| Part One: Effect of calcitriol on angiotensinⅡ-induced renal tubulointerstitial inflammation Objective:To explore the effect of calcitriol on tubulointerstitial inflammation and NF-κB pathway in angiotensinⅡ-induced chronic renal injury.Methods:1.Healthy male C57BL/6 mice aged 7-8 weeks were were randomly assigned to control group(n=5),Ang Ⅱ group(n=5),Ang Ⅱ +calcitriol group(n=5).All C57BL/6 mice underwent uninephrectomy surgery(left)and allowed to recover for 1 week.Then all mice were subcutateously implanted with osmotic micropumps The mice were treated with subcutenous 0.9% sterile saline infusion,subcutenous AngⅡ infusion and subcutenous Ang Ⅱ infusion combined with calcitriol intragastric administration respectively for 14 days.2.Effect of calcitriol on urine albumin/creatinine ratio,serum BUN,serum Cr and pathologic changes in tubulointerstitium were assessed.Infiltration by CD3(+)lymphocytes and F4/80(+)macrophages was assessed through immunohistochemistry.3.Effect of calcitriol on m RNA and protein expression of TNF-α and IL-6 was determined by q PCR and Western Blot.4.Effect of calcitriol on IKKβ、IκBα、p65 was assessed by Western Blot and immunohistochemistry.Results:1.Pathological change of chronic kidney injury was observed in the right kidney tissue of Ang Ⅱ-infused mice.Calcitriol alleviated tubulointerstitial injury,reduced inflammatory cell infiltration and improved urine albumin/creatinine ratio,serum BUN,serum Cr.2.Administration of calcitriol reduced AngⅡ-induced m RNA and protein of TNF-α and IL-6 in right kidneys.3.Less phosphorylated IKKβ and phosphorylated IκBα were observed in calcitriol-treated mice kidney than in AngⅡ group.In AngⅡ+calcitriol group less translocated p65 in nucleus leads to lower ratio of nuclear p65/cytoplasmic p65.4.Immunohistochemical results: in control group,p65 was sited in cytoplasm of renal epithelial cells;in AngⅡ+vehicle group,almost all p65 was presented in nucleus;in AngⅡ+calcitriol group,p65 was sequestered in cytoplasm in a portion of renal epithelial cells.Conclusions:1.Calcitriol ameliorated Ang Ⅱ-inducded renal tubulointerstitial inflammation.2.The suppression of calcitriol on tubulointerstitial inflammation was through inhibiting NF-κB signalling.Part Two:Effect of calcitriol on A20 expression in kidney of angiotensinⅡ-induced CKD mice Objective:Zinc finger protein A20 is a potent anti-inflammatiory regulator which has been shown to exert protective effects in inflammation,autoimmune diseases.Although A20 has been widely studied in acute organ injury such as acute kidney injury(AKI),its role in chronic kidney disease(CKD)is less reported.Therefore,we aimed to explore the effect of calcitriol on A20 expression in an angiotenisnⅡ-induced chronic kidney injury model.Methods:1.Healthy male C57BL/6 mice aged 7-8 weeks were randomly assigned to control group(n=5),AngⅡgroup and AngⅡ+calcitriol group(n=10).All mice underwent uninephrectomy surgery(left)and allowed to recover for 1 week.Then all mice were subcutateously implanted with osmotic micropumps.The mice were treated with subcutenous 0.9% sterile saline infusion,subcutenous Ang Ⅱ infusion and subcutenous Ang Ⅱ infusion combined with calcitriol intragastric administration respectively for 14 days.2.Expressions of A20 in the right kidney were determined by q PCR,Western Blot and immunohistochemistry.Results:1.A20 was mainly presented in tubular epithelial cells.In control and AngⅡ groups A20 m RNA and protein expression level was low.2.In AngⅡ+calcitirol group q PCR and Western Blot results showed that A20 expression was elevated significantly compared with control and AngⅡ groups.Conclusions:1.Administration