| Part Ⅰ The role of PA-induced inflammation in podocyte injuries of obesity-related glomerulopathyObjectiveTo investigate the effects of palmitic acid(PA)on glomerular podocytes and identify the potential role of PA-induced inflammation in obesity-related glomerulopathy(ORG).Methods1 MPC5 mouse podocytes were treated with different concentrations(50,100,150,200,250,300μM)of PA for 24 hrs or 48 hrs.CCK-8 was used to determine the cell viability of podocytes.To assess lipid deposition in MPC5 cells,cells treated with different concentrations(50,100,150 μM)of PA for 24 hrs were stained with Oil Red O dye or detected using a boron-dipyrromethene-based fluorescent lipid probe.Apoptosis was detected by flow cytometry using Annexin V-FITC/PI kit.Quantitative reverse transcription polymerase chain reaction(qRT-PCR)and Western blot were used to determine mRNA and protein levels of the MPC5 podocyte markers Nephrin and Podocin,and podocyte injury associated protein Desmin.2 MPC5 cells were treated with 150μM PA for 24 hrs,after which mRNA and protein levels of the NLRP3 inflammasome markers NLRP3,caspase-1,and ASC were detected by qRT-PCR and Western blot.Levels of the inflammatory cytokines IL-1β and IL-18 were detected by ELISA.The levels of reactive oxygen species(ROS)were determined using mitoSOX fluorescence staining.Results1 The viability of MPC5 cells was reduced treated by PA in a dose-dependent and timedependent manner.The deposition of intracellular lipid and apoptosis were gradually elevated with increased PA concentrations.Compared to the control group,the expression of the podocyte markers Nephrin and Podocin were decreased at both the mRNA and protein level in PA group,and expression of the podocyte injury related protein Desmin were significantly increased at both the mRNA and protein level.2 Compared to the control group,the expression levels of NLRP3 inflammasome markers in podocytes were upregulated in PA group.Secretion of the inflammatory factors IL-1β and IL-18 and the production of ROS were also increased in PA group.ConclusionPA can activate NLRP3 inflammasome,promote the secretion of the inflammatory factors IL-1β and IL-18,promote the production of ROS,and induce podocyte injury.Part Ⅱ Adiponectin improves palmitic acid-induced podocyte injuryObjectiveTo investigate the protective effects of adiponectin on podocyte injury induced by palmitic acid(PA).Methods1 In vitro experiments:MPC5 podocytes were treated either with PA alone,PA combined with different concentrations of adiponectin(1,2,4,8μg/mL)or PA after transfection with adiponectin siRNA.CCK-8 was used to determine the cell viability of podocytes.Flow cytometry using Annexin V-FITC/PI kit was used to evaluate cell apoptosis.Quantitative reverse transcription polymerase chain reaction(qRT-PCR)and Western blot were used to assess the expression levels of Nephrin,Podocin,Desmin and the NLRP3 inflammasome markers NLRP3,Caspase-1 and ASC.Expression levels of the inflammatory factors IL-1β and IL-18 were detected by ELISA.The levels of reactive oxygen species(ROS)were observed by mitoSOX fluorescence staining.2 In vivo experiments:A mouse model of obesity-related glomerulopathy was established.Twenty-four male mice were divided into ordinary diet(n=6),ordinary diet+globular adiponectin(gAd)injections(n=6),high-fat diet(HFD,n=6),or high-fat diet+gAd injections(n=6).Serum samples were collected to measure levels of fasting blood glucose,low density lipoprotein cholesterol,high density lipoprotein cholesterol,triglyceride,total cholesterol,and serum creatinine.Urine samples were collected to detect urinary microalbumin and creatinine and calculate ACR.H&E staining was used to observe the pathological changes of kidney and electron microscope was used to observe the ultrastructure changes of kidney under different feeding conditions,and immunofluorescence staining was used to analyze expression levels of Podocin and NLRP3 in the kidney tissue.Results1 In vitro experiments:Increased concentration of exogenous adiponectin supplementation(1,2,4,8 μg/mL)can reduce podocyte injury and ameliorate intracellular inflammation in the presence of PA,as demonstrated by increased podocyte activity,reduced podocyte apoptosis,up-regulated expressions of MPC5 podocyte markers(Nephrin and Podocin),down-regulated expressions of the podocyte injury related protein Desmin and NLRP3 inflammasome markers(NLRP3,ASC and caspase-1),down-regulated expressions of the inflammatory factors IL-1β and IL-18,as well as decreased ROS production,compared to