| Acute kidney injury(AKI)is a group of common clinical syndromes marked by a rapid decline in kidney function over a short period of time.The common causes are nephrotoxic drugs(e.g.cisplatin),ischemia,hypoxia,infection,etc.,with high incidence rate and high mortality rate.The damage of Renal tubular epithelial cells(RTEC)is the pathologic basis of AKI.When cells are injured,the normal structure of the cells was destroyed,the cytoskeleton lost its normal arrangement,the microvilli structure on the lumen side of the cells disappeared,and the basement membrane was destroyed,leading to cell degeneratio or death.The function and structure of mitochondria are abnormal in AKI.The renal tubular epithelial cells are rich in mitochondria.The function of cell physiology and cell repair and regeneration can not be separated from the energy supply of mitochondria.The stability of structure and function of mitochondria is of great significance for the maintenance of renal function.Mitochondrial dysfunction can cause apoptosis and necrosis of renal tubular epithelial cells and induce renal diseases.However,the exact pathogenic mechanism leading to AKI is not yet clear,and there is no no specific and satisfactory therapies in clinic.Pregnane X receptor(PXR)belongs to the NR1 I subfamily members in the nuclear receptor(NR)superfamily,mainly expressed in liver,kidney,small intestine and other tissues,plays an important role in biological regulation and "detoxification" function in the body.As a ligand dependent transcription factor,it regulates the transcription and expression of a variety of target genes,participating in the regulation of drug metabolic enzymes,glycolipid metabolism,inflammation and other signal pathways,and plays an important role in the occurrence and development of liver and intestinal diseases.However,the role of PXR in acute renal injury has not been reported.In this study,we studied the role of PXR in AKI and its mechanism.Aldo-keto reductase(AKRs)is a kind of NADPH dependent oxidoreductases,which can reduce harmful peroxides.AKRs have a wide range of substrates including carbohydrates,fatty aldehydes,steroids,prostaglandins and carcinogens.AKR1 B is the largest family of AKRs,including aldose reductase(AR)and AR-like protein.Aldo-keto reduce family 1,member B7(AKR1B7)plays an important role in the process of detoxification of lipid peroxide.In the liver and colon,PXR specific agonist pregnane-16α-carbonitrile(PCN)can significantly upregulate AKR1B7 expression.AKR1B7 is also confirmed as a downstream target gene of PXR in Hep G2 cells,suggesting that there may be a regulatory relationship between PXR and AKR1B7.Based on the above research background,this study speculates that the expression of PXR in kidney decreased under the effects of various injury factors of AKI,such as nephrotoxic drugs or renal ischemia-reperfusion injury,leading to the reduce of its target gene AKR1B7 expression,this leads to the injury of renal tubular epithelial cells and the occurrence of AKI by mediating mitochondrial dysfunction.In order to explore the mechanism,we carried out the following research,the experiment is divided into four parts.Part Ⅰ Expression of PXR in renal tissue of patients with acute kidney injuryObjective: To observe the location and quantitative expression of PXR in renal tissue during acute renal injury,and analyze the correlation between PXR and renal function.Method: 1.The renal biopsies of 20 patients with AKI and the normal renal tissues of6 patients with nephrectomy were selected as the control.The clinical parameters were collected.2.In vivo,the model of AKI was established by single intraperitoneal injection(I.P)of cisplatin(CP)into wild C57BL/6J mice.The mice were killed one day,two days and three days after injection of CP respectively,and the renal tissue was preserved.3.In vitro,the mice proximal