Supplementary MaterialsS1 Fig: Augmenting effect of high glucose on AGT mRNA in HK-2 cells, with the threshold effective concentration of 8 mM, could not be affected by valsartan. used to compare values between two groups and one-way ANOVA followed by the Tukeys test was used to compare values among more than two groups. = 0.069) (Fig 1B). In the additional experiment, we also found that 8 mM, the marginal level of postprandial blood glucose in healthy subjects, is the threshold glucose concentration that can stimulate increased AGT (S1A Fig). To exclude the influence of osmotic stress, mannitol was used to equalize the total osmotic tension, that could become induced by both mannitol and blood sugar, in each combined group. Needlessly to say, mannitol treatment for 48 h didn’t impact AGT mRNA amounts in HK-2 cells (Fig 1C). Consequently, the result of high blood sugar on AGT mRNA level had not been because of osmotic tension. Taken collectively, these data recommended that high blood sugar straight augmented AGT mRNA degrees of HK-2 cells inside a period- and dose-dependent way. Open in another home window Fig 1 Large blood sugar augments AGT mRNA level in HK-2 cells inside a period- and dose-dependent way.(A) AGT mRNA levels measured at different period points in HK-2 cells respectively treated with regular glucose (5.5 mM) and high blood sugar (15.0 mM). Weighed against regular blood sugar treatment, high blood sugar considerably augmented AGT mRNA level from 39 h to 63 h. (B) AGT mRNA levels measured in HK-2 cells respectively treated with different glucose concentrations for 48 h. Compared purchase PRI-724 with normal glucose treatment, high glucose augmented AGT mRNA level with the most significant effects by 15.0 or 20.0 mM glucose. (C) The effect of high glucose on AGT mRNA level was not due to osmotic stress which was further balanced with mannitol treatment for 48 h. Data are expressed as relative values to the 0h group (A) or normal glucose group (B and C). Values are presented as mean SEM. *studies conducted in our lab have shown that the augmentation of intrarenal AGT was inhibited by treatment with an Ang II receptor blocker (ARB) in diabetes [4, 8]. Additional experiments were performed to examine the effect of an ARB (valsartan) on high glucose-induced augmentation of AGT in HK2 cells. We used valsartan at a concentration of 10 M, as previously described [20, 21]. However, high glucose-induced augmentation of AGT was not affected by treatment with valsartan (S1B Fig). We further investigated the effects of high glucose purchase PRI-724 on AGT secretion into the cell culture medium. Compared with normal glucose, high Rabbit Polyclonal to ZAR1 glucose (15.0 mM) treatment for 48 h significantly augmented secreted AGT levels (40.812.69 = 0.68) (Fig 4D). Different from the mutation of HNF-5 binding sites, mutations in the binding sites of CREB (588 vs. 1066, relative ratios to empty vector transfection group under the same glucose purchase PRI-724 concentration; = 0.10) (Fig 4G). Similar data were observed for AGT secretion levels. Compared with normal glucose (5.5 mM), high glucose (15 purchase PRI-724 mM) treatment for 48 h significantly augmented AGT protein levels in the medium of HK-2 cells transfected with either empty vector (35.380.83 vs. 75.236.54 ng/mg total protein; = 0.12) (Fig 4H). Open in a separate window Fig 4 Mutation in HNF-5 binding sites reduces the effects of high glucose on AGT promoter activity as well as AGT mRNA and secretion levels.(A, B and C) Constructs with respective mutation of HNF-5 (A), CREB (B) or MEF2 (C) binding sites in main human AGT promoter sequences (AGT_-4,358/+122). Within binding sites, the underlined base pairs were substituted by mutagenesis. Horizontal line represents human AGT_-4,358/+122 with relevant mutation. Open box represents the luciferase reporter. (D, E and F) Effects of high glucose treatment on AGT promoter activity in HK-2 cells, which were transfected with either intact or relevant binding sites mutated construct of human AGT_-4,358/+122. Compared with purchase PRI-724 normal glucose (5.5 mM) treatment, high glucose (15.0 mM) significantly augmented AGT promoter activity of HK-2 cells transfected with the intact construct of human AGT_-4,358/+122 (D, F) and E. Furthermore, the above mentioned impact was abolished in HK-2 cells with mutated HNF-5 binding sites (D), without suffering from the binding sites mutation of CREB (E) or.