Functional analysis of senescence related Klotho gene and its application for the therapy

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Klotho expression plasmid was produced by inserting Klotho cDNA into pCAGGS vector, and the production of Klotho protein was confirmed by western blotting after transfection into the cells. We incubated HUVEC cells with the medium taken from COS-1 cells into which Klotho plasmid was transacted., and it was revealed that Klotho has anti-oxidative effect from the result that SOD activity was activated, and the expression of MnSOD was enhainced. To confirm this anti-oxidative effect in vivo, we infused klotho plasmid into mouse tail vein. Klotho protein was produced in Liver and kidney, and it was found that SOD activity was activated, LPO concentration was reduced in both Liver and kidney.We investigated the effect of Klotho protein on blood pressure. Klotho plasmid was infused into the animal model of hypertension, SHR and SHR-SP, and the blood pressure was measured after 6weeks, 12 weeks, however, no effect was found on blood pressure. In spite of that, SOD activity was activated, and LPO concentration was reduced as same as normal mouse. Moreover this anti-oxidative effect was reduced when L-NAME, inhibitor of NO synthase, was administrated which shows that anti-oxidative effect by Klotho is partly through the NO pathway.We measured intracellular cAMP concentration and PKC activity by incubating with medium containing Klotho protein on various cells to investigate the signaling pathway of Klotho. The result was that cAMP concentration was high in most cells, however, PKC activity was only increased in testis and kidney cells where Klotho gene is expressed. It was revealed that Klotho signaling pathways are different depends on its gene expression.1 alpha-OHase, vitamin D activating enzyme, gene expression was suppressed when Klotho plasmid was inserted into COS-1 cell, which shower the relation of Klotho with Ca metabolism.