tubular epithelial cell lines(RTECS)were cultured and stimulated with different concentrations of cisplatin(2,4,6,8ug/ml)or different times(2,6,12,24h)to induce RTEC injury.The serum creatinine(Scr)and urea nitrogen(BUN)concentration were measured by blood biochemical instrument.Immunohistochemi-cal staining to detect PXR expression in kidney tissue.The expression of PXR in renal tissue and RTEC was detected by Real time PCR and Western blot respectively.Results: 1.In the control group,PXR was mainly expressed in epithelial cells of proximal renal tubules,but less in distal renal tubules and collecting tubules,and also in mesangial cells,endothelial cells and podocytes.However,the expression of PXR decreased in the kidney biopsy of AKI patients.In addition,PXR expression in renal tubular cells was negatively correlated with the peak concentration of blood urea nitrogen(BUN)(r=-0.6377,P<0.01)and serum creatinine(SCr)(r=-0.7819,P<0.001).2.The results of IHC were consistent with those of human kidney.PXR was found in normal mouse kidney,and it was the most in proximal tubular epithelial cells.while the expression of PXR in proximal tubular was significantly decreased in the kidney of AKI mice treated with CP for three days.In the kidney of cisplatin induced AKI mice,the The expression of PXR m RNA and protein was down-regulated in a time-dependent manner.3.In vitro cultured RTECs,Cisplatin inhibited PXR expression in a dose-dependent and time-dependent manner.Conclusion: PXR expression was down regulated in acute renal injury,which was negatively correlated with the peak concentration of blood urea nitrogen and creatinine.Part Ⅱ The role of PXR in cisplatin induced acute renal injuryObjective: To explore the role of PXR in cisplatin induced acute renal injuryMethods: 1.Model preparation:(1)Animal models: PXR knockout(PXR-/-)rats were injected with cisplatin(7.5mg/kg,I.P)to induce AKI model,which was divided into four groups: WT,WT + CP,PXR-/-,PXR-/-+CP.Rats were killed 72 hours after injection of cisplatin,and serum and kidney samples were taken;The C57/B6 J mice were pretreated with PXR agonist PCN for 2 days,then the AKI mice model induced by cisplatin was prepared,which was divided into three groups: sham,CP and CP+PCN.The mice were killed 72 hours later,and serum and kidney samples were collected;PXR-/-rats were pretreated with PCN for 2 days,then cisplatin induced acute renal injury model was given,and PCN was continued to be given,divided into six groups: WT,WT + CP,WT + CP+PCN,PXR-/-,PXR-/-+ CP,PXR-/-+ CP + PCN,rats were killed 72 hours later,serum and kidney samples were taken;The C57BL/6J mice were injected with overexpressed PXR plasmid/empty vector by tail vein hydrodynamic injection,then model of AKI induced by cisplatin was prepared 36 hours later,which was divided into three groups: control,NC + CP,CP + PXR,mice were killed 72 hours later,serum and kidney samples were taken.(2)Cell models:RTEC cells were stimulated with cisplatin(5ug/ml)to prepare renal tubular cell injury models after PCN pretreatment,which was divided into three groups: Ctrl,CP and CP + PCN,cells were collected 24 hours later for subsequent detection;After being transfected with PXR si RNA,CP was given,which were divided into six groups respectively: si NC(Vehicle,CP,CP+PCN),si PXR(Vehicle,CP,CP+PCN),and cells were collected 24 hours later for subsequent detection;Using the stable PXR overexpression RTECs,CP was given,which were divided into four groups: NC(transfected empty plasmid),NC + CP,PXR,PXR + CP,and cells were collected 24 hours after cisplatin treatment;PXR overexpressed RTECs were pretreated with BAFa1,then CP was given to induce cell injury,which was divided into 6 groups:NC(vehicle,CP,CP + BAFa1),PXR(vehicle,CP,CP + BAFa1),and cells were collected 24 hours after cisplatin treatment.2.Sample analysis: The serum creatinine(Scr)and