Supplementary MaterialsSuppl Fig1: Shape S1. to shear (A). Removal of syndecan-4 was confirmed using cell surface immunofluorescence (t test = 0.0073; N = 6 controls; N = 14 enzyme treated). Fold-change in in response to shear stress normalized to matched controls confirmed that enzymatic damage to the glycocalyx was associated with a significant impairment in cells ability to up-regulate in response to shear stress (paired t test; N = 7; = 0.045) (B). Significance relative to control. All error bars = SEM. * 0.05; ** 0.01; *** 0.005; **** 0.001. The checkmark indicates glycocalyx degrading enzymes with 2 hours of pretreatment and ongoing exposure during shear stress. GNF 2 Enzymes used were as follows: hyaluronidase (2.5 mg/ml), chondroitinase (1 mu/ml), and neuraminidase (1 mu/ml). X indicates enzyme used was vehicle only. NIHMS1018273-supplement-Suppl_Fig2.docx (62K) GUID:?87D19CEB-6B32-4D31-B2CB-E5E428AB49EE Suppl Fig3: Figure S3. Heparanase activity increased in glomeruli and urine but not in conditionally immortalized human glomerular endothelial cells (CiGEnCs) after salt and aldosterone exposure. Urine heparanase activity (normalized to urine creatinine) increased after 28 days of salt and aldosterone (t test; = 0.0012; n = 5) (A). Glomerular lysate heparanase activity increased GNF 2 after 28 days of salt and aldosterone (t test; = 0.0059; n = 6) (B). Conditioned media from CiGEnCs exposed to salt and aldosterone for 5 days remained equivalent to control amounts (C). Batimastat got no measurable influence on heparanase activity. NIHMS1018273-supplement-Suppl_Fig3.docx (315K) GUID:?5FA44003-C1D1-4432-AADE-37851BAD13A1 Suppl Fig4: Shape S4. Analysis from the glomerular purification hurdle using electron microscopy verified that glomerular cellar membrane (GBM) width and podocyte feet process width hadn’t altered. Consultant electron micrograph from the purification barrier of the control mouse. The white arrow indicates the glycocalyx tagged with Alcian Blue. Pub = 500 nmol/l (A). Representative electron micrograph from the filtration barrier of the mouse subjected to 28 times of aldosterone and salt. The white arrow indicates the glycocalyx tagged with Alcian Blue. Pub = 500 nmol/l (B). Glomeruli from 4 control mice and 3 mice subjected to sodium and RASGRP1 aldosterone had been examined (total, 20 glomeruli; 60 capillary loops). No factor in GBM width was recognized (t GNF 2 check = 0.72; N = 4+3) (C). Podocyte feet process width didn’t increase considerably (t check = 0.31) (D). Fenestration denseness didn’t alter considerably (t check = 0.7) (E). Checkmark shows sodium + aldosterone: 1% NaCl in normal water (= 0.0011; Tukey modification shown; = 4) n. (c) Syndecan-4 surface area manifestation on CiGEnCs reduced after 5 times of contact with sodium and aldosterone (evaluation of variance = 0.0021; Tukey modification demonstrated; n = 5). (d) Heparan sulfate (HS) surface area manifestation on CiGEnC decreased after 5 days of exposure to 145 mmol/l NaCl and combined 145 mmol/l NaCl and 0.1 nmol/l aldosterone exposure (n = 5) (analysis of variance = 0.012; Tukey correction shown; n GNF 2 = 5). Salt row: the checkmark indicates the NaCl concentration increased to 145 mmol; the X indicates mannitol was added to balance osmolarity. Aldosterone row: the checkmark indicates 0.1 nmol/l aldosterone was added to media; the X indicates vehicle alone was added to media. Spironolactone (Spiro) row: the checkmark indicates 0.1 m spironolactone was added to media; the X indicates vehicle alone was added to media. Significance was relative to control unless indicated. All error bars = SEM. * 0.05; ** 0.01; *** 0.005. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Exposing CiGEnC to salt and aldosterone resulted in matrix metalloproteinase up-regulation Matrix metalloproteinase (MMP) inhibition and MR inhibition were equally effective in preserving the glycocalyx. MMP2 and MMP9 cleave syndecan-4 from the endothelial cell surface and have been implicated in glomerular disease.32 We have shown that MMPs are important in glycocalyx shedding in response to tumor necrosis factor-.33 We therefore conducted time-course experiments to study changes at the mRNA level for these key enzymes. Under iso-osmotic conditions, 145 mmol/l NaCl and 0.1 nmol/l aldosterone for 10 hours increased MMP2 and MMP9 mRNA expression significantly (Determine 2a). A total of 145 mmol/l NaCl and 0.1 nmol/l aldosterone exposure for 5 days also significantly increased MMP2 activity in conditioned cell media. This effect was prevented by MR antagonism with spironolactone (Physique 2b). HS and syndecan-4 loss from the CiGEnC glycocalyx was prevented by MR antagonism or MMP inhibition with batimastat. The effects on glycocalyx preservation of these 2 remote drug classes were equivalent; suggesting that this MMP inhibition may GNF 2 be effective in preserving the glycocalyx from MR-mediated damage. Open in a.