Study On The Protective Mechanism Of VDR Agisnst Cisplatin-induced Acute Kidney Injury In Mice By Regulating HIF-1α/HO-1 Signaling Pathway

Acute kidney injury（AKI）,as a syndrome characterized by rapid decline of renal function,has high incidence and mortality.Failure to intervene in time,AKI can seriously threaten the health of human and animals.Cisplatin（CDDP）is a spectral anticancer drug and one of the common factors inducing AKI.Due to the complex pathogenesis of AKI and the lack of satisfactory intervention methods,it is still an urgent problem in medical and veterinary clinic to find safe and effective drugs for prevention and treatment of AKI.Vitamin D receptor（VDR）can be activated by vitamin D and vitamin D analogues to play antioxidant and anti-inflammatory functions.At the beginning of the experiment,we found that the level of VDR decreased significantly in CDDP-induced AKI mice,suggesting that activation of VDR in vivo may be an effective means to prevent and control AKI.However,the specific molecular mechanism remains unknown.Based on this,the following experiments were carried out in this study:（1）First,forty 8-week-old male C57BL/6 mice were randomly divided into two groups,control group（C group,intraperitoneal injection of normal saline）and model group（CDDP group,intraperitoneal injection of 20 mg/kg CDDP）.AKI mouse models were obtained after 72 h treatment with CDDP,and the key signaling pathways were screened by transcriptomic and metabolomics analysis.（2）Subsequently,sixty 7-week-old male C57BL/6 mice were randomly divided into four groups:C group,VDR group（intraperitoneal injection of 0.2μg/kg paricalcitol）,VDR+CDDP group（On day 6 of paricalcitol treatment,CDDP was injected intraperitoneally）and CDDP group.The animals were observed and weighted after CDDP treatment.The animals were euthanized to collect blood and tissue samples,and the renal function of mice was detected by biochemical kit and ELISA kit.The histopathological changes,oxidative stress,mitochondrial dynamics and energy metabolism related genes,inflammatory cytokines,apoptosis,ferroptosis and the key gene expression and colocalization of related signaling pathway in mice kidneys were detected by Hematoxylin&Eosin（H&E）stain,transmission electron microscope（TEM）,real-time PCR（q RT-PCR）,antioxidant kit,TUNEL staining,western blot（WB）,immunohistochemistry（IHC）,immunofluorescence（IF）.（3）Finally,thirty 6-week-old male C57BL/6 mice were randomly divided into two groups:VDR+CDDP group and critical signaling pathway intervention group（Znpp group,5 mg/kg Znpp）.After 72 h of CDDP treatment,samples were collected for relevant detection to determine the specific molecular mechanism of renal protection by VDR.The in vitro experiment was consistent with the in vivo experiment,that is,the Human kidney-2 cell line（HK2）was treated with paricalcitol,CDDP and Znpp,respectively.In order to elucidate the effect of VDR on CDDP-induced AKI and the molecular mechanism,the cell viability,ROS level,mitochondrial dynamics,energy metabolism,inflammation,apoptosis and ferroptosis were detected.The main results are as follows:（1）The combined analysis of transcriptomics and metabolism showed that CDDP induced differential transcription genes and different metabolism were co-enriched into 72 Kyoto Encyclopedia of Genes and Genomes（KEGG）pathways in kidneys of AKI mice.Among them,significantly different transcription genes and metabolites were co-enriched into four KEGG pathways,namely metabolic pathway,HIF-1 signaling,glutathione metabolic and butyric acid metabolic.（2）After the injection of CDDP,the body weight of mice in AKI model group decreased significantly and the mice showed extreme depression,anorexia,squinting and curling.The results of renal function showed that blood urea nitrogen（BUN）c,serum creatinine（SCr）,kidney injury molecule 1（Kim-1）,neutrophil gelatinase-associated lipocalin（Nagl）decreased significantly,suggesting that the mouse kidney injury model was successfully established.After HK2 cells were treated with CDDP,the cell viability decreased significantly,indicating that CDDP induced HK2cell damage.VDR pretreatment significantly increase the levels of BUN,SCr,Kim-1,Nagl and the viability of HK2 cells in mice,indicating that VDR can improve kidney function.（3）The results of histopathological examination showed that the renal tubular epithelial cells in the kidney of AKI mice were obviously exfoliated,and many inflammatory cells infiltrated in the interstitial space,while the inflammatory cells in the kidney of VDR+CDDP group decreased,and the renal tubular epithelial cells shed less or were exfoliated.Using WB to detect the expression of inflammatory cytokines in the tissue,it was found that the protein expressions of tumor necrosis factorα（TNF-α）,interleukin-1β（IL-1β）and nuclear transcription factor kappa B（NF-κB）in the mice kidneys and HK2 cells of CDDP group were significantly increased.The protein levels of inflammatory cytokines in the kidneys and HK2 cells of the VDR+CDDP group were markedly lower than those of the CDDP group,indicating that VDR can significantly improve the renal inflammatory injury induced by CDDP.