urea nitrogen(BUN)concentration were measured by blood biochemical instrument.The histopathological changes of kidney were observed by PAS staining.The morphology of mitochondria in renal tubular epithelial cells was observed by transmission electron microscopy.The release of Cyt-c and expression of macrophage marker F4/80 were observed by immunohistochemistry.TUNEL staining was used to detect the apoptosis of renal tissue.Annexin V/PI double staining combined with flow cytometry was used to detect apoptosis rate in vitro.Real-time PCR was used to detect the expression of KIM-1 and NGAL in renal cortex.Western blot,real time PCR or ELISA were used to detect the expression of inflammatory factors(IL-1 β,IL-6,TN-α),apoptosis related proteins(Bax,Bcl-2,caspase 3,cleaved caspase 3),mitophagy related genes(LC3II,P62,Atg3,Atg5,Atg7,PINK1,Parkin,fundc1,Nix),mitochondrial biosynthesis related genes(PGC-1α,TFAM,SOD2,Nrf2)and mitochondrial fusion related genes(mfn1,Mfn2,OPA1)in renal cortex or RTECs.Mitosox red mitochondrial superoxide indicator was used to detect ROS content in cells mitochondria.JC-1 method was used to detect MMP.Luciferase method was used to detect ATP content,Seahorse bioenergy analyzer was used to detect OCR.Laser scanning confocal microscope(LSCM)was used to observe the change of fluorescence intensity or signal change in RTECS after transfection of p Ds Red2-mito or mt-m Keima-cox8,to evaluate the morphology of mitochondria and the activity of mitophagy.Result: 1.In vivo:(1)The concentrations of Scr and BUN were significantly increased in cisplatin-treated wild type rats,PXR gene knockout can further aggravate the increase of Scr and BUN caused by cisplatin,while PCN treatment and PXR overexpression markedly improved renal function in AKI mice.(2)The renal tubular epithelial cells were swollen,denatured and necrotic,the renal tubules were dilated and formed,renal tubular injury markers KIM-1 and NGAL were significantly up-regulated in cisplatin-treated mouse.PXR deficiency aggravated cisplatin-induced renal tubular pathological injury,and the expression of KIM-1 and NGAL was further increased.PCN treatment and PXR overexpression significantly attenuated renal tubular pathological injury in cisplatin treated mice,and the expression of KIM-1 and NGAL decreased.The results of transmission electron microscopy showed that the damage of mitochondria in PXR-/-+ CP group rats was more serious than that in WT+ CP,while PCN treatment and PXR overexpression abrogated cisplatin induced damage of mitochondrial morphological structure in mouse kidney.(3)The number of TUNEL-positive cells and the expression of Bax and cleaved caspase-3 increased,and the expression of Bcl-2 decreased in cisplatin-treated mice,The expressions of Bax and cleaved caspase-3 and the number of TUNEL-positive cells in the kidneys of cisplatin-treated PXR-/-rats were higher than those in the kidneys of cisplatin-treated wild-type rats.While PCN treatment or PXR overexpression significantly reduced the number of TUNEL positive cells and pro-apoptotic protein expression,increased Bcl-2 m RNA expression.At the same time,immunohistochemistry showed that PCN treatment and PXR overexpression could reduce cisplatin induced Cyt-C release.(4)The infiltration of immune cells,the expression and secretion of inflammatory factors such as IL-1 β,IL-6 and TNF-α were increased in renal tissue of cisplatin-treated mouse,and these inflammatory cells and factors induced induced by cisplatin were further aggravated by PXR gene knockout,.PCN treatment or PXR overexpression could significantly attenuated the infiltration of macrophages and the expression of inflammatory factors induced by cisplatin.