REV-ERBα-and Ferroptosis-based Mechanistic Studies On Aristolochic Acid Ⅰ-induced Renal Injury

ObjectivesAristolochic acid I(AAI)is an organic acid compound,mainly derived from Aristolochiaceae plants.Whilst AAI possesses anti-inflammatory,anti-tumor,antibacterial,and anti-venom properties,it is also nephrotoxic.Cases of aristolochic acid(AAI and its analogues)-induced kidney injury(the so-called ’aristolochic acid nephropathy’)have been reported sporadically aroud the world.The main reason is the consumption of Chinese herbal medicines containing aristolochic acid or food and drinking water contaminated with aristolochic acid.However,there is still no effective therapy for aristolochic acid nephropathy,probably because little is known about its pathological mechanism.REV-ERBα(also known as NR1D1,nuclear receptor subfamily 1 group D member 1)is a member of the nuclear receptor superfamily,and is also an important component of the mammalian circadian clock system.REV-ERBα plays an important role in many diseases such as sleep disorders,inflammation,cardiovascular diseases,metabolic disorders and cancer.This study aimed to investigate the chrononephrotoxicity of AAI and the role of REV-ERBα in AAI nephrotoxicity.Methods1.Wild-type mice were intraperitoneally injected with AAI(2.5 and 5 mg/kg)at different time points(ZT 2 and 14)once daily for 5 days.Plasma and kidney tissue were collected 24 h after the last dose.Creatinine(CRE)and blood urea nitrogen(BUN)were determined by kits.qPCR assay was performed to detect the expression of renal injury biomarker genes Ngal and Kim-1.The pathological characteristics of renal tissue were observed by H&E staining.2.To explore the relationship between circadian clock genes and AAI nephrotoxicity,we used qPCR and Western blotting to determine the renal mRNA and protein levels of REV-ERBα,PER2,BMAL1,and CLOCK in the AAI and control group.The in vitro regulatory effects of AAI(10 μM and 25 μM)and its toxic metabolite ALI(10 μM and 25 μM)on REV-ERBα and BMAL1 were evaluated based on a mouse renal tubular epithelial cell(mRTECs)model.Running-wheel activity assay was used to investigate the effect of AAI on the locomoter activity rhythm of mice.The effects of AAI and ALI on molecular clocks were studied based on a rhythmic Per2-dLuc cell model.3.To explore the relationship between ferroptosis and AAI nephrotoxicity,we detected renal ferroptosis-related indicators(iron concentration,GSH,GPX4 and HO-1 expression)in the AAI and control groups.In addition,ferroptosis-related indicators in mRTECs cells(treated with ALI for 24 h)were also measured.4.Kidney-specific Rev-erba knockout(Rev-erbakKO)mice were generated by breeding Rev-erbαflox/flox mice with Cdh16-Cre mice.The genotypes of Reverbαflox/flox,Cdh16-Cre and Rev-erbakKO mice were identified by PCR amplification and agarose gel electrophoresis experiments.The expression of REV-ERBα in Rev-erbakKO mice kidney was detected by qPCR and Western blotting.Rev-erbakKO and Rev-erbαflox/flox mice were used as the experimental objects and to establish AAI kidney injury model.Renal injury-and ferroptosisrelated indicators were detected,and the effect of Rev-erba ablation on AAI nephrotoxicity was evaluated.5.Wild-type mice were intraperitoneally injected with SR8278(a REV-ERBαantagonist,50 mg/kg),DFO(a ferroptosis inhibitor,100 mg/kg)and blank solvent.The antagonistic effects of SR8278 and DFO on AAI nephrotoxicity were evaluated by detecting renal injury-and ferroptosis-related indicators.mRTECs were incubated with SR8278(5 μM)and DFO(50 μM)for 8 h,respectively,and then with ALI(25 μM)for for another 24 h.The protective effects of SR8278 and DFO against ALI toxicity were confirmed by detecting cytotoxicity and ferroptosis-related indicators.Results1.Circadian variations in AAI nephrotoxicity in mice Plasma CRE/BUN and the