Figure 2.

mRNA and total protein levels of Klotho were not downregulated in immortalized human podocytes that were cultured under high glucose (HG) conditions, but the levels of the membrane form of Klotho protein decreased under HG conditions, together with an increase in soluble Klotho shedding. (A) The mRNA expression of Klotho was detected in immortalized human podocytes. (B) The mRNA expression of Klotho was measured under standard glucose (SG; 11 mM) and high glucose (HG; 30 mM, 5 days) conditions, which did not significantly differ (p = 0.96, Mann–Whitney test, n = 20–21). (C) The total protein expression of Klotho did not differ between podocytes that were cultured under standard glucose and high glucose conditions (p = 0.48, unpaired t-test, n = 14). (D) A significant decrease in the levels of the membrane form of Klotho protein was detected in podocytes under HG conditions (* p = 0.013, vs. SG, Mann–Whitney test, n = 5–6). (E) The immunofluorescent labeling of human podocytes for Klotho (red) confirmed the decrease in membrane-bound Klotho protein levels under HG conditions. These results were explained by (F) a significant increase in the levels of the soluble form of Klotho under HG conditions (** p = 0.007, vs. SG, Mann–Whitney test, n = 8), that was cut off from the cell membrane and shed extracellularly. bp, base pairs; MW, molecular weight.