Methods

Isolation of glomeruli Glomeruli were isolated from kidneys of 270 to 350 g Wistar males (Charles River, France) or six-month-old Buffalo/Mna males rats (Central Institut of Experimental Animals, Japan) using the usual sieving method [14, 15]. Briefly, animals were anaesthetized with 60 mg/kg of pentobarbital (Sanofi Sante Ani-male, France). The renal cortex was cut into small cubes, which were then delicately crushed on 180, 125, and 100 jm meshes successively. Isolated glomeruli were recovered on a 75 im mesh. At each step, the tissue was abundantly rinsed in an isotonic medium: RPMI 1640 solution (Gibco, Eragny, France) contain-ing 6% bovine serum albumin (BSA)(Sigma Aldrich, L'lsle d'Abeau Chesnes, France). This solution was oxygenated with a gas mixture (95% 2 and 5% C02). The isolated glomeruli were first examined with a microscope to verify their purity (usually'95% of the suspension) and to assess the percentage of glomeruli without a Bowman's capsule. Only preparations containing a majority of uneapsulated glomeruli were used in this study.

Volume measurement To measure the capillary volume of a large number of glomeruli easily and rapidly, a Multisizer counter (Coulter, Hileah, FL, USA) was used [16]. In this apparatus, a suspension of the particles (isolated glomeruli in this case) in a conducting liquid are first placed in a container into which a platinum electrode is immersed. Another electrode is placed in a second container, at the bottom of which is a 280 rm aperture through which the particles are forced to flow. When a particle passes through the aperture, the impedance between the two electrodes varies as a direct function of the particle volume, which is then directly deduced. In each experiment with Wistar rats, 1500 events (that is, glomeruli passing through the aperture) were counted over some 60 seconds, in an analytic window ranging from 0.11 to 1.4 ni. For glomeruli isolated from Buffalo/Mna rats, at least 4000 events had to be counted under the conditions previously described, owing to a wider dispersion of glomerular volumes which was probably likely due to the rat strain.