| 摘要: |
| 目的 考察肾组织PHD锌指蛋白14(PHF14)的表达变化,探讨PHF14在急性损伤后肾纤维化中的作用。方法 选用IVC级C57BL/6小鼠20只,随机分为2组:对照组(n=5)和叶酸刺激实验组(n=15),实验组又分为叶酸作用后2、14和28 d 3个亚组,每个亚组5只小鼠。实验组单次给予叶酸腹腔注射(250 mg/kg,溶媒为0.3 mol/L碳酸氢钠溶液),对照组单次给予溶媒腹腔注射。每组小鼠在预定的时间点处死,留取血清检测肌酐(SCr)和尿素氮(BUN);留取肾组织,行Masson染色验证模型,蛋白质免疫印迹法研究PHF14在急性肾损伤纤维化进程中的表达变化。利用PHF14基因敲除鼠,制作与上述相同的急性肾损伤模型,比较PHF14敲除组和未敲除组肾脏在第2、14和28天的病理变化,并行α-平滑肌肌动蛋白(α-SMA)和collagen 1免疫组织化学染色,蛋白质免疫印迹法检测转化生长因子β(TGF-β)、α-SMA、collagen 1的表达规律。结果 与对照组相比,实验组各时间点小鼠SCr与BUN变化曲线提示急性肾损伤病程;肾脏病理变化显示,随叶酸作用时间的增加,肾脏逐渐进展到纤维化,提示模型制作成功。在急性肾损伤至慢性纤维化过程中,PHF14持续高表达。与野生型小鼠相比,PHF14敲除小鼠TGF-β、α-SMA、collagen 1表达较高,肾纤维化程度更重。结论 叶酸腹腔注射可成功建立急性肾损伤及损伤后肾小管间质纤维化模型,PHF14的表达在叶酸促纤维化刺激后持续上调,而PHF14敲除会加重急性损伤后肾纤维化的发生,提示PHF14对肾脏纤维化进程起抑制作用,可能是内源性肾纤维化保护因子。 |
| 关键词: 慢性肾功能不全 急性肾损伤 PHD锌指蛋白14 纤维化 |
| DOI：10.16781/j.0258-879x.2016.04.0429 |
| 投稿时间：2015-11-28修订日期：2016-03-19 |
| 基金项目:国家自然科学基金面上项目(81570621);中华肾脏病学会科研基金(13030340419);"十二五"国家科技支撑计划项目(2011BAI10B08). |
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| PHF14 knockout promotes renal fibrosis following acute kidney injury in mice |
| YANG Bo,CHEN Si-xiu,MAO Zhi-guo* |
(Department of Nephrology, Changzheng Hospital, Kidney Institute of PLA, Second Military Medical University, Shanghai 200003, China
*Corresponding author) |
| Abstract: |
| Objective To study the regulation of PHF14 expression in the process of acute kidney injury (AKI) and renal fibrosis, so as to evaluate the role of PHF14 in the renal fibrosis after AKI. Methods Twenty adult C57BL/6 mice were randomly divided into two groups:vehicle group (n=5) and folic acid group (n=15). Mice were given intraperitoneal injection of vehicle (0.3 mol/L NaHCO3 solution) alone (control group) or together with folic acid (folic acid group) at the dose of 250 mg/kg. Mice in folic acid group were further divided into three subgroups according to the length of follow-up (2 day, 14 day and 28 day, respectively). Mice were sacrificed at the planned time points and the kidney tissues and blood samples were collected for evaluating the renal function and the renal fibrosis parameters; Masson's trichrome staining was used to verify AKI model; and Western blotting analysis was used to study the expression profile of PHF14. After the verification of renal fibrosis model, the induced PHF14 knockout mice and wild type mice were given folic acid or vehicle and were studied following the same protocol mentioned above. Western blotting analysis was performed to examine the expression profile of transforming growth factor β (TGF-β),α-smooth muscle actin (α-SMA) and collagen 1 at the predesigned time points. Immunohistochemical staining was performed with anti-α-SMA and anti-collagen 1 antibodies. Results The folic acid nephropathy model was verified by serum creatinine (SCr), blood urine nitrogen (BUN) and renal fibrosis morphologic analysis in folic acid group. Pathological changes of the kidney showed gradual renal fibrosis with the increase of folic acid exposure time, indicating the successful establishment of AKI model. PHF14 expression was elevated persistently during the process of fibrogenesis after folic acid-induced injury. Compared with wild type mice, Western blotting analysis and immunohistochemical staining showed that PHF14 knockout mice had higher expression of TGF-β, α-SMA and collagen 1. PHF14 knockout significantly exacerbated the interstitial inflammation and fibrosis in renal tissue in mice. Conclusion Intraperitoneal folic acid injection can create AKI and renal fibrosis in mice. PHF14 expression is persistently elevated after the pro-fibrotic insults. Knock-out of PHF14 gene can exacerbate the renal fibrosis following AKI, indicating that PHF14 might be a protective factor of renal fibrosis. |
| Key words: chronic renal insufficiency acute kidney injury PHD finger protein 14 fibrosis |
