Role Of Estrogen-Related Receptor ? In Acute Kidney Injury

Acute kidney injury(AKI)is a clinical syndrome which is characterized by abrupt decline of renal function and urine output,electrolyte disturbances,metabolic acidosis,volume overload as well as cardiopulmonary dysfunction,which affects the prognosis and increases the mortality risk of the patients.As the major site for reabsorption and excretion,renal tubular epithelial cells is abundant with mitochondria.Under numerous insults(e.g.,ischemia,hypoxia,toxins,proteinuria,etc),the biogenesis and dynamics of mitochondria as well as the abnormality of the tricarboxylic acid(TCA)cycle,fatty acid oxidation(FAO)and electron transport chain(ETC)/oxidative phosphorylation(OXPHOS)all contribute to the eventual abnormal energy metabolism in the renal tubular epithelial cells.Therefore,early intervention of the tubular injury is vital significance for the treatment of AKI.Estrogen related receptor ?(ERR?)belongs to the estrogen receptor subfamily members but without its inner ligands.The transcriptional activity of ERR? depends on many factors,such as cellular stress,co-activators,translational modifications as well as exogenous chemical compounds,etc.ERR? has been found mainly in high energydemanding tissues or organs including the kidney,heart,brain,muscle and liver.Recent evidence suggests that ERR? is involved in the regulation of a series of energy metabolism processes such as FAO,TCA cycle and OXPHOS.Provided the aforementioned background,we propose the hypothesis that the by enhancing the transcriptional activity of ERR? might protect against AKI via improving the mitochondrial energy metabolism.The first part presented by the current study focused on the expression and distribution of ERR? in the kidneys from normal control participants as well as the kidneys from different AKI mouse models.In the meantime,hydrodynamics injection techniques was employed to deliver the plasmid containing either vector or ERR? through the tail veins of the mice to explore the role of ERR? in AKI.The second part presented by the study mainly examined the efficacy of enhancing the transcriptional activity of ERR? by its agonist DY131 in different AKI models to further investigate the potential role of ERR?.The third part presented by the study harnessed the untargeted metabolomics method and dual-luciferase reporter gene assay to further investigate the mechanism of the potential role of ERR? in AKI.Part ? Expression and Effect of ERR? in AKIObjective: To determine the expression and distribution of ERR? in the kidney from normal control participants.To analyze the correlations between ERR? and clinical indexes of AKI patients.To observe the expression of ERR? in different AKI models induced by cisplatin,ischemia reperfusion(IR)and lipopolyssachride(LPS).To investigate the potential effect of ERR? overexpression within the AKI mice.Methods:(1)The paracancerous tissues of patients with renal carcinoma were used as controls.The expression of ERR? in the kidney was detected by immunohistochemical staining(IHC).(2)The correlations between ERR? and clinical indicators from AKI patients were analyzed via the online data from Nephromine database.(3)Wild type male mice(SPF grade,C57BL/6,7-8 weeks old,weight approx.25g)were randomly allocated into the following groups: control group(n=8): intraperitoneal injection of 5%Tween 80 or normal saline.Model group(n=8): AKI was induced by intraperitoneal injection of cisplatin(20 mg/kg).After 72 hours from the start of cisplatin injection,the mice were sacrificed and samples were collected.Renal ERR? expression was detected using IHC,Q-PCR and Western blot.(4)Mice were randomly grouped into: Sham group(n=10): abdomen opened and sutured,without clamping renal artery;IR group(n=10): abdomen opened,careful separation of connective tissue around the renal pedicle,renal arteries clamped quickly,with the kidney turning from bright red to purple black,suggesting complete clamping of the renal arteries.After 28 minutes,renal artery blood flow was restored by removing the clamps with the abdomen sutured.All mice should be kept in an environment with a temperature of 37°C during and after IR operation.The mice were sacrificed and samples were collected 36 hours after IR.IHC,Q-PCR and Western blot were used to detect the expression of ERR? in the kidney.