An ELISA for the detection of type IV collagen in human urine—Application to patients with glomerulonephritis

Abstract

Type IV collagen is the principal component of glomerular basement membrane (GBM) [1]. It renders structural support, provides a matrix for cell adhesion [2,3] and contributes to glomerular permselectivity for macromolecules [3]. Normally there is a constant remodelling of GBM, as a result of balanced degradation by a number of glomerular cell derived proteinases, and synthesis, principally by glomerular epithelial cells [4]. Alteration of the integrity of the glomerular filtration barrier is widely believed to be the cause of the proteinuria and haematuria associated with a number of different glomerulonephritides. In vitro studies of GBM degradation quantified the release of hydroxyproline rich, soluble type IV collagen-derived peptides as a marker for GBM damage, and have demonstrated that small, soluble peptide fragments are released from the insoluble GBM by the action of proteinases [5—8]. A number of in vivo studies have identified glomerular basement antigens in the urine of normal animals which increase in concentration during acute experimental nephritis [9]. Analysis of the urine of patients with rapidly progressive glomerulonephritis (RPGN) showed that immunoreactive GBM antigens were detectable in much higher concentration than in normal urine [10]. This study, however, did not identify or accurately quantify specific antigens but did attempt to define glomerular damage more specifically than did proteinuria. Other studies using immunoassays for both type IV collagen and its NC1 region in patients with diabetic nephropathy have yielded conflicting data [11—13]. An investigation in patients with active nephritis, however, has reported elevated urinary IV collagen [14}. In the present study the development of a competition ELISA for human type IV collagen in urine is detailed. Analysis of the urine of patients with a number of differing glomerulonephritides, including RPGN, and diabetic nephropathy is compared with normals. In addition the effects of conventional therapy on the excretion of type IV collagen in patients with RPGN is examined.