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).