Abstract
Alterations of the biochemical composition were studied in glomerular basement membrane (GBM) which were isolated from normal and experimental nephrotic rats. Experimental nephrotic syndrome was induced by intravenous injection of daunomycin or nephrotoxic serum obtained from rabbits. Preparation of GBM was obtained by sonication of isolated glomeruli using various stainless steel sieves. The purity of the GBM preparations was 98-100% in the test by a phase contrast microscopy. Immune complexes on the GBM were removed by incubation in 0.02M citrate buffer at pH 3.2.
Amino acids of GBM were analysed by JLC-5AH automatic amino acid analyser after hydrolysis. Amino sugars were analysed by KLA-5 automatic amino acid analyser, using eluate of 0.5M sodium citrate buffer at pH 6.09 with 8 % methanol. For the determination of carbohydrate, gas chromatography, Perkin Elmer Model 900B and Shimazu GC-4BM PFE, was used.
The kidney of daunomycin induced nephrotic rats (DMN rat) showed microscopically minimal changes and the kidney of nephrotoxic serum induced nephritic rats (NTN rat) showed slightly proliferative change with local thickening of GBM.
The GBM of normal rats contained quantitatively less hydroxyproline, proline, glycine, alanine, lysine and arginine, and more aspartic acid, threonine, serine, glutamic acid, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, hydroxylysine, histidine and half-cystine than in bovine tendon collagen (BTC). In the GBM of normal rats, however, increases in hydroxyproline/proline ratio and hydroxylysine/lysine ratio were demonstrated. It might suggest the active hydroxylation of proline and lysine in normal GBM. Normal GBM contained carbohydrate in 8%.
In the GBM of DMN rats, a decrease in the amount of 4-hydroxyproline, glycine, methionine and hydroxylysine, and increase in the contens of serine, glutamic acid, valine, leucine and arginine were detected. Among carbohydrate compositions in the GBM of DMN rats, only N-acetyl glucosamine was increased in amount. However, no changes were found in total amount of carbohydrates. On the other hand, in the GBM of NTN rats, a significant decrease in the amount of 4-hydroxyproline, glycine, isoleucine and half-cystine, and a significant increase in the contents of aspartic acid, alanine, leucine, tyrosine, hydroxylysine and lysine were demonstrated. In carbohydrate compositions, fucose, N-acetylglucosamine and N-acetylgalactosamine were decreased in amount, and N-acetylneuraminic acid was significantly increased. However, no changes were noted in total carbohydrates.
