Zhang Shouhua, Liu Queling, Xiao Juhua, Lei Jun, Liu Yi, Xu Hongyan, Hong Zhengdong
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.
Department of Oncology, the Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi Province, China.
Int Immunopharmacol. 2016 May;34:32-36. doi: 10.1016/j.intimp.2016.01.026. Epub 2016 Feb 23.
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.
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.
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).
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.
精密切割肾片（PCKS）模型似乎是一种有用的肾纤维化体外模型。然而，对PCKS模型的深入分子研究较少。因此，本研究的目的是调查和验证该模型的分子有效性。
在雄性C57BL/6小鼠中构建PCKS模型。为诱导肾纤维化，将PCKS在重组人转化生长因子-β1中孵育48小时。使用蛋白质印迹法测量磷酸化Smad2（p-Smad2，胞质和核）、Smad7、磷酸化ERK1（p-ERK1）、磷酸化ERK2（p-ERK2）和磷酸化p38丝裂原活化蛋白激酶（p-p38 MAPK）的蛋白表达。为评估Smad2/3异源复合物的形成和磷酸化Smad3（p-Smad3）的表达，在蛋白质印迹之前分别用抗Smad2或抗Smad3抗体进行免疫沉淀。
相对于对照组，PCKS模型中p-Smad2和p-Smad3显著上调（p<0.05）。然而，我们发现PCKS模型与对照组之间Smad7表达无显著差异（p>0.05）。相对于对照组，PCKS模型显示出显著更强的Smad2/3复合物形成和核转位（p<0.05）。相对于对照组，PCKS模型显示p-ERK1、p-ERK2和p-p38 MAPK的表达显著更高（p<0.05）。
PCKS模型显示出几种已确立的肾纤维化分子标志物。然而，相对于已确立的肾纤维化体内单侧输尿管梗阻模型，PCKS模型未能显示Smad7下调，似乎显示出p-Smad2和p-Smad3的“过度激活”以及ERK1/2和p38 MAPK信号的“激活不足”。
