Journal: Frontiers in Immunology

Article Title: The Long Pentraxin PTX3 Is an Endogenous Inhibitor of Hyperoxaluria-Related Nephrocalcinosis and Chronic Kidney Disease

doi: 10.3389/fimmu.2018.02173

Figure Lengend Snippet: Recombinant human PTX3 inhibits supersaturation-induced CaOx crystal aggregation. (A) Calcium oxalate crystals were generated in chemico in the presence of increasing concentrations of rhPTX3 and crystal size was measured microscopically 24 h later. (B) The effect of 1 μM rhPTX3 on microscopically measured calcium oxalate crystal size was compared to equimolar bovine serum albumin (BSA) and PBS. (C,D) Calcium oxalate crystals generated in the presence of 1 μM BSA or equimolar rhPTX3 were compared for their forward scatter (FSC) signal intensity by flow cytometry and displayed for (C) relative frequency of FSC and (D) overall FSC intensity. Data are from three independent experiments and represent means ± SEM in (A) . n.s., not significant; * p < 0.05, *** p < 0.001 between groups as indicated.

Article Snippet: The membranes were then incubated overnight at 4°C with a primary antibody for PTX3 (clone 2C3, Hycult Biotechs, Plymouth Meeting, USA) followed by incubation with a secondary rabbit anti-mouse IgG HRP-labeled antibody.

Techniques: Recombinant, Generated, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: The Long Pentraxin PTX3 Is an Endogenous Inhibitor of Hyperoxaluria-Related Nephrocalcinosis and Chronic Kidney Disease

doi: 10.3389/fimmu.2018.02173

Figure Lengend Snippet: Hyperoxaluria-induced nephrocalcinosis is associated with tubular PTX3 secretion. Female PTX3-competent ( Ptx3 +/+ ) and -deficient ( Ptx3 −/− ) animals with 10–12 weeks of age received either control or oxalate chow for 3 weeks ( n = 5). (A) Tubular expression of PTX3 was analyzed by immunohistochemical staining in frozen kidney sections and quantified as positive stained area per section in percent. (B) Detection of PTX3 in whole urine samples by immunoblotting (25 μg protein/lane, upper right). Ponceau Red staining shows major urinary proteins (mainly a2u-globulins, ~20 kDa, lower right) as a loading control. Optical density of the immunoreactive and Ponceau Red stained bands was quantified by ImageJ (NIH, Bethesda, MD). Data are from two independent experiments. n.s., not significant; * p < 0.05, *** p < 0.001 between groups as indicated.

Article Snippet: The membranes were then incubated overnight at 4°C with a primary antibody for PTX3 (clone 2C3, Hycult Biotechs, Plymouth Meeting, USA) followed by incubation with a secondary rabbit anti-mouse IgG HRP-labeled antibody.

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot

Journal: Frontiers in Immunology

Article Title: The Long Pentraxin PTX3 Is an Endogenous Inhibitor of Hyperoxaluria-Related Nephrocalcinosis and Chronic Kidney Disease

doi: 10.3389/fimmu.2018.02173

Figure Lengend Snippet: Lack of PTX3 induces hyperoxaluria-induced nephrocalcinosis in non-susceptible B6;129SV mice. Female PTX3-competent ( Ptx3 +/+ ) and -deficient ( Ptx3 −/− ) animals with 10–12 weeks of age received oxalate chow for 3 weeks ( n = 9). (A) Oxalic acid levels in urine at baseline and after 3 weeks of oxalate chow. (B) Quantification of calcium oxalate crystal deposition was conducted using Pizzolato's method on formalin fixed kidney sections as shown in (C) . (D) Analysis of PTX3 −/− kidneys by X-ray diffraction consistent with calcium-oxalate monohydrate crystals. Data are from four independent experiments. n.s., not significant; * p < 0.05, ** p < 0.01 vs. wild type and respective #, ##, and ### vs. week 1 as indicated.

Article Snippet: The membranes were then incubated overnight at 4°C with a primary antibody for PTX3 (clone 2C3, Hycult Biotechs, Plymouth Meeting, USA) followed by incubation with a secondary rabbit anti-mouse IgG HRP-labeled antibody.

Techniques:

Journal: Frontiers in Immunology

Article Title: The Long Pentraxin PTX3 Is an Endogenous Inhibitor of Hyperoxaluria-Related Nephrocalcinosis and Chronic Kidney Disease

doi: 10.3389/fimmu.2018.02173

Figure Lengend Snippet: Lack of PTX3 induces hyperoxaluria-induced progressive CKD in non-susceptible B6;129 mice. Ptx3 +/+ and Ptx3 −/− female littermates (10–12 weeks of age) were fed with oxalate chow for 3 weeks ( n = 9). (A) Quantification of tubular injury was conducted using PAS-stained kidney sections as shown in (B) . (C) Phase contrast image of crystal plugs in H&E-stained kidney section in 200× (left) and 400× (right) magnification. (D) Quantification of αSMA-positive area in kidney sections after 3 weeks of either control or oxalate chow feeding as shown in (E) . (F) Percentage of CD45 + cells within whole kidney cell suspension assessed by flow cytometry. (G) GFR assessed by transcutaneous measurement of FITC-sinistrin from subcutaneous capillaries at weeks 0, 1, 2, and 3 after onset of high oxalate diet in both genotypes. Data are from four independent experiments and represent means ± SEM in (G) . n.s., not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. wild type and respective #, ##, and ### vs. week 1 as indicated.

Article Snippet: The membranes were then incubated overnight at 4°C with a primary antibody for PTX3 (clone 2C3, Hycult Biotechs, Plymouth Meeting, USA) followed by incubation with a secondary rabbit anti-mouse IgG HRP-labeled antibody.

Techniques: Staining, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: The Long Pentraxin PTX3 Is an Endogenous Inhibitor of Hyperoxaluria-Related Nephrocalcinosis and Chronic Kidney Disease

doi: 10.3389/fimmu.2018.02173

Figure Lengend Snippet: PTX3 abolishes crystal adhesion molecule expression in vitro and in vivo . (A,B) Primary murine proximal tubular cells were isolated from Ptx3 +/+ and Ptx3 −/− littermate mice with 3 weeks of age and stimulated with PBS and 500 μg/ml CaOx crystals, respectively ( n = 4). Total RNA was isolated and transcribed to cDNA. Subsequent qPCR for (A) Cd44 and (B) AnxaII expression was conducted. (C–F) Ptx3 +/+ and Ptx3 −/− female littermates (10–12 weeks of age) were fed with oxalate chow for 3 weeks and formalin kidney sections were stained for (C,D) CD44 and (E,F) Annexin II, respectively ( n = 5). n.s., not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. wild type and respective ## and ### vs. PBS as indicated.

Article Snippet: The membranes were then incubated overnight at 4°C with a primary antibody for PTX3 (clone 2C3, Hycult Biotechs, Plymouth Meeting, USA) followed by incubation with a secondary rabbit anti-mouse IgG HRP-labeled antibody.

Techniques: Expressing, In Vitro, In Vivo, Isolation, Staining