绒毛蛋白1在单侧肾切除Habu肾炎小鼠模型中的作用

doi:10.3969/j.issn.1674-5817.2020.01.001

摘要/Abstract

摘要： 目的探讨单侧肾切除(UNX)小鼠Habu肾炎模型与正常小鼠Habu肾炎模型在肾脏功能、肾脏病理表现及肾小球蛋白绒毛蛋白1(VIL 1)表达等方面的异同及其分子机制。方法将24只6周龄SPF级雄性C57BL/6小鼠(18~20 g)随机分为2组, 实验组(n=12)接受UNX后通过尾静脉注射竹叶青蛇毒(HSV)构建Habu肾炎模型(HSV-UNX组), 对照组(n=12)接受假手术后行蛇毒尾静脉注射构建Habu肾炎模型(HSV组); 通过血液生化检测2组小鼠肾功能水平, 并采用过碘酸-雪夫染色观察2组小鼠肾组织病理改变; 进一步通过蛋白质组学对二者肾小球蛋白表达量进行分析, 筛选出2组间的表达差异蛋白, 于体外小鼠系膜细胞中研究差异蛋白对细胞增殖及凋亡的作用及机制。结果 HSV-UNX组小鼠的血肌酐和尿素氮水平较HSV组显著升高(均P<0.01),其肾组织系膜溶解水平高于HSV组(P<0.0001),系膜增殖水平低于HSV组(P<0.001); 蛋白组学分析及Western blotting证实VIL1在HSV-UNX组小鼠的表达量低于HSV组;在小鼠系膜细胞中, 敲低VIL1表达可通过上调caspase 3、Bax、 p21及 p27蛋白表达,促进细胞细胞凋亡(P<0.01),抑制细胞增殖(P<0.05)。结论 VIL1在单肾切除Habu肾炎小鼠模型中表达下调,VIL1下调可通过抑制细胞增殖和促进细胞凋亡导致肾小球自我修复能力下降, 加重肾功能损害。

关键词: 单侧肾切除(UNX), Habu肾炎, 绒毛蛋白1(VIL1), 小鼠模型

Abstract: Objective To investigate the differences of renal function, renal pathological manifestations and proteins expression between the Habu nephritis mice model with or without unilateral nephrectomy (UNX), as well as the molecular mechanism leading to these differences. Methods Twenty- four male SPF C57BL/6 mice (18-20 g) at the age of 6 weeks were randomly divided into two groups. One group (n=12) received UNX and then received tail vein injection of Habu snake venom (HSV-UNX group) after 1 week, and the other group (n=12) received sham surgery and then received tail vein injection of Habu snake venom (HSV group). The renal function of mice in the two groups was detected by blood biochemistry, and the renal histopathological changes of two groups were measured through periodinate-schiff staining. Furthermore, the expression levels of protein in glomeruli of the two groups were analyzed by comparative proteomics, and the differentially expressed proteins between the two groups were screened out. The levels of proteins, such as villin-1(IL1), caspase 3, Bax, p21 and p27, were validated by Western blotting. Results The renal function injury of the HSV-UNX group was worse than that of the HSV group (P<0.01), and the renal mesangiolysis level was higher than that of the HSV group (P<0.0001), while the mesangial proliferation was milder than that of the HSV group (P<0.001). It is confirmed by proteomic analysis and western blotting that VIL1 expression level in the HSV-UNX group was lower than that of the HSV group. In mouse mesangial cells (MMCs), cell apoptosis (P<0.01) and inhibiting cell proliferation (P<0.05) were protomed by VIL1 knockdown through up-regulating the expressions of caspase 3, Bax, p21 and p27 proteins. Conclusions VIL1 is down-regulated in the HSV-UNX mouse model, which impairs the self-repair ability of glomerular and aggravates the renal function damage by inhibiting cell proliferation and promoting cell apoptosis.

Key words: Unilateral nephrectomy (UNX), Habu nephritis, Villin-1(VIL1), Mouse model

中图分类号:

Q95-33

蒋红利, 马红叶, 薛瑾虹, 孙凌霜, 陈蕾. 绒毛蛋白1在单侧肾切除Habu肾炎小鼠模型中的作用[J]. 实验动物与比较医学, 2020, 40(1): 1-8.

JIANG Hongli, MA Hongye, XUE Jinhong, SUN Lingshuang, CHEN Lei. The Role of Villin-1 in Model of Habu Nephritis Mice with Unilateral Nephrectomy[J]. Laboratory Animal and Comparative Medicine, 2020, 40(1): 1-8.

