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The dysregulation of the metabolism of glycosaminoglycan and protein components of extracellular matrix (ECM) is a typical feature of diabetic complications. High glucose-induced enrichment of ECM with hyaluronan (HA) not only affects tissue structural integrity, but influences cell metabolic response due to the variety of effects depending on the HA polymer molecular weight. TSP-1-dependent activation of TGFβ1 axis is known to mediate numerous matrix disorders in diabetes, but its role concerning HA has not been studied so far. In this work we demonstrated that 30 mM d-glucose increased the incorporation of [3H]glucosamine in high-molecular-weight ( > 2000 kDa) HA of medium and matrix compartments of human mesangial cultures. Simultaneously, the synthesis of HA with lower molecular weight and HA degradation were not altered. The cause of the increased high-molecular-weight HA synthesis consisted in the up-regulation of hyaluronan synthase (HAS) 2 mRNA without alterations of the expression of HAS3, which generates HA of lower molecular weight. d-Glucose at 30 mM also stimulated the production of transforming growth factor β1 (TGFβ1), the excessive activation of which was determined by the up-regulation of thrombospondin-1 (TSP-1). The blockage of TGFβ1 action either by neutralizing anti-TGFβ1 antibodies or by quenching the TGFβ1 activation (with TSP-1-derived synthetic GGWSHW peptide) abolished the effect of high glucose on HAS2 mRNA expression and normalized the synthesis of HA. Exogenous human TGFβ1 had the same effect on HAS2 expression and HA synthesis as high glucose treatment. Therefore, we supposed that TSP-1-dependent TGFβ1 activation is involved in the observed high glucose effect on HA metabolism. Since high-molecular-weight HA polymers, unlike middle- and low-molecular weight HA oligosaccharides, are known to possess anti-inflammatory and anti-fibrotic functions, we suppose that the enrichment of mesangial matrix with high-molecular-weight HA may represent an endogenous mechanism to limit renal injury in diabetes.
Objectives: There is a need to replace liver biopsy with non-invasive markers that predict the degree of liver fibrosis in fatty liver disease related to obesity. Therefore, we studied four potential serum markers of liver fibrosis and compared them with histopathological findings in liver biopsy in children with non-alcoholic fatty liver disease (NAFLD). Methods: We determined fasting serum level of hyaluronic acid (HA), laminin, YKL-40 and cytokeratin-18 M30 in 52 children (age range 4-19, mean 12 years, 80 % of them were overweight or obese) with biopsy-verified NAFLD. Viral hepatitis, autoimmune and metabolic liver diseases (Wilson's disease, alpha-1-antitrypsin deficiency, cystic fibrosis) were excluded. Fibrosis stage was assessed in a blinded fashion by one pathologist according to Kleiner. Receiver operating characteristics (ROC) analysis was used to calculate the power of the assays to detect liver fibrosis (AccuROC, Canada). Results: Liver fibrosis was diagnosed in 19 children (37 %). The levels of HA and CK18M30 were significantly higher in children with fibrosis compared to children without fibrosis (p=0.04 and 0.05 respectively). The ability of serum HA (cut-off 19.1 ng/ml, Se=84 %, Sp=55 %, PPV=52 %, NPV=86 %) and CK18M30 (cut-off 210 u/l, Se=79 %, Sp=60 %, PPV=56 %, NPV=82 %) to differentiate children with fibrosis from those without fibrosis was significant (AUC=0.672 and 0.666, respectively). The combination of both markers was superior (AUC=0.73, p=0.002). Laminin and YKL-40 levels did not allow a useful prediction. Conclusions: Cytokeratin-18 and hyaluronic acid are suitable serum markers predicting liver fibrosis in children with NAFLD. Studying these markers may identify patients at risk of disease progression.
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