| Part one:Xenon preconditioning protects against renal ischemia-reperfusion injury via upregulation of miR-21Background and Objective:As a severe and devastating clinical syndrome, acute kidney injury (AKI) incidence is increasing year by year. The major cause of it is ischemia-reperfusion (I/R). Up to now, there is no specific treatment for this syndrome which possesses high morbidity and mortality. Pharmacological preconditioning can activates endogenous protective mechanisms and produces endogenous protective materials in body via direct or indirect pharmacological effects, then renders an organ more tolerant to subsequent injury. It is well documented that many agents can induce preconditioning effects, such as adenosine and volatile anesthetics. As an inhalational anesthetic, xenon (Xe) has many of the properties of the ideal anesthetic, and many studies have demonstrated that Xe also provided substantial organoprotective effects in acute injury of the brain, heart and kidney, independent of anesthesia. We utilized a mouse model of renal IR injury to test the hypothesis that xenon preconditioning might provide protective effect against renal IRI, and to discern the mechanism underlying xenon preconditioning. Furthermore, we examined the role of microRNA miR-21in renal protection conferred by the xenon preconditioning using in vivo knockdown of miR-21and analysis of miR-21target pathways.Materials and Methods:Male C57BL/6J mice weighing22-25g were used in this study, and randomly divided into different groups as follow:sham operation group, mice only received isolation of renal bilateral pedicles, without clamping pedicles. I/R group,30min ischemia was induced by clamping bilateral pedicles followed by reperfusion. N2+IR group and Xe+IR group, mice were exposed for2h to either70%xenon or70%N2balanced with30%oxygen24h before the induction of renal IR injury. Blood sample and kidneys from mice in sham, I/R, N2+IR and Xe+IR group were collected24h after renal IR (n=6in each group). N2group and Xe group, mice were only exposed for2h to either70%xenon or70%N2, kidneys were collected at2,12,24and48hrs after gas exposure. Xe+anti-miR-21+IR grorp and Xe+anti-scramble+IR group, locked nucleic acid (LNA) modified anti-miR-21or anti-scramble was given30min before gas mixture exposure, and renal IR was performed24h after gas exposure. Blood sample and kidneys were collected at12,24, and48hrs after renal reperfusion. Serum creatinine (Scr) and blood urea nitrogen (BUN) were examined. The kidney tissue was stained by HE. Malondialdehyde (MDA) concentration in serum was measured. Apoptosis was confirmed by terminal deoxynueleotidyl ffansferase(TdT)-mediated dUTP-biotin llick end labeling (TUNEL) assay. Expression and distribution of tumor necrosis factor alpha (TNF-a) and ED-1in the kidney were determined by immunohistochemical analysis. Expression levels of several mRNAs and miR-21were quantified by real time RT-PCR. The relative abundance of programmed cell death protein4(PDCD4), phosphatase and tensin homolog deleted on chromosome10(PTEN), protein kinase B (Akt), p-Akt, hypoxia inducible factor lalpha (HIF-la)and vascular endothelial growth factor (VEGF) was analyzed by western blot.Results:1) Levels of Scr and BUN were significantly increased24h after renal IR in I/R group and N2+IR group (Scr,1.60±0.44mg/dl and1.05±0.30mg/dl; BUN,173.05±8.68mg/dl and127.14±23.46mg/dl) when compared with sham group (0.15±0.02mg/dl and25.05±3.71mg/dl, p<0.05) and Xe+IR group (0.28±0.06mg/dl and45.86±12.97mg/dl, p<0.05).2) Extensive tubular necrosis was found at24h after renal IR injury, accompanied by proein casts and inflammatory cell infiltration in I/R group and N2+IR group. Kidney tissue in Xe+IR group mice only presented mild to moderate tubular injury and histologic injury score of the Xe+IR group was substantially lower than that of I/R and N2+IR group.3) Many apoptotic tubular cells were found at24h after renal IR injury in I/R group and N2+IR group, which mainly concentrated in corticomedullary injunction and outer medullar. Xe preconditioning significantly decreased the tubular cell apoptosis (4.80±1.23/HPF vs.11.10±2.33/HPF and10.10±1.23/HPF, P<0.05).4) Renal tissue MDA concentration in I/R group and N2+IR group mice was higher than that in Xe+IR group (1.00±0.09μmol/L vs.1.43±0.42μmol/L and1.35±0.25μmol/L, P<0.05).5) Renal protein expression of TNF-a was significantly elevated and ED-1+cells markedly increased and accumulated in the tubulointerstitium24h after renal IRI in I/R group, N2+IR group and Xe+IR group mice, and there was no significant difference between the three groups.6) Time-course analysis indicated that miR-21expression was upregulated from2h after xe preconditioning compared to N2pretreatment, and reached the peak at24h, then deceased gradually at48h. miR-21was significantly upregulated24h after renal IRI in Xe+IR group