of angiotensinⅡ after 14 days had no significant impact on A20 expression in renal tubular epithelia.2.In angiotensinⅡ-induced renal injury,administration of calcitriol could upregulate A20 expression level in renal tubular epithelial cells.Part Three: Calcitriol suppressed inflammatory response in rat renal tubular epithelial cells partly by stably upregulating A20 Objective:Calcitriol suppressed NF-κB activation in chronic Ang Ⅱ-induced tubulointerstial injury,which was accompanied by upregulated A20 in vivo.Considering that A20 is a key regulator in NF-κB pathway due to its dual roles in ubiquitination and deubiquitination,it needs elucidating whether A20 participates in calcitriol’s suppression on AngⅡ-induced inflammatory response in renal tubular epithelial cells.Methods:1.Wild rat tubular epithelial cells NRK-52 E were treated with calcitriol alone to choose the optimal concentration and best time point for subsequent experiments.Protein levels of A20 and m RNA of TNF-α and IL-6 were determined.2.Lentivirus containing sh RNA targeting A20 was purchased from Shanghai Genechem Co.,Ltd.A20-knockdown rat tubular epithelial cells were developed by transfecting lentivirus containing sh RNA into wildtype epithelial cells.Knockdown efficacy was assessed by q PCR and Western Blot analysis.sh CTRL and A20-knockdown NRK-52E(sh A20)were pretreated with calcitriol at 10-7M then stimulated with Ang Ⅱ for the following 24 hours.A20 expression was determined with q PCR、Western Blot.3.NRK-52 E cells with negative control lentivirus(sh CTRL)and A20-targeting lentivirus(sh A20)were divided into control groups,sh CTRL+AngⅡgroup,sh CTRL+AngⅡ+calcitriol group,sh A20+AngⅡ+ calcitriol group.Cells in each group were pretreated with vehicle or 10-7M calcitriol and then stimulated with 10-6M AngⅡfor 24 hours.Expressions of TNF-α and IL-6 in sh CTRL and sh A20 were assessed by q PCR,ELISA.4.In control group,sh CTRL+AngⅡgroup,sh CTRL+AngⅡ+calcitriol group and sh A20+AngⅡ+ calcitriol,NF-κB signaling was assessed by Western Blot analysis in sh CTRL and sh A20 cells.5.In control,sh CTRL+AngⅡgroup,sh CTRL+AngⅡ+calcitriol group,sh A20+AngⅡ+ calcitriol,A20 expression,p65 nucleus translocation was detected by laser confocal microscopy.Results:1.A20 increased in concentration-dependent and time-dependent manners.Calcitriol above 10-7M could not further induce A20 expression.The m RNA expression levels of TNF-α and IL-6 at 24 hour was still high at 24 hour.Thus,10-7M calcitriol was selected to treat cells for 24 hours.2.A20-knockdown NRK-52 E with above 70% knock-down efficacy was developed.3.After 24 h AngⅡ stimulation,expressions of TNF-α and IL-6 were elevated.Calcitriol reduced TNF-α and IL-6.However,the expressions of TNF-α and IL-6 in sh A20 cells were significantly higher than those in sh CTRL cells.4.After 24-hour Ang Ⅱ stimulation,phosphorylated IKKβ and phosphorylated IκBα and more translocated p65 in nucleus were significantly higher than those in the control group.Calcitriol reduced phosphorylated IKKβ and phosphorylated IκBα and sequestered p65 in cytoplasm.However,more phosphorylated IKKβ and phosphorylated IκBα and nuclear p65 were observed in sh A20+AngⅡ+calcitriol group than in sh CTRL +AngⅡ+calcitriol group.5.Immunofluorescent results: after 24 h treatment in control group,p65 was presented in cytoplasm,A20 expression level was low;in sh CTRL+AngⅡ group,most p65 was observed in nucleus,but A20 expression level was still low;in sh CTRL+ AngⅡ+calcitriol group,calcitriol upregulated A20 and sequestered p65 in cytoplasm;in sh A20+ AngⅡ+calcitriol group,more p65 was observed in nucleus.Conclusions:1.After 24 h treatment,calcitriol