Study On The Mechanism Of Renal Toxicity Induced By Tacrolimus By Inducing Mitochondrial Dysfunction

BackgroundTacrolimus(Tac)is a powerful immunosuppressant,which is widely used in organ transplantation to prevent rejection.However,the proportion of chronic renal toxicity in patients with long-term use of Tac is as high as17%-44%,mainly manifested as progressive irreversible structural renal damage,which seriously affects the renal function and long-term survival of patients after organ transplantation.Therefore,it is of great clinical significance to further study the mechanism of Tac nephrotoxicity and explore effective prevention and treatment methods for developing new drugs and further improving the survival of transplanted organs and patients.Long-term clinical use of Tac is often accompanied by some metabolic complications,such as new-onset diabetes after transplantation,hyperlipidemia,etc.Therefore,metabolic changes are closely related to the toxic effects of Tac.The kidney plays a key role in maintaining metabolic homeostasis through the filtration and reabsorption of the nephron,and the renal tubular epithelial cells are an important part of the nephron.It is well known that one of the targets of Tac in the kidney is renal tubular epithelial cells,which are rich in mitochondria to maintain important physiological functions,and mitochondria are important metabolic centers for cell physiological activities.Therefore,the kidney toxic effects of Tac may be related to mitochondrial dysfunction of renal tubular epithelial cells.Mitochondria synthesize ATP for the physiological activities of cells through oxidative phosphorylation.Oxidative phosphorylation depends on the normal structure and function of the oxidative respiratory chain complex,and mitochondrial genes can encode some of the important subunits.The replication and Transcription of mitochondrial genes are regulated by the proteins encoded by nuclear genes.In particular,Transcription factor A mitochondrial(TFAM)plays an important role in the regulation of mitochondrial gene expression.Therefore,we will further explore in depth whether Tac inhibits mitochondrial function leading to kidney injury by affecting the expression of TFAM and explore the mechanism.Objectives:1.Human renal tubular epithelial cells(HK-2)and C57/BL6 mice were used to establish Tac nephrotoxicity model to determine whether Tac can cause mitochondrial damage.2.To explore the mechanism of Tac induced mitochondrial damage by multi-omics sequencing.3.In vitro cell experiment,we further explored whether Tac inhibited mitochondrial function by inhibiting the phosphorylation of eukaryotic translation initiation factor 4E(e IF4E)-binding protein 1(4ebp1);The specific agonist was given to observe whether it could improve the toxic damage of Tac to mitochondria.Methods1.Effect of Tac on mitochondrial function in renal tubular epithelial cells(1)After HK-2 cells were stimulated with different concentrations of Tac for 24 h,morphological changes of cells were observed,and Epithelial mesenchymal transition(EMT)was detected by Western blot(WB).EMT)related proteins(E-cadherin,vimentin,α-smooth actin)expression,and real-time quantitative polymerase chain reaction(RT-q PCR)was used to detect the gene transcription levels of collagen and fibronectin.(2)Mitochondrial fluorescence probe was used to detect the changes of mitochondrial morphology after Tac stimulation.(3)The changes of intracellular Reactive oxygen species(ROS),mitochondrial membrane potential and ATP production after Tac stimulation were detected by mitochondrial membrane potential,ROS and ATP detection kits.(4)C57BL/6 mice were intraperitoneally injected with Tac(2mg/kg,intraperitoneal injection,once a day)for 8 weeks.PAS and Masson were used to detect the degree of renal injury and fibrosis at 4 and 8 weeks,and transmission electron microscopy was used to detect the mitochondrial morphology of renal tubular epithelial cells.The expression of Neutropil gelatinase-associated lipocalin(NGAL),a marker of kidney injury,was detected by immunohistochemistry.2.To explore the mechanism of Tac inhibition on mitochondrial function by multi-omics and bioinformatics(1)After HK-2 cells were stimulated with Tac(40μM)for 24 h,metabolomics,transcriptome and proteome sequencing were performed to screen differential metabolites and differentially expressed genes and proteins.