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Biglycan, a small leucine-rich proteoglycan, acts as a danger signal and is classically thought to promote macrophage recruitment via Toll-like receptors (TLR) 2 and 4. We have recently shown that biglycan signaling through TLR 2/4 and the CD14 co-receptor regulates inflammation, suggesting that TLR co-receptors may determine whether biglycan-TLR signaling is pro- or anti-inflammatory. Here, we sought to identify other co-receptors and characterize their impact on biglycan-TLR signaling. We found a marked increase in the number of autophagic macrophages in mice stably overexpressing soluble biglycan. In vitro, stimulation of murine macrophages with biglycan triggered autophagosome formation and enhanced the flux of autophagy markers. Soluble biglycan also promoted autophagy in human peripheral blood macrophages. Using macrophages from mice lacking TLR2 and/or TLR4, CD14, or CD44, we demonstrated that the pro-autophagy signal required TLR4 interaction with CD44, a receptor involved in adhesion, migration, lymphocyte activation, and angiogenesis. In vivo, transient overexpression of circulating biglycan at the onset of renal ischemia/reperfusion injury (IRI) enhanced M1 macrophage recruitment into the kidneys of Cd44+/+ and Cd44−/− mice but not Cd14−/− mice. The biglycan-CD44 interaction increased M1 autophagy and the number of renal M2 macrophages and reduced tubular damage following IRI. Thus, CD44 is a novel signaling co-receptor for biglycan, an interaction that is required for TLR4-CD44-dependent pro-autophagic activity in macrophages. Interfering with the interaction between biglycan and specific TLR co-receptors could represent a promising therapeutic intervention to curtail kidney inflammation and damage.

Renal ischemia reperfusion injury (IRI) is a serious cause of acute kidney injury (AKI). Danger-associated–molecular pattern molecules (DAMPs) are thought to promote IRI by initiating immune cell infiltration and driving disease progression, but the underlying pathophysiological mechanisms are mainly unclear. Poluzzi et al. demonstrate that soluble biglycan is a bimodal DAMP that both recruits proinflammatory macrophages and initiates resolution of inflammation and tissue remodeling in IRI, identifying a potential therapeutic target.

Hepatocyte growth factor regulates proteoglycan synthesis in interstitial fibroblasts.BackgroundHepatocyte growth factor (HGF) is a clinically important growth factor with therapeutic potential for the treatment of interstitial fibrosis and chronic renal failure. Proteoglycans are components of the renal interstitium, which have multiple actions, including growth regulation. In this study, we examined the effects of HGF on proteoglycan synthesis in interstitial fibroblasts, and the mechanisms of these effects.Methods and ResultsExpression and agonist-induced activation of the HGF receptor c-Met was detected in rat renal interstitial fibroblasts (NRK-49F) by reverse transcription-polymerase chain reaction (RT-PCR) analysis and immune complex/immunoblot assay. Moreover, stimulation of the cells with HGF resulted in a marked increase (five- to tenfold) in phosphorylation of extracellular signal-related protein kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK), but not of c-Jun NH2 terminal kinase (JNK). Treatment with HGF resulted in a time- and dose-dependent increase (P < 0.01) in both cell-associated and secreted proteoglycan synthesis to two- to fourfold of control levels. This effect was attenuated by the MAPK/ERK kinase (MEK) inhibitor PD98059 and the p38MAPK inhibitor SB203580. Ion-exchange chromatography suggested that chondroitin sulfate/dermatan sulfate proteoglycans were up-regulated after HGF treatment. Northern blot, RT-PCR, Western blot, and promoter activity assays revealed that HGF caused a significant increase in decorin mRNA and protein, as well as in biglycan mRNA, protein, and promoter activity, suggesting transcriptional control of gene expression. Since the effects of biglycan on fibroblast proliferation are still unclear, the effects of biglycan were examined by thymidine assay, and biglycan was found to attenuate transforming growth factor-β (TGF-β)–induced changes in cell proliferation.ConclusionThese results suggest that HGF causes an increase in the small leucine-rich proteoglycans biglycan and decorin by ERK1/2- and p38MAPK-mediated pathways in fibroblasts. These findings may be relevant for understanding potential mechanisms by which HGF can exert TGF-β inhibitory actions in the kidney.

