Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Parietal epithelia cells in the urine as a marker of disease activity in glomerular diseases.

doi: 10.1093/ndt/gfn235

Figure Lengend Snippet: Fig. 1. (A) Morphology of podocalyxin positive cells cultured from urine sediments of nephrotic patients. Three different shapes of podocalyxin positive cells were observed after overnight culture. Cells had either long thin cytoplasmic processes (white arrowheads, panel a), or cells with primary and secondary processes (panel b) or exhibited a rounded cobblestone shaped pattern with lamellopodia (white arrowheads, panel c). All forms can be visible in parallel in a single patient sediment. (B) Number of podocalyxin positive cells (black bars) and albumin/creatinine ratio (white bars) from the sediment and spot urine of healthy controls (n = 10), patients with active FSGS (n = 11), FSGS in partial remission (n = 19), FSGS in full remission (n = 6), active MCD (n = 6), MCD in partial remission (n = 6), MCD in full remission (n = 7), active MGN (n = 12), MGN in partial remission (n = 10), MGN in full remission (n = 3), active MPGN (n = 5) and MPGN in partial remission (n = 2). ∗∗P < 0.0001 FSGS active versus partial remission; ∗P = 0.0017 MGN active versus partial remission; †P = 0.04 MPGN active versus partial remission. (C) Correlation of podocalyxin positive cells with the total number of cells quantified by DAPI in cell containing samples pooled (All) (r2 = 0.875), patients with FSGS (r2 = 0.8992), MGN (r2 = 0.9958) and MPGN (r2 = 0.5269).

Article Snippet: Primary antibodies used were goat anti-human podocalyxin (PDX) (R&D Systems, Minneapolis, MN, USA), mouse anti-human PDX (gift from Dontscho Kerjaschki, Medical University of Vienna), mouse anti-human CK8–18 (Neomarker, CA, USA), rabbit anti-WT-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-human podocin (Alpha Diagnostic International, San Antonio, TX, USA) and mouse anti-synaptopodin (Progen Biotechnik, Heidelberg, Germany).

Techniques: Cell Culture

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Parietal epithelia cells in the urine as a marker of disease activity in glomerular diseases.

doi: 10.1093/ndt/gfn235

Figure Lengend Snippet: Fig. 2. Podocalyxin positive cells exhibit markers of proliferation and are double positive for parietal cell markers. (A) Cultured sediments of an FSGS patient were stained for podocalyxin (panels b, f, j) double labelled for Ki-67 (panels a–d), CK8–18 (panels e–h) and PGP 9.5 (panels i–l). White open arrows depict nuclear Ki-67 and podocalyxin double positive cells (panels a–d), the green arrow depicts CK8–18 single positive cell (panel e, merged in h) and the red arrow depicts podocalyxin only positive cell (panel f, merged in h). (B) Quantification of podocalyxin and CK8–18 positive cells in urine sediments of five patients with active FSGS. About 60% of the cultured cells are positive for podocalyxin. Thirty percent are positive for CK8–18. Almost all of the CK8–18 positive cells are positive for podocalyxin. About 30% of cells were podocalyxin only positive; <1% were CK8–18 only positive. (C) Quantification of podocalyxin and CK8–18 positive cells in urine sediments of five patients with active membranous glomerulonephritis. On average 50% of the cultured cells are positive for podocalyxin. Fourty-five percent are positive for CK8–18. Almost all of the CK8–18 positive cells are positive for podocalyxin. About 15% of cells were podocalyxin only positive, <10% were CK8–18 only positive.

Article Snippet: Primary antibodies used were goat anti-human podocalyxin (PDX) (R&D Systems, Minneapolis, MN, USA), mouse anti-human PDX (gift from Dontscho Kerjaschki, Medical University of Vienna), mouse anti-human CK8–18 (Neomarker, CA, USA), rabbit anti-WT-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-human podocin (Alpha Diagnostic International, San Antonio, TX, USA) and mouse anti-synaptopodin (Progen Biotechnik, Heidelberg, Germany).