(5)Mice were randomly allocated into two groups: control group(n=10): intraperitoneal injection of 5%Tween 80 or normal saline;model group(n=10): AKI induced by intraperitoneal injection of LPS(10 mg/kg).After 24 hours,the mice were sacrificed and samples were collected.The expression of ERR? in the kidney was detected by Q-PCR and Western blot.(6)The overexpression of ERR? in mice using hydrodynamic injection technique was confirmed by Q-PCR and Western blot.(7)Mice were randomly allocated into the following groups: Control group(n=10): mice were injected with empty plasmid through tail vein using hydrodynamic injection technique;Model group(n=10): mice were injected with empty plasmid through tail veins,and injected with cisplatin(20 mg/kg)intraperitoneally 36 hours later to induce AKI.Intervention group(n=10): mice were injected with ERR? plasmid through tail veins,and injected with cisplatin(20mg/kg)intraperitoneally 36 hours later to induce AKI.The mice were sacrificed and samples were harvested 72 hours after the injection of cisplatin.Blood Serum creatinine(Cr)and blood urea nitrogen(BUN)were measured.The expression of renal neutrophil gelatinase-associated lipocalin(NGAL)was detected by Q-PCR and Western blot.Hematoxylin-eosin staining(HE)and periodic acid-schiff staining(PAS)were used to evaluate the pathological changes of the kidney.The apoptosis related genes such as Bax and Bcl-2 were detected by Q-PCR.TUNEL staining of renal paraffin sections was used to evaluate renal cellular apoptosis.(8)Mouse proximal tubular cells(MPTCs)were stimulated with different concentrations of cisplatin(0,2.5,5,10 ?g/m L)for 24 h,and the expression of ERR? was detected by Western blot.MPTCs were transfected with si-NC/si-ERR?,and the expression of ERR? was detected by Q-PCR and Western blot.Furthermore,MPTCs were transfected with si-NC/si-ERR? for 24 h,and then treated with cisplatin at the dose of 5 ?g/m L for 12 h.The expressions of IL-6 and Bax were detected by Q-PCR,and MPTCs apoptosis was detected by flow cytometry.Results:(1)In renal tissues from normal controls,ERR? was widely expressed in renal tubular epithelial cells,but hardly expressed in glomeruli or renal interstitium.(2)The expression of renal ERR? was positively correlated with glomerular filtration rate(r=0.7053,P=0.0002),and negatively correlated with serum creatinine(r=-0.6117,P=0.0025).(3)IHC results showed that ERR? was widely expressed in renal tubular epithelial cells(mainly located in the nucleus)of mice,and the expression of ERR? was significantly reduced after cisplatin challenge.Q-PCR and Western blot results showed that,compared with the control group,the m RNA and protein levels of renal ERR? were decreased by 62.81%(P<0.0001)and 44.35%(P<0.001)respectively in cisplatin group.(4)Q-PCR results showed that the expression of ERR? in the kidney from mice in the IR group was 52.84% lower than that in the sham group(P<0.0001).Western blot results showed that the expression of ERR? in the kidney from mice in the IR group decreased by 26.69% compared with that in the sham group(P<0.01).IHC results also indicated that the same expression pattern of ERR?.(5)Q-PCR results showed that the expression of ERR? in the kidney from mice in the LPS group decreased by 86.02%(P<0.0001)compared with the control group.Western blot also confirmed that renal expression of ERR? decreased by 20.66%(P<0.05)after LPS administration.(6)The results of Q-PCR and Western blot showed that,compared with those injected with empty plasmid,the m RNA and protein levels of ERR? were increased by 4.58 fold(P<0.05)and 1.87 fold(P<0.001)respectively in renal tissues of mice injected with ERR? plasmid through tail vein.