ObjectiveTo assess the effect of plasmid-mediated short hairpin RNA (shRNA) on Klotho gene in mice medullary collecting duct (IMCD3) cells. MethodsThree 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. ResultsThe 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). ConclusionPlasma-mediated shRNA can highly inhibit the expression of Klotho, which suggests that it may be potential to study the pathogenesis in kidney disease.