(5)The m RNA expression of autophagy related genes(LC3II,P62,Atg3,Atg5,Atg7)and mitochondrial biosynthesis related genes(PGC-1α,TFAM,SOD2,Nrf2)in the renal cortex of cisplatin-treated mouse was down regulated,PXR gene knockout further down-regulated the expression of these genes related to mitochondrial function induced by cisplatin.2.In vitro:(1)When RTECs were stimulated by cisplatin,the apoptosis rate,the expression apoptosis related proteins(Bax,caspase 3,cleaved caspase)and inflammatory related factors(IL-1β,IL-6,TNF-α)were increased,and the anti-apoptosis protein Bcl-2 decreased.However,PXR-si RNA could eliminate the protective effect of PCN on cisplatin induced cell damage.(2)When RTECs were stimulated by cisplatin,the ROS in mitochondria increased,mt DNA copy number and ATP content decreased,OCR and maximal respiratory capacity reduced.PCN treatment or PXR overexpression appreciably reduced cisplatin-induced accumulation of mitochondrial ROS.Moreover,the MMP,copy number of mt DNA,ATP synthesis,OCR and maximal respiratory capacity was reversed upon exposure to PCN or in PXR overexpression RTECs.(3)When RTECS were stimulated by cisplatin,the expression of genes related to mitochondrial quality control decreased,PCN pretreatment and PXR overexpression increased the expression of mitochondrial biosynthesis related genes(PGC-1α,TFAM,SOD2,Nrf2),mitochondrial fusion related genes(mfn1,Mfn2,OPA1)and mitophagy related genes(LC3II,Atg3,Atg5,Atg7,PINK1,Parkin,fundc1,Nix),decreased the expression of expression of p62.(4)When RTECs were transfected with the p Ds Red2-mito plasmid,the red fluorescence intensity was decreased in cisplatin-treated RTECs but was markedly recovered by PCN pretreatment or overexpressing PXR.Cisplatin-treated stably expressing mt-m Keima-cox8 RTECs exhibited increased green fluorescence,whereas green fluorescence was converted to red fluorescence in PCN-treated cells or PXR-overexpressing cells,indicating an increase in mitophagy.(6)Flow cytometry showed that BAFa1 partially eliminated the protective effect of PXR overexpression on cisplatin induced apoptosis.Conclusion: Knockout of PXR gene aggravated cisplatin induced cisplatin induced renal function damage,tubular cell injury,apoptosis,inflammatory response and mitochondrial dysfunction,while activation or overexpression of PXR gene could significantly improved cisplatin induced mitochondrial dysfunction and renal pathological damage.Part Ⅲ Role of PXR in renal ischemia reperfusion induced acute renal injuryObjective: To investigate the role of PXR in rena ischemia reperfusion induced AKI.Method: 1.Animal models:(1)The Wild-type rats and PXR-/-rats were divided into four groups: WT+sham,WT+I/R,PXR-/-+sham,PXR-/-+I/R.ischemia/reperfusion(I/R)induced AKI model by clamping bilateral renal artery for 35 min,reperfusion for24 hours,then rats were killed and serum and kidney samples were taken;(2)The male C57BL/6J mice were divided into three groups: sham,I/R,I/R+PCN.After PCN pretreatment for two days,AKI mice model induced by renal ischemia reperfusion was prepared.The mice were killed 24 hours after modeling,and serum and kidney samples were taken.kidneys were taken.2.Sample analysis:Scr and BUN concentra-tion were measured by blood biochemical instrument.The histopathological changes of kidney were observed by PAS staining.TUNEL staining was used to detect the apoptosis of renal cells.Real time PCR was used to detect the m RNA expression of KIM-1 and NGAL.Result:(1)After renal ischemia-reperfusion injury,the concentration of Scr and BUN were significantly increased in WT rats,knockout PXR further increased the concentration of Scr and BUN,while the activation of PXR by PCN could reduce the increase of Scr and BUN induced by ischemia-reperfusion.