| Background:Acute kidney injury(AKI)is a common disease with high morbidity and mortality,and often develops into chronic kidney disease or even end-stage renal disease.In various clinical practices,such as surgery,shock,sepsis,trauma and renal transplantation,renal ischemia reperfusion is commonly caused by renal blood flow interruption,which leads to renal tubular and endothelial cell injury and secondary renal dysfunction,thus leading to acute renal injury[1].It is a challenge for clinicians to diagnose and treat AKI early in order to improve prognosis and reduce mortality.Studies have shown that AKI induced by ischemia reperfusion(IR)has a complex pathogenesis[2].Autophagy is a mechanism targeting a variety of stress responses.It promotes cell adaptation to stimuli by eliminating and recycling damaged macromolecules and organelles,and plays a role in cell protection[3-5].Autophagy plays an important role in ischemia-reperfusion-induced AKI in vivo and in vitro[6?7].Under stress conditions including cell starvation,hypoxia,nutrient and growth factor deficiency,endoplasmic reticulum stress and oxidative damage,autophagy pathways are up-regulated,most of which are related to the pathogenesis of AKI[8].Enhanced autophagy has been shown to protect renal tubular epithelial cells by reducing ischemia-reperfusion-induced apoptosis.However,its specific regulatory mechanism remains unclear[9].Recent studies suggest that renal underlying autophagy is critical in maintaining normal homeostasis of the proximal tubule.In the AKI model,the loss of autophagy in proximal tubules leads to tubular damage and worsening of renal function,suggesting that autophagy has a protective effect on the kidney in the AKI model[9].The process of autophagy in mammalian cells is characterized by the formation of autophagosomes and autophagosolysosomes.The formation of autophagosomes is dependent on the coordination of autophagy-related proteins,including[10]:?uncoor-dinated 51-like kinase(ULK)1/2 complex;? Beclin-1/class ?phosphatidylinositol-3-kinase complex;? Autophagy related gene(ATG)9 and vesicle protein 1;?Atg12-Atg5-Atg16 ubiquitinated protein system and LC3-?formed by microtubule-associated protein 1 light chain 3(LC3)transformation.The formation of LC3-? by LC3 involves the participation of Atg4,Atg7 and Atg3,and LC3-? is located on the membrane of autophagosome and is a biological marker of autophagy[11].Mammalian target of rapamycin(mTOR)is inhibited and activated ina classical autophagy regulatory pathway.The upstream signaling pathways of mTOR include growth factor signaling,phosphatidyl-nositide-3 kinase/protein kinase B(PI3K/Akt),mitogen-activated protein kinase(MAPK),adenosine Monophosphate activated protein kinase(AMPK)[12],small triphosphate guanosine,triphosphate kinase and calcium signal all participate in the regulation of autophagy.Beclin-1,an autophagy marker protein,is the most important positive regulator in the process of autophagy signal regulation.Together with the PI3K-? complex,Beclin-1 regulates the localization of other ATG proteins in the autophagy precursor structure and participates in the formation of autophagosomes.At the same time,autophagy can be activated by hypoxia,ATP depletion and organelle injury during renal ischemia.In renal reperfusion stage,reactive oxygen species(ROS)can directly stimulate autophagy formation through oxidative modification of autophagy,and can also induce autophagy through changes in mitochondrial membrane permeability through Bcl-2/adenovirus E1B binding protein 3[13].Specificity protein 1(SP1)is a common transcription factor,which is associated with a variety of basic biological processes,and has been proved to be of great significance in cell growth,differentiation,autophagy and apoptosis[14-16].Studies have shown that increased SP1 expression plays a crucial role in the protection of renal IR injury[17],but the mechanism of SP1 in AKI remains unclear.More and more evidence shows that there is an interesting interaction between SP1 and microRNAs[18,19].In addition,it has been found that miR-205 has a targeting relationship with phosphatase and tensin homolog deleted onchromosome ten(PTEN).MiR-205 can activate the Akt/autophagy pathway to promote angiogenesis of endothelial progenitor cells by targeting PTEN,and miR-205 can also directly target PTEN to determine radiological resistance of human nasopharyngeal carcinoma[20,21].In renal ischemia/reperfusion injury,some scholars have found that the PTEN/Akt pathway is involved in the regulation of apoptosis of renal tubular epithelial cells[22].However,it is still unclear whether overexpression of SP1 can mediate the activation