Effect of Plasmid-mediated Short Hairpin RNA on the Klotho Gene in Medullary Collecting Duct Cells in Mice_West China Medical Journal

Authors：

 CaiYingying ¹ , WangHan ² ,  DengJuelin ³

1. Department of Geriatrics, the First Affiliated Hospital of Chengdu Medical College, Chengdu, Sichuan 610500, P. R. China;
2. Department of Cardiovascular Medicine, the Third People'Hospital of Chengdu, Chengdu, Sichuan 610001, P. R. China;
3. Department of Geriatrics, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;

Corresponding author：

DengJuelin, Email: juelind2012@163.com

Keywords：

Klotho; Aging; Plasmid-mediated short hairpin RNA; Kidney

DOI：

10.7507/1002-0179.201600329

Video：

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Objective To assess the effect of plasmid-mediated short hairpin RNA (shRNA) on Klotho gene in mice medullary collecting duct (IMCD3) cells. Methods Three pairs of shRNA for Klotho (the first, second, and third pairs of shRNA) were designed and pRNAU6-Klotho were constructed, which were transfected into IMCD3 cells by Lipofactine2000. The negative control group and untreated group were set up at the same time. After 24 hours, the expressions of Klotho mRNA and protein levels were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Results The second pairs of shRNA had the best interference effect compared with the control group according to RT-PCR (P<0.01). The results of Western blotting showed that the Klotho protein levels in the second pairs of shRNA group differed much from all the other 4 groups (P<0.001). Conclusion Plasma-mediated shRNA can highly inhibit the expression of Klotho, which suggests that it may be potential to study the pathogenesis in kidney disease.

Citation： CaiYingying, WangHan, DengJuelin. Effect of Plasmid-mediated Short Hairpin RNA on the Klotho Gene in Medullary Collecting Duct Cells in Mice. West China Medical Journal, 2016, 31(7): 1205-1208. doi: 10.7507/1002-0179.201600329 Copy

1.	Kuro-o M, Matsumura Y, Aizawa H, et al. Mutation of the mouse klotho gene leads to a syndrome resembling ageing[J]. Nature, 1997, 390(6655):45-51.
2.	唐荣, 周巧玲, 舒金勇, 等. 冬虫夏草提取液对肾小管上皮细胞Klotho表达和凋亡的影响[J]. 中南大学学报:医学版, 2009, 34(4):300-307.
3.	Xiao L, Naganawa T, Lorenzo J, et al. Nuclear isoforms of fibroblast growth factor 2 are novel inducers of hypophosphatemia via modulation of FGF23 and KLOTHO[J]. J Biol Chem, 2010, 285(4):2834-2846.
4.	Fire A, Xu SQ, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature, 1998, 391(6669):806-811.
5.	Hammond M. Dicing and slicing:the core machinery of the RNA interference pathway[J]. FEBS Lett, 2005, 579(26):5822-5829.
6.	Perico N, Remuzzi G, Benigni A. Aging and the kidney[J]. Curr Opin Nephrol Hypertens, 2011, 20(3):312-317.
7.	Clapp WL, Madsen KM, Verlander JW, et al. Morphologic heterogeneity along the rat inner medullary collecting duct[J]. Lab Invest, 1989, 60(2):219-230.
8.	Rauchman MI, Nigam SK, Delpire E, et al. An osmotically tolerant inner medullary collecting duct cell line from an SV40 transgenic mouse[J]. Am J Physiol, 1993, 265(3 Pt 2):F416-F424.
9.	夏前明, 黄坚, 肖政亮, 等. 家兔肾脏内髓集合管细胞的分离与原代培养[J]. 中国应用生理学杂志, 2000, 16(1):92-95.
10.	夏前明, 钱桂生. 肾脏内髓集合管细胞培养及其应用的研究进展[J]. 成都军区医院学报, 2000, 2(4):26-29.
11.	Oz OK, Hajibeigi A, Howard K, et al. Aromatase deficiency causes altered expression of molecules critical for calcium reabsorption in the kidneys of female mice[J]. J Bone Miner Res, 2007, 22(12):1893-1902.
12.	Koh N, Fujimori T, Nishiguchi S, et al. Severely reduced production of klotho in human chronic renal failure kidney[J]. Biochem Biophys Res Commun, 2001, 280(4):1015-1020.
13.	Aizawa H, Saito Y, Nakamura T, et al. Downregulation of the klotho gene in the kidney under sustained circulatory stress in rats[J]. Biochem Biophys Res Commun, 1998, 249(3):865-871.
14.	Manya H, Inomata M, Fujimori T, et al. Klotho protein deficiency leads to overactivation of mu-calpain[J]. J Biol Chem, 2002, 277(38):35503-35508.
15.	Takeshita K, Yamamoto K, Ito M, et al. Increased expression of plasminogen activator inhibitor-1 with fibrin deposition in a murine model of aging, "Klotho" mouse[J]. Semin Thromb Hemost, 2002, 28(6):545-554.
16.	Ishizaka N, Mitani H, Nagai R. Angiotensin Ⅱ regulates klotho gene expression[J]. Nippon Rinsho, 2002, 60(10):1935-1939.
17.	Utsugi T, Ohno T, Ohyama Y, et al. Decreased insulin production and increased insulin sensitivity in the Klotho mutant mouse, a novel animal model for human aging[J]. Metabolism, 2000, 49(9):1118-1123.

