Hyperglycemia and diabetes patient-derived serum do not alter Klotho production in TECs in vitro . (A, B) Representative immunofluorescence images of TECs stained for Klotho and corresponding scatterplots produced by quantitative TissueFAXS analysis. (A) Negative control staining (primary antibody omitted) with corresponding scatterplot. (B) Immunofluorescence image of TECs cultured in the presence of 30 mM glucose, stained for Klotho, with corresponding scatterplot. (C) The total number of TECs in vitro was significantly lower when cells were incubated with medium containing 30 mM glucose compared with lower glucose concentrations. However, there was no effect of hyperglycemic culture conditions on the percentage of Klotho+ cells (D) or the Klotho staining intensity (E). In addition, serum derived from diabetic subjects did not affect the total number of cells (F), percentage of Klotho+ cells (G), the Klotho staining intensity (H) or the relative level of Klotho mRNA expression (I) compared with serum derived from healthy control subjects. (J) No difference in renal Klotho mRNA expression was observed between hyperglycemic Ins2Akita and normoglycemic control mice. However, a significant reduction in renal Klotho mRNA expression was detected in 8 months old mice compared with 3 months old mice, independent of the presence of diabetes. Data are expressed as scatterplots with the horizontal bar representing the median; *p < 0.05.