Glycosaminoglycan and proteoglycan inhibit the depolymerization of β 2 -microglobulin amyloid fibrils in vitro. Background Although several kinds of evidence suggest that glycosaminoglycans (GAGs) and proteoglycans (PGs) may contribute to the development of β 2 -microglobulin–related (Aβ 2 m) amyloidosis, the precise roles of these molecules for the development of Aβ 2 m amyloidosis are poorly understood. Methods We investigated the effects of GAGs and PGs on the depolymerization of Aβ 2 m amyloid fibrils at a neutral pH, as well as on the formation of the fibrils at an acidic pH in vitro, using fluorescence spectroscopy with thioflavin T and electron microscopy. Results Depolymerization of Aβ 2 m amyloid fibrils at pH 7.5 at 37°C was inhibited dose-dependently by the presence of some GAGs (heparin, dermatan sulfate, or heparan sulfate) or PGs (biglycan, decorin, or keratan sulfate proteoglycan). Electron microscopy revealed that a significant amount of Aβ 2 m amyloid fibrils remained in the reaction mixture with some lateral aggregation. Second, when monomeric β 2 m was incubated with aggrecan, biglycan, decorin, or heparin at pH 2.5 at 37°C for up to 21days, the thioflavin T fluorescence increased depending on dose and time. Electron microscopy revealed the formation of rigid and straight fibrils similar to Aβ 2 m amyloid fibrils in β 2 m incubated with biglycan for 21days. Conclusion These results suggest that some GAGs and PGs could enhance the deposition of Aβ 2 m amyloid fibrils in vivo, possibly by binding directly to the surface of the fibrils and stabilizing the conformation of β 2 m in the fibrils, as well as by acting as a scaffold for the polymerization of β 2 m into the fibrils.

Glucocorticoid regulation of proteoglycan synthesis in mesangial cells.BackgroundProteoglycans are integral components of the mesangial matrix and glomerular permeability barrier. Recent studies have shown that changes in glomerular proteoglycan expression may play a major role in the pathogenesis of renal disease. Steroid hormones are used as first-choice therapy for the treatment of glomerular diseases, however, the effects of glucocorticoids on expression of glomerular proteoglycans are unknown.MethodsThis study examined the effects of in vitro and in vivo administration of dexamethasone on proteoglycan synthesis and gene expression of proteoglycan core proteins using rat (RMC) and human (HMC) mesangial cells.ResultsTreatment of cultured RMC with dexamethasone resulted in a dose- and time-dependent decrease (P < 0.05) in both cell-associated and secreted proteoglycan synthesis to approximately 50% of control levels. This effect was inhibited by the glucocorticoid antagonist mifepristone, and mimicked by prednisolone or corticosterone treatment. Separation of proteoglycans by ion-exchange and gel permeation chromatography suggested that chondrotin sulfate/dermatan sulfate proteoglycans were down-regulated after steroid treatment. Northern blot analysis, RT-PCR, Western blot, and promoter activity assays revealed that dexamethasone caused a significant decrease in decorin mRNA (to 61 ± 8% of controls), whereas biglycan expression and promoter activity were increased after steroid treatment. A similar trend was found in glomeruli isolated from rats treated in vivo with dexamethasone.ConclusionsThese results demonstrate that treatment of mesangial cells with steroids results in a decrease in total proteoglycan synthesis, as well as subtype-specific changes in proteoglycan core protein gene expression by transcriptional control, furthering our understanding of the effects of steroid treatment on the renal glomeruli.

