Effect of castration on renal glycosaminoglycans and their urinary excretion in male and female rats with chronic renal failure

Glycosaminoglycans (GAGs) participate in a variety of processes in the kidney,and evidence suggests that gender-related hormones participate in renalfunction. The aim of this study was to analyze the relationship of GAGs, gender,and proteinuria in male and female rats with chronic renal failure (CRF). GAGswere analyzed in total kidney tissue and 24-h urine of castrated (c), male (M),and female (F) Wistar control (C) rats (CM, CMc, CF, CFc) and after 30 days ofCRF induced by 5/6 nephrectomy (CRFM, CRFMc, CRFF, CRFFc). Total GAGquantification and composition were determined using agarose and polyacrylamidegel electrophoresis, respectively. Renal GAGs were higher in CF compared to CM.CRFM presented an increase in renal GAGs, heparan sulfate (HS), and proteinuria,while castration reduced these parameters. However, CRFF and CRFFc groups showeda decrease in renal GAGs concomitant with an increase in proteinuria. Ourresults suggest that, in CRFM, sex hormones quantitatively alter GAGs, mainlyHS, and possibly the glomerular filtration barrier, leading to proteinuria. Thelack of this response in CRFMc, where HS did not increase, corroborates thistheory. This pattern was not observed in females. Further studies of CRF areneeded to clarify gender-dependent differences in HS synthesis.     

Chondroitin-6-sulfate attenuates inflammatory responses in murine macrophages via suppression of NF-κB nuclear translocation

Inflammation is a host protective response to noxious stimuli, and excessive production of pro-inflammatory mediators by macrophages (mφ) can lead to numerous pathological conditions. In this study, immunomodulatory effects of immobilized and soluble glycosaminoglycans (GAGs) on mouse-bone-marrow-derived mφ were compared by measuring nitric oxide (NO). We demonstrate here that all GAGs studied except for heparin were able to modulate interferon-γ/lipopolysaccharide (IFN-γ/LPS)-induced NO release by mφ to varying extents after 24 h of incubation. In particular, the modulatory activities of soluble chondroitin-6-sulfate (C6S), hyaluronic acid and heparan sulfate altered markedly after covalent immobilization. Of these, soluble C6S exhibited the strongest NO inhibitory activity, and the inhibition was dose- and time-dependent. Moreover, C6S significantly reduced pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α production by IFN-γ/LPS- or LPS-activated mφ. Specifically, the C6S-mediated suppression of mφ pro-inflammatory phenotype was accompanied by an increase in the IL-10 level, suggesting a possible switch towards anti-inflammatory/wound healing M2 state. In addition, the highest magnitude of inhibitory effects was obtained when cells were pre-treated with C6S prior to IFN-γ/LPS or LPS challenge, suggesting an additional role for C6S in protection against microbial infection. Further investigations reveal that the anti-inflammatory effects of C6S on activated mφ may be ascribed at least in part to suppression of NF-κB nuclear translocation.     

Extracellular matrix and wound healing

Extracellular matrix has been known for a long time as an architectural support for the tissues. Many recent data, however, have shown that extracellular matrix macromolecules (collagens, elastin, glycosaminoglycans, proteoglycans and connective tissue glycoproteins) are able to regulate many important cell functions, such as proliferation, migration, protein synthesis or degradation, apoptosis, etc., making them able to play an important role in the wound repair process. Not only the intact macromolecules but some of their specific domains, that we called “Matrikines”, are also able to regulate many cell activities. In this article, we will summarize main findings showing the effects of extracellular matrix macromolecules and matrikines on connective tissue and epithelial cells, particularly in skin, and their potential implication in the wound healing process. These examples show that extracellular matrix macromolecules or some of their specific domains may play a major role in wound healing. Better knowledge of these interactions may suggest new therapeutic targets in wound healing defects.     

