Claudin-14在纳米细菌肾结石形成中表达的动态研究
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摘要
目的本实验通过尾静脉注射用肾结石患者尿液培养的纳米细菌构建肾结石模型检测肾脏组织中Claudin-14(紧密连接蛋白-14)蛋白的表达情况,初步探讨Claudin-14在纳米细菌形成结石中的作用。方法 60只Wistar雄性大鼠随机分为正常对照组和纳米细菌组,每组30只。正常对照组Wistar雄性大鼠一次性给予尾静脉注射0.9%氯化钠注射液1.2mL,纳米细菌组Wistar雄性大鼠一次性给予尾静脉注射等量的纳米细菌悬浊液。两组Wistar雄性大鼠注射后的10周内每周处死3只。每周采用病理组织学方法评估每组3只Wistar雄性大鼠成石情况,并通过实时荧光定量PCR(qRT-PCR)、免疫组化方法连续10周检测紧密连接蛋白-14(Claudin-14)mRNA和蛋白的表达。结果 (1)病理组织学检查结果:第4周以后纳米细菌组Wistar雄性大鼠肾小管内发现晶体颗粒,且主要分布于近、远曲小管,1～10周对照组Wistar雄性大鼠未见晶颗粒。(2)实时荧光定量聚合酶链式反应(qRT-PCR)和免疫组化结果:3周内Claudin-14mRNA和蛋白在纳米细菌组中几乎未见表达,从第4周起Claudin-14mRNA和蛋白在纳米细菌组中表达水平增高并且对照组明显增高,1～10周间Claudin-14mRNA和蛋白在对照组中几乎未见表达。结论 Claudin-14表达的增加可能在纳米细菌形成结石中起重要作用。
Objective To explore the role of Claudin-14 in the formation of nanobacteria renal stones.Methods A total of 60 Wistar male rats were randomly divided into normal control group and nanobacteria group,with 30 rats in either group.The rats in the control group were given a one-time intravenous injection of 1.2 mL 0.9%sodium chloride solution via the tail vein,while rats in the nanobacteria group were given one-time intravenous injection of 1.2 mL nanobacteria suspension.In the following 10 weeks,3 rats were sacrificed in both groups every week.The stone formation was evaluated with histopathological method,and the mRNA and protein expressions of Claudin-14 were detected with qRT-PCR and immunohistochemical methods.Results The histopathological results showed that,from the 4th to 10th week,crystal particles were found in the renal tubules of rats in the nanobacteria group,which were mainly distributed in the proximal and distal convoluted tubules,while no crystal granules were observed in the control group from the 1st to 10th week.The qRT-PCR and immunohistochemical results showed that Claudin-14 was not expressed in the nanobacteria group from the 1st to 3rd week,which gradually increased from the 4th to 10th week,while Claudin-14 was hardly expressed in the control group from the 1st to 10 th week.Conclusion The increased expression of Claudin-14 may play an important role in the formation of nanobacteria renal stones.
Objective To explore the role of Claudin-14 in the formation of nanobacteria renal stones.Methods A total of 60 Wistar male rats were randomly divided into normal control group and nanobacteria group,with 30 rats in either group.The rats in the control group were given a one-time intravenous injection of 1.2 mL 0.9%sodium chloride solution via the tail vein,while rats in the nanobacteria group were given one-time intravenous injection of 1.2 mL nanobacteria suspension.In the following 10 weeks,3 rats were sacrificed in both groups every week.The stone formation was evaluated with histopathological method,and the mRNA and protein expressions of Claudin-14 were detected with qRT-PCR and immunohistochemical methods.Results The histopathological results showed that,from the 4th to 10th week,crystal particles were found in the renal tubules of rats in the nanobacteria group,which were mainly distributed in the proximal and distal convoluted tubules,while no crystal granules were observed in the control group from the 1st to 10th week.The qRT-PCR and immunohistochemical results showed that Claudin-14 was not expressed in the nanobacteria group from the 1st to 3rd week,which gradually increased from the 4th to 10th week,while Claudin-14 was hardly expressed in the control group from the 1st to 10 th week.Conclusion The increased expression of Claudin-14 may play an important role in the formation of nanobacteria renal stones.