Small proteoglycans of normal adult human kidney: Distinct expression patterns of decorin, biglycan, fibromodulin, and lumican. Background Among the members of the small leucine-rich proteoglycan family, decorin, biglycan, and fibromodulin have been proposed to be potent modulators of transforming growth factor-β (TGF-β) activity, thereby playing an important role in the pathogenesis of fibrotic kidney diseases. Furthermore, decorin expression influences the expression of p21 WAF1/CIP1 , which has been related to kidney hypertrophy and hyperplasia. However, none of the members of this proteoglycan family have been investigated in normal adult human kidney cortex, thus making it impossible to correlate disease-mediated alterations of their expression with the normal situation in vivo. Methods The chondroitin/dermatan sulfate proteoglycans, decorin and biglycan, and the keratan sulfate proteoglycans, fibromodulin and lumican, were investigated in normal human adult renal cortex by immunohistochemistry on the light and electron microscopic level and by in situ hybridization. Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) methods were used to get an estimate of their expression in isolated glomeruli. Decorin excretion with the urine was measured by Western blotting. Results Two bands of decorin and a single band of biglycan mRNA were identified in Northern blots of isolated glomeruli. Amplification by RT-PCR was required to detect the signals for fibromodulin and lumican. All four proteoglycans were preferentially expressed in the renal interstitium with accumulations around tubules. Weak expression was found in the mesangial matrix. Biglycan was expressed by glomerular endothelial cells and, together with fibromodulin, was synthesized and deposited in distal tubular cells and collecting ducts. Immunogold labeling indicated the presence of the proteoglycans in the glomerular basement membrane, which was interpreted as a result of glomerular filtration. Indirect evidence suggested tubular reuptake of decorin after glomerular filtration. Conclusion The data indicate that the different cells of the adult human kidney are characterized by a distinct expression pattern of the four small proteoglycans. It is suggested that these proteoglycans may have distinct pathophysiological roles depending upon whether they are expressed by mesangial cells, endothelial cells, epithelial cells, or cells of the tubulointerstitium.

Effect of nitric oxide modulation on TGF-β1 and matrix proteins in chronic cyclosporine nephrotoxicity. Background Chronic cyclosporine (CsA) nephrotoxicity is characterized by interstitial fibrosis and afferent arteriolar hyalinosis. L-arginine (L-Arg), the substrate for nitric oxide (NO) synthase and N-nitro-L-arginine-methyl ester (L-NAME), the NO synthase inhibitor, were shown to modulate acute CsA nephrotoxicity. However, the mechanism of fibrosis in chronic CsA nephrotoxicity remains unclear. Thus, we examined the effect of NO modulation on fibrosis and the expression of transforming growth factor-β1 (TGF-β1) and matrix proteins in chronic CsA nephrotoxicity. Methods Rats were administered CsA (7.5 mg/kg), CsA + L-Arg (1.7 g/kg), CsA + L-NAME (3.5 mg/kg), vehicle (VH), VH + L-Arg, and VH + L-NAME, and were sacrificed at 7 or 28 days. NO production, physiologic parameters, and histology were studied in addition to the mRNA expression of TGF-β1, plasminogen activator inhibitor-1 (PAI-1) and the matrix proteins biglycan and collagens type I and IV by Northern and the protein expression of PAI-1 and fibronectin by enzyme-linked immunosorbent assay. Results While L-NAME strikingly reduced NO biosynthesis and worsened the glomerular filtration rate and CsA-induced fibrosis, L-Arg had the opposite beneficial effect. In addition, the CsA-induced up-regulated expression of TGF-β1, PAI-1, and the matrix proteins biglycan, fibronectin, and collagen I was significantly increased with L-NAME and strikingly improved with L-Arg. Collagen IV expression was not affected. Also, NO modulation did not affect VH-treated rats. Conclusions Chronic CsA nephrotoxicity can be aggravated by NO blockade and ameliorated by NO enhancement, suggesting that NO maintains a protective function. NO modulation was associated with a change in TGF-β1 expression, which, in turn, was associated with alterations in matrix deposition and matrix degradation through its effect on PAI-1.