of autophagy by miR-205/PTEN/Akt signaling pathway to reduce acute kidney injury induced by ischemia reperfusion,which is worthy of further study.Therefore,this study aims to explore whether SP1 can regulate autophagy by mediating miR-205/PTEN and activating the Akt signaling pathway,so as to alleviate acute renal injury caused by ischemia/reperfusion,and to provide a new perspective and therapeutic target for the improvement of acute renal injury.Part ?:The role of SP1 in an in vitro model of ischemia-reperfusion-induced acute kidney injuryObjective:HK-2 cells were cultured in vitro,the hypoxia-reoxygenation injury model of HK-2 cells was established to investigate the changes in the expression levels of SP1,miR-205 and PTEN in hypoxia-reoxygenated HK-2 cells,and to explore the effects and impacts of overexpression of SP1 on hypoxia-reoxygenated HK-2 cells,and to observe the changes of autophagy-related proteins,cell proliferation,apoptosis rate and the secretion of inflammatory factors.Methods:1.Renal tubular epithelial cells(HK-2)were cultured in vitro and a hypoxia-reoxygenation injury HK-2 cell model was established.The cells were randomly divided into control group and hypoxia-reoxygenation group(I/R group),and the proliferation of HK-2 cells was detected by MTT.HK-2 cell apoptosis rate was determined by flow cytometry after Annexin V/PI staining.The expression levels of SP1,PTEN and miR-205 mRNA were detected by RT-qPCR.The protein expression levels of SP1 and PTEN were detected by Western blot.The secretion of TNF-? and IL-6 inflammatory cytokines in the supernatant of HK-2 cells was detected by ELISA.2.In order to further study the role of SP1 in hypoxia-induced renal cell injury,HK-2 cells were transfected with SP1 overexpression vector and SP1 overexpression model was established,which were randomly divided into control group,hypoxia-reoxygenation group(I/R group),I/R+vector group and I/R+SP1 group.The mRNA expressions of SP1,miR-205 and PTEN were detected by RT-qPCR.The protein expression changes of PTEN,Akt,p-Akt and autophagy-related proteins(P62,Beclin-1,LC3?/?)were detected by Western blot.The expression and localization of autophagy protein LC3 were detected qualitatively by immunofluorescence staining.Cell proliferation was detected by MTT.HK-2 cell apoptosis rate was determined by flow cytometry after Annexin V/PI staining.The secretion of TNF-?and IL-6 inflammatory cytokines in the supernatant of HK-2 cells was detected by ELISA.Results:1.We detected the proliferation changes of HK-2 cells by MTT,and the results showed that the activity of HK-2 cells decreased gradually with the extension of time;Compared with the control group,hypoxia-reoxygenation injury at 72h significantly inhibited the proliferation of HK-2 cells.Cells were collected at 72h time point for flow cytometry,RT-qPCR,Western blotting and the supernatant of cells was collected for ELISA.Cell apoptosis was detected by flow cytometry.Compared with pre-hypoxia,the apoptosis rate of HK-2 cells increased significantly 72h after hypoxia-reoxygenation.RT-qPCR was used to detect the expression levels of SP1,PTEN and miR-205 mRNA in HK-2 cells after 72 hours of hypoxia-reoxygenation injury.Compared with the control group,the expression levels of SP1 mRNA and miR-205 mRNA in HK-2 cells with hypoxia-reoxygenation injury were decreased,while the expression of PTEN mRNA was significantly increased.Meanwhile,the protein expression levels of SP1 and PTEN were analyzed by Western blotting.The results showed that the expression of SP1 protein decreased and the expression of PTEN protein increased in HK-2 cells after 72 hours of hypoxia-reoxygenation injury.The secretion of inflammatory cytokines IL-6 and TNF-? in the supernatant of HK-2 cells increased significantly after 72 hours of hypoxia-reoxygenation injury.2.In all experimental groups,cells were collected at 72h time point for flow cytometry,RT-qPCR,Western blotting and the supernatant of cells was collected for ELISA.After transfection with SP1 overexpression vector,the expression of SP1 mRNA and miR-205 mRNA in HK-2 cells with hypoxia-reoxygenation injury for 72h in I/R+vector group was significantly increased,while the expression of PTEN mRNA was significantly decreased in I/R+SP1 group compared with I/R+vector group.Western blotting detected the expressions of PTEN,p-Akt/Akt,Beclin-1,P62 and LC3II/I in HK-2 cells exposed to hypoxia-reoxygenation injury for 72 hours.The results showed that the protein levels of PTEN and P62 in IPR group were increased,while the protein levels of p-Akt/Akt,Beclin-1 and LC3II/I were decreased compared with the control group.Compared with I/R+Vector group,I/R+SP1 group decreased the protein expressions of PTEN and P62 and increased the protein levels of p-Akt/Akt,Beclin-1 and LC3?