(7)Blood biochemical results showed that compared with the empty plasmid group,serum Cr and BUN in the empty plasmid+cisplatin group increased by 5.94 fold(19.20±6.14 vs.114.0±54.62 ?mol/L,P<0.0001),6.29 fold(10.20±1.03 vs.64.12±10.23 mmol/L,P<0.0001)respectively.The serum Cr and BUN of mice in the ERR? plasmid+cisplatin group decreased by 58.60%(114.0±54.62 vs.47.20±16.95,P=0.0152)and 21.15%(64.12±10.23 vs.50.56±11.20,P=0.0025)respectively,compared with that in the empty plasmid+cisplatin group.(6)Q-PCR results showed that the m RNA level of NGAL in the kidney of mice from the empty plasmid+cisplatin group was 341.83 fold higher than that from the empty plasmid group(P<0.0001),while the expression of NGAL in the kidney of mice from ERR? plasmid+cisplatin group decreased by 51.08% compared with those from empty plasmid+cisplatin group(P<0.01).Western blot also indicated that renal ERR? overexpression could significantly inhibit the upregulation of NGAL induced by cisplatin.Renal HE and PAS staining suggested that cisplatin could induce renal tubular injury(degeneration and necrosis of renal tubular epithelial cells,dilation of tubular lumen,formation of casts,loss of brush border,etc.)and that renal overexpression of ERR? could effectively alleviate the above pathological changes.The results of renal tubular injury were further confirmed by pathological scores(empty plasmid+cisplatin group vs.ERR? plasmid+cisplatin group: 3.8 vs.2.8,P<0.05).Q-PCR results showed that compared with the empty plasmid group,renal expression of Bax in mice from empty plasmid+cisplatin group increased by 2.38 fold(P<0.01),Bcl-2 decreased by 35.66%(P<0.01),and Bax/Bcl-2 ratio increased by 4.07 fold(P<0.0001).Compared with empty plasmid+cisplatin group,renal Bax expression decreased by 12.41%,Bcl-2 increased by 43.42%(P<0.05),and Bax/Bcl-2 ratio decreased by 39.37%(P<0.05)in ERR? plasmid+cisplatin group.The TUNEL staining of renal sections further indicated that the number of TUNEL positive cells in the ERR? plasmid+cisplatin group was significantly lower than that in the empty plasmid+ cisplatin group.(8)Western blot showed that the expression of ERR? in MPTCs stimulated by cisplatin at 5 and 10 ?g/m L was decreased.Q-PCR and Western blot results confirmed that the expression of ERR? in MPTCS was decreased after being transfected with si-ERR?.Q-PCR results showed that the level of IL-6 in si-ERR? group was 9.77-fold than that in si-NC group(P<0.01).Compared with si-NC+cisplatin group,the level of IL-6 in si-ERR?+cisplatin group increased by 2.08 times(P<0.01).Compared with si-NC group,the level of Bax in si-ERR? group was 1.86-fold higher(P<0.05).Compared with si-NC+cisplatin group,Bax level in si-ERR?+cisplatin group increased by 1.28 times(P<0.05).The apoptosis of MPTCs was detected by flow cytometry.Compared with the si-NC group,the percentage of cellular apoptosis in si-ERR? group was 3.14-fold higher(P<0.01).Compared with si-NC+cisplatin group,the percentage of cellular apoptosis in siERR?+cisplatin group increased by 2.19 times(P<0.001).Conclusions: ERR? was widely and abundantly distributed in tubular epithelial cells but not glomeruli or interstitium within the kidney from normal control group.The renal expression of ERR? was positively correlated with the glomerular filtration rate and negatively correlated with serum creatinine.ERR? was also decreased in the kidneys from AKI mice treated with cisplatin,IR and LPS.Overexpression of ERR? within the mouse ameliorated cisplatin-induced AKI.In vitro experiments suggested that silencing of ERR? aggravated cisplatin-induced MPTCs injury.Part ? Effects of ERR? agonist DY131 in AKIObjective: To investigate the role of ERR? in AKI by employing ERR? agonist DY131 in different AKI animal models.Methods:(1)The safety of DY131 in mice at the dose of 5mg/kg was evaluated.Wild type male mice were randomly allocated into the following groups: control group(n=5): intraperitoneal injection of the same amount of normal saline(once a day,for three consecutive days).Drug group(n=5): intraperitoneal injection of DY131(once a day,for three consecutive days).The mice were sacrificed and samples were collected 72 hours afterwards.Serum Cr,BUN,alanine aminotransferase(ALT),aspartate aminotransferase(AST),lactate dehydrogenase(LDH)and creatine isoenzyme(CKMB)were detected in both groups.HE staining results of the kidney,liver and heart were evaluated.