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| 米扎格列净通过抑制钠-葡萄糖共转运体1的功能抑制常染色体显性多囊肾细胞增殖和纤维化 |
| 刘雯瑜1,2,吴双成1,张天琛3,付莉莉1,解良瑜1,胡菀芊1,郁胜强1* |
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(1. 海军军医大学(第二军医大学)第二附属医院肾脏病科, 上海 200003;
2. 中国人民解放军联勤保障部队北戴河康复疗养中心肾脏病科, 秦皇岛 066000;
3. 海军军医大学(第二军医大学)第一附属医院病理科, 上海 200433
*通信作者) |
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| 摘要: |
| 目的 探究钠-葡萄糖共转运体1（SGLT1）抑制剂米扎格列净（MIZA）在常染色体显性多囊肾病（ADPKD）中的作用。方法 用蛋白质印迹法、qPCR、免疫荧光染色测定PKD1-/-小鼠和PKD1+/+小鼠肾脏组织、人肾癌旁组织和人ADPKD组织中SGLT1的表达和分布。用MIZA处理囊肿衬里上皮细胞OX161和肾小管上皮细胞UCL93，37 ℃孵育24、48和72 h后通过MTT实验和集落形成实验观察细胞增殖情况。以100 μmol/L MIZA处理OX161细胞48 h后，通过qPCR测定细胞中α1-Ⅰ型胶原蛋白、α1-Ⅲ型胶原蛋白和纤连蛋白1 的mRNA表达量。用犬肾细胞MDCK 3D囊肿形成实验验证MIZA对囊肿形成的作用。通过mRNA-seq数据分析筛选UCL93细胞和OX161细胞、OX161细胞和100 μmol/L MIZA处理48 h后的OX161细胞的差异表达基因，利用京都基因与基因组百科全书（KEGG）数据库进行通路富集分析。结果 SGLT1在ADPKD患者和PKD1-/-小鼠多囊肾组织中的表达水平较正常肾脏组织升高（P＜0.05，P＜0.01），免疫荧光染色发现SGLT1主要表达在囊肿衬里上皮细胞。在体外实验中，MIZA呈浓度和时间依赖性地抑制多囊肾细胞的增殖和纤维化，3D形成实验表明MIZA抑制了囊肿的形成。mRNA-seq数据分析和KEGG富集分析结果显示，OX161细胞和100 μmol/L MIZA处理48 h的OX161细胞的差异表达基因主要富集在PI3K-Akt、MAPK等信号通路，与OX161细胞和UCL93细胞的差异表达基因富集通路相同。结论 SGLT1抑制剂MIZA可能通过PI3K-Akt、MAPK等通路抑制多囊肾细胞的增殖和纤维化，延缓多囊肾的生长，是ADPKD的一个潜在治疗靶点。 |
| 关键词: 常染色体显性多囊肾病 钠-葡萄糖共转运体1 细胞增殖 纤维化 磷脂酰肌醇3-激酶 蛋白激酶B 丝裂原活化蛋白激酶 信号通路 |
| DOI：10.16781/j.CN31-2187/R.20240144 |
| 投稿时间：2024-02-28修订日期：2024-07-01 |
| 基金项目: |
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| Mizagliflozin inhibits proliferation and fibrosis of autosomal dominant polycystic kidney cells by inhibiting function of sodium-glucose cotransporter 1 |
| LIU Wenyu1,2,WU Shuangcheng1,ZHANG Tianchen3,FU Lili1,XIE Liangyu1,HU Wanqian1,YU Shengqiang1* |
(1. Department of Nephrology, The Second Affiliated Hospital of Naval Medical University (Second Military Medical University), Shanghai 200003, China;
2. Department of Nephrology, Beidaihe Rest and Recuperation Center, Joint Logistics Support Force of PLA, Qinhuangdao 066000, Hebei, China;
3. Department of Pathology, The First Affiliated Hospital of Naval Medical University (Second Military Medical University), Shanghai 200433, China
*Corresponding author) |
| Abstract: |
| Objective To investigate the role of sodium-glucose cotransporter 1 (SGLT1) inhibitor mizagliflozin (MIZA) in autosomal dominant polycystic kidney disease (ADPKD). Methods Western blotting, quantitative polymerase chain reaction (qPCR), and immunofluorescence staining were used to determine the expression and distribution of SGLT1 in kidney tissues of PKD1-/- and PKD1+/+ mice, human renal cancer adjacent tissue and ADPKD tissue. Renal cyst lining epithelial cells OX161 and renal tubular epithelial cells UCL93 were treated with MIZA, incubated at 37 ℃ for 24, 48, and 72 h, and then were subjected to methyl thiazolyl tetrazolium and colony formation assay to observe cell proliferation. The qPCR method was used to determine the mRNA levels of collagen 1α1, collagen 3α1, and fibronectin 1 in OX161 cells treated with 100 μmol/L MIZA for 48 h. The Madin-Darby canine kidney (MDCK) cell 3D cyst formation assay verified the effect of MIZA on cyst formation. The mRNA-seq technology was used to detect differentially expressed genes between UCL93 cells and OX161 cells, and between OX161 cells and OX161 cells treated with 100 μmol/L MIZA for 48 h, and then the differentially expressed genes were analyzed with Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Results The expression level of SGLT1 was significantly increased in the tissues of ADPKD patients and PKD1-/- mice compared to those in normal kidney tissues (P＜0.05, P＜0.01). Immunofluorescence staining revealed that SGLT1 was mainly expressed in the cystic lining epithelial cells. Additionally, MIZA inhibited the proliferation and fibrosis of polycystic kidney cells in a concentration- and time-dependent manner, and also inhibited cyst formation in 3D formation assay in vitro. The mRNA-seq analysis and KEGG enrichment analysis showed that differentially expressed genes between OX161 cells and OX161 cells cultured in 100 μmol/L MIZA for 48 h were mainly enriched in the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) signaling pathways, which were the same as those between OX161 cells and UCL93 cells. Conclusion The SGLT1 inhibitor MIZA may inhibit the proliferation and fibrosis of polycystic kidney cells through signaling pathways such as PI3K-Akt and MAPK, delaying the growth of polycystic kidney, and it is a potential therapeutic target for ADPKD. |
| Key words: autosomal dominant polycystic kidney disease sodium-glucose cotransporter 1 cell proliferation fibrosis phosphatidylinositol 3-kinase protein kinase B mitogen-activated protein kinase signaling pathway |
