title: Molecular validation of the precision-cut kidney slice (PCKS) model of renal fibrosis through assessment of TGF-β1-induced Smad and p38/ERK signaling.
abstract: BACKGROUND
  The precision-cut kidney slice (PCKS) model appears to be a useful ex vivo model of renal fibrosis. However, little in-depth molecular investigation on the PCKS model has been performed. Therefore, the aim of this study will be to investigate and validate the molecular validity of this model.
  
  
  METHODS
  The PCKS model was constructed in male C57BL/6 mice. To induce renal fibrosis, PCKS were incubated in recombinant human TGF-β1 for 48 h. Protein expression of phosphorylated Smad2 (p-Smad2, cytosolic and nuclear), Smad7, phosphorylated ERK1 (p-ERK1), phosphorylated ERK2 (p-ERK2), and phosphorylated p38 MAPK (p-p38 MAPK) was measured using Western blotting. To assess Smad2/3 heteromeric complex formation and phosphorylated Smad3 (p-Smad3) expression, immunoprecipitation was performed with an anti-Smad2 or an anti-Smad3 antibody, respectively, prior to Western blotting.
  
  
  RESULTS
  p-Smad2 and p-Smad3 were significantly upregulated in the PCKS model relative to control (p<0.05). However, we found no significant difference in Smad7 expression between the PCKS model and control (p>0.05). The PCKS model demonstrated significantly greater Smad2/3 complex formation and nuclear translocation relative to control (p<0.05). The PCKS model showed significantly greater expression of p-ERK1, p-ERK2, and p-p38 MAPK relative to control (p<0.05).
  
  
  CONCLUSIONS
  The PCKS model displays several well-established molecular markers of renal fibrosis. However, the PCKS model failed to display Smad7 downregulation and appears to display "over-activation" of p-Smad2 and p-Smad3 as well as "under-activation" of ERK1/2 and p38 MAPK signaling vis-à-vis the well-established in vivo unilateral ureteric obstruction model of renal fibrosis.
authors.affiliation: Department of General Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi Province, China; Department of General Surgery, Jiangxi Children's Hospital, Nanchang, Jiangxi Province, China.
authors.forename: Shouhua
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Department of Oncology, the Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi Province, China.
authors.forename: Queling
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Department of Ultrasound, Jiangxi Provincial Maternal and Child Health Hospital, Nanchang, Jiangxi Province, China.
authors.forename: Juhua
authors.initials: J
authors.lastname: Xiao
authors.affiliation: Department of General Surgery, Jiangxi Children's Hospital, Nanchang, Jiangxi Province, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Lei
authors.affiliation: Department of Urology, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi Province, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of General Surgery, Jiangxi Children's Hospital, Nanchang, Jiangxi Province, China.
authors.forename: Hongyan
authors.initials: H
authors.lastname: Xu
authors.affiliation: Department of Urology, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi Province, China. Electronic address: ndefy01@126.com.
authors.forename: Zhengdong
authors.initials: Z
authors.lastname: Hong
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D002478:Cells, Cultured; D004195:Disease Models, Animal; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005355:Fibrosis; D006801:Humans; D007668:Kidney / Q000378:metabolism / Q000473:pathology*; D007674:Kidney Diseases / Q000175:diagnosis*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009924:Organ Culture Techniques; D015398:Signal Transduction; D051899:Smad2 Protein / Q000235:genetics / Q000378:metabolism*; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000276:immunology; D015854:Up-Regulation; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D053773:Transforming Growth Factor beta1; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases
keywords: ERK; Precision-cut kidney slice, PCKS; Renal fibrosis; Smad, Smad2, Smad3, Smad7; p38