Optical imaging of articular cartilage degeneration using near-infrared dipicolylamine probes

Articular cartilage is the hydrated tissue that lines the ends of long bones in load bearing joints and provides joints with a smooth, nearly frictionless gliding surface. However, the deterioration of articular cartilage occurs in the early stages of osteoarthritis (OA) and is clinically and radiographically silent. Here two cationic near infrared fluorescent (NIRF) dipicolylamine (DPA) probes, Cy5-DPA-Zn and Cy7-DPA-Zn, were prepared for cartilage degeneration imaging and OA early detection through binding to the anionic glycosaminoglycans (GAGs). The feasibility of NIRF dye labeled DPA-Zn probes for cartilage degeneration imaging was examined ex vivo and in vivo. The ex vivo studies showed that Cy5-DPA-Zn and Cy7-DPA-Zn not only showed the high uptake and electrostatic attractive binding to cartilage, but also sensitively reflected the change of GAGs contents. In vivo imaging study further indicated that Cy5-DPA-Zn demonstrated higher uptake and retention in young mice (high GAGs) than old mice (low GAGs) when administrated via local injection in mouse knee joints. More importantly, Cy5-DPA-Zn showed dramatic higher signals in sham joint (high GAGs) than OA side (low GAGs), through sensitive reflecting the change of GAGs in the surgical induced OA models. In summary, Cy5-DPA-Zn provides promising visual detection for early cartilage pathological degeneration in living subjects.     

Glycosaminoglycan antigens in peripheral nerve: Studies with antibodies from a patient with neuropathy and monoclonal gammopathy

An immunoblot staining procedure was developed for the detection of antibody binding to glycosaminoglycans (GAGs). The method was used to study the binding of a human monoclonal antibody (M-protein) from a patient with peripheral neuropathy, previously found to react with proteoglycans (PGs). GAGs prepared from human peripheral nerve were separated by electrophoresis on cellulose acetate membranes, transferred onto nitrocellulose sheets, and immunostained with the M-protein. The M-protein bound to a single GAG band with intermediate mobility which eluted with 1.25 N NaCl on ion-exchange chromatography and was chondroitinase sensitive. The M-protein appeared to bind to chondroitin sulfate containing proteoglycans in peripheral nerve.     

Clomerular and urinary heparan sulphate in congenital nephrotic syndrome

Studies using cationic probes have suggested that a reduction in glomerular anionic sites, composed principally of the glycosaminoglycan heparan sulphate, is responsible for the abnormal glomerular permeability in the congenital nephrotic syndrome (CNS). We therefore analysed the glycosaminoglycan content of the glomerular basement membrane (GBM) from an infant who died of CNS and from an infant who died of unrelated causes. We also measured the urinary excretion of glycosaminoglycans in children with nephrotic syndrome, both congenital and acquired, and in healthy children. Heparan sulphate constituted 59% of the glycosaminoglycan content of the GBM in the normal infant, the other principal glycosaminoglycan being chondroitin sulphate. In the GBM from the infant with CNS the heparan sulphate was greatly reduced, constituting only 3% of total glycosaminoglycans. The urinary excretion of heparan sulphate was significantly increased in CNS (expressed both in relation to creatinine and to chondroitin sulphate) compared with normal children and to those with acquired nephrotic syndrome. Diminished GBM content of heparan sulphate may be responsible for the abnormal glomerular permeability in CNS and may be a consequence of defective incorporation of heparan sulphate into the GBM with subsequent loss into the urine.     

Los glicosaminoglicanos se encuentran implicados en la adherencia de Candida albicans y Malassezia spp. a queratinocitos,pero no a fibroblastos dérmicos

Background and objectiveSuperficial mycoses are some of the most common diseases worldwide. The usual culprits — yeasts belonging to the genera Malassezia and Candida — are commensal species in the skin that can cause opportunistic infections. We aimed to determine whether these yeasts use glycosaminoglycans (GAGs) as adhesion receptors to mediate binding to epithelial cells.Material and methodsIn keratinocyte and dermal fibroblast cultures, we used rhodamine B and genistein to inhibit GAG synthesis to study the role these molecules play in the adhesion of Candida albicans (C. albicans) and Malassezia species to cells. We also analyzed GAG involvement by means of enzyme digestion, using specific lyases.ResultsRhodamine B partially inhibited the adhesion of both fungi to keratinocytes but not to fibroblasts. Selective digestion of heparan sulfate enhanced the binding of Malassezia species to keratinocytes and of both fungi to fibroblasts. Chondroitin sulfate digestion decreased C. albicans adhesion to keratinocytes, but increased the adhesion of the filamentous forms of this species to fibroblasts.ConclusionsCell surface GAGs appear to play a role in the adhesion of C albicans and Malasezzia species to keratinocytes. In contrast, their adhesion to fibroblasts appears to be enhanced by GAG inhibition, suggesting that some other type of receptor is the mediator.