Decorin, biglycan and their endocytosis receptor in rat renal cortex. Background Among the small proteoglycans, biglycan and decorin have been proposed to be potent modulators of TGF-β-mediated inflammatory kidney diseases. They were considered to become induced during glomerulonephritis and to subsequently inactivate the cytokine. Methods Decorin and biglycan as well as their endocytosis receptor were investigated in normal rat renal cortex, in anti-Thy-1 glomerulonephritis, in polycystic kidneys, in the remnant kidney following 5/6-nephrectomy, and in kidneys from the Milan normotensive strain by immunohistochemistry and in situ hybridization. Northern blots were used for the detection of mRNA expression for decorin and biglycan in isolated glomeruli. Functional aspects of the endocytosis of decorin and biglycan were studied in cultured mesangial cells. Results In the normal adult rat kidney decorin was expressed preferentially by Bowman's capsule and by interstitial connective tissue cells, but only in trace amounts by mesangial cells. In contrast, biglycan was found in tubular epithelial cells, in association with glomerular capillaries, podocytes and occasionally in the mesangium. In the tubulointerstitium of diseased kidneys (polycystic kidneys, 5/6-nephrectomy, kidneys from the Milan normotensive strain) there was a general up-regulation of decorin expression, while biglycan was localized only in distinct foci of fibrotic lesions. Glomerulosclerosis (5/6-nephrectomy, Milan normotensive strain) was associated with an increased staining for both decorin and biglycan within glomeruli. However, even in the anti-Thy-1 model of an acute mesangioproliferative glomerulonephritis where the greatest accumulation of decorin was found there was only a slight enhancement of decorin mRNA in isolated glomeruli. Decorin and biglycan become degraded upon receptor-mediated endocytosis. Immunohistochemical investigations indicated that the pattern of expression of the receptor protein correlated well with the immunolocalization of both decorin and biglycan. In vitro experiments with cultured mesangial cells provided direct evidence for the expression of the receptor and for the cell's capability to endocytose decorin as well as biglycan. Conclusions Decorin and biglycan are characterized by a distinct expression pattern in the normal rat kidney, whereas the presence of their endocytosis receptor protein correlates with the expression of both proteoglycans. Decorin is almost completely absent in the normal mesangium. Both proteoglycans become up-regulated in various models of renal disease. The mesangial accumulation of decorin in the anti-Thy-1 glomerulonephritis that is observed in spite of the only slightly enhanced mRNA expression could result from decreased decorin turnover and/or increased mesangial retention.

Rat mesangial cells in vitro synthesize a spectrum of proteoglycan species including those of the basement membrane and interstitium. Accumulation of extracellular matrix within the mesangium is an important event in the development of glomerular disease. In this report we have used indirect immunofluorescence to positively identify a number of constituents of the mesangial matrix synthesized by rat mesangial cells (RMC) in vitro including laminin, fibronectin, type IV collagen and the basement membrane heparan sulphate proteoglycan (BM-HSPG) known as perlecan. In addition, using Mab 2B5 we demonstrate that RMC synthesize a specific basement membrane chondroitin sulfate (BM-CSPG), a matrix component that in normal animals is localized in the mesangium but is not found in the pericapillary glomerular basement membrane (GBM). Further characterization of the proteoglycans synthesized by RMC in vitro revealed: (i) a second large CSPG, identified as versican; (ii) two small dermatan sulphate proteoglycans identified as biglycan and decorin, which together account for the majority Of the proteoglycans; (iii) a large HSPG-I, probably related to perlecan; and (iv) a small HSPG-II. The cell layer proteoglycans can be sub-divided into a class that are probably free in the membrane, and a class of anchored molecules of the extracellular matrix or stabilized by cytoskeletal elements.

Up-regulation of extracellular matrix proteoglycans and collagen type I in human crescentic glomerulonephritis. Background The pathogenesis of crescentic glomerulonephritis (CGN) involves cellular migration and proliferation in the urinary space, frequently followed by fibrous organization. Extracellular matrix proteoglycans (PGs) may regulate these events via effects on cellular migration, interactions with growth factors, including transforming growth factor-β (TGF-β), and control of collagen fibrillogenesis. The expression of PG in human CGN is unknown. Methods Renal tissues from 18 patients with CGN were examined immunohistochemically for versican, decorin, biglycan and collagen type I, and were compared with morphologically normal tissues from six tumor nephrectomies. Synthesis of decorin, biglycan, and procollagen type I mRNAs was evaluated by in situ hybridization. Results Versican was strongly expressed in cellular crescents and periglomerular areas, whereas decorin and biglycan accumulated in collagen type I-enriched regions, including fibrocellular and fibrous crescents, and interstitial fibrosis. PG and collagen type I accumulation colocalized with myofibroblasts in crescents, periglomerular areas, and interstitium. Conclusions The temporal and spatial patterns of expression demonstrated in this study provide evidence to support pathogenic roles for PG in the evolution of CGN. Based on known biological properties of this molecule, versican may facilitate migration of cells in developing crescents. Decorin and biglycan may contribute to progression of CGN, perhaps via interactions with collagen type I in the remodeled extracellular matrix.