(2)The signaling pathways of differential metabolites and related proteins were enriched by GO,KEGG and other databases to screen the pathways and mechanisms of Tac affecting mitochondrial function.3.Role of 4EBP1/TFAM in regulating mitochondrial function in renal tubular epithelial cells(1)RT-q PCR detection of Mitochondrially encoded cytochrome c oxidase III,a subunit of the mitochondrial respiratory chain complex in HK-2 cells stimulated with different concentrations of Tac MT-CO3,Mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1,MT-ND1,Mitochondrially encoded ATP synthase membrane subunit 8,MT-ATP8,Mitochondrially encoded cytochrome b,MT-CYB);(2)Western blotting was used to detect the expression of MT-CO3,MT-ND1,MT-ATP8,MT-CYB,TFAM,4EBP1,and 4EBP1 phosphorylation in HK-2 cells stimulated with different concentrations of Tac.(3)The expression of TFAM and phosphorylated 4EBP1 in kidney was detected by immunohistochemistry.(4)HK-2 cells were pretreated with NV-5138,a specific agonist of m TORC1 upstream of 4EBP1,and then stimulated with Tac.WB was used to detect the expression of MT-CO3,MT-ND1,MT-ATP8,and MT-CYB in HK-2 cells treated with different concentrations of Tac.And the phosphorylation of TFAM and 4EBP1.(5)HK-2 cells were pretreated with m TORC1 specific agonist NV-5138 and then stimulated with Tac.Mitochondrial membrane potential(MMP),cellular reactive oxygen species(ROS),and ATP production were detected by mitochondrial membrane potential,cellular reactive oxygen species(ROS),and ATP production assay kits.Results1.Tac promotes mitochondrial dysfunction in renal tubular epithelial cells(1)Tac reduced the expression of E-Cad,increased the expression of Vim and α-SMA,and increased the transcription of FN and COl-1 genes in HK-2 cells,which indicated that epithelial-mesenchymal transition occurred in HK-2 cells.(2)Tac increased the expression of Fis1 and Mff,OPA1 and Mfn2,and decreased the expression of Mfn1 in HK-2 cells.Mitotracker fluorescent probe showed that the mitochondrial network became fragmented and strong fluorescence.(3)Tac can lead to the increase of mitochondrial membrane potential and intracellular ROS in HK-2 cells,which further causes mitochondrial oxidative phosphorylation disorder and leads to the decrease of ATP production.(4)In the mouse model of chronic nephrotoxicity,Tac caused vacuolar degeneration,lumen enlargement and cell atrophy of renal tubular epithelial cells,which further led to tubulointerstitial fibrosis.At the same time,it was found that the mitochondria in renal tubular epithelial cells changed from normal rod and rod to small round and globular,the mitochondrial crest was reduced,and the mitochondrial outer membrane was destroyed.2.Metabolic and proteomic studies revealed that Tac inhibited mitochondrial gene expression(1)Metabonomics analysis showed that Tac caused energy metabolic conversion in HK-2 cells,namely,the increase of amino acid and nucleotide catabolism,the accumulation of lipid metabolic intermediates,and the decrease of glucose metabolism and glycerophospholipid metabolism.(2)Proteomics analysis showed that Tac decreased the transcription and translation of mitochondrial genes in HK-2 cells,and inhibited the expression of proteins encoded by mitochondrial genes.(3)Tac decreased the expression of mitochondrial gene transcription regulator TFAM.(4)KEGG enrichment analysis showed that Tac may affect mitochondrial energy metabolism through 4EBP1/TFAM pathway.3.Tac inhibits mitochondrial function in renal tubular epithelial cells through 4EBP1/TFAM pathway(1)The expression levels of mitochondrial genes encoding oxidative respiratory chain proteins(MT-CO3,MT-ND1,MT-CYB,MT-ATP8)in HK-2 cells were further verified by WB.(2)Tac reduced the phosphorylation of 4EBP1 and the protein expression of TFAM.(3)NV-5138 increased the phosphorylation of 4EBP1 and TFAM protein expression by activating m TORC1,which further increased the expression of mitochondrial genes encoding oxidative respiratory chain proteins(MT-CO3,MT-ND1,MT-CYB,MT-ATP8).(4)NV-5138 has a protective effect on TAC-induced mitochondrial dysfunction in HK-2 cells by up-regulating 4EBP1/TFAM,that is,reducing intracellular ROS,increasing mitochondrial membrane potential and promoting ATP synthesis.(5)Downregulation of 4EBP1/TFAM pathway was found in TAC-induced chronic nephrotoxicity by IHC.ConclusionThrough in vivo and in vitro experiments,our research found that:1.Tac can cause morphological changes of renal tubular cells,and then cause tubulointerstitial fibrosis,which is related to the changes in the morphology and function of mitochondria in tubular epithelial cells.Tac leads to energy metabolism conversion of renal tubular cells,which may affect cell energy metabolism by down-regulating mitochondrial gene expression through 4EBP1/TFAM pathway.Tac can down-regulate the transcription and translation of mitochondrial genes by inhibiting the 4EBP1/TFAM pathway,and activation of the pathway can inhibit the toxic damage of Tac to the mitochondria of renal tubular cells.