| BackgroundCKD which brings a big huge pain to patients and family and a huge economic burden to Community is currently a burden for the public health system.Chronic low-grade inflammation is a common feature of CKD and may cause atherosclerotic CVD through various pathogenetic mecha-nisms.Evidence suggests that persistent inflammation may also be a risk factor for progression of CKD, which may result in a vicious inflammation-driven circle.Chronic low-grade inflammation, as evidenced by increased levels of pro-inflammatory cytokines, such asinterleukin1(IL-1), interleukin6(IL-6), tumor necrosis factor (TNF-a) and high sensitivity C-reactive protein(hsCRP), are common features of CKD.The mechanism of chronic inflammation under the condition of CKD has not yet been fully elucidated.Mitochondria are defined as the "powerhouse of the cell" because of their ability to produce the vast majority of energy necessary for cellular metabolism through the oxidative phosphor-ylation system (OXPHOS). Mitochondrial biogenesis and degradation (mitophagy) usually occur in balance within healthy cells,AMPK and Peroxisome proliferator-activated receptor y coactivator-1α (PGC-la) are strong promoters of mitochondrial biogenesis and oxidative metabolism, both SIRT1and AMPK together or independently activate PGC-1a to induce the expression of downstream genes involved in mitochondrial biogenesis,such as NRF-1/NRF-2、 TFAM, a direct regulator of mitochondrial DNA replication/transcription and Oxidative phosphorylation enzymes such as COX、ATPsynthase, and their imbalance may be a major contributor to oxidative stress and cellular metabolic decline。Given the central role for mitochondria in fuel utilization and energy production, disordered mitochondrial function at the cellular level can impact whole-body metabolic homeostasis and has been implicated in the pathogenesis of several diseases.The renewal of mitochondria through the process of biogenesis is vital for maintaining mitochondrial integrity, and a diminished capacity for organelle biogenesis has been implicated in the pathogenesis of several diseases such as aging, neurode-generation, as well as type2diabetes.Macrophages are important innate immune cells that are associated with two distinct phenotypes:a pro-inflammatory (or classically activated) subset with prototypic macrophage functions such as inflammatory cytokine production and bactericidal activity, and an anti-inflammatory (or alternatively activated (AAM)) subset linked with wound healing and tissue repair processes. Many studies have shown that macrophage inflammation play an important role in metabolic disease such as obesity and type2diabetes and other metabolic pathologies. The AMP-activated protein kinase (AMPK) lies at the crossroads of metabolically driven macrophage inflammation and exerts control over mitochondrial metabolism, and therefore is vital for dictating the inflammatory status of macrophages. The latest study also found that mitochondrial biogenesis plays an important role in macrophage activation.Mitochondrial Dysfunction can lead to excessive macrophages activation,resulting in the occurrence of many inflammatory diseases.Many studies have shown that Macrophage infiltration is one of the most striking features of glomerular and tubulointerstitial disease and macrophage inflammation plays an important role in the development and progress of kidney diease, For example:M1-like macrophage-derived TNFa, IL-6, have been identified as important mediators in renal architecture and function injury,but the mechanism of macrophage acivation under the condition of CKD is unclear. Current research depicts specific modes of immunity and energy metabolism as being interrelated at the molecular, cellular, organ and organism level. Chronic kidney disease also belong to the scope of metabolic diseases, this mechanism of macrophage inflammation may be associated with mitochondrial biogenesis pathway. So exploreing the activation of immune