mice, when compared to the N2+IR group.7) Administration of LNA-anti-miR-21before Xe preconditioning markedly decreased the expression of miR-21in mouse kidneys in Xe+anti-miR-21group and Xe+anti-miR-21+IR group. Time-coures assay showed that knockdown of miR-21substantially increased the level of Scr24and48hrs after renal IRI in Xe+anti-miR-21+IR group when compared with Xe+scramble+IR group (24h,1.37±0.28mg/dl vs.0.81±0.14mg/dl;48h,0.78±0.10mg/dl vs.0.47±0.08mg/dl, p<0.05), exacerbated renal function and histologic damage.8) Knockdown of miR-21induced significant upregulation of PDCD4and PTEN, two pro-apoptotic target effectors of miR-21in protein level but not in mRNA level, and resulted in significant downregulation of p-Akt expression while no influence in Akt. Mice receiving the anti-miR-21treatment showed a substantial increase of apoptotic cells in kidneys24h after renal IRI when compared with mice receiving the scrambled anti-miR (p<0.05).9) Time-course analysis indicated that the expression of HIF-lα and its target effector VEGF was upregulated from2h after xe preconditioning compared to N2pretreatment, and reached the peak at24h, then deceased gradually at48h. Mice receiving scrambled anti-miR showed higher HIF-1α protein expression in kidneys compared with mice receiving anti-miR-21(p<0.05).Conclusions:1) Xenon preconditioning protected renal function against IRI, characterized by attenuation of tubular cell damage, apoptosis, and oxidative stress, but not a reduction of inflammation.2) Xenon preconditioning upregulated miR-21, HIF-la protein and its target effector VEGF in time dependent manner.3) A locked nucleic acid-modified anti-miR-21given before xenon preconditioning knocked down miR-21effectively and exacerbated subsequent renal IRI functionally and morphologically.4) Knockdown of miR-21induced significant upregulation of PDCD4and PTEN, and resulted in significant downregulation of p-Akt expression, eventually leading to increasing of tubular cell apoptosis. Part two:Intermittent exposure to xenon protects against gentamicin-induced nephrotoxicityBackground and Objective:Drug nephrotoxicity is one of the most common causes of acute kidney injury. It occurs in10-20%of patients treated with the therapeutic doses of aminoglycosides, especially gentamicin. The mechanisms involved in gentamicin-induced nephrotoxicity are multifaceted, including tubular epithelial cell necrosis and apoptosis, oxidative stress, inflammatory response and hemodynamic. In spite of undesirable side effect, gentamicin is widely used against severe infections of Gram-negative bacteria due to its excellent antibacterial profile and efficacy. Given the largely ineffective therapeutic strategies in clinic, the development of new and efficient therapeutic interventions preventing the incidence of gentamicin-induced nephrotoxicity is clearly needed. We utilized a rat model of gentamicin-induced nephrotoxicity to test the hypothesis that xenon treatment might provide protective effect against nephrotoxicity, and to discern the mechanisms underlying renal protection exerted by xenon.Materials and Methods:Male Wistar rats weighing22-25g were used in this study, and randomly divided into different groups as follow:control group:rats received a daily intraperitoneal (i.p.) injection of1ml saline for7days; gentamicin group (gent): received a daily i.p. injection of gentamicin sulfate solution at a dose of100mg/kg/BW for7days; N2+gentamicin group (N2+gent) and Xe+gentamicin group (Xe+gent):rats pretreated with70%nitrogen or70%xenon balanced with30%oxygen for2hrs, followed by7days of gentamicin treatment initiated24hrs later, and repeatedly treated with70%nitrogen for30minutes on day2,4, and6during the7-day administration of gentamicin. Blood, urine and kidneys were collected respectively on days2,4, and7after the last gentamicin injection for assay (n=6at each time point in each group). N2pretreatment group (N2) and xenon pretreatment group (Xe), rats were only exposed for2h to either70%xenon or70%N2, kidneys were collected at24h after gas exposure. Serum creatinine (Scr) and blood urea nitrogen (BUN) were examined. The kidney tissue was stained by HE. Malondialdehyde (MDA) concentration in serum was measured by TBARS assay. Apoptosis was confirmed by terminal deoxynueleotidyl ffansferase(TdT)-mediated dUTP-biotin Hick end labeling (TUNEL) assay. Expression and distribution of tumor necrosis factor alpha (TNF-a) and ED-1in the kidney were determined by immunohistochemical analysis. Expression levels of several mRNAs and miR-21were quantified by real time RT-PCR. The relative abundance of hypoxia inducible factor1alpha (HIF-1α), vascular endothelial growth factor (VEGF), three HIF prolyl hydroxylases (PHDs) were analyzed by western blot.Results:1) Levels of Scr and BUN were significantly increased and creatinine clearance (Ccr) was significantly