(2)To evaluate the effect of DY131 on cisplatin-induced AKI.Mice were randomly allocated into three groups: control group(n=8): intraperitoneal injection of 5%Tween 80 or normal saline.Model group(n=8): AKI was induced by intraperitoneal injection of cisplatin(20 mg/kg).In the intervention group(n=8),DY131 was intraperitoneally injected(5mg/kg),and cisplatin(20 mg/kg)was injected intraperitoneally 2 hours later to induce AKI.The intervention group was given another intraperitoneal injection of DY131 at 24 and 48 hours respectively,while the control and model groups were given 5%Tween 80.After 72 hours from the start of cisplatin injection,the mice were sacrificed and samples were collected.Serum Cr,BUN,ALT,AST and LDH were measured.Western blot was used to detect the expression of ERR? in the kidneys of mice.Q-PCR was used to detect the expression of NGAL and kidney injury molecule-1(KIM-1)in the kidney.The expression of NGAL protein was detected by Western blot.HE and PAS staining were used to evaluate the pathological changes of the kidney.Enzyme linked immunosorbent assay(ELISA)was used to detect the circulatory levels of TNF and IL-6 in mice.Q-PCR was used to detect the expression of TNF,IL-1?,MCP-1 and IL-6 in mouse kidneys.The apoptosis-related genes Caspase3 and Bax were detected by Q-PCR.TUNEL staining of renal paraffin sections was used to evaluate renal cellular apoptosis.(3)To evaluate the effect of DY131 on the renal ischemia-reperfusion(IR)-induced AKI.Mice were randomly grouped into: Sham group(n=10): abdomen opened and sutured,without clamping renal artery;IR group(n=10): abdomen opened,careful separation of connective tissue around the renal pedicle,renal arteries clamped quickly,with the kidney turning from bright red to purple black,suggesting complete clamping of the renal arteries.After 28 minutes,renal artery blood flow was restored by removing the clamps with the abdomen sutured.DY131+IR group(n=10): mice were intraperitoneally injected with DY131(5mg/kg)1 and 2 hours before IR,respectively,and DY131 was given again 12 hours after IR surgery.All mice should be kept in an environment with a temperature of 37°C during and after IR operation.The mice were sacrificed and samples were collected 36 hours after IR.Serum Cr,BUN,ALT,AST and LDH were detected.The expression of NGAL in the kidney was detected by Q-PCR and Western blot.HE and PAS staining were used to evaluate the pathological changes of the kidney.Serum levels of TNF? and IL-6 were detected by ELISA.TUNEL staining of renal paraffin sections was used to evaluate renal cellular apoptosis.(4)To evaluate the effect of DY131 on lipopolysaccharide(LPS)-induced AKI.Mice were randomly allocated into three groups: control group(n=10): intraperitoneal injection of 5%Tween 80 or normal saline;model group(n=10): AKI induced by intraperitoneal injection of LPS(10 mg/kg);intervention group(n=10): mice were pretreated with DY131(5 mg/kg/day)for two consecutive days before LPS challenge.After 24 hours,the mice were sacrificed and samples were collected.Serum Cr and BUN levels were measured.The expression of NGAL in the kidney was detected by Q-PCR and Western blot.HE staining was used to evaluate the pathological changes of the kidney.The apoptosis-related genes Caspase3,Bax and Bcl-2 were detected by Q-PCR.Results:(1)Safety assessment of DY131(5mg/kg)in vivo(saline group vs.DY131 group): Cr 20.0±4.47 vs.15.0±7.75 ?mol/L,P=0.2960;BUN 11.0±1.30 vs.9.70±1.12 mmol/L,P=0.1692;ALT 27.0±4.0 vs.23.0±4.0 U/L,P=0.1950;AST 150.0±54.13 vs.113.0±39.70 U/L,P=0.3023;LDH 370.0±137.19 vs.289.0±89.58 U/L,P=0.3517;CKMB 303.0±78.52 vs.271.0±55.80 U/L,P=0.5251.HE staining of the kidney,liver and heart showed no obvious pathological changes.