Gareth J. Thomas

The potential role of human kidney cortex cysteine proteinases in glomerular basement membrane degradation

(1) The degradation of glomerular basement membrane and some of its constituent macromolecules by human kidney lysosomal cysteine proteinases has been investigated. Three cysteine proteinases were extracted from human renal cortex and purified to apparent homogeneity. These proteinases were identified as cathepsins B, H and L principally by their specific activities towards Z-Arg-Arg-NHMec, Leu-NNap and Z-Phe-Arg-NHMec, respectively, and their Mr on SDS-polyacrylamide gel electrophoresis under reducing conditions. (2) Cathepsins B and L, at acid pH, readily hydrolysed azocasein and degraded both soluble and basement membrane type IV and V collagen, laminin and proteoglycans. Their action on the collagens was temperature-dependent, suggesting that they are only active towards denatured collagen. Cathepsin L was more active in degrading basement membrane collagens than was cathepsin B but qualitatively the action of both proteinases were similar, i.e., at below 32 degrees C the release of an Mr 400,000 hydroxyproline product which at 37 degrees C was readily hydrolysed to small peptides. (3) In contrast, cathepsin H had no action on soluble or insoluble collagens or laminin but did, however, hydrolyse the protein core of 35S-labelled glomerular heparan sulphate-rich proteoglycan. (4) Thus renal cysteine proteinases form a family of enzymes which together are capable of degrading the major macromolecules of the glomerular extracellular matrix.

Structural and Functional Changes in Heparan Sulfate Proteoglycan Expression Associated with the Myofibroblastic Phenotype

The principal cells implicated as the source of the extracellular matrix in areas of progressive fibrosis are fibroblasts with the phenotypic appearance of myofibroblasts. This report describes differences in heparan sulfate proteoglycan expression between myofibroblasts and normal fibroblasts, associated with impaired responses to fibroblast growth factor-2 (FGF-2). Although both cell types responded to platelet-derived growth factor, myofibroblasts, unlike fibroblasts, did not proliferate to FGF-2. A response was acquired, however, when myofibroblasts were incubated with FGF-2 in the presence of heparan sulfate (HS) and heparin. Selective digestion with pronase, NaOH/NaBH(4), heparinase I, or low pH nitrous acid showed that each HS-glycosaminoglycan region comprised a pronase-resistant peptide separating two HS chains. The HS-glycosaminoglycan chains from myofibroblasts were larger (K(av), 0.32; molecular weight, 50 kd) than those from fibroblasts (K(av), 0.4; molecular weight, 33 kd), although their disaccharide composition was identical. The chains from myofibroblasts, however, contained three, compared to two, heparinase 1-resistant sequences separated by larger contiguous areas of low sulfation. Furthermore, although there was no difference in FGF-2-binding affinity between the two cell types, the chains secreted by myofibroblasts had twice the binding capacity of those from fibroblasts. Thus, it is likely that the difference in response to FGF-2 is because of a difference in FGF-2 sequestration and receptor interaction with FGF-2-HS complexes. A comparative investigation into HS fine structure is being undertaken to examine these findings in more detail.

Evidence that kidney lysosomal proteinases degrade the collagen of glomerular basement membrane.

Three thiol-dependent tissue proteinases have been isolated from human kidney cortex by methods using detergent extraction, gel chromatography, affinity chromatography and separation by ion-exchange. The properties of each enzyme toward several chromogenic low molecular weight substrates, azocasein and collagen indicate that they are similar to cathepsins B, H and L. All three enzymes degrade glomerular basement membrane at acid pH. Cathepsin B and L were particularly active against glomerular basement membrane. It has been demonstrated that in liver these enzymes exist together in lysosomes. These properties indicate that the kidney thiol proteinases may be an important factor in the turnover of glomerular basement membrane.

Bikunin Present in Human Peritoneal Fluid Is in Part Derived from the Interaction of Serum with Peritoneal Mesothelial Cells

We recently reported that peritoneal fluid mainly contains two proteoglycans; one is the interstitial proteoglycan referred to as decorin, and the other an uncharacterized small chondroitin sulfate proteoglycan. In the present study, we have used a two-step process to isolate the small chondroitin sulfate proteoglycan free of decorin. The purified molecule ran as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent molecular mass 50 kd made up of a chondroitin-4-sulfate glycosaminoglycan chain and a 30-kd core protein. NH2-terminal analysis of the core protein showed significant sequence homology with bikunin, a component of the human inter-alpha-trypsin inhibitor (IalphaI) family. A Western blot analysis using anti-human inter-alpha-trypsin inhibitor confirmed the identity of the small chondroitin sulfate proteoglycan as bikunin, and a trypsin inhibitor counterstain assay confirmed its anti-trypsin activity. Examination of serum from patients receiving continuous peritoneal dialysis suggests that free bikunin in peritoneal fluid may be the result of leakage of serum proteins into the peritoneum. Our findings further show that the interaction of serum with peritoneal mesothelial cells offers a new and novel explanation for the presence of bikunin in peritoneal fluid.

The Binding of Human Lysosomal Elastase to Glomerular Basement Membrane

The interaction of purified elastase with glomerular basement membrane and its antigens was studied. There appears to be at least two types of interaction. The first is a weak interaction and the enzyme can be removed simply by extended washing with neutral buffer. The second, stronger interaction is desorbed with high salt concentration at alkaline pH. The different interactions probably represent the binding of isoenzymes. Further experiments demonstrate that all isoenzymes degrade the membrane. Serum proteins prevent binding and inhibit the action of elastase on glomerular basement membrane.

Mechanical injury of human peritoneal mesothelial cells (HPMC) is accompanied by an increase in hyaluronan synthesis

ABSTRACT Hyaluronan (HA) has been implicated in tissue repair but its role during injury to the peritoneum is poorly understood. To delineate its possible role we have investigated several aspects of HA metabolism in an in vitro model of peritoneal injury. Confluent, growth arrested HPMC were wounded and the synthesis of HA during the repair process determined by [3H]-glucosamine labelling. After an initial lag period of 12 h de novo synthesis of HA was up-regulated 1.75±0.15-fold (n=3) above controls and remained so until the re-establishment of the monolayer. Cytochemical staining for UDP-glucose dehydrogenase was localised to the cells at the margin of the wound and also to those cells migrating into the wound. Using RT-PCR, mRNA for HAS II was not detected in non-injured cells, but on wounding the monolayer, mRNA for HAS II was induced, maximal 12-24h after injury and remained elevated for up to 144h. In contrast mRNA for HAS III was constitutive before injury but its expression was reduced during the restitution of the wound. These findings suggest that HAS II and HAS III are differentially regulated in response to mesothelial damage. In a separate study, wounded cells were co-cultured with endotoxin-free HA (Mr 250,000 Da). The inclusion of HA (range 0-3.3 μg/ml) increased the rate of cell migration in a dose-dependent manner. This response to HA was abrogated by prior digestion of the HA to disaccharides with Streptomyces hyaluronidase. These findings suggest that HA plays a critical role in the response of the mesothelium to injury.