Urine samples of patients with renal stones and biopsy tissue samples from patients with nephrocalcinosis were tested for miR-93-5p expression. A renal stone mouse model was established with intraperitoneal injection of glyoxylic acid, during which mice were treated with PUFAs and/or an miR-93-5p inhibitor adenovirus. Periodic acid-Schiff staining, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling staining, oil red O staining, triacylglycerol assay, and colorimetry testing were performed to assess glycogen deposition, apoptosis, lipid accumulation, blood urea nitrogen, and serum creatinine levels, respectively. Renal proximal tubular epithelial cells (human kidney 2 [HK-2]) were subjected to gain- and loss-of-function assays before calcium-oxalate monohydrate (COM) induction and PUFA treatment. Cell counting kit 8, flow cytometry, and lactate dehydrogenase activity assays were used to examine cell viability, apoptosis, and damage. A luciferase reporter gene assay verified the interaction between miR-93-5p and Pknox1, and miR-93-5p and Pknox1 levels were assessed using a reverse transcription-quantitative polymerase chain reaction and Western blot analysis.
miR-93-5p was downregulated in clinical samples with renal stones and negatively targeted Pknox1. PUFAs increased miR-93-5p expression and reduced apoptosis, glycogen deposition, and lipid accumulation in mice with renal stones, which were annulled by miR-93-5p downregulation. PUFAs increased proliferation and diminished apoptosis, lipid accumulation, and lactate dehydrogenase activity in COM-induced HK-2 cells, which were negated by miR-93-5p inhibition. Pknox1 overexpression reversed the effect of miR-93-5p upregulation on COM-induced HK-2 cells.
PUFAs repressed renal stone-induced renal tubular damage via the miR-93-5p/Pknox1 axis.
方法:检测肾结石患者的尿液样本和肾钙质沉着患者的活检组织样本的miR-93-5p表达。腹腔注射乙醛酸建立肾结石小鼠模型,在此期间用PUFAs和/或miR-93-5p抑制剂腺病毒治疗小鼠。周期性酸性希夫染色,末端脱氧核苷酸转移酶脱氧尿苷三磷酸缺口末端标记染色,油红O染色,三酰甘油测定,进行比色法测试以评估糖原沉积,凋亡,脂质积累,血尿素氮,和血清肌酐水平,分别。在草酸钙一水合物(COM)诱导和PUFA处理之前,对肾近端肾小管上皮细胞(人肾2[HK-2])进行功能增益和功能丧失测定。细胞计数试剂盒8,流式细胞术,和乳酸脱氢酶活性测定用于检查细胞活力,凋亡,和损坏。荧光素酶报告基因测定验证了miR-93-5p和Pknoxl之间的相互作用,并且使用逆转录定量聚合酶链反应和Western印迹分析评估了miR-93-5p和Pknoxl水平。
结果:miR-93-5p在肾结石和阴性靶向Pknox1的临床样本中下调。PUFAs增加miR-93-5p表达并减少细胞凋亡,糖原沉积,和肾结石小鼠的脂质积累,通过miR-93-5p下调而无效。PUFA增加增殖和减少凋亡,脂质积累,和COM诱导的HK-2细胞中的乳酸脱氢酶活性,通过miR-93-5p抑制而否定。Pknox1过表达逆转了miR-93-5p上调对COM诱导的HK-2细胞的影响。
结论:PUFA通过miR-93-5p/Pknox1轴抑制肾结石诱导的肾小管损伤。