Journal: Kidney International

Article Title: Blocking matrix metalloproteinase-mediated syndecan-4 shedding restores the endothelial glycocalyx and glomerular filtration barrier function in early diabetic kidney disease

doi: 10.1016/j.kint.2019.09.035

Figure Lengend Snippet: Syndecan-4 ( S dc 4 ) mRNA and protein are altered in early diabetic kidney disease (DKD). Glomerular endothelial cells (GECs) were sorted by fluorescence-activated cell sorting, and ( a ) S dc 4 and ( b ) S dc 1 mRNA expression were determined. The 2 −ΔΔCT method of quantification was used to calculate the fold change, normalized to glyceraldehyde-3-phosphate dehydrogenase, at week 8 post-streptozotocin (STZ), ( S dc 4 : control, 1.000 ± 0.2565, n = 9 mice; diabetes, 2.470 ± 0.6580, n = 8 mice; * P < 0.05; S dc 1 : control, 1.000 ± 0.1112, n = 5 mice; diabetes, 1.663 ± 0.4289, n = 5 mice; nonsignificant [NS]). Control and diabetic kidney sections were labeled with SDC4 and endothelial membrane label R18. ( c ) Peak-to-peak assessment of the glomerular endothelial glycocalyx using SDC4 labeling at week 9 post-STZ showed a significant reduction in glycocalyx SDC4 in diabetes (control, 182.5 ± 12.04, n = 5 mice; diabetes, 64.31 ± 6.513, n = 5 mice; *** P < 0.0001). Isolated glomerular lysates from control and diabetic mice were used to determine ( d ) SDC4 and ( e ) SDC1 concentration using SDC4 and SDC1 ectodomain enzyme-linked immunosorbent assay (ELISA). The data were then normalized to total protein content. SDC4 (control, 1.327 ± 0.1439, n = 6 mice; diabetes, 0.8059 ± 0.1218, n = 6 mice; * P < 0.05); SDC1 (control, 9.671 ± 1.742, n = 6 mice; diabetes, 9.699 ± 1.313, n = 6 mice; NS) at week 8 post-STZ. SDC4 shedding in the ( f ) plasma and ( g ) urine was determined in DKD using the SDC4 ectodomain ELISA at week 8 post-STZ. For plasma: control, 2.631 ± 0.2860, n = 6 mice; diabetes, 4.801 ± 0.3793, n = 6 mice; ** P < 0.005. Urine SDC4 was normalized to creatinine (control, 3.343 ± 0.9410, n = 4 mice; diabetes, 39.32 ± 13.03, n = 4 mice; * P < 0.05; Mann-Whitney test at week 8 post-STZ). Each dot or square on the graph represents a mouse. Data are expressed as the mean ± SEM, and unpaired Student t test was used for statistical analysis unless specified.

Article Snippet: The concentrations of SDC4 (ectodomain) in mouse glomerular lysate, plasma, and urine were quantified using a sandwich enzyme immunoassay (mouse SDC4 ELISA kit, CSB-EL020891MO; Cusabio, Houston, TX) according to the manufacturer’s instructions.

Techniques: Fluorescence, FACS, Expressing, Control, Labeling, Membrane, Isolation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, MANN-WHITNEY

Journal: Kidney International

Article Title: Blocking matrix metalloproteinase-mediated syndecan-4 shedding restores the endothelial glycocalyx and glomerular filtration barrier function in early diabetic kidney disease

doi: 10.1016/j.kint.2019.09.035

Figure Lengend Snippet: Blockade of matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9) ameliorates diabetes-induced changes in syndecan-4 (SDC4) and reduces plasma MMP activity. Diabetic (Dia) + MMP2 and MMP9 inhibitor (MMPI) or vehicle (Veh)-treated kidney sections were labeled with SDC4 and endothelial membrane label R18. ( a ) Peak-to-peak assessment of the glomerular endothelial glycocalyx (eGLX) using SDC4 labeling at week 9 post-streptozotocin showed a significant restoration in glycocalyx thickness (Dia Veh, 109.2 ± 9.003, n = 5 mice; Dia MMPI, 246.0 ± 22.36, n = 5 mice; *** P = 0.0005). ( b ) Isolated glomerular lysate from diabetic + MMPI or vehicle-treated mice was used to determine S dc 4 mRNA expression. The 2 −ΔΔCT method of quantification was used to calculate the fold change, normalized to glyceraldehyde-3-phosphate dehydrogenase and relative to Dia Veh (Dia Veh, 1.000 ± 0.089, n = 11 mice; Dia MMPI, 0.6658 ± 0.04849, n = 12 mice; ** P = 0.0029). ( c ) Isolated glomerular lysate from diabetic + MMPI or vehicle-treated mice was used to determine SDC4 concentration with previously used SDC4 ectodomain enzyme-linked immunosorbent assay. The data were then normalized to total protein content (Dia Veh, 0.6426 ± 0.06780, n = 5 mice; Dia MMPI, 1.150 ± 0.1634, n = 7 mice; * P = 0.0322). MMPI attenuated SDC4 shedding in the ( d ) plasma (Dia Veh, 1.000 ± 0.1029, n = 11 mice; Dia MMPI, 0.5449 ± 0.09749, n = 9 mice; ** P = 0.0054), but not in the ( e ) urine (Dia Veh, 1.000 ± 0.1895, n = 13 mice; Dia MMPI, 0.8501 ± 0.1290, n = 8 mice; nonsignificant [NS]) in diabetic kidney disease. The fold change of diabetic MMPI relative to diabetic vehicle was calculated to enable pooling of results from different experiments. ( f,g ) Plasma MMP2 (Dia Veh, 3.001 ± 0.3977, n = 13 mice; Dia MMPI, 1.528 ± 0.4924, n = 7 mice; * P = 0.0366) and MMP9 (Dia Veh, 1.150 ± 0.07157, n = 9 mice; Dia MMPI, 0.9266 ± 0.03281, n = 6 mice; * P = 0.0314) activities, using MMP2 and MMP9 Biotrak Activity Assays (GE Healthcare Life Sciences, Buckinghamshire, UK), were reduced in the diabetic + MMPI group when compared with the diabetic + vehicle group. Each dot or square on the graph represents a mouse. Data are expressed as the mean ± SEM, and unpaired Student t test at week 9 post-streptozotocin was used for statistical analysis unless specified.

Article Snippet: The concentrations of SDC4 (ectodomain) in mouse glomerular lysate, plasma, and urine were quantified using a sandwich enzyme immunoassay (mouse SDC4 ELISA kit, CSB-EL020891MO; Cusabio, Houston, TX) according to the manufacturer’s instructions.

Techniques: Clinical Proteomics, Activity Assay, Labeling, Membrane, Isolation, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay