Fig. 3. Insulin activation targeting the Orai1 channel induces albuminuria in mice.

a, b mRNA (a) and protein (b) expression levels of Orai1 in the isolated glomeruli and medulla of wild-type (WT), Cre+;Orai1^fl/+, and Cre+;Orai1^fl/fl mice. n = 3 independent experiments. c Immunofluorescence image displaying Orai1 (green) and synaptopodin (red; Synap.) expression in the isolated glomeruli of Cre+;Orai1^fl/+ and Cre+;Orai1^fl/fl mice. Scale bar = 25 μm. All images were selected among 3 mice in each group by independent experiments. d H & E staining of the kidney sections of the WT, Cre+;Orai1^fl/+, and Cre+;Orai1^fl/fl mice. e Representative traces of SOCE with the images of bright-field and fura-2 fluorescence (upper panel) microscopy carried out in situ on acutely isolated mouse glomeruli from WT, Cre+;Orai1^fl/+, and Cre+;Orai1^fl/fl mice. f Quantification of SOCE in e. n = 30 (WT), 25 (Cre+;Orai1^fl/+), and 29 (Cre+;Orai1^fl/fl) glomeruli per group. g Representative traces of fura-2 ratio showing insulin effects on SOCE in acutely isolated mouse glomeruli from Cre+;Orai1^fl/+ and Cre+;Orai1^fl/fl mice. h Quantification of SOCE in g. n = 60, 26, 24, and 48 glomeruli per each group, respectively. i Effects of insulin administration (i.p.; 5 U/kg) on 24 h urinary albumin/creatinine ratio (Alb/Cr Ratio) (left) and albumin excretion (right) in Cre+;Orai1^fl/+ and Cre+;Orai1^fl/fl mice. n = 8, 7, 8, and 7 mice per group. j Immunoblotting analysis showing insulin effects on synaptopodin in isolated glomeruli from Cre+;Orai1^fl/+ and Cre+;Orai1^fl/fl mice. k Densitometry of relative (Rel.) synaptopodin expression in j. n = 8, 7, 8, and 7 mice per each group. l Immunoblotting analysis showing the expression of exogenous Orai1 protein (detected by anti-Flag antibody) 48 h after in vivo gene delivery of Flag-tagged-Orai1. m Immunostaining of mCherry-tagged Orai1 (red; mCh-Orai1) and synaptopodin (green; Synap.) in mice injected with Orai1 transgene. Scale bar = 25 μm. Immunostaining was repeated two times from 3 mice each. n Effect of transgenic Orai1 overexpression and insulin administration (i.p.; 5 U/kg) on urinary Alb/Cr ratio (left) and albumin excretion. n = 5 mice each. o Immunoblotting analysis showing the effect of transgenic Orai1 overexpression and insulin inoculation (i.p.; 5 U/kg) on synaptopodin expression. p Densitometry of relative (Rel.) synaptopodin levels in o. n = 5 mice each. q Representative TEM images of foot processes from in vivo gene delivery of Flag-tagged-Orai1 with/without administration of insulin. EM repeated in 3 mice each. Magnification is ×30,000. Scale bar = 1 μm. Bar graphs are expressed as mean ± SEM and analyzed by two-way ANOVA followed by Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns not significant (a, f, h, i, k, n, p).