Inhibition of heparin-binding epidermal growth factor-like growth factor increases albuminuria in puromycin aminonucleoside nephrosis.

BACKGROUND: Our previous work in the acute puromycin aminonucleoside nephrosis (PAN) model has demonstrated up-regulation of heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA and protein within glomerular epithelial cells (GECs) prior to the onset of proteinuria. METHODS: To determine whether increased HB-EGF expression in the acute PAN model contributes to the pathogenesis of proteinuria, a monoclonal antibody (DE10) was produced against recombinant human HB-EGF. RESULTS: The specificity of DE10 for human HB-EGF was confirmed by enzyme-linked immunosorbent assay, immunohistochemical staining, and flow cytometry of transfected cells expressing human and rat HB-EGF, and inhibition of cell proliferation. DE10 also reacted with cells transfected with rat HB-EGF cDNA. Administration of 0.5 mg affinity-purified DE10 to normal rats did not cause significant albuminuria compared with controls. Five days after the induction of the acute PAN model, albuminuria was significantly greater in animals treated with 0.5 mg DE10 than a control mAb (162.6 +/- 32.4 vs. 64.8 +/- 10.2 mg/day, respectively, P < 0.01). Rats treated with DE10 had an earlier onset of severe albuminuria, but no increase in maximal albuminuria at later time points. Electron microscopy showed marked podocyte effacement in both DE10-treated and control animals, but no obvious difference between groups. However, adhesion of the human GEC line 56/10 A1 to laminin and fibronectin, but not to collagens I or IV, was reduced by DE10. CONCLUSIONS: This study suggests that HB-EGF contributes to the integrity of the glomerular filtration barrier, particularly when the podocyte has been injured. Following podocyte injury, adhesion to laminin in the glomerular basement membrane by HB-EGF may be important in reducing albuminuria.

CD44 in glomerulonephritis: expression in human renal biopsies, the Thy 1.1 model, and by cultured mesangial cells.

CD44 is a transmembrane proteoglycan that serves as a cell adhesion receptor and is involved in cell-cell and cell-matrix interactions, both key events in the pathogenesis of clinical and experimental glomerulonephritis. In addition, recent evidence suggests that the binding of cytokines to proteoglycans could regulate cytokine function. We have, therefore, studied the expression of CD44 by mesangial cells in culture and in experimental (Thy 1.1 model) and human glomerulonephritis. Mesangial expression of CD44 detected by immunohistochemistry was markedly increased four days after induction of the Thy 1.1 model, coinciding with the peak of mesangial cell proliferation and macrophage infiltration. Analysis of 92 human renal biopsies by immunohistochemistry showed that CD44 expression by infiltrating cells within the glomerulus, in focal interstitial infiltrates and within the interstitium (interstitial fibroblasts, and extracellular matrix), was significantly increased in biopsies with a greater degree of histological damage. There was, however, no increase in mesangial staining in diseased kidneys as compared with control sections. In contrast, cultured human mesangial cells expressed CD44 strongly when assayed by immunohistochemistry, immunoprecipitation and Northern blotting. CD44, therefore, is an example of a protein strongly expressed by mesangial cells in vitro and weakly or not at all in vivo, but which is up-regulated in a disease model. In human disease, however, little expression was detected within the glomerular mesangium, which may be related to the greater proliferation and more profound disruption of mesangial architecture seen in the Thy 1.1 model. CD44 expression by infiltrating cells and by components of the interstitium could, however, play an important role in the pathogenesis of chronic progressive renal disease in humans.

Protein synthesis and secretion by cultured retinal pigment epithelia.

Protein synthesis and secretion by post-natal sheep and calf retinal pigment epithelial (RPE) cells was investigated following labelling of choroidal pieces, isolated RPE cells and RPE cells in tissue culture with L-[U-14C] leucine. We show that RPE cells secrete a specific set of proteins that includes retinol binding protein (RBP) and transthyretin (TTR), which are both involved in retinol transport in blood. Using a two-chambered culture system we show that protein secretion by the post-natal RPE cells occurs predominantly across the apical pole of the cells, i.e., across the surface of the cells which, in vivo, faces the retina. In agreement with results of others using foetal RPE cells (Ong, D.E., Davis, J.T., O'Day, W.T. and Bok, D. (1994) Biochemistry 33, 1835-1842) we show that RBP and, to a lesser extent, TTR are also secreted predominantly across the apical pole of the cell. We have developed a cell culture model for the RPE that may be used as an in vitro model for studying transport across the blood-retinal barrier.

Cloning, sequencing and bacterial expression of human glycine tRNA synthetase.

The human glycine tRNA synthetase gene (GlyRS) has been cloned and sequenced. The 2462 bp cDNA for this gene contains a large open reading frame (ORF) encoding 685 amino acids with predicted M(r) = 77,507 Da. The protein sequence has approximately 60% identity with B. mori GlyRS and 45% identity with S. cerevisiae GlyRS and contains motifs 2 and 3 characteristic of Class II tRNA synthetases. A second ORF encoding 47 amino acids is found upstream of the large ORF. Translation of this ORF may precede the expression of GlyRS as a possible regulatory mechanism. The enzyme was expressed in E. coli as a fusion protein with a 13 kDa biotinylated tag with an apparent M(r) = 90 kDa. The fusion protein was immunoprecipitated from crude bacterial extract with human EJ serum, which contains autoantibodies directed against GlyRS, and with rabbit polyclonal serum raised against a synthetic peptide derived from the predicted amino acid sequence of human GlyRS. Bacterial extract containing the fusion protein catalyses the aminoacylation of bovine tRNA with [14C]-gly at 10-fold increased level above normal bacterial extract and confirms that the cDNA encodes human GlyRS.

The effect of transforming growth factor beta 1 on mesangial cell fibronectin synthesis: increased incorporation into the extracellular matrix and reduced pI but no effect on alternative splicing.

Fibronectin is a multidomain glycoprotein which accumulates in mesangial proliferative glomerulonephritis (MPGN). Recent evidence has implicated transforming growth factor beta (TGF-beta) in the pathogenesis of experimental MPGN. We have, therefore, examined the influence of TGF-beta 1 on mesangial cell fibronectin synthesis. Considering, first, levels of mRNA, TGF-beta 1 increased steady-state fibronectin RNA in cultured mesangial cells by 1.9 times 24 hr after treatment of cycling mesangial cells and by 11.8 times in growth-arrested cells. There was, however, no alteration in fibronectin pre-mRNA splicing in either the EIIIA or IIICS regions. Fibronectin protein concentrations in cell culture supernatants, determined by immunoprecipitation of supernatants from cells labeled with [35S]methionine and by ELISA, were not increased by treatment with TGF-beta 1. Western blots and immunoprecipitation of metabolically labeled cells showed that fibronectin was increased, however, in the deoxycholate-insoluble extracellular matrix (ECM) of cells stimulated with TGF-beta 1. TGF-beta 1 altered the physicochemical properties of fibronectin in ECM and supernatant such that the isoelectric point of fibronectin, determined from Western blots of 2D SDS-PAGE gels, was reduced so that both became more acidic. These studies demonstrate, therefore, that in addition to increasing its synthesis, TGF-beta 1 increases incorporation of fibronectin into the ECM. Because fibronectin possesses binding sites for other ECM proteins, greater incorporation of fibronectin following TGF-beta 1 treatment may be an important pathogenetic mechanism in mesangial sclerosis. Moreover, the altered charge of fibronectin may increase localization of serum immunoglobulins to the mesangium.