(2)The renal tubules show varying degrees of swelling,degeneration,necrosis,and cast formation in mouse after renal ischemia-reperfusion injury,knockout PXR further exacerbated I/R induced renal tubular injury,along with Kim-1 and NGAL m RNA expressions in the kidneys of PXR-/-rats after I/R challenge were higher than those in the kidneys of wild-type rats subjected to I/R.Moreover,compared to the WT rats,PXR-/-rats exhibited an increased number of TUNEL positive cells after I/R challenge.In contrast to the results in PXR-/-rats,PCN treatment significantly alleviated the pathological damage of renal tubules and the m RNA expressions of Kim-1 and Ngal induced by I/R.Conclusion: Knockout of PXR effectively aggravated ischemia-reperfusion induced AKI,while activation of PXR attenuated ischemia-reperfusion induced AKI.Part Ⅳ AKR1B7 mediates the protective effect of PXR in acute renal injury and Its MechanismObjective: To screen the target gene of PXR in AKI renal tissue,clarify the regulation of PXR on AKR1B7 expression,and explore the role of AKR1B7 in AKI and its effect on mitochondrial function.Method: 1.Screening and identification of PXR target genes: Quantitative proteomic analysis based on i TRAQ was used to evaluate the differential protein expression in renal cortex of WT rats and PXR-/-rats.The expression of AKR1B7 in PXR-/-rats and PXR overexpressed RTECs was detected by Real time PCR.The binding activity of PXR and AKR1B7 promoter was detected by double luciferase reporter gene method.RNA fluorescence in situ hybridization(FISH)was used to detect the location and expression of AKR1B7 in RTECs.2.Study on the role of PXR target gene AKR1B7(1)Animal models: The C57BL/6J mice were injected with cisplatin(20mg / kg,i.p), then the mice were killed on the first day,the second day and the third day after injection of cisplatin respectively,and the kidney tissue was retained;The C57BL/6J mice were injected with overexpressed AKR1B7 plasmid/empty vector by tail vein hydrodynamic injection,then model of AKI induced by cisplatin was prepared 36 hours later,which was divided into three groups: control,NC + CP,AKR1B7+CP,mice were killed 72 hours later,serum and kidney samples were taken.(2)Cell models: RTECS were cultured and stimulated with different concentrations of cisplatin(2,4,6,8ug/ml)or different times(2,6,12,24h)to induce RTEC injury;Using the stable AKR1B7 overexpression RTECs,cisplatin was given,which were divided into four groups: NC(transfected empty plasmid),NC + CP,AKR1B7,AKR1B7 +CP,and cells were collected 24 hours after cisplatin treatment;AKR1B7overexpressed RTECs were pretreated with BAFa1,then CP was given to induce cell injury,which was divided into 6 groups: NC(vehicle,CP,CP+BAFa1),AKR1B7(vehicle,CP,CP+BAFa1),and cells were collected 24 hours after cisplatin treatment.3.Sample analysis: The Scr and BUN concentration were measured by blood biochemical instrument.The histopathological changes of kidney were observed by PAS staining.The morphology of mitochondria in renal tubular epithelial cells was observed by transmission electron microscopy.The release of Cyt-c and expression of macrophage marker F4/80 were observed by immunohistochemistry.TUNEL staining was used to detect the apoptosis of renal tissue.Annexin V/PI double staining combined with flow cytometry was used to detect apoptosis rate in vitro.Real-time PCR was used to detect the expression of KIM-1 and NGAL in renal cortex.Western blot,Real time PCR or ELISA were used to detect the expression of AKR1B7,inflammatory factors(IL-1 β,IL-6,TN-α),apoptosis related proteins(Bax,Bcl-2,caspase 3,cleaved caspase 3),mitophagy related genes(LC3II,P62,Atg3,Atg5,Atg7,PINK1,Parkin,fundc1,Nix),mitochondrial biosynthesis related genes(PGC-1α,TFAM,SOD2,NRF2)and mitochondrial fusion related genes(Mfn1,Mfn2,OPA1)in renal cortex or RTECs.The expression of AKR1B7 in mitochondria was detected by Mito tracer staining,FISH and mitochondrial separation.RNA sequence was used to analyze the changes of gene transcription level in RTECs after overexpression of AKR1B7.Mitosox red mitochondrial superoxide indicator was used to detect ROS content in cells mitochondria.JC-1 method was used to detect MMP.Luciferase method was used to detect ATP content,Seahorse bioenergy analyzer was used to detect OCR.LSCM was used to observe the morphological changes of mitochondria in RTECS transfected with p Ds Red2-mito.Results: 1.