1. Kuro-o M, Matsumura Y, Aizawa H, et al. Mutation of the mouse klotho gene leads to a syndrome resembling ageing[J]. Nature, 1997, 390(6655):45-51.
2. 唐荣, 周巧玲, 舒金勇, 等. 冬虫夏草提取液对肾小管上皮细胞Klotho表达和凋亡的影响[J]. 中南大学学报:医学版, 2009, 34(4):300-307.
3. Xiao L, Naganawa T, Lorenzo J, et al. Nuclear isoforms of fibroblast growth factor 2 are novel inducers of hypophosphatemia via modulation of FGF23 and KLOTHO[J]. J Biol Chem, 2010, 285(4):2834-2846.
4. Fire A, Xu SQ, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans[J]. Nature, 1998, 391(6669):806-811.
5. Hammond M. Dicing and slicing:the core machinery of the RNA interference pathway[J]. FEBS Lett, 2005, 579(26):5822-5829.
6. Perico N, Remuzzi G, Benigni A. Aging and the kidney[J]. Curr Opin Nephrol Hypertens, 2011, 20(3):312-317.
7. Clapp WL, Madsen KM, Verlander JW, et al. Morphologic heterogeneity along the rat inner medullary collecting duct[J]. Lab Invest, 1989, 60(2):219-230.
8. Rauchman MI, Nigam SK, Delpire E, et al. An osmotically tolerant inner medullary collecting duct cell line from an SV40 transgenic mouse[J]. Am J Physiol, 1993, 265(3 Pt 2):F416-F424.
9. 夏前明, 黄坚, 肖政亮, 等. 家兔肾脏内髓集合管细胞的分离与原代培养[J]. 中国应用生理学杂志, 2000, 16(1):92-95.
10. 夏前明, 钱桂生. 肾脏内髓集合管细胞培养及其应用的研究进展[J]. 成都军区医院学报, 2000, 2(4):26-29.
11. Oz OK, Hajibeigi A, Howard K, et al. Aromatase deficiency causes altered expression of molecules critical for calcium reabsorption in the kidneys of female mice[J]. J Bone Miner Res, 2007, 22(12):1893-1902.
12. Koh N, Fujimori T, Nishiguchi S, et al. Severely reduced production of klotho in human chronic renal failure kidney[J]. Biochem Biophys Res Commun, 2001, 280(4):1015-1020.
13. Aizawa H, Saito Y, Nakamura T, et al. Downregulation of the klotho gene in the kidney under sustained circulatory stress in rats[J]. Biochem Biophys Res Commun, 1998, 249(3):865-871.
14. Manya H, Inomata M, Fujimori T, et al. Klotho protein deficiency leads to overactivation of mu-calpain[J]. J Biol Chem, 2002, 277(38):35503-35508.
15. Takeshita K, Yamamoto K, Ito M, et al. Increased expression of plasminogen activator inhibitor-1 with fibrin deposition in a murine model of aging, "Klotho" mouse[J]. Semin Thromb Hemost, 2002, 28(6):545-554.
16. Ishizaka N, Mitani H, Nagai R. Angiotensin Ⅱ regulates klotho gene expression[J]. Nippon Rinsho, 2002, 60(10):1935-1939.
17. Utsugi T, Ohno T, Ohyama Y, et al. Decreased insulin production and increased insulin sensitivity in the Klotho mutant mouse, a novel animal model for human aging[J]. Metabolism, 2000, 49(9):1118-1123.

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Effect of Plasmid-mediated Short Hairpin RNA on the Klotho Gene in Medullary Collecting Duct Cells in Mice

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