/?.Immunofluorescence staining results showed that the fluorescence intensity of autophagy protein LC3 in HK-2 cells decreased 72h after hypoxia-reoxygenation injury,while the overexpre-ssion of SP1 could increase the fluorescence intensity of autophagy protein LC3.MTT results showed that overexpression of SP1 could increase the proliferation of hypoxia-reoxygenated HK-2 cells 72h after hypoxia-reoxygenation injury.Flow cytometry showed that overexpression of SP1 could reduce the apoptosis rate of HK-2 cells 72h after hypoxia-reoxygenation.ELISA results showed that overexpression of SP1 could reduce the secretion of inflammatory cytokines IL-6 and TNF-? in the supernatant of HK-2 cells induced by hypoxia-reoxygenation injury for 72 hours.Conclusion:1.In hypoxia-reoxygenated HK-2 cells,the expression of SP1 and miR-205 was down-regulated,while the expression of PTEN was up-regulated.Hypoxic-reoxygenation injury significantly inhibited the proliferation of HK-2 cells and increased the apoptosis of HK-2 cells,and significantly increased the secretion of inflammatory cytokines IL-6 and TNF-?.2.In hypoxia-reoxygenated HK-2 cells,overexpression of SP1 can alleviate hypoxia-reoxygenated HK-2 cell injury by activating autophagy,increasing proliferation of HK-2 cells,reducing apoptosis of HK-2 cells,and reducing the secretion of inflammatory cytokines IL-6 and TNF-?.Part ?:The role and mechanism of SP1-mediated miR-205/PTEN/Akt axis regulation of autophagy in an in vitro model of acute kidney injury induced by ischemia and reperfusionObjective:HK-2 cells were cultured in vitro to verify the targeting binding relationship between miR-205 and PTEN.To verify that SP1 mediates miR-205/PTEN/Akt axis to regulate autophagy to alleviate acute kidney injury induced by ischemia/reperfusionMethods:1.HK-2 cells were transfected with miR-205 inhibitor or mimic,and randomly divided into control group,hypoxia-reoxygenation group(I/R group),I/R+miR-205 inhibitor group,and I/R+miR-205 mimic group.Cells were collected 72h after hypoxia-reoxygenation in all experimental groups for various tests.The expressions of miR-205 and PTEN mRNA were detected by RT-qPCR.The protein expression of PTEN was detected by Western blot2.By using the bioinformatics analysis tool(TargetScan),the 3' UTR of PTEN gene was predicted to be the target of miR-205 online,and the target relationship between PTEN and miR-205 was further verified by dual luciferase reporter gene assay.PGL3-luciferase reporter vectors of wild-type(PTEN-WT)and mutant-type(PTEN-LUT)3' UTR of PTEN gene were constructed and transfected into HK-2 cells,respectively.They were randomly divided into NC mimic+PTEN-WT group,miR-205 mimic+PTEN-WT group,NC mimic+PTEN-MUT group,miR-205 mimic+PTEN-MUT group.The luciferase activity of PTEN 3'-UTR plasmid in HK-2 cells was detected by dual luciferase reporter genes.3.SP1 overexpression vector was constructed and HK-2 cells were transfected with miR-205 inhibitor and randomly divided into control group(control group),hypoxia-reoxygenation group(I/R group),I/R+vector+inhibitor NC group,I/R+SP1 group,I/R+miR-205 inhibitor group,and I/R+SP1+miR-205 inhibitor group.Cells or cell supernatants in each experimental group were collected 72h after hypoxia-reoxygenation for various tests.The expressions of miR-205 and PTEN mRNA were detected by RT-qPCR.The protein expression changes of PTEN,Akt,p-Akt and autophagy-related proteins(P62,Beclin-1,LC3?/?)were detected by Western blot.The expression and localization of autophagy protein LC3 were detected qualitatively by immunofluorescence staining.Cell proliferation was detected by MTT.HK-2 cell apoptosis rate was determined by flow cytometry after Annexin V/PI staining.The secretion of TNF-? and IL-6 inflammatory cytokines in the supernatant of HK-2 cells was detected by ELISA.Results:1.MiR-205 inhibitor or mimic was transfected into HK-2 cells,and the transfection efficiency was confirmed by RT-qPCR.The level of miR-205 was significantly down-regulated in the cells transfected with miR-205 inhibitor,while the expression of miR-205 was up-regulated in the cells transfected with miR-205 mimic.The expression level of PTEN was detected by RT-qPCR and Western blot,and miR-205 knockdown promoted the expression of PTEN,while miR-205 overexpression inhibited the expression of PTEN.2.After successfully constructing PGL3-luciferase reporter vectors of PTEN wild-type(PTEN-WT)and mutant(PTEN-MUT)3'UTR,dual luciferase reporter gene detection showed that compared with the mutant plasmid transfection group,the luciferase activity of PTEN-WT 3'UTR plasmid could be significantly reduced by miR-205 mimic.3.SP1 overexpression vector and miR-205 inhibitor were transfected into HK-2 cells,and the expression levels of miR-205 and PTEN were measured by RT-qPCR.The results showed that SP1 overexpression could promote the expression of miR-205 mRNA and inhibit the expression of PTEN mRNA in the hypoxia-reoxygenation-induced HK-2 cells.MiR-205 inhibitor could reverse the expression changes of miR-205 mRNA and PTEN mRNA caused by SP1 overexpression.The protein levels of PTEN,Akt,p-Akt,Beclin-1,P62 and LC3?