Isolation and characterization of rat tubular basement membrane

Abstract

Tubules were isolated by the passage of coarsely homogenized kidney parenchyma through a series of stainless steel sieves of varying pore sizes. The isolated tubules, which were free of glomeruli or any contaminating tissue, based on light microscopic examination, served as starting material for the isolation of tubular basement membrane (TBM). The tubules were sonically disrupted, and the subsequent isolation procedure was conceptually similar to that of the isolation of glomerular basement membrane (GBM). The chemical composition of TBM was compared to that of GBM and collagen. 3-hydroxyproline, a marker for basement membrane protein, was present in higher amount in TBM (10 residues per 1,000 amino acid residues) than in GBM (7 residues per 1,000). Glycine and hydroxylysine content was also higher in TBM (255 and 27 residues per 1,000, respectively, in contrast to 204 and 18 residues in GBM). The total carbohydrate content was 7.6 mg per 100 mg of dry weight (neutral sugars, 6.3; GlcN, 0.9; GalN, 0.1; and sialic acid, 0.3). By electron microscopic examination, the isolated TBM was similar to GBM, except for greater variability in diameter and serrated appearance of one surface of the tubular basement membrane fragments. The method is suitable for the isolation of TBM in sufficient quantity for subsequent chemical analyses and immunologic studies.

Molecular Sieve in Renal Glomerular and Tubular Basement Membranes as Revealed by Electron Microscopy

“…One method was the ordinary way of obtaining TBM by ultrasonic disruption [11], Isolated tubuli were suspended in I M NACI solution. The sus pension was sonicated in a bath using a Kubota ultrasonicator (model 200).…”

“…Tubuli were prepared from renal cortices obtained from 20 Wister rats weighing about 300 g and from one fourth of a bovine kidney with stainless steel sieves of varying pore sizes [11]. Homogenized renal cortices were forced through 60-, 80-and 120-mesh sieves in rats and 60-, 120-, 150-, 170-and 250-mesh sieves in cattle.…”

Alterations in Glomerular Anionic Sites in the Autologous Phase of Canine Anti-Glomerular Basement Membrane Nephritis

“…GBMs were prepared from the perfused kidneys of young beagle dogs by the method of Krisko et al (8) as modified by Shirota et al (18). The kidneys were decapsulated, and the cortices were finely minced.…”

Murine Autoimmune Interstitial Nephritis and Associated Antigen: Purification of a Soluble Tubular Basement Membrane Antigen from Mice Kidneys

“…These pieces were passed through a rough mesh and then a 120 stainless screen. The fiber-rich material which remained on the 120 mesh was then suspended in saline and centrifuged at 4'C for 30 min at 1.500 g on a Ficoll discontinuous gradient (10,20,30,40, and 50%). The portion most rich in fibers, which was obtained in 20% Ficoll, was washed with saline several times and sonicated to ex clude fragments of renal tubular epithelia attached to fibers.…”

“…To clarify the mechanisms it is impor tant to study the immunological characters of the TBM antigen. Mahieu and Winand [7] in 1970 first isolated and determined the chemical properties of human TBM, fol lowed by similar studies on animal TBM [8][9][10], These investigations contributed to the development of techniques for isolation and chemical analysis of tubular fibers, but as far as purity of TBM antigen is concerned, much remains to be desired. Using several successive procedures, we have been able to purify TBM antigen in a soluble form and produce a typical autoimmune interstitial nephritis in BALB/c mice after immuniza tion with it.…”