Function Of Selenium And Zinc On Relieve Ochratoxin Ainduced Renal Injury

Ochratoxin A(OTA),a natural occurring mycotoxin produced by fungi of Aspergillus and Penicillium genera,can contaminate feed and food materials.Because of the wild distribution of Aspergillus and Penicillium,humans and animals are at a high risk of exposure through ingestion of contaminated feed and foodstuff.It has been reported that OTA induced a variety of toxicological effects including neurotoxicity,hepatotoxicity and nephrotoxicity.Kidney is considered to be the target organ of OTA,but the mechanisms of its toxicity have not been clearly determined.Studies have shown that OTA exerts its toxic effects by mediating apoptosis,generating reactive oxygen species(ROS),and interfering the synthesis of antioxidant enzymes.Pyroptosis is one of the most popular research areas and is a proinflammatory programmed cell death.However,whether OTA-induced nephrotoxicity via pyroptosis pathway need to be explored.Given the widespread and harmful nature of OTA,there is an urgent need to find safe and effective mitigation methods.As a new technology with safety,convenience and few side effects,nutritional regulation has received more and more attention.Selenium(Se),an antioxidant trace element for humans and animals,has many important biological functions,including enhancing the antioxidant function and antitoxic elements,and so on.It has been reported that Se exerts its biological functions through selenoproteins.Zinc is an essential trace element in human body.It is widely involved in the regulation of antioxidant system and the stability of DNA.However,the role of nutrients in reducing the toxicity of OTA and its mechanism remain to be further studied.Therefore,in this study,MDCK cells and kidney tissues were used to study the protective mechanism of nutrient on OTA-induced nephrotoxicity and kidney injury.Experiment 1:OTA induces nephrotoxicity via NLRP3 inflammasome and Caspase-1dependent pyroptosisWe conducted this study to determine the nephrotoxicity of continuous administration of OTA and the potential mechanisms both in vitro and in vivo.In vivo tests,mice were intraperitoneal injected with different doses of OTA(1.0 mg/kg·d-1,2.0 mg/kg·d-1)every other day and lasted for 14 days.We found that 2.0 mg/kg·d-1 OTA administration significantly(P<0.05)triggered the mice renal injury as indicated by increasing histological injury and renal fibrosis molecules(α-SMA,Vimentin,TGF-β)compared with the control group;Besides,2.0 mg/kg·d-1 treatment also actived the NLRP3 inflammasome and induced pyroptosis.In vitro tests,a canine kidney epithelial cell lines(MDCK)was applied in this study.We found that OTA caused cytotoxicity in a dose-dependent manner and significantly(P<0.05)caused the up-regulation of renal fibrosis genes(α-SMA,Vimentin,TGF-β).Afterwards,compared with the control group,2.0 μg/ml OTA treatment were showed to elicit NOD-like receptor protein 3(NLRP3)inflammasome activation and Caspase-1-dependent pyroptosis,leading to the expression and secretion of pro-inflammatory cytokines(IL-6,TNF-α)and upregulation of pyroptosis related genes(GSDMD,IL-1β,IL-18)in MDCK cells.The pyroptosis-elicited renal fibrosis was significantly(P<0.05)abrogated by inhibiting NLRP3 activity with inhibitor MCC950 and silencing NLRP3 with siRNA.Furthermore,knockdown of Caspase-1 also ameliorated pyroptosis-elicited renal fibrosis.In summary,the data indicated that OTA induces nephrotoxicity via NLRP3 inflammasome and Caspase-1dependent pyroptosis.Our study provided novel insights into the nephrotoxicity and the underlying mechanisms of OTA.Experiment 2:Function of selenium on relieving OTA-induced canine nephrotoxicityPrevious studies have shown that OTA induces in vitro and in vivo nephrotoxicity via NLRP3 inflammasome activated and Caspase-1-dependent pyroptosis.In this study,the protective effects of selenomethionine against OTA-induced nephrotoxicity were investigated using the canine kidney epithelial cells(MDCK)as a model.We measured the cell viability,LDH activity and the expression of renal fibrosis,NLRP3 inflammasome and pyroptosis related genes.MTT and LDH results indicated that SeMet supplementation could significantly(P<0.05)inhibited the 2.0 μg/ml OTA induced cytotoxicity in MDCK cells.Then,the expressions of α-SMA,Vimentin and TGF-β were detected by RT-PCR.The results indicated 8 μM SeMet supplementation could significantly(P<0.05)down-regulate the expression of OTA-induced renal fibrosis related genes.In addition,the up-regulation of OTA-induced NLRP3 inflammasomes and pyroptosis-related genes were also significantly inhibited by SeMet at a concentration of 8 μM(P<0.05).In summary,selenomethionine can alleviate OTA-induced nephrotoxicity and inhibit NLRP3 inflammasome and Caspase-1dependent cell pyroptosis.Therefore,selenomethionine supplementation may be an attractive strategy for protecting humans and animals from risk of kidney damage induced by OTA.Experiment 3:Zinc supplementation alleviates OTA-induced oxidative stress and apoptosis in MDCK cells by up-regulating metallothioneinsThe aim of the present study was to investigate the protective effects of Zn supplementation on OTA-induced oxidative stress and apoptosis of Madin-Darby canine kidney(MDCK)epithelial cells and explore the potential mechanisms.Initially,results of MTT and LDH activity revealed that Zn supplementation significantly(P<0.05)inhibited OTA-induced cytotoxicity in MDCK cells.Then,it was observed that Zn supplementation alleviated OTA-induced ROS accumulation by using a confocal microscope.Given the important role of apoptosis in OTA-induced cytotoxicity,we found that Zn supplementation markedly prevented OTA-induced apoptosis(P<0.05).Interestingly,OTA treatment slightly increased the levels of Metallothionein-1(MT-1)and Metallothionein-2(MT-2)in MDCK cells compared with the control group(P<0.05);While Zn supplementation further improved the increase of MT-1 and MT-2 induced by OTA(P<0.05).However,the inhibitive effects of Zn supplementation on OTA-induced ROS accumulation and apoptosis were significantly blocked after double knockdown of MT-1 and MT-2(P<0.05).These results suggested that MT-1 and MT-2 may act as an adaptive protective response to OTA-induced renal cytotoxicity and oxidative stress.In summary,our present study determined that OTA could induce cytotoxicity,oxidative stress,and apoptosis in MDCK cells.Zn supplementation attenuated OTA-induced ROS production and apoptosis through the upregulation of MTs in MDCK cells.