decreased48h after last gentamicin injection in gent group, N2+gent group and Xe+gent group (Scr,1.42±0.21mg/dl,1.24±0.52mg/dl,1.00±0.28mg/dl; BUN,29.72±6.85mmol/L,22.40±5.90mmol/L,21.59±5.11mmol/L; Ccr,1.61±0.38ml/min/kg.bw,2.03±0.69ml/min/kg.bw,2.28±0.36ml/min/kg.bw, P<0.05) when compared with control group (Scr,0.21±0.04mg/dl; BUN,6.01±0.96mmol/L; Ccr,6.15±1.18ml/min/kg.bw), while there was no significant difference between N2+gent group and Xe+gent group. At4days after the final gentamicin administration, there were significant improvements in renal functional parameters in the Xe+gentamicin group when compared with the gentamicin or N2+gentamicin control groups (Scr,0.59±0.07mg/dl vs.1.18±0.27mg/dl and0.80±0.11mg/dl; BUN,12.47±5.40mmol/L vs.18.80±5.62mmol/L and24.25±13.21mmol/L; Ccr,4.06±0.62ml/min/kg.bw vs.2.47±0.38ml/min/kg.bw and2.37±0.40ml/min/kg.bw, p<0.05). N-acetyl-β-D-glucosaminidase (NAG) had higher activities in the urine of gentamicin administered animals2and4days after the final injection compared to the control (p<0.01). The enzyme activities were significantly reduced in Xe+gent group when compared with the gent group and N2+gent group at the two time points. On7th day, renal function ameliorated obviously in all experimental groups, while the urine NAG activities were significantly reduced in Xe+gent group when compared with the gent group and N2+gent group.2) Extensive tubular necrosis was found at2days after the final gentamicin administration, which were prominent in the cortical proximal segments, accompanied by casts and inflammatory cell infiltration in gent group and N2+gent group. Kidney tissue in Xe+gent group rats only presented mild to moderate tubular injury and histologic injury score of the Xe+gent group was substantially lower than that of gent group and N2+gent group. On4th day, histologic injury ameliorated obviously in all experimental groups, while histologic score of the Xe+gent group was substantially lower than that of gent group and N2+gent group (p<0.05).3) Apoptotic tubular cells were found at2and4day after the final gentamicin administration, while TUNEL-positive cells were significantly fewer in the Xe+gent group than the gent group and N2+gent group in kidneys (P<0.05).4) MDA concentration in renal cortical tissues was significantly reduced in Xe+gent group at4day after the last administration of gentamicin compared with the gent group and N2+gent group (1.87±0.02μmol/L vs.2.13±0.10μmol/L and2.06±0.05μmol/L; P<0.05).5) Renal protein expression of TNF-a was significantly elevated and ED-1+cells markedly increased and accumulated in the tubulointerstitium2and4day after the final gentamicin administration in gent group, N2+gent group and Xe+gent group rats, and there was no significant difference between the three groups (P>0.05).6) Quantitative RT-PCR analysis of mRNA expression showed that HIF-2a and VEGF were significantly higher in Xe-pretreatment group than in the N2-pretreatment group and control (P<0.05), while the HIF-la mRNA expression was not affected. At4day after the last gentamicin administration, the expression levels of HIF-2a and VEGF mRNA were significantly higher in Xe+gent group than in the gent group and N2+gent groups (P<0.05). mRNA expression of PHD1significantly decreased while PHD2increased, and PHD3was not affected24h after gas exposure in renal cortex in Xe-pretreatment group, when compared with N2-pretreatment group. Xe pretreatment significantly upregulated miR-21expression in adult rat kidney at24hr after70%Xe exposure, and Xe+gent group showed higher miR-21expression than either the gent group or N2+gent group (P<0.05).7) Parallel to alteration of the mRNA levels, Xe pretreatment upregulated the protein expression of HIF-2a and VEGF in kidney, but not HIF-la. At4day after the last gentamicin administration, HIF-2a and VEGF protein expressions were significantly higher in Xe+gent group than in the gent group and N2+gent group (P<0.05). In all experimental groups, HIF-1α was unaffected. The protein expressions of PHD1, PHD2, and PHD3in the cortex paralleled to patterns of the mRNAexpression.Conclusions:1) Xe provided morphological and functional renoprotection, characterized by attenuation of renal tubular damage, apoptosis, and oxidative stress, but not a reduction in inflammation.2) Xe pretreatment upregulated HIF-2a and its downstream effector VEGF in kidney, but not HIF-1α. HIF-2α and its targt effectors might contribute to xenon protection against gentamicin-induced renal injury.3) Xe pretreatment upregulated PHD2, suppressed PHD1, and had no influence on PHD3in the rat kidneys, which might be related to the upregulation of HIF-2α.4) Xe Pretreatment also increased the expression of miR-21, a strong anti-apoptotic factor, which might contribute to xenon protection via inhibiting cell apoptosis. |
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