Complement Membrane Attack Complex C5b-9 Mediates Ferroptosis Of Tubular Epithelial Cells In Trichloroethylene Sensitized Mice Through Inducing IP3R-Dependent Mitochondrial Disorder

Background and subject Occupational medicamentose-like dermatitis due to trichloroethylene(OMDT)is a systemic disease characterized by extensive mucocutaneous hypersensitivity reactions with multiorgan failure following occupational exposure to TCE.OMDT can be subdivided into exfoliative dermatitis,erythema multiforme,measles-like dermatitis,and bullous epidermal necrolysis based on the characteristics and severity of the lesions.Although the prevalence of OMDT in the occupational population exposed to TCE has been reported to be in the range of 0.2512.5%,while the clinical characteristics of OMDT are acute onset,rapid progression and high mortality,and more than 9%of OMDT patients die due to secondary severe infection,multiorgan failure and hepatic encephalopathy.All these have made OMDT one of the most prominent problems in the field of occupational dermatology.Clinical studies have shown that in addition to mucocutaneous lesions,OMDT patients are often accompanied by multiple organ damage,including immune kidney injury.Moreover,our previous results from the TCE transdermal sensitization mouse model,which can well mimic the condition of OMDT patients,showed that TCE sensitization could cause immune kidney injury in mice,and a large number of membrane attack complex(MAC)C5b-9 deposits were found in sensitized positive mouse renal tubular epithelial cells(TECs),suggesting that C5b-9 may play an important role in TCE-induced kidney injury.However,the exact mechanism by which C5b-9 causes renal injury in TCE-sensitized mice is unclear.The aim of our project is to investigate the role of C5b-9 deposition in TCE-sensitizationinduced TECs injury using a TCE-sensitization mouse model and an in vitro cell model.Methods Mouse Model:Six-to eight-week-old female BALB/c mice were used to establish the TCE transdermal sensitization mouse model.The dorsal coat of all mice was shaved to expose the dorsal skin after one week of adaptive feeding.All healthy mice were divided into blank control group(Blank),vehicle control group(Vehicle),and TCE treatment group(TCE).On the first day,100 μL of 25%TCE(TCE:olive oil:acetone:FCA=5:2:3:10,v-v-v-v)was injected subcutaneously into the dorsal skin of the TCE group,100 μL of vehicle(olive oil:acetone:FCA=2:3:5,v-v-v-v)was injected subcutaneously into the mice in the Vehicle group,and 100 μL of normal saline(NS)was injected subcutaneously into the mice in the Blank group.The 4th,7th,and 10th days are the sensitization stage.On days 4,7,and 10,100 μL of 50%TCE(TCE:olive oil:acetone=5:2:3,v-v-v),100 μL of vehicle(olive oil:acetone=2:3,v-v),and 100 μL of normal saline were applied to the dorsal skin of mice in the TCE,Vehicle,and Blank groups,respectively.Days 17 and 19 were the challenge period.On days 17 and 19,100 μL of 30%TCE(TCE:olive oil:acetone=3:2:5,v-v-v),100 μL of vehicle(olive oil:acetone=2:5,v-v),and 100 μL of normal saline were applied to the dorsal skin of mice in the TCE,Vehicle,and Blank groups,respectively.Twenty-four hours after the last challenge,the mice in the TCE group were divided into TCE sensitization negative group(TCE-)and TCE sensitization positive group(TCE+)according to the score of the dorsal skin.On the 21st day,the mice were anesthetized and then sacrificed by cervical dislocation after collecting ocular venous blood and urine.Dorsal skin and kidney tissues were collected for subsequent molecular studies.Animal experiments consisted of two parts.1)Based on the TCE-sensitized mouse model,we added a ferroptosis inhibitor group,co-treating mice with TCE and Fer-1.The TCE+Fer-1 group mice were subjected to intraperitoneal injection with Fer-1(5 