cells by metabolic factors are important for the prevention and treatment of metabolic inflammation.We hypothesized that mitochondrial dysfunction in macrophage may play important role in the pathogenesis of inflammation in chronic kidney disease. We investigated the mitochondrial biogenesis in the condition of chronic kidney diseasewe observe the effects of CRF on mitochondria biogenesis in peritoneal macrophages derived from5/6NX rat. we also observed the effects of uremic serum on mitochondria biogenesis in uremic serum treated peritoneal macrophages derived from rat and THP-1cells. In addition that,we observe the effects of AICAR and AC-AMPK on mitochondria biogenesis about US-stimulate peritoneal macrophages from sham rats.MethodsThe first part:Studies in peritoneal macrophages from5/6NX rat1. Animal model preparationsSD male rats were maintained on a12/12h light/dark cycle at25℃and fed adlibitum a standard laboratory chow for one week. Subsequently, the animals were randomly assigned to two groups:sham (n=8).chronic renal failure (n=24). Surgical five-sixth nephrectomy (5/6Nx) and sham operations in rats were performed. Briefly, under pentobarbital anesthesia, two-thirds of the left kidney was removed in the first stage of the procedure, and a week later the right kidney was totally excised (totally5/6Nx). Control animals were sham-operated with only decapsulation of the kidney.2. The collection of blood, urineAt the10weeks after opertation, the consecutive24-hour urine samples were collected in metabolic cages before the animals were sacrificed. The animals (n=6in each group) were anesthetized with sodium pentobarbital. The blood was collected.3.Isolation and cultivation of peritoneal macrophagesThe rats were sacrificed under ether anesthesia, and then soaked in75%alcohol5minutes. Rats of each group were intraperitoneally injected with10mL of RPMI1640. Then their abdominal cavities were opened under sterile conditions to retrieve the buffer solutions. After that, the buffer solutions were centrifuged at800(xg) for5min. The supernatants were removed and the cells were then suspended with RPMI1640complete media containing10%of fetal bovine serum for culturing.4.Measurement of mtDNA Copy NumberThe mtDNA content is the mtDNA copy number normalized to the copy number of a nuclear gene. After extraction of macrophage DNA, quantitative PCR reactions were performed using CytB, for mtDNA copy estimation, and β-actin for the nuclear genome.5.Measurement of mitochondrial massTo measure mitochondrial mass, cells were stained with100nM of MitoTracker Green FM (Invitrogen)(30min at37℃). Cells were washed with PBS, trypsinized and resuspended in PBS containing1%heat inactive FBS. Data were acquired with a BD FACS Canto II flow cytometer (BD Biosciences) and analyzed with FlowJo analytical software (Treestar).6.Determination of NRF1mRNA, NRF2mRNA, TFAM mRNA,CO mRNA,COX mRNA,ATPsynthase by mRNA real-time PCR and determination of the express of CO、COX by western blotting.Total RNA of macrophage was extracted with TRIZOL reagent. NRF1mRNA, NRF2mRNA, TFAM mRNA,CO mRNA,COX mRNA.ATPsynthaseby mRNA were detected by real-time PCR according to the manufacturer’s protocol.Macrophages were homogenized in ice-cold fractionation buffer. The lysate was incubated on ice for15min and then centrifuged at12,000xg for10min at4℃. Samples were diluted4:1(v/v) with5×Laemmli buffer, heated to100℃and then the the express of CO、 COX was detected by western blotting.7. Determination activation of AMPKMacrophages were homogenized in ice-cold fractionation buffer. The lysate was incubated on ice for15min and then centrifuged at12,000xg for10min at4℃. Samples were diluted4:1(v/v) with5×Laemmli buffer, heated to100℃and then the activation of AMPK was detected by western blotting.8. Determination of the activation of PGC1α The protein of macrophages was extracted as mentioned above,the activation of PGC1α was detected by immunoprecipitation and immunoblotting.9. StatisticsAll