（4）Ultrastructural observation found that VDR can improve CDDP-induced AKI mouse kidney mitochondrial cristae rupture.Using q RT-PCR and WB for further detection,we found that VDR can reduce the m RNA and protein expression of mitochondrial dynamic-related protein 1（Drp1）and mitochondrial fission protein 1（fis1）in the kidney of CDDP-induced AKI mice,and remarkably up-regulated the expressions of optic atrophy 1（Opa1）,mitochondrial fusion protein 1（Mfn1）and Mfn2.In addition,fluorescent probes and IF method were used to detect mitochondrial activity and Drp1/fis1/Opa1 levels in CDDP exposed HK2 cells.The result showed that mitochondrial activity was positively correlated with Opa1 levels and negatively correlated with Drp1 and fis1 levels,suggesting that CDDP induces excessive fission of renal mitochondria and in turn leads to mitochondrial damage.Moreover,the levels of Drp1 and fis1 in the mice kidneys and HK2 cells in the VDR+CDDP group were significantly lower than those in the CDDP group,and the levels of Opa1,Mfn1 and Mfn2 were significantly increased,indicating that VDR can reduce CDDP-induced mitochondrial damage by maintaining the balance of mitochondrial dynamics in the kidney.（5）Using the kit to detect ATPase,we found that VDR significantly increased the activities of Na⁺-K⁺-ATPase,Ca²⁺-Mg²⁺-ATPase and Ca²⁺-ATPase in the kidneys of CDDP-induced AKI mice.The expression of energy metabolism related genes in kidney was detected by q RT-PCR and WB methods,and we found that VDR significantly up-regulated the m RNA and protein expressions of lactate dehydrogenase（LDH）,pyruvate kinase（PK）and pyruvate dehydrogenase complex component X（PDHX）in renal tissues of mice exposed to CDDP.The in vitro experiment results are consistent with the in vivo experiment,that is,the protein levels of LDH,PK and PDHX increased in HK2 cells exposed to CDDP after VDR pretreatment,suggesting that VDR can eliminate the negative impact of CDDP on renal energy metabolism to a certain extent.（6）The detection of oxidative stress kit found that VDR significantly increased the levels of glutathione（GSH）and catalase（CAT）in the kidneys of AKI mice,and inhibited the accumulation of hydrogen peroxide（H₂O₂）and malondialdehyde（MDA）.Using IF analysis the superoxide dismutase 2（SOD2）in the kidneys of mice,we found that VDR significantly increased the fluorescence intensity of SOD2 in the kidneys of AKI mice.Besides,VDR significantly weakened the fluorescent intensity of reactive oxygen species（ROS）in CDDP treated mouse kidneys and HK2cells.These results indicate that VDR can increase the antioxidant level and inhibit the accumulation of oxides,thereby alleviating the oxidative damage of the kidney induced by CDDP.In addition,using kit,IF,fluorescent probe and WB analysis the levels of ferrous ion(Fe²⁺)and glutathione peroxidase 4（GPX4）,we found that VDR significantly inhibited CDDP-induced accumulation of Fe²⁺in the mouse kidney and HK2 cells,and increased the expression of GPX4.Combined with the results of GSH and MDA,VDR can improve CDDP-induced ferroptosis in kidney.（7）The results of TUNEL staining showed that VDR significantly reduced the number of renal apoptosis induced by CDDP in mice.Using q RT-PCR,IHC and WB methods to detect the expression of apoptosis-related genes,we found that VDR markedly up-regulated the expressions of B-cell lymphoma-2（Bcl-2）in the renal tissue of AKI mice,and down-regulated the expressions of cytochrome C（Cyt C）,cysteinyl aspartate specific proteinase 3（Caspase-3）,Caspase-9 and Bcl-2-associated X protein（Bax）.AO/EB staining found that VDR reduced the apoptosis of CDDP-exposed HK2 cells,and the expression of apoptosis-related proteins in the cells was consistent with the in vivo test,indicating that VDR alleviated CDDP-induced AKI to a certain extent by reducing renal apoptosis.（8）Combined KEGG enrichment analysis of transcriptomics and metabolomics found that HIF-1 signaling pathway was significantly enriched in AKI mouse kidneys.Using q RT-PCR analysis the key genes of HIF-1 signaling pathway,we found that the m RNA expressions of HIF-1αand HO-1 in the kidneys of CDDP-induced AKI mice were significantly higher than those in the C group,but lower than VDR+CDDP group.The IF method was used to detected the co-localization of HIF-1αand HO-1 in kidney and HK2 cells,we found that HIF-1αobviously entered the nucleus in the mice kidney of in the CDDP group and VDR+CDDP group,but the level of HO-1 in the cytoplasm of CDDP group was significantly less than that of VDR+CDDP group.The in vitro test results were similar to those in vivo,that is,HIF-1αin CDDP-exposed HK2 cells rarely or no entered the nucleus,and the fluorescence intensity of HIF-1αand HO-1 in CDDP group cells was higher than that of C group and weaker than that of VDR+CDDP group.These results suggest that under CDDP stimulation,HIF-1αand HO-1 may be highly expressed in the kidney as hypoxia-sensing factors and stress proteins,respectively.Moreover,VDR can promote the entry of HIF-1αinto the nucleus to regulate HO-1,thereby exerting anti-oxidation,anti-inflammation and anti-apoptosis effects.In addition,using the HIF-1α/HO-1 signaling inhibitor protoporphyrin IX zinc（Znpp）to pretreat the mice and HK2 cells in the VDR+CDDP group,and we found that the renoprotective effect of VDR was weakened,further indicating that VDR attenuated CDDP-induced renal injury by regulating HIF-1α/HO-1 signaling pathway.In summary,VDR alleviates CDDP-induced renal tissue structural damage and dysfunction,the specific molecular mechanism is that VDR up-regulates the HIF-1α/HO-1 signaling pathway to improve mitochondrial dynamic,renal energy metabolism and prevent excessive mitochondrial fission induces a large amount of ROS accumulation,thereby attenuating renal oxidative damage,inflammation,apoptosis and ferroptosis,and ultimately mitigate CDDP-induced AKI.This study explored the protective mechanism of VDR on CDDP-induced AKI from the perspective of VDR regulating HIF-1α/HO-1 signaling pathway,which not only provides new ideas and experimental data for the prevention and treatment of AKI in humans and animals,but also enriches the biological knowledge of VDR.