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. 2021 Mar 10;22(6):2809. doi: 10.3390/ijms22062809

Figure 1.

Figure 1

Danegaptide prevents TGFβ1-evoked increases in hemichannel-mediated dye uptake. In panel (A), human kidney 2 (HK2) cells were cultured in low glucose (5 mM) ± TGFβ1 (10 ng/mL) ± danegaptide (50, 100 and 1000 nM) for 48 h and cell viability assessed. Results are presented as the mean ± SEM (n = 3). Incubation with TGFβ1 (10 ng/mL ± danegaptide (50–1000 nM)) did not alter 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake, lactate dehydrogenase (LDH) release or crystal violet (CV) staining. In panels (B) and (C), carboxyfluorescein dye uptake was used to assess hemichannel activity in HK2 cells and human proximal tubule epithelial cells (hPTECs), with the degree of dye loading being directly proportional to opening. Cells were cultured in low glucose (5 mM) ± TGFβ1 (10 ng/mL) ± danegaptide (50 or 100 nM) for 48 h. Danegaptide prevented TGFβ1-evoked increases in carboxyfluorescein dye uptake in HK2 cells (panel (B)) and hPTECs (panel (C)). Minimal dye loading occurred in control cells, whilst dye loading significantly increased in cells treated with TGFβ1. Addition of danegaptide (50 or 100 nM) reduced dye uptake, returning the fluorescence intensity to control levels. Intensity is expressed as a percentage compared to low-glucose controls and is representative of 3 separate experiments. Data are presented as the mean ± SEM (n = 3), with key significances indicated (** p < 0.01, *** p < 0.001; one-way ANOVA and Tukey’s post-test).