values are mean±SEM. The significance of differences among mean values was determined by Idependent-Samples T Test. Statistical comparison of the control group with treated groups was performed using statistical soft ware SPSS13.0.The accepted level of significance was p<0.05.The second part:Studies in the effect of uremic serum on peritoneal macrophages and THP-11. Preparation of uremic serum1.1Uremic serum from SD ratsThe whole blood specimen from5/6Nx rat was centrifuged at3000rpm for10min at4℃,And the upper of the specimens was what we want.1.2Uremic serum from uremia patientsThe whole blood specimen from uremia patients was centrifuged at3000rpm for10min at4℃,And the upper of the specimens was what we want.2. Cell culture2.1Isolation and cultivation of peritoneal macrophagesAfter the blood was taken, rats of each group were intraperitoneally injected with10mL of RPMI1640. Then their abdominal cavities were opened under sterile conditions to retrieve the buffer solutions. After that, the buffer solutions were centrifuged at800(xg) for5min. The supernatants were removed and the cells were then suspended with RPMI1640complete media containing10%of fetal bovine serum for culturing.2.2THP-1were obtained from American Type Culture Collection (NO.CL-173) and were cultured in high glucose1640with10%fetal bovine serum,100U/ml penicillin and0.lmg/ml streptomycin at37℃in a5%CO2incubator. After confluence reached 80%,THP-1were growth-arrested in20ng/ml PMA for24hours and then treated with Uremic Serum24hours for the indicated concentration.3. Measurement of mtDNA Copy Number3.1MtDNA Copy Number of rat peritoneal macrophagesPeritoneal macrophages were exposed to0%,10%,20%uremic serum24hours,The mtDNA content is the mtDNA copy number normalized to the copy number of a nuclear gene. After extraction of macrophage DNA, quantitative PCR reactions were performed using CytB for mtDNA copy estimation, and β-actin for the nuclear genome.3.2MtDNA Copy Number of THP-1THP-1were exposed to0%,10%,20%uremic serum24hours,The mtDNA content is the mtDNA copy number normalized to the copy number of a nuclear gene. After extraction of macrophage DNA, quantitative PCR reactions were performed using ND1for mtDNA copy estimation, and p-actin for the nuclear genome.4. Measurement of mitochondrial mass4.1Mitochondrial mass of rat peritoneal macrophagesPeritoneal macrophages were exposed to0%,10%,20%uremic serum24hours, then cells were stained with100nM of MitoTracker Green FM (Invitrogen)(30min at37℃). Cells were washed with PBS, trypsinized and resuspended in PBS containing1%heat inactive FBS. Data were acquired with a BD FACS Canto II flow cytometer (BD Biosciences) and analyzed with Flow Jo analytical software (Treestar).4.2Mitochondrial mass of THP-1THP-1were exposed to0%,10%,20%uremic serum24hours,then cells were stained with100nM of MitoTracker Green FM (Invitrogen)(30min at37℃). Cells were washed with PBS, trypsinized and resuspended in PBS containing1%heat inactive FBS. Data were acquired with a BD FACS Canto II flow cytometer (BD Biosciences) and analyzed with FlowJo analytical software (Treestar).5. Determination of NRF1mRNA, NRF2mRNA, TFAM mRNA,CO mRNA,COX mRNA,ATPsynthaseby mRNA real-time PCR 5.1Determination of NRF1mRNA, NRF2mRNA, TFAM mRNA,CO mRNA,COX mRNA,ATPsynthaseby mRNA of rat peritoneal macrophagesPeritoneal macrophages were exposed to0%,10%,20%uremic serum24hours,Total RNA of macrophage was extracted with TRIZOL reagent. NRF1mRNA, NRF2mRNA, TFAM mRNA,CO mRNA,COX mRNA,ATPsynthaseby mRNA were detected by real-time PCR according to the manufacturer’s protocol.5.2Determination of NRF1mRNA, NRF2mRNA, TFAM mRNA,CO mRNA,COX mRNA,ATPsynthaseby mRNA of THP-1THP-1were exposed to0%,10%,20%uremic serum24hours, Total RNA of macrophage was extracted with TRIZOL reagent. NRF1mRNA, NRF2mRNA, TFAM mRNA,CO mRNA,COX mRNA,ATPsynthaseby mRNA were detected by real-time PCR according to the manufacturer’s protocol.6. Determination of the express of CO,COX and