(1)Quantitative proteomic analysis showed that there were 152 differentially expressed proteins(up-regulated ≥ 1.5 times or down regulated ≤ 0.67 times,P<0.05)in the kidney of PXR-/-rats compared with WT rats.Among the identified mitochondrial related differentially expressed proteins,AKR1B7 was significantly down regulated in the kidney of PXR-/-rats.Real time PCR showed that AKR1B7 was downregulated in PXR-/-rats.while upregulated.in RTECs stably expressing PXR.Double luciferase reporter gene assay showed that PXR had high binding activity with AKR1B7 promoter sequence.(3)When stimulated by cisplatin,consistent with PXR changes,the expression of AKR1B7 m RNA decreased in WT rats and RTECs.In addition,cisplatin induced the down-regulation of AKR1B7 expression in kidney in a time and dose-dependent manner.FISH results showed that AKR1B7 was primarily distributed in the cytoplasm and the expression decreased in cisplatin-treated RTECs but recovered upon treatment with PCN.2.In vivo:(1)The expression of AKR1B7 in the kidney cortex markedly increased after 36 h post-injection of the AKR1B7 plasmids.(2)Compared with NC+CP mice, tail vein high-pressure injection of AKR1B7 plasmids significantly reduced the concentration of Scr and BUN,improved renal function in AKI mice.(3)AKR1B7overexpression significantly attenuated renal tubular pathological injury,decreased the expression of KIM-1 and NGAL,and ameliorated mitochondrial abnormalities in cisplatin treated mice.(4)Compared to cisplatin treatment alone,overexpression of AKR1B7 with cisplatin treatment reduced the number of TUNELpositive cells and F4/80 positive cells,inhibited the release of mitochondrial Cyt-c,decrease the expression of Bax,IL-1 β,IL-6 and TNF-α,and increase the expression of Bcl-2.3.In vitro:(1)AKR1B7 overexpression noticeably prevented cisplatin-induced cell injury-as evidenced by the decrease in cell apoptosis and restored expression of BAX,cleaved caspase-3,Cyt-c,and Bcl-2-and inhibited the inflammatory response in cisplatin treated RTECs.(2)Overexpression of AKR1B7 significantly reduced cisplatin induced ROS accumulation in mitochondria.Moreover,reduced MMP,copy number of mt DNA,ATP synthesis,OCR and maximal respiratory capacity was reversed in AKR1B7 overexpression RTECs.(3)Mito tracer,FISH and mitochondrial isolation showed that AKR1B7 m RNA expression could be detected in mitochondria.The RNA-seq analysis exhibited that AKR1B7 overexpression enhanced the expression of mitochondrial protective genes including LARS2 and EYA2,indicating that AKR1B7 has mitochondrial protective function.(4)AKR1B7 overexpression restored or increased the expression of genes related to mitochondrial quality control,increased mitochondrial biosynthesis related genes(PGC-1α,TFAM,SOD2,Nrf2),mitochondrial fusion related genes(mfn1,Mfn2,OPA1)and mitophagy related genes(LC3II,Atg3,Atg5,Atg7,PINK1,Parkin,fund-c1,Nix),decreased the expression of p62.(5)RTECs were transfected with the p Ds Red2-mito plasmids.LSCM observed that mitochondrial morphological damage upon cisplatin treatment but recovered in the AKR1B7 overexpressing RTECs.(6)The results of Real time PCR showed that BAFa1 partially eliminated the protective effect of AKR1B7 on the cisplatin-induced RTEC apoptosisConclusion: AKR1B7 is a transcription target of PXR in the kidney.Overexpression of AKR1B7 could alleviate cisplatin induced mitochondrial dysfunction by participating in mitochondrial quality control,and then attenuate cisplatin induced renal failure,tubular injury,apoptosis and inflammation. |
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