNF-α,MCP-1,IL-6,IL-1β and IL-18 were significantly reduced in Rhbdf2-/-and KSP-cre/Rhbdfl/fl mice.In vitro NRK-52E cells were used in OGD treatment.WB results showed siRNA-Rhbdf2 inhibit the expression iRhom2 by 60%.The results of lactate dehydrogenase and Caspase-3 activity showed that Rhbdf2 silencing could significantly reduce the death of NRK-52E induced by OGD.Meanwhile,RT-qPCR results showed that expression of relevant inflammatory factors were significantly decreased after Rhbdf2 silencing,which verified the conclusion in vivo.These results suggest that Rhbdf2 deficiency alleviates renal ischemia-reperfusion injury by reducing inflammatory cell infiltration and inflammatory factor expression.3.2 iRhom2 promots renal ischemia-reperfusion injury by stimulating in endoplasmic reticulum stress and regulating the cleavage of ATF6Endoplasmic reticulum stress plays an important role in inflammatory injury of acute kidney injury.WB results showed iRhom2 regulated endoplasmic reticulum stress signaling pathway especially ATF6 signaling pathway.WB results also indicated that iRhom2 could regulated the cleavage of ATF6.CO-IP results showed that iRhom2 could combined with ATF6.Dual immunofluorescence staining found that iRhom2 translocated from endoplasmic reticulum to Golgi after NRK-52E cells were stimulated by OGD.Whether the translocation of iRhom2 could assist ATF6 to transfer from endoplasmic reticulum to Golgi remains to be further verified.These results suggest that iRhom2 may participate in endoplasmic reticulum stress signaling pathway and aggravate renal ischemia-reperfusion injury mainly through regulating the cleavage of ATF6,and the specific mechanism needs to be further studied.Conclusions and Innovation:1.The expression of iRhom2 was significantly increased in clinical specimens and animal models of AKI.The elevated iRhom2 aggravated kidney injury caused by AKI by increasing inflammatory cell infiltration and promoting the expression of inflammatory factors.It was also found that conditional knockout Rhbdf2 mice significantly alleviated inflammatory response and improved acute kidney injury,with no significant difference compared with Rhbdf2-/-mice,suggesting that iRhom2 in renal tubular epithelial cells might play an important role in AKI.2.This study provided new insights into the biological functions of iRhom2 and found that iRhom2 might regulate the endoplasmic reticulum stress signaling pathway especially ATF6 signaling pathway in renal ischemia-reperfusion injury.Finally,our study provided a potential therapeutic target and important experimental foundations for the treatment of AKI.