activation of AMPK6.1Determination of the express of CO,COX and activation of AMPK of rat peritoneal macrophagesPeritoneal macrophages were exposed to0%,10%,20%uremic serum24hours, Macrophages were homogenized in ice-cold fractionation buffer. The lysate was incubated on ice for15min and then centrifuged at12,000xg for10min at4℃. Samples were diluted4:1(v/v) with5×Laemmli buffer, heated to100℃and then the activation of CO,COX,AMPK was detected by western blotting.6.2Determination of the express of CO、COX and activation of AMPK of THP-1THP-1were exposed to0%,10%,20%uremic serum24hours, Macrophages were homogenized in ice-cold fractionation buffer. The lysate was incubated on ice for15min and then centrifuged at12,000xg for10min at4℃. Samples were diluted4:1(v/v) with5×Laemmli buffer, heated to100℃and then the activation of CO,COX, AMPK was detected by western blotting.7.StatisticsAll values are presented as mean±SEM. The significance of differences among mean values was determined by One-way ANOVA. Statistical comparison of the control group with treated groups was performed using statistical soft ware SPSS13.0.The accepted level of significance was P<0.05.The three part:Studies in AICAR and AC-AMPK on activation of US-stimulate peritoneal macrophages from sham rat1. Measurement of mtDNA Copy Number1.1AICAR on mtDNA Copy Number from US-stimulate peritoneal macrophagesPeritoneal macrophages were pretreated wit1Mm AICAR4hours,then were exposed to20%uremic serum for24hours,The mtDNA content is the mtDNA copy number normalized to the copy number of a nuclear gene. After extraction of macrophage DNA, quantitative PCR reactions were performed using CytB for mtDNA copy estimation, and β-actin for the nuclear genome.1.2AC-AMPK on Copy Number from US-stimulate peritoneal macrophagesPeritoneal macrophages were transfected with AC-AMPK24hours,then were exposed to20%uremic serum for24hours,The mtDNA content is the mtDNA copy number normalized to the copy number of a nuclear gene. After extraction of macrophage DNA, quantitative PCR reactions were performed using CytB for mtDNA copy estimation, and β-actin for the nuclear genome.2. Measurement of mitochondrial mass2.1AICAR on mitochondrial mass from US-stimulate peritoneal macrophagesPeritoneal macrophages were pretreated wit1Mm AICAR4hours,then were exposed to20%uremic serum for24hours, then cells were stained with100nM of MitoTracker Green FM (Invitrogen)(30min at37℃). Cells were washed with PBS, trypsinized and resuspended in PBS containing1%heat inactive FBS. Data were acquired with a BD FACS Canto Ⅱ flow cytometer (BD Biosciences) and analyzed with FlowJo analytical software (Treestar).2.2AC-AMPK on mitochondrial mass from US-stimulate peritoneal macrophagesPeritoneal macrophages were transfected with AC-AMPK24hours,then were exposed to20%uremic serum for24hours, then cells were stained with100nM of MitoTracker Green FM (Invitrogen)(30min at37℃). Cells were washed with PBS, trypsinized and resuspended in PBS containing1%heat inactive FBS. Data were acquired with a BD FACS Canto Ⅱ flow cytometer (BD Biosciences) and analyzed with FlowJo analytical software (Treestar).3.Determination of the protein expression of CO,COX3.1AICAR on the protein expression of CO,COX from US-stimulate peritoneal macrophagesPeritoneal macrophages were pretreated wit1Mm AICAR4hours,then were exposed to20%uremic serum for24hours, Macrophages were homogenized in ice-cold fractionation buffer. The lysate was incubated on ice for15min and then centrifuged at12,000×g for10min at4℃. Samples were diluted4:1(v/v) with5×Laemmli buffer, heated to100℃and then the activation of CO,COX was detected by western blotting3.2AC-AMPK on the protein expression of CO,COX from US-stimulate peritoneal macrophagesPeritoneal macrophages were transfected with AC-AMPK24hours,then were exposed to20%uremic serum for24hours, Macrophages were homogenized in ice-cold fractionation buffer. The lysate was incubated on ice for15min and then centrifuged