Gene therapy by skeletal muscle expression of decorin prevents fibrotic disease in rat kidney.

There are currently no effective therapies for progressive fibrotic diseases. Recent evidence has implicated overproduction of transforming growth factor-beta1 (TGF-beta1) as a major cause of tissue fibrosis. Furthermore, this evidence implies that inhibitors of TGF-beta1 may be clinically useful as antifibrotic agents. The proteoglycan decorin is a known inhibitor of TGF-beta1. In a rat model of glomerulonephritis we have shown that fibrosis is mediated by TGF-beta1. We report here that transfer of decorin cDNA into rat skeletal muscle increases the amount of decorin messenger RNA and protein present in skeletal muscle and decorin present in kidney, where it has a marked therapeutic effect on fibrosis induced by glomerulonephritis. Transfected glomerulonephritic rats showed a significant reduction in levels of glomerular TGF-beta1 mRNA and TGF-beta1 protein, extracellular matrix accumulation and proteinuria. These results demonstrate the potential of gene therapy as a novel treatment for fibrotic diseases caused by TGF-beta1.

Molecular characterization of EAP-300: a high molecular weight, embryonic polypeptide containing an amino acid repeat comprised of multiple leucine-zipper motifs.

In this study we report the biochemical and initial molecular characterization of EAP-300, a developmentally regulated embryonal protein that has been shown previously to be expressed by radial glia in various regions of the CNS, including putative glial barriers. In the present study we have shown that the 300 kDa EAP-300 polypeptide is developmentally regulated in all tissues expressing the protein, which include various PNS and CNS tissues and muscle. In neural tissue the protein is readily detected during early embryogenesis, subsequently down-regulated at later stages, and is not detected in the adult. In contrast to neural tissue, small amounts of the protein are expressed in heart, consistent with earlier studies which showed that EAP-300 expression was maintained in the Purkinje cells of the heart conduction system. Metabolic labeling demonstrates that EAP-300 is a phosphoprotein, and is fatty acylated based on incorporation of [3H]palmitate. We also show that the normal developmental down-regulation of EAP-300 by glia does not occur in vitro, and these data therefore suggest that the signal(s) that regulates EAP-300 gene expression during development in vivo is absent in dissociated cell cultures. We have also initiated molecular studies of EAP-300 by screening embryonic brain cDNA expression libraries with a mixture of EAP-300 monoclonal antibodies. Sequence analysis of partial EAP-300 cDNAs indicate that the protein is related, if not identical, to IFAPa-400, a developmentally regulated intermediate filament-associated protein in chick that is proposed to participate in cell differentiation. These studies also indicate that EAP-300 mRNA is developmentally regulated and is expressed by glial cells in putative CNS barrier structures. Our studies also suggest that two pools of EAP-300 may exist in cells, implying that unlike IFAPa-400 the EAP-300 protein may not always be associated with intermediate filaments. Interestingly, our studies demonstrate that EAP-300 contains a novel repeat amino acid domain comprised of multiple leucine-zipper motifs, which may contribute to its function during glial differentiation.

Laminin and fibronectin content of mouse glomerular and tubular basement membrane.

Because the glomerular basement membrane (GBM) is subject to damage in a multitude of renal diseases, a model of basement membrane permeability properties would be useful for learning more about this important barrier. Isolated, perfused tubular basement membrane (TBM) allows measurement of permeability, but it is not known whether TBM is similar enough to GBM for data to be extrapolated from this model to the glomerulus. As a first approach to assessing differences between GBM and TBM, we looked at composition. Renal glomeruli and tubules were isolated from Swiss-Webster mice by sucrose-gradient centrifugation. GBM and TBM were isolated by sonication in 1% deoxycholate and then subjected to a sequential extraction procedure. Analysis of the solubilized basement membranes by electrophoresis revealed a complex mixture of proteins. Immunoblot analysis demonstrated that, among the proteins, laminin and fibronectin were found exclusively in the guanidine and guanidine/dithiotreitol extracts. The total amount of laminin extracted in GBM, 1.8 +/- 0.001 micrograms/mg dry weight (n = 2 groups animals, by inhibitory ELISA), was significantly less than in TBM, 3.4 +/- 0.1 micrograms/mg dry weight (n = 2); however, the total amount of fibronectin extracted did not differ between GBM and TBM, 8.2 +/- 0.8 and 7.7 +/- 1.0 micrograms/mg dry weight (n = 2) respectively. Examination of deoxycholate supernatants was carried out to see if components of GBM or TBM were solubilized during isolation of basement membranes. Immunoblot analysis revealed loss of some laminin and fibronectin occurred during the detergent isolation of GBM and TBM. We conclude that GBM and TBM are qualitatively similar in that they have the same protein components, but differ significantly in content of laminin and probably other macromolecular components.

L-arginine metabolism in immune-mediated glomerulonephritis in the rat.

Low-protein diets slow the progression of some renal diseases. We recently found that dietary restriction of L-arginine markedly ameliorates disease in antithymocyte serum-induced glomerulonephritis in the rat, suggesting that L-arginine may play a key role in the beneficial effects of low-protein diets. L-arginine is metabolized by nitric oxide synthases to nitric oxide and L-citrulline or by arginase to urea and L-ornithine. L-ornithine is a precursor for polyamines, which are required for cell proliferation and for proline, an essential component of collagen. In a time course of disease, we found that inducible nitric oxide synthase gene expression and nitric oxide production were increased very early. Arginase activity was significantly increased until 5 days of disease. Ornithine decarboxylase, the rate-limiting step for polyamine synthesis, was increased at 3 days coincident with the onset of cell proliferation. Gene expression of ornithine aminotransferase, a proline synthetic enzyme, was increased from day 1, paralleling increased collagen synthesis. Thus, the three pathways of L-arginine metabolism are upregulated in a manner consistent with their possible roles in the cell lysis, cell proliferation, and collagen deposition, which characterize this model of glomerulonephritis.

Differential effects of diabetes and glomerulonephritis on glomerular basement membrane composition.

The hallmark of renal diseases involving the glomerulus is the presence of proteinuria. While the routes of pathogenesis of proteinuria have not been established, alterations in the barrier function of the glomerular basement membrane (GBM) have been implicated. We evaluated the effect of streptozotocin diabetes and passive Heymann nephritis (PHN) over time on the macromolecular composition of rat GBM to determine if changes in composition correlate with proteinuria. Six to twelve rats from each group (control, diabetic, and PHN) were sacrificed 1, 5, 28, 56, or 84 days after induction of disease. Identical amounts of GBM were subjected to a sequential extraction procedure, and type IV collagen, entactin, laminin, fibronectin, and anionic charge content were quantitated in the extracts. Type IV collagen and entactin content did not change with time or disease. Both laminin and fibronectin contents increased with time in GBM in all groups, but this increase was significantly greater in diabetic GBM. A significant decrease in anionic charge content of GBM coincided with the onset of albuminuria at Day 28 in diabetes, but no change was seen in PHN. In diabetic rats, the increase in laminin content over control preceded the onset of albuminuria, while the increase in fibronectin was not apparent until after albuminuria was present. In PHN, no differences in type IV collagen, entactin, laminin, fibronectin, or anionic charge content of GBM were found compared with control, even though profound albuminuria was evident from Day 5 through 84. Thus, while alterations in laminin and fibronectin content may contribute to the loss of glomerular permselectivity in streptozotocin diabetes, such changes apparently are not involved in PHN.