引文
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[2]汪越,张力,郝宗耀,等.纳米细菌及其诱发泌尿系结石形成的研究进展[J].临床泌尿外科杂志,2015,30(7):665-668.
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[5]孙春,黎承杨,邓耀良,等.尿钙对含钙肾结石患者尿液MCP-1、TFF1及HMGB1生成的影响[J].广东医学,2015,36(13):2017-2021.
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[8]褚浩,王勤章,吴双,等.纳米细菌大鼠肾结石模型肾脏结石形成时间的动态研究[J].中国全科医学,2017,20(21):2613-2618.
[9]安瑞华,冯陶,郭应禄,等.香豆素对实验性大鼠草酸钙结石形成的影响[J].中华泌尿外科杂志,1994,15(3):209-212.
[10]BRIKOWSKI TH,LOTAN Y,PEARLE MS.Climate-related increase in the prevalence of urolithiasis in the United States[J].Proc Nati Acad Sci U S A,2008,105(28):9841-9846.
[11]CURHAN GC.Epidemiology of stone disease[J].Urol Clin North Am,2007,34(3):287-293.
[12]吴敬彰,赵津津,张占五,等.TRPV5和OPN在泌尿系结石患者肾组织中的表达及意义[J].广东医学,2016,37(20):3101-3104.
[13]李扬,张士青.Randall斑块在草酸钙肾结石形成中的作用[J].现代泌尿外科杂志,2015,20(9):679-682.
[14]魏勃,安瑞华.肾结石危险因素的研究进展[J].现代泌尿外科杂志,2014,19(12):832-835.
[15]HU WG,WANG XF,XU T,et al.[Establishment nephrolithiasis rat model induced by nanobacteria and analysis of stone formation][J].Beijing Da Xue Xue Bao Yi Xue ban,2010,42(4):433-435.
[16]SHIEKH FA,KHULLAR M,SINGH SK.Lithogenesis:induction of renal calcifications by nanobacteria[J].Urol Res,2006,34(1):53-57.
[17]CIFTCIOGLU N,HADDAD RS,GOLDEN DC,et al.A potential cause for kidney stone formation during space flights:enhanced growth of nanobacteria in microgravity[J].Kid Int,2005,67(2):483-491.
[18]刘志立.单侧上尿路结石患者双侧肾盂尿纳米细菌的检测及意义[D].石河子大学,2015:1-40.
[19]粟宏伟,王杰,朱永生,等.钙化性纳米微粒致大鼠肾结石模型的构建[J].重庆医学,2016(3):310-312.
[20]KAJANDER EO,CIFTCIOGLU N,AHO K,et al.Characteristics of nanobacteria and their possible role in stone formation[J].Urol Res,2003,31(2):47-54.
[21]ELKOUBY-NAOR L,ABASSI Z,LAGZIEL A,et al.Double gene deletion reveals lack of cooperation between claudin 11and claudin 14tight junction proteins[J].Cell Tissue Res,2008,333(3):427-438.
[22]HOU J,GOODENOUGH DA.Claudin-16and claudin-19function in the thick ascending limb[J].Curr Opin Nephrol Hypertens,2010,19(5):483-488.
[23]HOU J,RENIGUNTA A,KONRAD M,et al.Claudin-16and claudin-19interact and form a cation-selective tight junction complex[J].J Clin Invest,2008,118(2):619-628.
[24]KONRAD M,SCHALLER A,SEELOW D,et al.Mutations in the tight-junction gene claudin 19(CLDN19)are associated with renal magnesium wasting,renal failure,and severe ocular involvement[J].Am J Hum Genet,2006,79(5):949-957.
[25]THORLEIFSSON G,HOLM H,EDVARDSSON V,et al.Sequence variants in the CLDN14gene associate with kidney stones and bone mineral density[J].Nat Genet,2009,41(8):926-930.
[2]汪越,张力,郝宗耀,等.纳米细菌及其诱发泌尿系结石形成的研究进展[J].临床泌尿外科杂志,2015,30(7):665-668.
[3]SHIEKH FA,MILLER VM,LIESKE JC.Do calcifying nanoparticles promote nephrolithiasis?a review of the evidence[J].Clin Nephrol,2009,71(1):1-8.