at12,000×g for10min at4℃. Samples were diluted4:1(v/v) with5xLaemmli buffer, heated to100℃and then the activation of CO、COX was detected by western blotting.4. Determination of the activation of AMPK4.1AICAR on the activation of AMPK from US-stimulate peritoneal macrophagesPeritoneal macrophages were pretreated wit1Mm AICAR4hours,then were exposed to20%uremic serum for24hours, Macrophages were homogenized in ice-cold fractionation buffer. The lysate was incubated on ice for15min and then centrifuged at12,000×g for10min at4℃. Samples were diluted4:1(v/v) with5×Laemmli buffer, heated to100℃and then the activation of AMPK was detected by western blotting 4.2AC-AMPK on the activation of AMPK from US-stimulate peritoneal macrophagesPeritoneal macrophages were transfected with AC-AMPK24hours,then were exposed to20%uremic serum for24hours AC-AMPK on the activation AMPK from US-stimulate peritoneal macrophages Macrophages were homogenized in ice-cold fractionation buffer. The lysate was incubated on ice for15min and then centrifuged at12,000×g for10min at4℃. Samples were diluted4:1(v/v) with5×Laemmli buffer, heated to100℃and then the activation of AMPK was detected by western blotting.5. StatisticsAll values are presented as mean±SEM. The significance of differences among mean values was determined by One-way ANOVA. Statistical comparison of the control group with treated groups was performed using statistical soft ware SPSS13.0.The accepted level of significance was P<0.05.ResultsThe first part:Studies in peritoneal macrophages from5/6NX rat1. General dataData are summarized in Table1.As expected, the CRF group exhibited a significant increase in serum creatinine and BUN concentration (p<0.05), and a significant lower body weight (p<0.05) when compared with the sham-operated normal control group.2. MtDNA Copy Number in peritoneal macrophagesThe mtDNA Copy Number from CRF rats were decreased relative to sham-operated animals (P<0.05)3. Mitochondrial mass in peritoneal macrophagesThe mitochondrial mass from CRF rats were decreased relative to sham-operated animals (P<0.05)4.NRF1, NRF2, TFAM,CO,COX,ATPsynthase gene expression in peritoneal macrophages The gene expressions of NRF1, NRF2, TFAM,CO,COX,ATPsynthase in peritoneal macrophages from CRF rats were decreased relative to sham-operated animals (P<0.05)5. Determination of the protein express of CO、COX and activation of AMPKThe protein expression of CO、COX and P-AMPK In peritoneal macrophages from CRF rats were decreased relative to sham-operated animals (P<0.05).The expression of AMPK have no change.6. Determination of the activation of PGC1αThe protein expression of AC-PGC1α In peritoneal macrophages from CRF rats were increased relative to sham-operated animals (P<0.05).The expression of PGC1α have no change. Conclusion:In the present study,we tested the hypothesis that mitochondrial biogenesis was inhibited in chronic renal failureThe second part:Studies in the effect of uremic serum on peritoneal macrophages and THP-11.Measurement of mtDNA Copy Number1.1The effect of uremic serum on mtDNA Copy Number in peritoneal macrophagesAfter of stimulation of peritoneal macrophages by0%,10%,20%uremic serum,the mtDNA Copy Number reduced gradually with the rise of concentration(p<0.05),The effect was maximal at20%uremic serum treatment.1.2The effect of uremic serum on mtDNA Copy Number in THP-1After of stimulation of THP-1by0%,10%,20%uremic serum,the mtDNA Copy Number reduced gradually with the rise of concentration(p<0.05),The effect was maximal at20%uremic serum treatment.2.Measurement of mitochondrial mass2.1The effect of uremic serum on mitochondrial mass in peritoneal macrophages After of stimulation of peritoneal macrophages by0%,10%,20%uremic serum,mitochondrial mass reduced gradually with the rise of concentration(p<0.05),The effect was maximal at20%uremic serum treatment.2.2The effect of uremic serum on mitochondrial mass in THP-1After of stimulation of THP-1by0%,10%,20%uremic serum,mitochondrial