METTL10表达下调与足细胞损伤的关系

doi:10.3969/j.issn.1674-8115.2021.10.003

摘要/Abstract

摘要：

目的·分析足细胞损伤中的差异基因表达，并验证其中关键基因METTL10的蛋白表达。方法·从NCBI_GBO数据库下载足细胞损伤相关RNA芯片数据集（GSE108629），包括正常足细胞组（n=4）和损伤足细胞组（n=4）；采用limma 软件包和affy软件包进行2组间差异表达基因分析，以基因表达倍数变化（fold change，FC）对数的绝对值| log FC |≥2且校正P值（q值）<0.05为筛选条件。收集于上海交通大学医学院附属仁济医院进行肾组织活检并诊断为局灶节段性肾小球硬化或者微小病变的大量蛋白尿患者，以肾组织活检结果为轻微病变或者良性肾小动脉硬化症的患者为对照组；收集患者的基本资料和临床资料，利用免疫荧光染色检测患者肾脏病理标本中METTL10蛋白表达水平。BAL/BC小鼠随机分为2组：阿霉素（adriamycin，ADR）处理组按10 mg/kg尾静脉注射ADR构建小鼠肾病模型（n=6）；对照组等体积注射磷酸盐缓冲液（n=6）。利用代谢笼收集小鼠的24 h尿液，分别检测注射前和注射14 d时2组小鼠尿白蛋白肌酐比值（urinary albumin creatinine ratio，UACR）；在第15日处死小鼠，收集小鼠肾脏组织标本，通过蛋白印迹法检测ADR诱导小鼠肾病模型中METTL10和足细胞标志物包括synaptopodin、nephrin、podocin和Wilms’ tumor 1 （WT1）的表达水平。结果·在损伤足细胞中发现5 353个差异表达基因（3 402个显著上调，1 951个显著下调），METTL10是其中一个显著下调的基因。免疫荧光检测结果显示METTL10在人正常肾小球组织中表达，并且METTL10与足细胞标志物podocalyxin存在共定位；与对照组相比，METTL10在大量蛋白尿患者肾小球组织中表达明显下降。与对照组相比，注射14 d时ADR处理组小鼠UACR明显升高（P=0.005）；蛋白印迹法检测结果显示，与对照组小鼠相比，ADR处理组小鼠肾脏中METTL10表达下调（P=0.001），同时伴随足细胞标志物synaptopodin、nephrin、podocin和WT1表达下降，差异均具有统计学意义（对应P值分别为0.001、0.005、0.000和0.006）。结论·METTL10表达下调可能与足细胞损伤有关。

关键词: 足细胞损伤, 蛋白尿, METTL10, 差异表达基因

Abstract:

Objective·To identify the differentially expressed genes (DEGs) in podocyte injury and validate the expression of METTL10, one of these critical genes.

Methods·The microarray expression profile of a podocyte injury model, dataset GSE108629, was downloaded from the GEO database, containing 4 normal podocyte samples and 4 injured podocyte samples, and the DEGs were diagnosed using the limma software package and affy software package (|log FC| ≥ 2 and adjusted P value<0.05). Patients with significant proteinuria who underwent renal biopsy and were diagnosed as focal segmental glomerulosclerosis or minimal change disease were retrospectively enrolled. Control group was patients who underwent renal biopsy and were proven as slight pathological changes or benign arteriolar neophrosclerosis. The general and clinical data were collected, and the expression of METTL10 in renal tissue was detected by immunofluorescence staining. Adriamycin (ADR)-induced nephropathy mouse model was established by a single intravenous injection of ADR into BAL/BC mouse. Mice were randomly divided into two groups: control group (treated with the same volume of PBS) and ADR group (treated with ADR 10 mg/kg body weight). On Day 0 and Day 14, urine was collected for 24 h using a metabolic cage and urinary albumin creatinine ratio (UACR) of mice were detected; on Day 15, mice were sacrificed and kidney tissue was collected. The expression of METTL10 and podocyte marker proteins like synaptopodin, nephrin, podocin, and Wilms' tumor 1 (WT1) in kidneys from ADR-treated mouse model was analyzed by Western blotting.