/? were detected by Western blot.The results showed that SP1 overexpression inhibited the protein expressions of PTEN and P62,and promoted the protein expressions of p-Akt/Akt,Beclin-1 and LC3II/I.MiR-205 inhibitor could reverse the effect of SP1 overexpression,thereby promoting the expression of PTEN and P62 proteins,and inhibiting the expression of p-Akt/Akt,Beclin-1 and LC3?/? proteins.MTT assay showed that SP1 overexpression promoted the cell proliferation inhibited by hypoxia-reoxygenation injury,while miR-205 inhibitor reversed the effect of SP1 overexpression and thus inhibited the cell proliferation of HK-2 cells after hypoxia-reoxygenation injury.The cell apoptosis rate was detected by flow cytometry,and the results showed that SP1 overexpression inhibited cell apoptosis,and miR-205 inhibitor reversed the effect of SP1 overexpression and promoted the hypoxia-reoxygenation induced apoptosis of HK-2 cells.SP1 overexpression detected by ELISA can inhibit the secretion of IL-6 and TNF-?,and miR-205 inhibitor can reverse the effect of SP1 overexpression and promote the secretion of IL-6 and TNF-? in HK-2 cells induced by hypoxia-reoxygenation.Conclusion:1.PTEN is a direct target of miR-205 in HK-2 cells.2.SP1 overexpression can restore autophagy through miR-205/PTEN/Akt axis to alleviate hypoxia-reoxygenation-induced HK-2 cell injury.Part ?:The role of SP1 in rat kidney I/R injury model and itsmechanismObjective:In order to observe the effect of SP1 on renal ischemia reperfusion injury in vivo and its mechanism,a rat model of renal ischemia reperfusion(I/R)injury was established and treated with SP1 overexpression vector.Methods:Kidney I/R model was established in SD rats,and the rats were divided into model group(Model group,3,6,12,24,48 h)and experimental group(SP1 group,3,6,12,24,48 h).The experimental group was injected with SP1 overexpression vector 20 ?mol/kg through tail vein 2 hours before surgery,and the ischemia reperfusion model group was injected with 0.9%NaCl in the same volume in the same way.At the same time,a sham operation group was set up,and the sham operation group was performed with sham operation without clamping bilateral pedicle.At 3,6,12,24 and 48 hours after renal ischemia reperfusion surgery,serum was collected to measure the creatinine level,renal tissue was collected for TUNEL staining to calculate the apoptosis rate of renal tubules,and renal tissue was collected for RT-qPCR to detect the expression of miR-205 mRNA in renal tissue.Kidney tissue samples were collected 24 hours after renal ischemia reperfusion in rats for immunohistochemical staining to understand the expression of SP1.The protein extracted from renal tissue samples of rats was collected at 48 h after renal ischemia reperfusion and Western Blotting was used to detect the protein expression changes of PTEN,Akt,p-Akt and autophagy related proteins(P62,Beclin-1,LC3?/?).Results:The level of serum creatinine was significantly increased after ischemia reperfusion and reached the peak at 48h.The overexpression of SP1 could significantly reduce the level of serum creatinine.The expression of miR-205 was significantly decreased after ischemia-reperfusion,while the overexpression of SP1 could significantly increase the expression of miR-205.The apoptotic index was significantly increased after ischemia reperfusion,and the overexpression of SP1 could inhibit apoptosis.Immunohistochemical analysis showed that the expression of SP1 in renal tissue was decreased after ischemia reperfusion,but increased after overexpression of SP1.Western blot analysis showed that the expressions of PTEN and P62 were increased,while the protein levels of p-Akt/Akt,Beclin-1 and LC3?/LC3? were significantly decreased after ischemia/reperfusion.Overexpression of SP1 can inhibit the expression of PTEN and P62 proteins in rat kidney tissue,and promote the protein expression of p-Akt/Akt,Beclin-1 and LC3?/LC3?.Conclusion:SP1 overexpression can alleviate renal ischemia-reperfusion injury in rats by restoring autophagy. |
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