Techniques: Cell Culture, Staining

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Parietal epithelia cells in the urine as a marker of disease activity in glomerular diseases.

doi: 10.1093/ndt/gfn235

Figure Lengend Snippet: Fig. 3. PAS, podocalyxin and CK8–18 staining in corresponding kidney biopsies from a patient with focal-segmental glomerulosclerosis (FSGS) (a–c), membranous glomerulonephritis (MGN) (d–f) and membranoproliferative glomerulonephritis (MPGN) (g–i). (a) FSGS with segmental attached scle- rosed capillary loops (PAS stain, 400× magnification). (d) Membranous glomerulonephritis with granular to linear IgG deposits (insert) in the thickened glomerular basement membranes (PAS stain and immunohistochemistry, 400× magnification). (g) Membranoproliferative glomerulonephritis with lobular accentuation of the glomerular tuft and C3 deposits (insert) in peripheral glomerular basement membranes (PAS) stain and immunohistochem- istry, 400× magnification). (panels b, e and h) Immunohistochemistry for podocalyxin with expression in visceral podocytes and parietal cells (400× magnification). (panels c, f and i) Immunohistochemistry for cytokeratin 8/18 with expression in some parietal cells (400× magnification).

Article Snippet: Primary antibodies used were goat anti-human podocalyxin (PDX) (R&D Systems, Minneapolis, MN, USA), mouse anti-human PDX (gift from Dontscho Kerjaschki, Medical University of Vienna), mouse anti-human CK8–18 (Neomarker, CA, USA), rabbit anti-WT-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-human podocin (Alpha Diagnostic International, San Antonio, TX, USA) and mouse anti-synaptopodin (Progen Biotechnik, Heidelberg, Germany).

Techniques: Staining, Immunohistochemistry, Expressing

Journal: eLife

Article Title: Cytotoxic T-cells mediate exercise-induced reductions in tumor growth

doi: 10.7554/eLife.59996

Figure Lengend Snippet: ( A ) Wild type (WT) and MMTV-PyMT (PyMT) mice on the FVB background were allowed to exercise voluntarily (in running wheels, runners) or left non-exercised (locked running wheels, non-runners) between 4 and 12 weeks of age. ( B ) Running distance (km/day) in WT and PyMT mice. *p<0.05, two-tailed unpaired t test. ( C ) Tumor volume measured twice weekly for PyMT running and non-running mice. Mean and SEM (n = 10–11). ( D ) Survival of PyMT mice, runners, and non-runners. Survival curve ns = not significant, Log-rank (Mantel-Cox) test, (n = 10–11). ( E ) Tumor initiation as age (day) of first indication of a palpable tumor in running (Runners) and non-running (Non-runners) PyMT mice. n = 11, ns = not significant, two-tailed t test. ( F ) Tumor stage from histological scoring (1-4) in mammary glands of running and non-running PyMT mice at 12 (n = 6–10) and 8 (n = 4) weeks of age, ns = not significant, two-tailed t test. ( G ) Individual body weight for WT and PyMT running and non-running mice. n = 7–10, ns = not significant, one-way ANOVA with Tukey’s multiple comparison test. ( H ) Immunohistological characterization of PyMT tumors from non-running and running mice using CD3, F4/80, PCNA, Podocalyxin (PODXL), and Granzyme B (GZMB) antibodies, respectively. n = 8–15, ns = not significant, *p<0.05, Two-tailed unpaired t test. ( I ) Flow-cytometry-based frequency of macrophages within I3TC tumor. Ns = not significant, two-tailed t-test. ( J ) Flow-cytometry-based frequency of neutrofiles within I3TC tumor. Ns = not significant, two-tailed t-test.

Article Snippet: Nonspecific protein interactions were blocked using 20% goat serum in PBS-T. Primary antibodies were used in mammary glands against CD3 (#ab5690, Abcam, UK) to assess lymphocytic infiltration, F4/80 (#MCA497, Abd Serotec, Germany) to assess macrophage density, podocalyxin (#AF1556, R and D Systems, Germany) to assess capillary density, PCNA (#M0879, Dako, Denmark) to identify proliferating cells, and Granzyme B (#ab4059, Abcam, UK) to identify effector lymphocytes.