参考文献

[1] Gao J, Wu L, Wang Y, et al.Knockdown of Cxcl10 inhibits mesangial cell proliferation in murine Habu nephritis via ERK signaling[J]. Cell Physiol Biochem, 2017, 42(5):2118-2129.
[2] Jefferson JA, Johnson RJ .Experimental mesangial proliferative glomerulonephritis (the anti-Thy- 1.1 model)[J]. J Nephrol, 1999, 12(5):297-307.
[3] Emancipator SN .Prospects and perspectives on IgA nephropathy from animal models[J]. Contrib Nephrol, 2011, 169:126-152.
[4] Haas CS, Campean V, Kuhlmann A, et al.Analysis of glomerular VEGF mRNA and protein expression in murine mesangioproliferative glomerulonephritis[J]. Virchows Arch, 2007, 450(1):81-92.
[5] Nag S, Larsson M, Robinson RC, et al.Gelsolin: The tail of a molecular gymnast[J]. Cytoskeleton, 2013, 70(7):360-384.
[6] Lu Y, Cai G, Cui S, et al.FHL2-driven molecular network mediated Septin2 knockdown inducing apoptosis in mesangial cell[J]. Proteomics, 2014, 14(21-22):2485-2597.
[7] Abe-Yoshio Y, Abe K, Miyazaki M, et al.Involvement of bone marrow-derived endothelial progenitor cells in glomerular capillary repair in Habu snake venom-induced glomerulonephritis[J]. Virchows Arch, 2008, 453(1):97-106.
[8] He F, Zhou M, Yu T, et al.Sublytic C5b-9 triggers glomerular mesangial cell apoptosis in rat Thy-1 nephritis via Gadd45 activation mediated by Egr-1 and p300-dependent ATF3 acetylation[J].J Mol Cell Biol, 2016, 8(6):477-491.
[9] Blantz RC, Steiner RW.Benign hyperfiltration after living kidney donation[J]. J Clin Invest, 2015, 125(3):972-744.
[10] Wormser C, Aronson LR.Perioperative morbidity and long-term outcome of unilateral nephrectomy in feline kidney donors: 141 cases (1998-2013)[J]. J Am Vet Med Assoc, 2016, 248(3):275-281.
[11] Rayner HC, Ward L, Walls J, et al.Cholesterol feeding following unilateral nephrectomy in the rat leads to glomerular hypertrophy[J]. Nephron, 1991, 57:453-459.
[12] Uil M, Scantlebery AML, Butter LM, et al.Combining streptozotocin and unilateral nephrectomy is an effective method for inducing experimental diabetic nephropathy in the ‘resistant' C57Bl/6J mouse strain[J]. Sci Rep, 2018, 8(1):5542.
[13] Bretscher A, Weber K .Villin is a major protein of the microvillus cystoskeleton which binds both G and F actin in a calcium-dependent manner[J]. Cell, 1980, 20(3):839-847.
[14] Roy S, Esmaeilniakooshkghazi A, Patnaik S, et al.Villin-1 and gelsolin regulate changes in actin dynamics that affect cell survival signaling pathways and intestinal inflammation[J]. Gastroenterology, 2017, 154(5):1405-1420.
[15] Xieraili M, Yasen M, Mogushi K, et al.Villin 1 is a predictive factor for the recurrence of high serum alpha-fetoprotein-associated hepatocellular carcinoma after hepatectomy[J]. Cancer Sci , 2012, 103(8):1493-1501.
[16] Wang Y, Srinivasan K, Siddiqui MR, et al.A novel role for Villin in intestinal epithelial cell survival and homeostasis[J]. J Biol Chem, 2008, 283(14):9454-9464.
[17] Nakamura E, Satoh T, Iwakawa M, et al.Villin1, a diagnostic marker for endometrial adenocarcinoma with high grade nuclear atypia.[J]. Cancer Biol Ther, 2011, 12(3):181-190.
[18] Decuypere JP, Ceulemans LJ, Wylin T, et al.Plasmatic villin 1 is a novel in vivo marker of proximal tubular cell injury during renal ischemia-reperfusion[J]. Transplantation, 2017, 101(11):1-25.
[19] Jing X, Huang WH, Tang YJ, et al.Eucommia ulmoides Oliv. (Du-Zhong) lignans inhibit angiotensin II-stimulated proliferation by affecting p21, p27, and Bax expression in rat mesangial cells[J]. Evid Based Complement Alternat Med,2015, 2015(3):1-8.
[20] Ding Y, Wang Y, Chen J, et al.p21 overexpression sensitizes osteosarcoma U2OS cells to cisplatin via evoking caspase-3 and Bax/Bcl-2 cascade[J]. Tumour Biol, 2014, 35(4):3119-3123.
[21] Wang Y, George SP, Srinivasan K, et al. Reorganization of cytoskeleton as basis for apoptosis[J]. Gastroenterology, 2008, 134(4)(suppl 1):A-70.
[22] Meng J, Vardar D, Wang Y, et al.High-resolution crystal structures of villin headpiece and mutants with reduced F-actin binding activity[J]. Biochemistry, 2005, 44(36):11963-11973.
[23] Heijden MVD, Versteilen AMG, Sipkema P, et al.Rho-kinase-dependent F-actin rearrangement is involved in the inhibition of PI3-kinase/Akt during ischemia-reperfusion-induced endothelial cell apoptosis[J]. Apoptosis, 2008, 13(3):404-412.
[24] Lockett S, Verma C, Brafman A, et al.Quantitative analysis of F-actin redistribution in astrocytoma cells treated with candidate pharmaceuticals[J]. Cytometry A, 2014, 85(6):512-521.
[25] Ye JY, Liang EY, Cheng YS, et al.Serotonin enhances megakaryopoiesis and proplatelet formation via p-Erk1/2 and F-actin reorganization.[J]. Stem Cells, 2015, 32(11):2973-2982.