Figure 5. High glucose and oxidative stress induce TRα1 reexpression and podocyte dedifferentiation in vitro that are reversed by T₃.

(A) Representative images of TRα1 (red) and F-actin (green) staining in human podocytes. Both high levels of glucose and H₂O₂ induced TRα1 overexpression and partial translocation from the nucleus to cytoplasm and increased cell size (top). T₃ treatment of stimulated podocytes decreased TRα1 expression and restored its nuclear and perinuclear localization, and reduced cell size, but had no effect on control cells (bottom). (B) A representative image of TRα1 (red) and F-actin (green) staining of injured podocytes. (C) Multinucleated podocytes were visible after glucose treatment (asterisks). (D) Densitometric analysis of TRα1 protein levels in podocytes by Western blot. Both glucose and H₂O₂ increased TRα1 expression significantly, and T₃ treatment markedly reversed this effect. (E) Quantification of podocytes with peripheral F-actin distribution. Diabetic stimuli caused F-actin rearrangements that were reversed by T₃. (F–J) Densitometric analysis of (F) α-actinin4, (G) nephrin, (H) α-SMA, (I) Ret, and (J) GFRα1 proteins by Western blot. Diabetic stimuli decrease the expression of adult podocyte markers (F and G), cause the reexpression of the mesenchymal marker α-SMA (H), and increase Ret (I), and GFRα1 (J) protein levels. T₃ treatment markedly reversed all these changes. (K) Representative images of DIO3 (red) and F-actin (green) staining in human podocytes. Both high glucose and H₂O₂ stimulation induced DIO3 upregulation mostly in the nuclear and perinuclear regions (top). T₃ treatment of injured podocytes decreased DIO3 expression while having no effect on control cells (bottom). (L) Densitometric analysis of DIO3 protein levels in podocytes by Western blot. Both glucose and H₂O₂ markedly increased DIO3 expression compared with control. T₃ treatment reversed this effect. Data from 3 independent experiments are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, 1-way ANOVA corrected with Tukey’s post hoc test. n = 3–15 samples per each condition. Tubulin protein expression (D and F) or ponceau red staining (G–J and L) was used as loading control. Scale bars: 50 μm.