mg/kg)1 hour before the initial challenge(day 17)and the last challenge(day 19).All mice were divided into Blank,Vehicle,TCE-,TCE+,TCE+Fer1 sensitization negative(Fer-1-)and TCE+Fer-1 sensitization positive(Fer-1+)groups according to the skin reaction.This part is to explore the role of TECs ferroptosis in the TCE sensitization induced kidney injury.2)Same as the part 1,we treated BALB/c female mice with CD59,a C5b-9 inhibitor,based on the TCE-sensitization model.The TCE+CD59 group mice were subjected to intraperitoneal injection with CD59(1.25 mg/kg)1 hour before the initial challenge and the last challenge.All mice were divided into Blank,Vehicle,TCE-,TCE+,TCE+CD59 sensitization negative(CD59-)and TCE+CD59 sensitization positive(CD59+)groups according to the skin reaction.This part was to investigate the role of tubule-specific C5b-9 deposition in the TCE sensitization-induced TECs ferroptosis.In vitro model:Complement C5b6 and normal human serum(NHS),the source of complement C7,C8 and C9,were used to assemble C5b-9 on the cytomembrane of HK2 cells to explore the mechanism of TECs ferroptosis induced by C5b-9.The cell experiments consisted of five parts.1)HK-2 cells were treated with different concentrations of C5b-9(0,0.05,0.1,0.2,0.4,0.8,1.6 μg/ml)and 10%NHS to establish a C5b-9-attacked cell model.2)HK-2 cells were treated with C5b-6 and NHS combined with 1 μM ferroptosis inhibitor Fer-1 to investigate whether C5b-9 could induce ferroptosis in HK-2 cells.3)HK-2 cells were treated with C5b-6 and NHS combined with IP3R siR to explore the mechanism of cytosolic Ca2+ overload caused by C5b-9.4)HK2 cells were treated with C5b6 and NHS combined with 2 μM intracellular Ca2+ chelator BAPTA-AM to explore the role of cytosolic Ca2+ overload in mitochondrial disorder induced by C5b-9.5)HK-2 cells were treated with C5b-6 and NHS in combination with 10 nM cyclosporin A(CsA),a mitochondrial membrane transport pore(mPTP)inhibitor,to investigate the role of mPTP opening in C5b-9-induced ferroptosis.Results Dorsal skin reactions in all mice were evaluated 24 h after the last challenge.No obvious edema or erythema was observed in mice in the Blank and Vehicle groups,and the sensitization rate was 0(0/7).Some mice in TCE,TCE+Fer-1,and TCE+CD59 groups showed allergic reactions such as edema or erythema on the dorsal skin,and the sensitization rate was 45.83%(11/24),43.47%(10/23),and 42.11%(8/19),respectively.There was no significant difference in sensitization rate among different groups.By analyzing the renal cortex of the TCE-sensitized mice,we found that the lipid peroxidation-associated proteins such as acyl-CoA synthetase long chain family member 4(ACSL4)and cyclooxygenase(COX2)were increased in the TCE-sensitized positive group,while the levels of glutathione peroxidase 4(GPX4)and glutathione(GSH)were decreased in the TCE-sensitized positive group compared with those in the Blank,Vehicle and TCE-sensitized negative groups.Consistently,kidney non-heme iron,ROS and lipid peroxides such as malondialdehyde(MDA)and 4-hydroxynonenal(4HNE)levels were also significantly higher in the TCE-sensitization-positive group,and the 4HNE were mainly localized in renal tubular epithelial cells.In addition,transmission electron microscopy(TEM)analysis showed that the mitochondrial ridges of renal tubular epithelial cells were decreased or even disappeared in TCE-sensitized-positive mice.Interestingly,pretreatment with the ferroptosis inhibitor Fer-1 can alleviate these abnormal changes caused by TCE sensitization.These results suggest that TCE sensitization can cause renal tubular ferroptosis in mice.Analysis of renal tubular injury indicators showed that pretreatment with Fer-1 significantly attenuated renal tubular vacuolar degeneration caused by TCE sensitization and reduced the levels of