[4]ROBERTSON WG,PEACOCK M,HEYBURN PJ,et al.Risk factors in calcium stone disease of the urinary tract[J].Br J Urol,1978,50(7):449-454.
[5]孙春,黎承杨,邓耀良,等.尿钙对含钙肾结石患者尿液MCP-1、TFF1及HMGB1生成的影响[J].广东医学,2015,36(13):2017-2021.
[6]DIMKE H,DESAI P,BOROVAC J,et al.Activation of the Ca(2+)-sensing receptor increases renal claudin-14expression and urinary Ca(2+)excretion[J].Am J physiol Renal physiol,2013,304(6):F761-769.
[7]孙稳,王勤章,丁国富,等.钙敏感受体及紧密连接蛋白-14的表达对肾草酸钙结石形成的影响[J].医学研究生学报,2014,27(11):1143-1147.
[8]褚浩,王勤章,吴双,等.纳米细菌大鼠肾结石模型肾脏结石形成时间的动态研究[J].中国全科医学,2017,20(21):2613-2618.
[9]安瑞华,冯陶,郭应禄,等.香豆素对实验性大鼠草酸钙结石形成的影响[J].中华泌尿外科杂志,1994,15(3):209-212.
[10]BRIKOWSKI TH,LOTAN Y,PEARLE MS.Climate-related increase in the prevalence of urolithiasis in the United States[J].Proc Nati Acad Sci U S A,2008,105(28):9841-9846.
[11]CURHAN GC.Epidemiology of stone disease[J].Urol Clin North Am,2007,34(3):287-293.
[12]吴敬彰,赵津津,张占五,等.TRPV5和OPN在泌尿系结石患者肾组织中的表达及意义[J].广东医学,2016,37(20):3101-3104.
[13]李扬,张士青.Randall斑块在草酸钙肾结石形成中的作用[J].现代泌尿外科杂志,2015,20(9):679-682.
[14]魏勃,安瑞华.肾结石危险因素的研究进展[J].现代泌尿外科杂志,2014,19(12):832-835.
[15]HU WG,WANG XF,XU T,et al.[Establishment nephrolithiasis rat model induced by nanobacteria and analysis of stone formation][J].Beijing Da Xue Xue Bao Yi Xue ban,2010,42(4):433-435.
[16]SHIEKH FA,KHULLAR M,SINGH SK.Lithogenesis:induction of renal calcifications by nanobacteria[J].Urol Res,2006,34(1):53-57.
[17]CIFTCIOGLU N,HADDAD RS,GOLDEN DC,et al.A potential cause for kidney stone formation during space flights:enhanced growth of nanobacteria in microgravity[J].Kid Int,2005,67(2):483-491.
[18]刘志立.单侧上尿路结石患者双侧肾盂尿纳米细菌的检测及意义[D].石河子大学,2015:1-40.
[19]粟宏伟,王杰,朱永生,等.钙化性纳米微粒致大鼠肾结石模型的构建[J].重庆医学,2016(3):310-312.
[20]KAJANDER EO,CIFTCIOGLU N,AHO K,et al.Characteristics of nanobacteria and their possible role in stone formation[J].Urol Res,2003,31(2):47-54.
[21]ELKOUBY-NAOR L,ABASSI Z,LAGZIEL A,et al.Double gene deletion reveals lack of cooperation between claudin 11and claudin 14tight junction proteins[J].Cell Tissue Res,2008,333(3):427-438.
[22]HOU J,GOODENOUGH DA.Claudin-16and claudin-19function in the thick ascending limb[J].Curr Opin Nephrol Hypertens,2010,19(5):483-488.
[23]HOU J,RENIGUNTA A,KONRAD M,et al.Claudin-16and claudin-19interact and form a cation-selective tight junction complex[J].J Clin Invest,2008,118(2):619-628.
[24]KONRAD M,SCHALLER A,SEELOW D,et al.Mutations in the tight-junction gene claudin 19(CLDN19)are associated with renal magnesium wasting,renal failure,and severe ocular involvement[J].Am J Hum Genet,2006,79(5):949-957.
[25]THORLEIFSSON G,HOLM H,EDVARDSSON V,et al.Sequence variants in the CLDN14gene associate with kidney stones and bone mineral density[J].Nat Genet,2009,41(8):926-930.
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