Na k atpase α1

Human primary CECs or iPSC-derived CECs were fixed with 4% paraformaldehyde overnight at 4 °C, washed with PBS/0.05% Tween 20 (PBST) buffer, permeabilized in 0.5% Triton X-100, and blocked in PBST containing 1% bovine serum albumin (Sigma-Aldrich). Samples were incubated with primary antibodies against human anti-ZO-1 (1:250; Life Technologies), Na⁺/K⁺ ATPase α1 (1:200, Santa Cruz Biotechnology, Dallas, TX, USA), SLC4A11 (1:250, Novus, Centennial, CO, USA), and N-cadherin (1:200, Cell signaling technology, Danvers, MA, USA) overnight at 4 °C, followed by goat anti-mouse IgG Alexa Fluor 488-conjugated and donkey anti-rabbit IgG Alexa Fluor 647-conjugated secondary antibodies (1:500; Abcam, Cambridge, UK). Nuclei were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for 10 min and fluorescence signals detected with either a fluorescence microscope (EVOS, Life Technologies) or a laser-scanning confocal microscope (Olympus, Tokyo, Japan).

The protein concentration of kidney tissue samples or HK-2 cells was determined with the Enhanced BCA Protein Assay Kit (Beyotime, Shanghai, China). Equal amounts of protein (50 μg) were separated on 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA), and were subsequently blocked by 5% non-fat milk powder in TBST (10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.6) for 1 h. After blocking, membranes were incubated with primary antibodies against AQP1 (1:500), AQP2, Na,K-ATPase α1, NKCC2, NHE3, β-actin (1;1000) (Santa Cruz Biotechnology) and AQP3 (1:1000, Alomone Labs, Jerusalem, Israel). Then the membranes were washed with TBST and the primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). The bands were visualized with enhanced chemiluminescence reagent (Pierce Biotech, IL, USA). Protein expression levels were determined by analyzing the signals captured on the membranes using the ImageJ software (NIH, Maryland, USA).

The cryopreserved iPSC-derived CECs were also analyzed by immunocytochemistry. Briefly, the cells were fixed with 4% paraformaldehyde (PFA) for 15 minutes followed by blocking with 5% bovine serum albumin (MilliporeSigma). The cells were first incubated with 1:100 zona occludens-1 (ZO-1; Cell Signaling Technology), 1:50 N-cadherin (Cell Signaling Technology), and 1:40 ATPase sodium/potassium subunit alpha1 (Na⁺/K⁺ATPase α1; Santa Cruz Biotechnology) primary antibodies overnight at 4°C. The cells were next treated with 1:100 FITC-conjugated goat anti-rabbit IgG (against ZO-1; MillporeSigma), 1:75 FITC-conjugated goat anti-mouse IgG (against N-cadherin; MilliporeSigma), and 1:75 FITC-conjugated goat anti-mouse IgG (against Na⁺/K⁺ATPase α1; MilliporeSigma) secondary antibodies for 2 hours at room temperature. The nuclei were counterstained with 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI; MilliporeSigma). The images of mounted cells were captured using a microscope (Olympus LX81; Olympus, Tokyo, Japan) equipped with software (Slidebook Software 3i; Denver, CO, USA) and prepared using image-editing software (Adobe Photoshop CS5; Adobe Systems, Inc., San Jose, CA, USA).