expression levels of renal Ngal/Kim-1 were increased in AAI-treated mice.H&E staining indicated that there were massive necrosis of renal tubular epithelial cells,cell rupture,mesangial hyperplasia and a large number of protein casts in the kidneys of AAI-treated mice.These results indicated that AAI induced severe renal injury in mice.In addition,renal injury at ZT2 was more severe than at ZT14.Therefore,AAI nephrotoxicity displays a significant circadian rhythm.2.Circadian clock genes were dysregulated in AAI-treated mice The protein levels of REV-ERBα increased,while the protein levels of BMAL1 and PER2 decreased,in the kidneys of AAI-treated mice compared with control mice.By contrast,CLOCK protein showed no significant change.Nevertheless,the mRNA levels of these clock genes were consistently decreased in AAI-treated mice.In mRTEC cells,ALI(but not AAI)significantly up-regulated REV-ERBαprotein and down-regulated BMAL1 protein expression.In addition,AAI altered the wheel-running activity of mice under dark-dark condition,and ALI altered the rhythm of Per2-dLuc cells.The above results suggested that AAI nephrotoxicity is related to abnormal expression of circadian clock genes(such as REV-ERBα).3.Ferroptosis was involved in AAI nephrotoxicityCompared with control mice,the iron level,HO-1 mRNA and protein levels was increased,and the GSH level,GPX4 mRNA and protein levels were decreased in the kidneys of the AAI kidney injury model.Similarly,ALI increased iron and HO-1 expression,and decreased GSH and GPX4 expression in mRTECs.These results suggested that AAI nephrotoxicity is related to ferroptosis.4.Kidney-specific loss of Rev-erba relieved AAI-induced renal injury and ferroptosis in miceThe expression levels of REV-ERBα mRNA and protein were almost absent in the kidneys of Rev-erbαkKO mice,while they showed no significant change in other tissues(heart,liver and lung).AAI treatment led to increased levels of plasma BUN/CRE and renal Ngal in Rev-erbαflox/flox mice.H&E and PAS staining confirmed that AAI caused severe renal damage in Rev-erbαflox/flox mice.Compared with Rev-erbαflox/flox mice,Rev-erbαkKO mice had lower plasma CRE/BUN and renal Ngal levels and less kidney damage after AAI treatment.In addition,AAI increased iron level and decreased GSH and GPX4 levels in the kidneys of Rev-erbαflox/flox mice.The changes of these ferroptosis markers in the kidneys of Rev-erbαkKO mice were smaller.Furthermore,Rev-erba silencing increased GSH and GPX4 levels and cell viability in ALI-treated mRTECs.These results suggested that REV-ERBα plays an important role in AAI nephropathy through modulating ferroptosis.5.Small molecule antagonism of REV-ERBa alleviated AAI-induced renal injury in micePlasma CRE/BUN and renal Ngal expression were significantly decreased in SR8278-treated mice compared to control mice.H&E and PAS staining also confirmed that SR8278 could reduce the degree of renal injury in mice treated with AAI.Moreover,SR8278 also decreased the iron level and increased the GSH and GPX4 levels in the kidneys of AAI-treated mice.In ALI-treated mRTECs,SR8278 significantly increased the cell viability and GPX4 levels.Similar to the effects of SR8278,DFO also attenuated AAI/ALI-induced renal injury and ferroptosis.In summary,the REV-ERBα antagonist SR8278 can reduce AAI nephrotoxicity by inhibiting ferroptosis.ConclusionFirstly,we found that AAI nephrotoxicity shows a diurnal rhythmicity,accompanied with abnormal expression of core clock genes in mouse kidneys.Secondly,REV-ERBα plays an important role in AAI nephropathy through regulating ferroptosis.Finally,targeted inhibition of REV-ERBα alleviated AAI nephrotoxicity in mice.This study deepens the understanding of the pathological mechanism of AAI nephrotoxicity and provides new strategies for the treatment of aristolochic acid nephropathy.