cells treated with PA alone.Inhibition of the expression of adiponectin in podocytes via siRNA can increase levels of podocyte injury and intracellular inflammation,as demonstrated by reduced podocyte activity,increased podocyte apoptosis,down-regulated expressions of MPC5 podocyte marker proteins(Nephrin and Podocin),up-regulated expressions of the podocyte injury associated protein Desmin and NLRP3 inflammasome markers(NLRP3,caspase-1 and ASC),up-regulated expression of the inflammatory factors IL-1β and IL-18 and increased production of ROS,compared to treatment with siRNA control.PA further exacerbated the effects of adiponectin inhibition on MPC5 podocytes,and these effects could be reversed by the addition of adiponectin.2 In vivo experiments:The podocyte diameter(assessed by light microscope)of mice fed with a HFD was significantly larger than that of mice fed with ordinary chow.Mice fed with a HFD also showed decreased podocyte density(assessed by electron microscope),increased urinary ACR,and podocyte fusion.Compared with mice fed with ordinary chow,mice fed with a HFD had higher levels of body weights,fasting blood glucose,low-density lipoprotein cholesterol,triglycerides,total cholesterol,serum creatinine and urinary ACR.The concentration of HDL cholesterol in HFD fed mice was significantly lower than that of mice fed with ordinary chow.However,in mice receiving HFD+gAd injections,the above changes induced by HFD alone were reversed.The expression level of serum adiponectin in mice fed with high-fat diet was lower than that in mice fed with ordinary chow.Immunofluorescence staining showed that compared with HFD fed mice,mice receiving HFD+gAd injections showed increased expression of Podocin and decreased expression of NLRP3.These results suggested that adiponectin might perform a protective effect against renal injury in vivo induced by HFD.ConclusionAdiponectin can protect against podocyte injury induced by palmitic acid and HFD.Part Ⅲ Adiponectin downregulates the NF-κB/NLRP3 inflammasome signaling pathway and improves podocyte injuryObjectiveTo investigate whether adiponectin protects against ORG podocyte injuries by inhibiting the NF-κB/NLRP3 inflammasome signaling pathway.Methods1 Podocytes were transfected with or without adiponectin siRNA,and then treated with PA either alone or in combination with the NLRP3 inhibitor MCC950(100 nM)(control group,PA group and PA+ MCC950 group).The cell viability of podocytes was evaluated by CCK-8.qRT-PCR and Western blot were used to measure expression levels of the podocyte marker proteins(Nephrin and Podocin),the podocyte injury associated protein Desmin,NLRP3 inflammasome markers(NLRP3,caspase-1 and ASC)and NF-κB.The level of apoptosis of podocytes in each group was detected by flow cytometry.Expression levels of the inflammatory cytokines IL-18 and IL-1β were detected by ELISA.The levels of ROS in MPC5 podocytes were detected by mitoSOX fluorescence staining.2 Podocytes were treated with PA,and then treated with either adiponectin alone or in combination with the NF-κB inhibitor PDTC:control group,PA group,PA+gAd group,PDTC group,PDTC+PA group and PDTC+PA+gAd group.The expression levels of the podocyte marker proteins(Nephrin and Podocin),the podocyte injury related protein Desmin,NLRP3 inflammasome markers(NLRP3,caspase-1 and ASC)and NF-κB were determined by Western blot.Results1 NLRP3 inhibitor MCC950 reduced MPC5 podocyte injury and intracellular oxidative stress,and improved PA or adiponectin siRNA-induced podocyte injury,as demonstrated by increased podocyte activity,decreased podocyte apoptosis,up-regulated expression of the podocyte marker proteins(Nephrin and Podocin),down-regulated expression of podocyte injury related protein Desmin,downregulated expressions of the NLRP3 inflammasome markers(NLRP3,caspase-1 and ASC)and the inflammatory factors IL-1β,and IL-18,as well as decreased ROS production,compared to cells treatment with PA or adiponectin siRNA alone.2 PDTC significantly reduced the expression of p-NF-κB in the presence of PA,inhibited the reverse of adiponectin on the decreased expression level of Nephrin and Podocin by PA,on the increased expression level of Desmin by PA,and on the NLRP3 inflammasome activation by PA.ConclusionAdiponectin can reduce podocyte inflammatory responses,improve podocyte injuries and reduce levels of podocyte oxidative stress by inhibiting the NF-κB/NLRP3 inflammasome signaling pathway,suggesting adiponectin may be useful for the prevention and treatment of ORG. |
PDF Full Text Request