(2)To evaluate the effect of DY131 on cisplatin-induced AKI.Serum biochemical results showed that compared with the control group,serum BUN of mice increased by 4.55 fold(11.60±0.94 vs.52.75±18.82 mmol/L,P<0.0001)and serum Cr increased 15.23 fold(6.50±4.44 vs.99.0±53.81 ?mol/L,P<0.0001)in the cisplatin group.The above two indexes in DY131 group were decreased by 45.97%(52.75±18.82 vs.28.50±9.82 mmol/L,P<0.01)and 66.16%(99.0±53.81 vs.33.50±16.24 ?mol/L,P<0.01),respectively.Western blot results showed that the expression of ERR? in the kidney from mice in cisplatin group was decreased by 52.76%(P<0.05)compared with the control group,while the expression of ERR? in the kidney from mice in DY131 group was 54.03% higher than that in the cisplatin group(P=0.2827).Q-PCR results showed that the m RNA levels of NGAL and KIM-1 in the kidney from mice in cisplatin group were 958.20 fold higher(P<0.0001)and 2004.46 fold higher(P<0.001)compared with that in the control group.The m RNA levels of NGAL and KIM-1 in kidney from mice in DY131 group decreased by 53.54%(P<0.01)and 26.34%(P=0.3169)compared with that in cisplatin group.Western blot further confirmed the effect of DY131 on suppressing NGAL at the protein level.HE and PAS staining results suggested that cisplatin induced renal pathological damages(such as degeneration,swelling and necrosis of tubular epithelial cells,loss of brush border,tubular dilation,cast formation,etc.)were attenuated by DY131 intervention.The pathological score of renal tubular injury further suggested that DY131 alleviated renal tubular injury induced by cisplatin(cisplatin group vs.DY131+cisplatin group: 4.0 vs.2.4,P<0.01).ELISA results revealed that the circulatory levels of TNF and IL-6 increased by 10.13 fold(7.15 vs.72.45 pg/m L,P<0.0001)and 11.88 fold(13.37 vs.158.80 pg/m L,P<0.0001)compared with the control group,respectively.Compared with the cisplatin group,the serum levels of the above inflammatory cytokines in the DY131 intervention group decreased by 62.11%(72.45 vs.27.45 pg/m L,P<0.0001) and 70.48%(158.80 vs.46.88pg/m L,P<0.0001),respectively.Q-PCR revealed that the m RNA levels of TNF,IL-1?,MCP-1 and IL-6 in the kidney from mice in the control group were 79.87(P<0.001),13.63(P<0.001),32.82(P<0.0001)and 178.98(P<0.01)fold higher than that in the control group,respectively.Compared with the cisplatin group,the m RNA expression of the above cytokines in the kidney from mice treated with DY131 decreased by 75.45%(P<0.01),76.30%(P<0.01),43.71%(P<0.05)and 84.18%(P<0.01),respectively.Q-PCR results showed that the expression of caspase3 and Bax in kidney from mice in the cisplatin group increased by 4.85(P<0.0001)and 15.04 fold(P<0.0001),respectively compared with that in the control group.However,the expression of caspase3 and Bax in the kidney from mice in the DY131 group was 44.08%(P<0.05)and 49.46%(P<0.01)lower than that in the cisplatin group.TUNEL staining of the kidney further suggested that cisplatin significantly induced apoptosis of renal cells while DY131 intervention could significantly reduce the number of apoptotic cells.(3)To evaluate the effect of DY131 on renal IR-induced AKI.Serum biochemical results showed that serum BUN and Cr in the IR group increased by 6.03(7.60±0.76 vs.45.80±9.63 mmol/L,P<0.0001)and 6.65(14.80±8.21 vs.98.40±51.23 ?mol/L,P<0.0001)fold compared with that in the sham group.The above two indexes in DY131 group decreased by 49.00%(45.80±9.63 vs.23.36±14.74 mmol/L,P=0.0002)and 51.63%(98.40±51.23 vs.47.60±31.21,?mol/L,P=0.0126)compared with that in IR group,respectively.Q-PCR results showed that renal NGAL expression in the IR group was 390.86 fold higher than that in the sham group(P<0.0001).However,the expression of NGAL in the kidney from mice in DY131 group decreased by 51.93% compared with the IR group(P<0.01).Western blot further confirmed the protective effect of DY131 on the kidney.HE and PAS staining results suggested that DY131 alleviated renal pathological damages induced by IR,such as necrosis of tubular epithelial cells,loss of brush border,focal luminal dilation,casts,etc.The pathological score of tubular injury also suggested that DY131 alleviated renal IRI(IR group vs.DY131+IR group: 4.0 vs.2.8,P<0.01).ELISA results showed that serum TNF? and IL-6 levels in the IR group were 7.86(6.38 vs.55.84 pg/m L,P<0.0001)and 170.91(11.57 vs.1977.14 pg/m L,P<0.05)fold higher than that in the sham group.Compared with the IR group,serum levels of the above inflammatory cytokines in DY131 group decreased by 57.15%(55.84 vs.23.93 pg/m L,P<0.01)and 93.17%(1977.14 vs.134.99 pg/m L,P=0.0652).TUNEL staining of the kidney indicated that DY131 alleviated the apoptosis of renal cells induced by IRI.