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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Abstract Full text Figures/Tables Video References Cited by

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.	唐榮, 周巧玲, 舒金勇, 等. 冬蟲夏草提取液對腎小管上皮細胞Klotho表達和凋亡的影響[J]. 中南大學學報:醫學版, 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, 黃堅, 肖政亮, 等. 家兔腎臟內髓集合管細胞的分離與原代培養[J]. 中國應用生理學雜志, 2000, 16(1):92-95.
10.	夏前明, 錢桂生. 腎臟內髓集合管細胞培養及其應用的研究進展[J]. 成都軍區醫院學報, 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. 唐榮, 周巧玲, 舒金勇, 等. 冬蟲夏草提取液對腎小管上皮細胞Klotho表達和凋亡的影響[J]. 中南大學學報:醫學版, 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, 黃堅, 肖政亮, 等. 家兔腎臟內髓集合管細胞的分離與原代培養[J]. 中國應用生理學雜志, 2000, 16(1):92-95.
10. 夏前明, 錢桂生. 腎臟內髓集合管細胞培養及其應用的研究進展[J]. 成都軍區醫院學報, 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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West China Medical Journal

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

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