mass reduced gradually with the rise of concentration(p<0.05),The effect was maximal at20%uremic serum treatment.3. Measurement of NRF1, NRF2, TFAM,CO,COX,ATPsynthase gene expression3.1The effect of uremic serum on NRF1, NRF2, TFAM,CO,COX,ATPsynthase in peritoneal macrophages.After of stimulation of peritoneal macrophages by0%,10%,20%uremic serum, NRF1, NRF2, TFAM,CO,COX,ATPsynthase mRNA reduced gradually with the rise of concentration(p<0.05),The effect was maximal at20%uremic serum treatment.3.2The effect of uremic serum on NRF1, NRF2, TFAM,CO,COX,ATPsynthase in THP-1After of stimulation of THP-1by0%,10%,20%uremic serum, NRF1, NRF2, TFAM,CO,COX,ATPsynthase mRNA reduced gradually with the rise of concentration(p<0.05),The effect was maximal at20%uremic serum treatment.4. Determination of the protein expression of CO, COX and activation of AMPK4.1The effect of uremic serum on protein expression of CO、COX P-AMPK in peritoneal macrophages.After of stimulation of peritoneal macrophages by0%,10%,20%uremic serum, protein expression of CO,COX,P-AMPK reduced gradually with the rise of concentration (p<0.05),The effect was maximal at20%uremic serum treatment. The expression of AMPK have no change.4.2The effect of uremic serum on NRF1, NRF2, TFAM,CO,COX,ATPsynthase in THP-1After of stimulation of THP-1by0%,10%,20%uremic serum, protein expression of CO,COX,P-AMPK reduced gradually with the rise of concentration (p<0.05),The effect was maximal at20%uremic serum treatment. The expression of AMPK have no change.Conclusion:Uremic serum inhibit mitochondrial biogenesis in rats and humanThe three part:Studies in AICAR and AC-AMPK on activation of US-stimulate peritoneal macrophages from sham rat1. Measurement of mtDNA Copy Number1.1AICAR on mtDNA Copy Number from US-stimulate peritoneal macrophagesUremic serum inhibited mtDNA Copy Number relative to control group (P<0.05).AICAR increased mtDNA Copy Number in US-stimulate (P<0.05)1.2AC-AMPK on mtDNA Copy Number from US-stimulate peritoneal macrophagesUremic serum inhibited mtDNA Copy Number relative to control group (P<0.05).AC-AMPK increased mtDNA Copy Number in US-stimulate (P<0.05)2. Measurement of mitochondrial mass2.1AICAR on mitochondrial mass from US-stimulate peritoneal macrophagesUremic serum inhibited mitochondrial mass relative to control group (P<0.05).AICAR increased mitochondrial mass in US-stimulate (P<0.05)2.2AC-AMPK on mitochondrial mass from US-stimulate peritoneal macrophagesUremic serum inhibited mitochondrial mass relative to control group (P<0.05).AC-AMPK increased mitochondrial mass in US-stimulate (P<0.05).3. Determination of the protein expression of CO,COX3.1AICAR on the protein expression of CO,COX from US-stimulate peritoneal macrophagesUremic serum inhibited the protein expression of CO,COX relative to control group (P<0.05).AICAR increased the protein expression of CO,COX in US-stimulate (P<0.05) 3.2AC-AMPK on the protein expression of CO,COX from US-stimulate peritoneal macrophagesUremic serum inhibited the protein expression of CO,COX relative to control group (P<0.05).AICAR increased the protein expression of CO,COX in US-stimulate (P<0.05)4. Determination of the activation of AMPK4.1AICAR on the activation of AMPK from US-stimulate peritoneal macrophagesUremic serum inhibited the protein expression of P-AMPK relative to control group (P<0.05).AC-AMPK increased the protein expression of P-AMPK in US-stimulate (P<0.05). The expression of AMPK have no change.4.2AC-AMPK on the protein expression of CO,COX from US-stimulate peritoneal macrophagesUremic serum inhibited the protein expression of P-AMPK relative to control group (P<0.05).AC-AMPK increased the protein expression of P-AMPK in US-stimulate (P<0.05). The expression of AMPK have no change.Conclusion:AICAR and AC-AMPK can activate mitochondria biogenesis in macrophages; AMPK-SIRT1-PGC1αmay play an important role in mitochondria biogenesis of macrophages... |
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