(4)To evaluate the effect of DY131 on LPSinduced AKI.Serum biochemical results showed that serum BUN and Cr in the LPS group increased by 5.28(7.95±0.73 vs.41.97±4.36 mmol/L,P<0.0001)and 2.69(13.50±3.07 vs.36.30±12.11 ?mol/L,P<0.0001)fold compared with that in the control group.The above two indexes in the DY131 group decreased by 31.95%(41.97±4.36 vs.28.56±15.72 mmol/L,P=0.0239)and 32.23%(36.30±12.11 vs.24.60±10.29 ?mol/L,P=0.0404)respectively compared with that in the LPS group.Q-PCR results showed that the expression of NGAL in the kidney from mice in the LPS group was 491.53 fold higher than that in the control group(P<0.0001).However,the expression of NGAL in the kidney from mice in the DY131 group was 28.18% lower than that in the LPS group(P<0.05).Western blot further suggested that DY131 alleviated LPS-induced renal tubular injury.HE staining of the kidney indicated that the swollen and degenerated epithelial cells of renal tubules in the LPS group were attenuated by DY131.The expressions of caspase3,Bcl-2 and Bax/Bcl-2 ratio in the kidney from mice in the LPS group detected by Q-PCR were 1.74 fold higher(P<0.01),48.90% lower(P<0.0001),2.12 fold higher(P<0.0001)than that in the control group.Compared with the LPS group,the expressions of the above indexes in the DY131 group were reduced by 29.71%(P<0.05),increased by 43.15%(P<0.05)and decreased by 37.64%(P<0.001),respectively.Conclusions: ERR? agonist DY131 at the dose of 5 mg/kg had no apparent side effects on the kidney,heart and liver.DY131 ameliorated cisplatin,IRI as well as LPS-induced AKI.Part ? Mechanism of the protective effect of ERR? in AKIObjective: To investigate the possible mechanism of the protective effect of ERR? against AKI.Methods:(1)The expression of key enzymes involved in the ?-oxidation process of mitochondrial fatty acids in the kidney tissues,such as acyl-Co A dehydrogenase short chain(ACADS),acyl-Co A dehydrogenase medium chain(ACADM)and carnitine palmityl transferase 1(CPT1),were detected by Q-PCR.(2)The expressions of key enzymes in the TCA cycle,such as pyruvate dehydrogenase(PDHB),malate dehydrogenase 1(MDH1),isocitrate dehydrogenase(IDH3B)and succinyl coenzyme A ligase(SUCLA2)in the kidney tissues,were detected by Q-PCR.(3)The expressions of NDUFB8,SDHC,UQCRB,COX6A2 and ATP5 B,representative markers of mitochondrial ETC complex I-V in the kidney tissues,were detected by Q-PCR.(4)The expressions of peroxisome proliferator-activated receptor ? co-activator 1?(PGC1?),PGC1? and mitochondrial transcription factor A(TFAM)in the kidney from mice in the control,cisplatin and DY131+cisplatin groups were detected by Q-PCR.The expression of TFAM was also detected by Western blot.The copy number of mitochondrial DNA(mt DNA)was detected by Q-PCR.(5)Vector/ERR? plasmid was transfected into MPTCs,and the transfection efficiency was verified by Q-PCR and Western blot.The expression of TFAM was detected by Western blot after the transfection of ERR? plasmid in MPTCs.The reporter gene plasmid of TFAM promoter was co-transfected into MPTCs with Vector/ ERR? plasmid.The transcription of TFAM promoter was detected by dual luciferase reporter assay system.The correlation between ERR? and TFAM in the kidney was analyzed using data from Nephromine database.(6)The expressions of 13 mitochondrial-encoding genes in the kidney were detected by Q-PCR.(7)Morphological structure of mouse kidney mitochondria was observed by transmission electron microscopy.