Results·A total of 5 353 genes were significantly differentially expressed in injured podocytes compared with intact podocytes, among which 3 402 DEGs were up-regulated and 1 951 DEGs were down-regulated. METTL10 was found to be one of those that overe significantly down-regulated. Immunofluorescence staining indicated that METTL10 was expressed in normal human glomeruli and METTL10 was colocalized with podocalyxin, one of podocyte specific markers. Additionally, METTL10 was low expressed in the renal biopsy specimens from patients with significant proteinuria compared with that in the control group. On Day 14 after administration of ADR, mice developed significant proteinuria, shown by UACR detection (P=0.005); Western blotting revealed that METTL10 was significantly down-regulated (P=0.001), along with the significantly decreased expression of synaptopodin (P=0.001), nephrin (P=0.005), podocin (P=0.000) and WT1 (P=0.006) in ADR-treated mice compared with that in normal mice. The differences of the expression of METTL10 and the above podocyte markers between these two groups were statistically significant.

Conclusion·The down-regulation of METTL10 may be involved in podocyte injury.

Key words: podocyte injury, proteinuria, METTL10, differentially expressed genes (DEGs)

中图分类号:

R692

包继文, 李子扬, 尧欢珍, 吴蓓, 周文彦, 顾乐怡, 王玲. METTL10表达下调与足细胞损伤的关系[J]. 上海交通大学学报(医学版), 2021, 41(10): 1290-1296.

Ji-wen BAO, Zi-yang LI, Huan-zhen YAO, Bei WU, Wen-yan ZHOU, Le-yi GU, Ling WANG. Relation of down-regulation of METTL10 and podocyte injury[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(10): 1290-1296.

图/表 8

图1 足细胞损伤中的DEGs火山图

Fig 1 Identification of DEGs in injured podocytes shown by the volcanoplot

图2 足细胞损伤中前50个差异表达基因聚类热图Note： Red areas represent high expressed genes, and blue areas represent low expressed genes of damaged podocytes compared with those of normal podocytes.

Fig 2 Heatmap for the top50 of DEGs in injured podocytes

图3 METTL10 mRNA在足细胞损伤中的表达变化

Fig 3 Changes of METTL10 mRNA level in damaged podocytes

表1 大量蛋白尿组和对照组的人口学及基线资料

Tab 1 Demographic and baseline characteristics of control group and patients with massive proteinuria

Item	Control group （n=3）	Significant proteinuria group（n=3）
Age/year	41.67±10.73	31.00±2.52
Gender/n
Male	2	2
Female	1	1
Blood pressure/mmHg
Systolic	125.70±9.39	126.70±10.04
Diastolic	80.33±5.04	82.67±3.71
sCr/（μmol·L^-1）	109.00±24.21	67.80±6.90
eGFR/［mL·（min·1.73m²）^-1］	71.00±19.86	113.70±0.88
Urine protein for 24 h/g	0.41±0.21	7.17±2.42^①

图4 免疫荧光染色检测患者肾小球组织中METTL10的表达(×400)Note：The arrow indicates colocalization of METTL10 (green) and podocalyxin (red) in podocytes.

Fig 4 Immunofluorescence staining for detection of METTL10 expression in glomerula (×400)

图5 2组小鼠UACR的变化

Fig 5 Change of UACR in the two groups

图6 小鼠肾脏组织足细胞标志物蛋白的表达 (n=6)Note：A. Immunoblotting analysis of the expression of podocyte markers. B. Immuno-quantification of podocyte markers.

Fig 6 Expression of podocyte markers in kidney tissue of mice (n=6)

图7 小鼠肾脏组织中METTL10蛋白表达（n=6）Note：A. Western blotting. B. Comparison of METTL10 expression between the two groups.

Fig 7 Expression of METTL10 protein in kidney tissue of mice (n=6)

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