stably upregulated A20.2.A20 participated in calcitriol’s suppression on Ang Ⅱ-induced NF-κB activation and downregulated downstream TNF-α and IL-6.3 Deficiency of A20 did not abrogate the renoprotection of calctiriol,but diminished calctiriol’s protective effects.Part Four: Effect and mechanism of calcitriol on angiotensinⅡ-induced EMT in NRK-52 E cell Objective:Ang Ⅱ not only induces inflammatory response in renal tubular epithelial cells but also is a potent EMT inducer.Studies show that Wnt/β-catenin signaling may be involved in AngⅡ-induced EMT.In this part,we explored whether calcitriol can protect NRK-52 E cells from AngⅡ-induced EMT and whether Wnt/β-catenin signaling is engaged.Methods:1.NRK-52 E cells were treated with AngⅡat different concentrations or for different durations.Vimentin and E-cadherin were determined by q PCR and Western Blot.2.Pyrvinium pamoate was used to block Wnt/β-catenin signaling.Cyclin D1,c-myc,p-serine9-GSK3β,p-β-catenin and β-catenin were determined by Western Blot to check whether Wnt/β-catenin is involved in AngⅡ-induced EMT.EMT markers such as vimentin and E-cadherin were also assessed by q PCR 、Western Blot.3.AngⅡ AT1 R blocker losartan and AT2 R blocker PD123319 were used to investigate by which receptor Ang Ⅱ induces EMT through activating Wnt/β-catenin pathway Cyclin D1,c-myc,p-serine9-GSK3β,p-β-catenin,β-catenin,Vimentin,E-cadherin were evaluated by q PCR,Western Blot.4.NRK-52 E cells were pretreated with 10-7M calcitriol and then challenged with 10-6M Ang Ⅱ for 72 hours.Vimentin 、 E-cadherin and Wnt/β-catenin signaling activation were determined by q PCR,Western Blot.Vimentin,E-cadherin and β-catenin were also detected by immunofluorescence.Results:1.Vimentin expression increased in Ang Ⅱconcentration-dependent and time-dependent manners,whereas E-cadherin expression decreased in AngⅡconcentration-dependent and time-dependent manners2.Phosphorylation of GSK3β at 9-serine promoted by AngⅡimpaired phosphorylation of β-catenin resulting in increase of active β-catenin.Pyrvinium pamoate blocked Wnt/β-catenin signaling.No significant differences were observed in c-myc 、 cyclin D1,p-serine9-GSK3β 、p-β-catenin and β-catenin between Ang Ⅱ +Pyr group and control group.However,vimentin expression was higher in AngⅡ+Pyr group than that in control group.E-cadherin expression was lower in AngⅡ+Pyr group than that in control group.3.After treatment with losartan,PD123319,vimentin and E-cadherin expressions in AngⅡ+losartan group were not significantly different from those in control group.No significant differences were observed in p-serine9-GSK3β 、 p-β-catenin expression between Ang Ⅱ+losartan and control group.expressions in Ang Ⅱ +PD123319 group were not significantly different from those in Ang Ⅱ group.No significant differences were observed in vimentin,E-cadherin p-serine9-GSK3β and p-β-catenin expression between Ang Ⅱ +PD123319 group and Ang Ⅱ group.4.Calcitriol decreased Ang Ⅱ-induced vimentin and restored E-cadherin expression.p-serine9-GSK3β was also decrease by calcitriol treatment,which led to dowregulation of β-catenin.Immunofluorescence results confirmed Western Blot results.Downregulation of β-catenin by calcitriol resulting in less β-catenin translocation into nucleus was also observed by immunofluorescence.Conclusions:1.Ang Ⅱ promoted EMT in concentration-dependent and time-dependent manners.2.Wnt/β-catenin signaling was involved in AngⅡ-induce EMT.3.AT1 R mediated AngⅡ-induced EMT.Blocking AT2 R probably had no impact on AngⅡ-induced EMT.4.Calciriol suppressed Ang Ⅱ-induced EMT by disrupting Wnt/β-catenin signaling. |
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