(8)Mouse kidney metabolites and intermediated were approached with nontarget metabolomics method and analyzed using the web-based tool suite.Results:(1)Q-PCR results showed that,compared with the control group,the expressions of ACADS,ACADM and CPT1 A in the kidney from mice in the cisplatin group were decreased by 22.32%(P<0.01),34.96%(P<0.001)and 25.65%(P<0.05),respectively.Compared with the cisplatin group,however,the expressions of the above genes in the kidney from mice in the DY131 group increased by 49.03%(P<0.0001),29.10%(P<0.05)and 73.92%(P<0.01),respectively.(2)Q-PCR results showed that the expressions of PDHB,MDH1,IDH3 B and SUCLA2 in the kidney from mice in the cisplatin group were decreased by 48.43%,58.61%,55.65% and 54.74% compared with the control group(P values <0.05).The expressions of the above genes in the kidney from mice in the DY131 group was 45.95%,74.49%,1.17 fold and 1.10 fold higher than that in the cisplatin group(P values <0.05).(3)Q-PCR results indicated that the expressions of mitochondrial electron transport chain complex I-V in the kidney from cisplatin-treated mice decreased by 75.59%(P<0.001),64.45%(P<0.01),66.19%(P<0.01),60.49%(P<0.01)and 39.75%(P=0.079).However,the expressions of the above genes in the kidney from DY131-treated mice were 2.23 fold(P<0.05),1.50 fold(P<0.05),1.33 fold(P<0.01),73.44%(P=0.279)and 69.36%(P<0.05)higher than that in the cisplatin group.(4)Q-PCR results showed that the expressions of PGC1?,PGC1? and TFAM were decreased by 84.38%(P<0.001),67.77%(P<0.01)and 57.00%(P<0.05)respectively in the cisplatin group compared with the control group.However,the expression of the above genes in the kidney from mice in DY131 group was 1.16(P<0.05),69.25%(P<0.05)and 1.50 fold(P<0.01)higher than that in the cisplatin group.Western blot further confirmed that DY131 could restore the expression of TFAM in the kidney of AKI mice.Compared with the control group,the mt DNA copy number in the kidney from mice in cisplatin group decreased by 29.44%(P<0.01).However,the mt DNA copy number in the kidney from mice in DY131 group was 22.25% higher than that in the cisplatin group(P<0.05).(5)The transfection efficiency of ERR? in MPTCs was verified by Q-PCR and Western blot,and the results indicated that ERR? was successfully overexpressed in MPTCs.Western blot indicated that the overexpression of ERR? could up-regulate the expression of TFAM.Dual luciferase reporter gene assay results indicated that the transcriptional activity of TFAM increased by 1.87 times after the overexpression of ERR? in MPTCs(P<0.01).Nephromine database analysis showed that the expression of ERR? in the kidney was positively correlated with TFAM(r=0.4875,P<0.0001).(6)Q-PCR results showed that the majority of the 13 mitochondrial genome-encoding genes were suppressed in the cisplatin group compared with that in the control group.The expressions of the above genes in the DY131 group were restored to various degrees.(7)The results of transmission electron microscopy demonstrated that the altered ultrastructural changes in the cisplatin group,such as swollen and fragmented mitochondria as well as vacuolar changes,were ameliorated by DY131 intervention.(8)Metabolomics profiles identified the prominently altered kidney metabolites with statistical significance and revealed the following enriched metabolic pathways: arginine/proline metabolism,histidine/alanine/aspartate/glutamate metabolism,glycine/serine/threonine metabolism,purine metabolism,nicotinate/nicotinamide metabolism,as well as other metabolic pathways(tryptophan,aminoacyl-transfer RNA biosynthesis,methionine,vitamin B2,cysteine metabolisms,etc.).Quantitative analyses of the intermediated of these metabolic pathways showed that the decreased metabolites in the kidney from mice in the cisplatin group could be restored by DY131 treatment to different degrees.Conclusions: ERR? mediated protective effects against AKI through regulating mitochondrial FAO,TCA cycle and ETC,thereby promoting the mitochondrial biogenesis,as well as correcting multiple dysregulated metabolic pathways.