α1microglobulin(α1-MG),β2-MG,Ngal,and Kim-1 in the serum and urine of TCE sensitization-positive mice.This indicated that renal tubular ferroptosis was involved in TCE sensitization-induced kidney injury.In this study,we found that TCE sensitization can cause C5b-9 specific deposition in renal tubules,and the changes in ACSL4,COX2,GPX4,non-heme iron,GSH,MDA,ROS,4HNE and mitochondrial damage caused by TCE sensitization were all significantly alleviated after pretreatment with complement regulatory protein CD59 to interfere with C5b-9 synthesis.Consistently,CD59 also attenuated the structural and functional renal tubular damage induced by TCE sensitization.This suggests that tubule-specific C5b-9 deposition may play an important role in ferroptosis and tubular injury in TCE-sensitized mice.Moreover,the in vitro C5b9-attacked HK-2 cell model also found that C5b-9 reduced cell viability,increased intracellular Fe2+ levels,and caused GSH depletion and lipid peroxide accumulation.Interestingly,pretreatment with the intracellular Ca2+ chelator BAPTA-AM attenuated C5b-9-induced ferroptosis in HK-2 cells.To investigate the role of Ca2+in C5b-9-induced renal tubular ferroptosis,we examined the activation of the endoplasmic reticulum Ca2+transporter IP3R.The results showed that IP3R was activated in the kidney of TCEsensitized mice,and pretreatment with CD59 blocked the activation of IP3R.In addition,IP3R activation was also found in C5b-9 challenged HK-2.Further investigation revealed that TCE sensitization caused increased cytosolic Ca2+levels and activation of the mitochondrial unidirectional Ca2+transporter MCU,resulting in depolarization of the mitochondrial membrane potential and reduction of mitochondrial oxidative phosphorylation-related proteins(OXPHOS),while CD59 pretreatment alleviated these abnormal changes.Furthermore,this phenomenon was also replicated in a C5b-9 attacked HK-2 cell model.To investigate the role of IP3R in this series of reactions,we used small interfering RNA(siR)to knock down the expression of IP3R.The results showed that mitochondrial Ca2+overload and mitochondrial damage caused by C5b-9 were alleviated after IP3R siR pretreatment.In addition,pretreatment with IP3R siR alleviated the decrease in cell viability,depletion of GSH,accumulation of lipid peroxides,and increase in intracellular Fe2+induced by C5b-9.These results suggest that cytosolic Ca2+overload and mitochondrial damage induced by IP3R activation may play an important role in C5b9-mediated renal tubular ferroptosis in TCE-sensitized mice.To further investigate the mechanism of cytosolic Ca2+overload causing mitochondrial disorder and ferroptosis,we examined mitochondrial mPTP openness.The results showed that mPTP was significantly activated in primary TECs from TCE-sensitized mice,and the activation of mPTP was effectively inhibited after CD59 pretreatment.Similarly,mPTP was significantly activated in C5b-9 challenged HK-2 cells.Additionally,pretreatment with BAPTA-AM not only blocked C5b-9-induced mPTP activation,but also alleviated the degree of mitochondrial depolarization.Further investigations revealed that C5b-9induced mitochondrial membrane potential depolarization and reduction of OXPHOSrelated proteins were significantly attenuated after mPTP activation was inhibited by CsA.In addition,the C5b-9-induced decrease in cell viability,depletion of GSH,accumulation of lipid peroxides,and increase in free Fe2+were also significantly ameliorated by CsA.These results suggest that mPTP opening may play an important role in mitochondrial dysfunction and ferroptosis induced by C5b-9.Conclusion In conclusion,TCE sensitization-induced renal tubular ferroptosis is mediated by tubule-specific C5b-9 deposition and IP3R-dependent cytosolic Ca2+overload and mitochondrial dysfunction may play a key role.