2.8 Determination of glomerular albumin permeability
Glomeruli from 8-10 week Sprague Dawley rats were isolated as previously described (Ilatovskaya, 2015) using a variable-sieving process (150µm, 106µm, then 75µm) after an in-vivo  renal perfusion of high-molecular weight FITC-labeled dextran (150kD, TdB Consultancy AB, Uppsala, Sweden). Each condition was tested with 3 or more rats, and a minimum of 9 glomeruli. Glomeruli were affixed to the optical window of poly-L-lysine coated culture dish (MatTek, Ashland, MA), and bathed in a 5% BSA solution containing (in mM): 145 NaCl, 2 CaCl2, 4.5 KCl, 2 MgCl2, 10 HEPES, TRITC-labeled dextran, pH 7.35 (adjusted with NaOH).  Only de-encapsulated glomeruli with detached afferent and efferent arteriole were included. Using an AR-1 confocal microscope, a Z-stack of 27 images (total thickness of ∼72 μm) was collected while in the initial 5% solution (baseline), which was followed by a repeated collected every 2 min following bath exchange to 1% albumin bath solution, for a total of 10 minutes. Glomeruli were either pre-incubated for 5 minutes in angiotensin II (10µM) to induce permeability in the presence and absence of PTUPB (1µM) to determine its effects on permeability. Following the conclusion of the experiment, volumetric recompositing of individual z-planes was carried out using Fiji image processing package (ImageJ 2.0.0, National Institutes of Health, USA) and Origin Pro 6.0 (Origin Lab, Northampton, MA).