Inflammatory mediatorsCLP significantly increased renal transcription of the proinflammatory cytokines TNF-a, IL-1b, and IL-6 (Fig. 5A). In mice pretreated (2 h) with NAH, no statistically GSK1363089MedChemExpress Foretinib significant increase in the expression of these mediators occurred 4 h after CLP, suggesting that heparanase inhibition attenuates renal inflammatory gene expression (in the absence of neutrophilic infiltration, Fig. 3A). Interestingly, urinary IL-6 was not altered by NAH (Fig. 5B), indicating that (at least early in sepsis) urine IL-6 may reflect circulating levels of this cytokine as opposed to renal production. We next sought to determine the mechanism by which heparanase inhibition suppressed renal inflammatory geneexpression. Once fragmented, glycosaminoglycans (such as hyaluronic acid) may function as a proinflammatory damage-associated molecular pattern (Jiang et al. 2005). We sought to determine if HS fragmentation similarly induced activation of cellular inflammatory responses, thereby explaining the renal-protective effects of heparanase inhibition. Interestingly, both full-length and heparinase-III-treated HS suppressed inflammatory gene expression in endothelial monolayers (Fig. 5C). These findings, which contrast the inflammatory phenotype of low-weight hyaluronic fpsyg.2016.01503 acid, are consistent with previous observations suggesting that heparanase-degraded HS suppresses inflammatory responses (Lider et al. 1995).NAH does not influence renal NO or angiotensin II during early sepsisHS degradation may alter endothelial NO signaling (Florian et al. 2003), potentially altering glomerular arteriolar tone and thus glomerular filtration. We sought to measure renal NO production 4 h after CLP and to determine if this production was altered by antecedent NAH treatment. CLP was not associated with a significant change in urine nitrate and nitrite (NOx, Fig. 6A) species,2013 | Vol. 1 | Iss. 6 | e00153 Page?2013 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.M. I. Lygizos et al.Heparanase Mediates Early Septic Renal DysfunctionABCDEFGFigure 3. Early septic renal dysfunction occurs in the absence of renal neutrophilic inflammation, renal vascular leak, or systemic hypotension. (A) Hematoxylin and eosin staining of kidneys harvested 4 h after cecal ligation and puncture (CLP) or sham surgery revealed no evidence of acute tubular necrosis or neutrophilic inflammation. Images representative of five mice per group. Scale bar: 100 lm. (B) The absence of neutrophilic renal inflammation 4 h after CLP was confirmed via immunofluorescence for the neutrophil marker Ly-6B.2 (n = 7 mice per group). Lungs harvested from mice 2 h after intravenous lipopolysaccharide (LPS) (20 lg/g body weight) serve as a positive control for neutrophil Ixazomib citrateMedChemExpress MLN9708 infiltration (n = 4 mice per group). There was no evidence of renal vascular hyperpermeability 4 h after CLP, as demonstrated by unchanged (C) urine protein/creatinine ratios (n = 5 per group), (D) kidney wet ry ratios (n = 7? per group), and (E) kidney Evans blue dye (EBD)-labeled albumin extravasation (n = 4? per group). (F) Left ventricular systolic pressures and (G) heart rates (measured via direct left ventricular puncture in anesthetized, ventilated, open-chest mice) were consistent with hemodynamically compensated, hyperdynamic sepsis 4 h after CLP. *P < 0.05 in (B ).similar to observations made in rats 24 h after CLP (Xie et al. 2008).Inflammatory mediatorsCLP significantly increased renal transcription of the proinflammatory cytokines TNF-a, IL-1b, and IL-6 (Fig. 5A). In mice pretreated (2 h) with NAH, no statistically significant increase in the expression of these mediators occurred 4 h after CLP, suggesting that heparanase inhibition attenuates renal inflammatory gene expression (in the absence of neutrophilic infiltration, Fig. 3A). Interestingly, urinary IL-6 was not altered by NAH (Fig. 5B), indicating that (at least early in sepsis) urine IL-6 may reflect circulating levels of this cytokine as opposed to renal production. We next sought to determine the mechanism by which heparanase inhibition suppressed renal inflammatory geneexpression. Once fragmented, glycosaminoglycans (such as hyaluronic acid) may function as a proinflammatory damage-associated molecular pattern (Jiang et al. 2005). We sought to determine if HS fragmentation similarly induced activation of cellular inflammatory responses, thereby explaining the renal-protective effects of heparanase inhibition. Interestingly, both full-length and heparinase-III-treated HS suppressed inflammatory gene expression in endothelial monolayers (Fig. 5C). These findings, which contrast the inflammatory phenotype of low-weight hyaluronic fpsyg.2016.01503 acid, are consistent with previous observations suggesting that heparanase-degraded HS suppresses inflammatory responses (Lider et al. 1995).NAH does not influence renal NO or angiotensin II during early sepsisHS degradation may alter endothelial NO signaling (Florian et al. 2003), potentially altering glomerular arteriolar tone and thus glomerular filtration. We sought to measure renal NO production 4 h after CLP and to determine if this production was altered by antecedent NAH treatment. CLP was not associated with a significant change in urine nitrate and nitrite (NOx, Fig. 6A) species,2013 | Vol. 1 | Iss. 6 | e00153 Page?2013 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.M. I. Lygizos et al.Heparanase Mediates Early Septic Renal DysfunctionABCDEFGFigure 3. Early septic renal dysfunction occurs in the absence of renal neutrophilic inflammation, renal vascular leak, or systemic hypotension. (A) Hematoxylin and eosin staining of kidneys harvested 4 h after cecal ligation and puncture (CLP) or sham surgery revealed no evidence of acute tubular necrosis or neutrophilic inflammation. Images representative of five mice per group. Scale bar: 100 lm. (B) The absence of neutrophilic renal inflammation 4 h after CLP was confirmed via immunofluorescence for the neutrophil marker Ly-6B.2 (n = 7 mice per group). Lungs harvested from mice 2 h after intravenous lipopolysaccharide (LPS) (20 lg/g body weight) serve as a positive control for neutrophil infiltration (n = 4 mice per group). There was no evidence of renal vascular hyperpermeability 4 h after CLP, as demonstrated by unchanged (C) urine protein/creatinine ratios (n = 5 per group), (D) kidney wet ry ratios (n = 7? per group), and (E) kidney Evans blue dye (EBD)-labeled albumin extravasation (n = 4? per group). (F) Left ventricular systolic pressures and (G) heart rates (measured via direct left ventricular puncture in anesthetized, ventilated, open-chest mice) were consistent with hemodynamically compensated, hyperdynamic sepsis 4 h after CLP. *P < 0.05 in (B ).similar to observations made in rats 24 h after CLP (Xie et al. 2008).