Techniques: Two Tailed Test, Comparison, Flow Cytometry

Journal: eLife

Article Title: Cytotoxic T-cells mediate exercise-induced reductions in tumor growth

doi: 10.7554/eLife.59996

Figure Lengend Snippet:

Article Snippet: Nonspecific protein interactions were blocked using 20% goat serum in PBS-T. Primary antibodies were used in mammary glands against CD3 (#ab5690, Abcam, UK) to assess lymphocytic infiltration, F4/80 (#MCA497, Abd Serotec, Germany) to assess macrophage density, podocalyxin (#AF1556, R and D Systems, Germany) to assess capillary density, PCNA (#M0879, Dako, Denmark) to identify proliferating cells, and Granzyme B (#ab4059, Abcam, UK) to identify effector lymphocytes.

Techniques: Transgenic Assay, Marker, Derivative Assay, Staining, Software, Plasmid Preparation, Avidin-Biotin Assay

Journal: The Journal of Biological Chemistry

Article Title: Loss of mucin-type O -glycans impairs the integrity of the glomerular filtration barrier in the mouse kidney

doi: 10.1074/jbc.M117.798512

Figure Lengend Snippet: Sialoproteins such as podocalyxin are targets of O-glycosylation in glomerulosclerosis. A, IHC of podocalyxin (PODXL) in WT and iC1galt1−/− adult mice after 4 weeks of tamoxifen-induced deletion. B, Western blotting of podocalyxin and evaluation of O-glycosylation/sialylation status of podocalyxin in WT and iC1galt1−/− mice. VVL is a lectin-recognizing Tn antigen. Sialylation of podocalyxin was probed with lectins: MALII recognizes N-acetylneuraminic acid (Neu5Ac)-linked α2,3-Gal; SNA recognizes α2,6-linked sialic acid. IB, immunoblot; IP, immunoprecipitation. C, podocalyxin–NHERF2 interaction was disrupted after loss of O-glycans. Kidney lysates from adult WT and iC1galt1−/− mice 8 weeks post-induction were immunoprecipitated with polyclonal anti-NHERF2 antibody or anti-podocalyxin, followed by immunoblotting. Arrows indicate podocalyxin proteins with differential degrees of O-glycosylation. Blue arrow indicates hypo-O-glycosylated portion of podocalyxin. D, immunofluorescent staining of pEzrin and podocalyxin in glomeruli from WT and iC1galt1−/− mice. Scale bars = 20 μm.

Article Snippet: After inactivation of endogenous peroxidase with 0.3% hydrogen peroxide and blocking with normal blocking serum, the sections were incubated with glycosylation-independent goat anti-podocalyxin antibody (R&D Systems) or biotinylated mAb against Tn antigen (mouse IgM, clone Ca3638) overnight at 4 °C.

Techniques: Glycoproteomics, Western Blot, Immunoprecipitation, Staining

Journal: medRxiv

Article Title: Factor H-related protein 3 (FHR-3) deposition in kidney allografts – localization and correlation with complement activation

doi: 10.1101/2023.10.24.23297478

Figure Lengend Snippet: Representative images from immunofluorescent double staining of factor H-related protein 3 (FHR-3) with (A-C) Podocalyxin, (D-F) Podocin, (G-I) Vimentin, (J-L) Desmin, and (M-O) complement receptor 1 (CR1) in kidney allografts prior to transplantation from organ donors after brain death. Nuclei were counterstained with DAPI (blue). Podocyte markers (green) were stained with fluorescein isothiocyanate (FITC)-conjugated antibodies (A, D, G, J and, M), and FHR-3 (red) was visualized using the TSA Tetramethylrhodamine (TRITC) System (B, E, I, K, N).

Article Snippet: Specific cell populations in the glomerulus were detected using a rabbit pAb to mouse CD31 that cross-reacts with human CD31 (ab28364; Abcam), a rabbit pAb to human platelet-derived growth factor receptor-β (PDGFR-β) (RB-1692, Thermo Fisher Scientific), a rabbit pAb to human podocin (P0372, Sigma-Aldrich), a goat pAb to human podocalyxin (AF1658, R&D systems), a rabbit pAb to human complement receptor 1 (CR1/CD35) (gifted by Mohamed R. Daha, generated previously in the laboratory of Nephrology, Leiden, The Netherlands), a goat pAb to human vimentin (AF2105, Novus Biologicals), and a pAb to human desmin (ab15200; Abcam).

Techniques: Double Staining, Transplantation Assay, Staining