Production of extracellular matrix by glomerular epithelial cells is regulated by transforming growth factor-β1. Transforming growth factor-β (TGF-β) has widespread effects on extracellular matrix production by many cultured cell lines and appears to play a role in the pathological accumulation of extracellular matrix that accompanies inflammatory and fibrotic diseases such as glomerulonephritis. Earlier experiments have shown that mesangial cells respond to TGF-β1 with a marked increase in the production of two chondroitin/dermatan sulfate proteoglycans, decorin and biglycan, but their production of other matrix components elevated in glomerulonephritis is not substantially affected by TGF-β1. Since the glomerular epithelial cells are also thought to contribute to matrix production in the glomerulus, we examined the ability of these cells to produce some of the nonproteo glycan matrix components in response to TGF-β1. Exposure of glomer ular epithelial cells to TGF-β1 increased the production of fibronectin and type IV collagen, in addition to biglycan. Enhancement of the cell layer accumulation of laminin was also observed. These results show that TGF-β1 has a differential effect on extracellular matrix production by epithelial and mesangial cells from glomeruli. TGF-β1 released in the glomerulus secondary to injury could thus affect both cell types and lead to increased intraglomerular production of proteoglycans, whereas the increased fibronectin, type IV collagen, and laminin may primarily originate from the epithelial cells.

Transforming growth factor-β regulates production of proteoglycans by mesangial cells. Accumulation of glomerular extracellular matrix is a prominent feature of most forms of progressive glomerular disease. Since some growth factors may play a role in extracellular matrix production, we examined the effects of transforming growth factor-β (TGF-β), interleukin 1, platelet derived growth factor, and tumor necrosis factor on the production of extracellular matrix components by cultured rat mesangial cells. In control experiments we found that mesangial cells produced two distinct proteoglycans identified as the small chondroitin/dermatan sulfate proteoglycans biglycan (PG I) and decorin (PG II) by showing that their mobility on SDS-PAGE changed upon digestion by chondroitinase ABC, and that they reacted with antibodies raised against synthetic peptides from the core protein sequence of human biglycan and decorin. Exposure to TGF-β for 48 hours stimulated an 8- to 10-fold increase in the biglycan and decorin bands, and induced a structural change detected as a shift in electrophoretic mobility. TGF-β did not demonstrably affect the production of other matrix proteins by the mesangial cells. The other growth factors tested had no comparable effect on the production of proteoglycans or other extracellular matrix components by these cells. Our results show that TGF-β is unique among growth factors in its regulatory effects on mesangial cell proteoglycan production. The release or activation of TGF-β during glomerular injury could mediate the accumulation of proteoglycans in the extracellular matrix and predispose the kidney to development of glomerulosclerosis.

Nonenzymatic glycation of mesangial matrix and prolonged exposure of mesangial matrix to elevated glucose reduces collagen synthesis and proteoglycan charge. Expansion of the mesangial matrix in diabetes occurs after prolonged exposure to the diabetic milieu. To mimic the long-term hyperglycemia of diabetes mellitus we developed tissue culture systems that might approximate the chronic state. This was accomplished in two ways: (1) by growing mesangial cells on extracellular matrix glycated and crosslinked in vitro and (2) by continuously growing cells on their own matrix on filters in elevated glucose medium (500 mg/dl) for up to eight weeks without passage. Synthesis of collagen and proteoglycans was evaluated in cells grown under these conditions. In both these situations, 3 H-proline incorporation into collagenase sensitive protein and 35 S incorporation into sulfated proteins were reduced compared to control cultures. Despite reduction in 35 S incorporation into proteoglycans in the high glucose cultures, total glycosaminoglycan content was unchanged. However, proteoglycans generated by mesangial cells grown in elevated glucose media were of a lower negative charge than controls. In mesangial cells continuously grown on filters, the levels of messenger RNA for collagen types I and IV, biglycan and TGF-β were not different in cells grown at elevated or standard glucose concentrations for two and four weeks. We conclude that crosslinking of mesangial matrix or continuous culture of cells for prolonged periods of time in high glucose medium, which may also crosslink matrix, suppresses collagen synthesis and reduces the negative charges on matrix proteoglycans without altering mRNA levels.

