Journal: bioRxiv

Article Title: Proteolytic Cleavage by Matriptase Exacerbating Kidney Injury: a Novel Therapeutic Target

doi: 10.1101/843607

Figure Lengend Snippet: (A) Microarray was performed with glomerular RNA of control mice (N=3), and ADR nephropathy mice at 2 weeks (N=3) and at 6 weeks (N=3). Statistical significance (p<0.05) was calculated using moderated student’s t-test followed by Benjamini-Hochberg false discovery rate correction on GeneSpring GX. Fold change cut-off of > 2 lead to extract 357 probes in ADR nephropathy mice at 6 weeks compared with control mice. Among those genes, 15 genes are categorized as serine protease while only Matriptase is a type II membrane anchored serine protease. Heatmap shows that all serine proteases are upregulated at 42 days in glomerulus of mice with ADR nephropathy. (B) Among 7 probes, which are categorized as membrane anchored serine proteinase and its cognate inhibitors, only Matriptase and HAI-1 are significantly upregulated in ADR-nephropathy mice compared with control mice (B, C, D). Hgfac, hepatocyte growth factor activator; Hpn, hepsin; Prss8, serine protease 8

Article Snippet: 15 ng/mL Recombinant Mouse Matriptase (R&D Systems) and the substrate were incubated in assay buffer including 50 mM Tris-HCL (pH 8.5), 50 mM NaCl and 0.01% Tween20 for 2hr.

Techniques: Microarray, Membrane

Journal: bioRxiv

Article Title: Proteolytic Cleavage by Matriptase Exacerbating Kidney Injury: a Novel Therapeutic Target

doi: 10.1101/843607

Figure Lengend Snippet: (A) Immunohistochemistry shows an increase in glomerular Matriptase protein expression, and mRNA expression and protein of Matriptase are significantly higher in glomeruli of ADR-induced nephropathy mice. (B) Glomerular serine protease activity is enhanced in ADR mice compared with control mice. (C) Matriptase (green) is co-localized (yellow) with Nephrin (red) in glomerulus of mouse with ADR nephropathy. (D) Matriptase protein expression is observed in human glomerulus of diabetic nephropathy (DN) and membranous nephropathy (MN) while it is negative in that of IgA nephropathy (IgAN). Bar: 20 μm

Article Snippet: 15 ng/mL Recombinant Mouse Matriptase (R&D Systems) and the substrate were incubated in assay buffer including 50 mM Tris-HCL (pH 8.5), 50 mM NaCl and 0.01% Tween20 for 2hr.

Techniques: Immunohistochemistry, Expressing, Activity Assay

Journal: bioRxiv

Article Title: Proteolytic Cleavage by Matriptase Exacerbating Kidney Injury: a Novel Therapeutic Target

doi: 10.1101/843607

Figure Lengend Snippet: (A) IC 50 of IN-1 for Matriptase activity is 1.3 nM in a protease activity assay whereas that of Nafamostat mesilate (NM) is 0.86 nM. (B) IC 50 of IN-1 for Thrombin activity is 2.8 μM whereas that of NM is 99 nM. (C) Urinary albumin/creatinine ratio (ACR) is higher in mice with ADR nephropathy, but it is significantly suppressed by the injection of 10 mg/kg IN-1 three times a week on day 28 and on day 42. (D) Glomerular sclerosis is higher in mice with ADR nephropathy compared to normal control mice. However, it is significantly reduced by IN-1 treatment on day 42. (E, F) Podocin positive area and the number of WT positive cells in glomerulus are significantly lower by ADR treatment, but such detrimental effect of ADR is slightly but significantly ameliorated by the chronic treatment with IN-1 on day 42.

Article Snippet: 15 ng/mL Recombinant Mouse Matriptase (R&D Systems) and the substrate were incubated in assay buffer including 50 mM Tris-HCL (pH 8.5), 50 mM NaCl and 0.01% Tween20 for 2hr.

Techniques: Activity Assay, Injection

Journal: bioRxiv

Article Title: Proteolytic Cleavage by Matriptase Exacerbating Kidney Injury: a Novel Therapeutic Target

doi: 10.1101/843607

Figure Lengend Snippet: (A) While wild type (WT) of Matriptase cleaves Podocin and produces its small fragment, the inactive form of Matriptase (G827R) fails to do it in HEK293 cells overexpressing mPodocin-FLAG and either WT-Matriptase or G827R Matriptase mutant. (B) Matriptase-induced Podocin cleavage occurs at the membrane fraction, but not in the cytoplasm of HEK293 cells overexpressing Matriptase-His and mPodocin-FLAG. (C) Matriptase cleavage of Podocin R50A mutant, as opposed to wild type, R45A and R54A mutants, is declined using in vitro cleavage assay. (D) Full length of HAI-1 or secreted type of HAI-1 (deleted transmembrane region (dTM)) blocks the cleavage of Podocin in response to Matriptase in HEK293 cells. (E) N-terminal fragment of human Podocin tagged with 3xFLAG is translocated into nucleus, particularly in nucleoli of U2OS cells.

Article Snippet: 15 ng/mL Recombinant Mouse Matriptase (R&D Systems) and the substrate were incubated in assay buffer including 50 mM Tris-HCL (pH 8.5), 50 mM NaCl and 0.01% Tween20 for 2hr.

Techniques: Mutagenesis, Membrane, In Vitro, Cleavage Assay