Fig 2: Albumin endocytosis enhances ORAI1 channel internalization and impairs SOCE. a TIRF microscopy images showing human proximal tubular epithelial cells (PTECs) after incubation with FITC-albumin (50 µg ml−1) for 4 h. PTECs were transfected with ORAI1-tagged with CFP. Lysosomes were stained using LysoBrite Red. b The PTECs without FITC-albumin treatment. Scale bars, 10 µm for (a) and (b). c Mean lysosome size and fluorescence intensity (ratio of CFP-ORAI1 in lysosomes (Lyso-ORAI) to LysoBrite Red (Lyso-Red) (n = 32–50). d FITC-albumin uptake in PTECs in 1.5 mM Ca2+ bath solution (n = 30) or in Ca2+-free solution with 0.4 mM EGTA (n = 30). e Pre-incubation with FITC-albumin for 30 min in 1.5 mM Ca2+ solution reduces store-operated Ca2+ influx in PTECs. Bar chart shows the mean data for the peak of SOCE in the FITC-albumin incubated (n = 12) and control PTECs (n = 16). f Unaffected SOCE when PTECs were pre-incubated with (n = 13) or without (n = 15) FITC- albumin for 30 min in Ca2+-free solution. Average data are presented as mean ± s.e.m. The data sets are compared by t test. Statistical significance is indicated by * P < 0.05, ** P < 0.01

Fig 3: ORAI channels determine albumin reabsorption in vitro and in vivo. a FITC-albumin uptake by human proximal tubular epithelial HK-2 cells after incubation with FITC-albumin (50 µg mL−1) for 60 min with or without 2-APB (100 µM) or DES (10 µM) or BTP2 (1 µM). Scale bar, 25 µm. b Mean ± s.e.m. data for the fluorescence of FITC-albumin uptake by PTECs in a. The fluorescence was normalized by the total lysed protein (n = 6 for each group). c HK-2 cells were transfected with ORAI siRNAs (si-ORAI1/2/3) and scramble siRNA (control) (n = 7 each group). d Male Akita type 1 diabetic mice (C57BL/6-Ins2Akita/J) and STZ-induced diabetic male C57BL/6 J mice were injected either with BTP2 (0.29 mg kg−1, i.p., b.i.d) for 3 days, or vehicle (saline) as control (n = 9–10 for each group). Age-matched non-diabetic wide-type male mice were also used (n = 13–14). Urine albumin-creatinine ratio (uACR) was measured on day 5. e Schematic representation of Cre-LoxP-mediated recombination and generation of a linear product contain one lox-P site and the target gene (EYFP-mOrai1E108Q mutant) via crossing the Cre+ mice with proximal tubule-specific expression driven by sodium-glucose cotransporter 2 (SGLT2) promoter. f Expression of EYFP-mOrai1E108Q mutant in tubules (Scale bar, 200 µm). g Urine albumin-creatinine ratio (uACR) was measured in male DN-Orai1 transgenic mice and littermates on day 32–38 after birth (n = 27, 34, 17 and 17 for Cre+, wild-type, Cre−-DN-Orai1 and Cre+-DN-Orai1 mice, respectively). Average data are presented as mean ± s.e.m. Data sets are compared by t test in (d) and by ANOVA in (b, c, and g). Statistical significance is indicated by * P < 0.05, ** P < 0.01, *** P < 0.001

Fig 4: Increased protein expression and interaction of TRPC channels and Orai1 with STIM1 in neointima from balloon-injured rat carotid artery. H&E-stained sections of (a) sham-operated (intact) and (b) balloon-injured arteries 14 days after injury. Prominent neointima is present and narrows the lumen. Photos are taken at 10× and 40× magnification. M = media; NI = neointima; L = lumen. Protein expression levels of TRPC1, TRPC3, TRPC4, TRPC6, Orai1, and STIM1 (c–h) are upregulated in injured (media + neointima) compared to uninjured VSMCs. Co-immunoprecipitation assay using anti-STIM1 antibody to immunoprecipitate STIM1 shows increased interaction of STIM1 with (c) TRPC1; (d) TRPC3; (e) TRPC4; (f) TRPC6; and (g) Orai1 more strongly in injured VSMCs.

Fig 5: Knockdown of ORAI1 protects from GSH depletion. HT22S cells were transfected with two independent siRNAs against STIM1, STIM2, ORAI1 and TRPM7 or with non-targeting control siRNA and 48 h later subjected to cell survival experiments. (a, d, f and h) RT-PCR analyses of successful knockdown of STIM1 (a), STIM2 (d), ORAI1 (h) and TRPM7 (f). Signals of genes of interest were normalized to hprt and plotted as expression in percentage relative to control-siRNA transfected samples. (b and i) Verification of successful knockdown by immunoblot. The membranes were simultaneously probed with antibody against either STIM1 (b) or ORAI1 (i) and GAPDH respectively actin as loading control and analyzed with the Licor infrared imaging system. (c, e, g and j) Survival experiments of siRNA-transfected cells. The indicated amounts of glutamate or BSO were added 48 h after transfection and viability was quantified with the CTB reagent 16 h after glutamate or BSO addition. The graphs show the mean fluorescence±S.E.M. of 15 replicates obtained in three independent experiments plotted against glutamate or BSO concentration