Expression of decorin, biglycan, and collagen type I in human renal fibrosing disease. Bacκound The extracellular matrix proteoglycans decorin and biglycan may have a pathogenic role in renal fibrosing disease via regulation of the activity of growth factors, such as transforming growth factor-β;, and effects on collagen type I fibrillogenesis. The expression of decorin and biglycan in human glomerular diseases characterized by mesangial sclerosis is unknown. Methods Decorin, biglycan, and collagen type I were localized immunohistochemically in human renal biopsy cases of amyloidosis ( N = 18), diabetic nephropathy ( N = 11), fibrillary glomerulonephritis ( N = 5), immunotactoid glomerulopathy ( N = 5), light-chain deposition disease ( N = 4), idiopathic mesangial sclerosis ( N = 4), and nephrosclerosis ( N = 6), and in morphologically normal tissues obtained from tumor nephrectomies ( N = 8). Decorin and biglycan mRNA synthesis was evaluated by in situ hybridization. Results Decorin and biglycan protein were not identified in normal glomeruli. Decorin accumulated in amyloid deposits, but not in deposits of fibrillary glomerulonephritis or immunotactoid glomerulopathy. Biglycan weakly accumulated in amyloid deposits, and both decorin and biglycan weakly stained mesangial nodules in cases of morphologically advanced light-chain deposition disease and diabetic nephropathy. In all analyzed cases, irrespective of the underlying disease, decorin and biglycan accumulated in glomeruli in areas of fibrous organization of the urinary space and in areas of tubulointerstitial fibrosis. Biglycan, but not decorin, accumulated in the neointima of arteriosclerotic blood vessels. Decorin and biglycan mRNA synthesis was detected at sites of proteoglycan accumulation in glomeruli, interstitium, and neointima. Collagen type I colocalized with decorin and biglycan deposits. Conclusions Differences in extracellular matrix proteoglycan composition may be diagnostically useful in distinguishing morphologically similar diseases. Distinct patterns of proteoglycan expression may be related to modulation of specific growth factor activity in different glomerular diseases.

Sirolimus increases transforming growth factor-β1 expression and potentiates chronic cyclosporine nephrotoxicity.BackgroundSirolimus (SRL) is increasingly being used to decrease cyclosporine (CsA) exposure. SRL is not known to be nephrotoxic and has a mechanism of action distinct from CsA. We investigated the effect of combining CsA and SRL on renal structure and function and on transforming growth factor-β1 (TGF-β1) and extracellular matrix (ECM) proteins in a model of chronic CsA nephrotoxicity.MethodsRats treated with vehicle, SRL 0.3mg/kg/day, CsA 5 or 10mg/kg/day, or CsA5+SRL were sacrificed at 7 or 28days. Physiologic and histologic changes were studied in addition to TGF-β1 mRNA and protein expressions, and mRNA expression of plasminogen activator inhibitor-1 (PAI-1) and ECM proteins biglycan and types I and IV collagen.ResultsWhile SRL alone did not alter renal function and structure, it potentiated the nephrotoxic actions of CsA when used in combination with low-dose CsA5 and resulted in significant changes similar to high-dose CsA10. In addition, SRL alone increased TGF-β1 by 44% to 49% (P < 0.05 vs. VH). When used in combination with low-dose CsA5, SRL potentiated TGF-β1 mRNA and protein by 121% and 176%, respectively (P < 0.05 vs. VH and CsA5), to levels achieved with high-dose CsA10. The expression of the ECM proteins followed that of TGF-β1; a similar effect was observed with PAI-1, suggesting a decrease in ECM degradation.ConclusionBecause SRL augments nephrotoxicity, caution should be exercised when it is used in combination with CsA. More studies are needed to determine the long-term clinical impact of SRL on nephrotoxicity and allograft function.