Journal: The American Journal of Pathology

Article Title: Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition

doi: 10.1016/j.ajpath.2018.07.025

Figure Lengend Snippet: Analysis of parietal epithelial cells (PECs) expressing apolipoprotein (APOL) 1 and synaptopodin (SYNAPT) in renal biopsy specimens of patients with HIV-associated nephropathy (HIVAN). A: Paraffin-fixed renal biopsy specimens of controls and patients with HIVAN co-labeled for cytokeratin (green fluorescence), synaptopodin (purple fluorescence), and APOL1 (red fluorescence). Representative fluoromicrographs are displayed. A glomerulus in a control patient does not show any expression of APOL1 by PECs, but a glomerulus in a patient with HIVAN displays APOL1 expression by PECs (yellow fluorescence in the co-labeled image). An occasional PEC also displays co-labeling for APOL1 and SYNAPT. B: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and cytokeratin (Cytok) using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence) and Cytok (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using the CellProfiler pipeline to analyze PECs that expressed APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. C: More than 20 glomeruli from eight biopsy samples from patients with HIVAN were analyzed, and data of area occupied by pixels expressing Cytok and APOL1 were collected. A dot plot is showing the number of pixels co-expressing Cytok and APOL1 between controls and patients with HIVAN. A dot plot is showing a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok and APOL1. D: A pipeline of modules used for the analysis of the PECs expressing APOL1 or APOL1 and Cytok using Broad Institute's CellProfiler suite. Representative original images of the glomeruli from controls and patients with HIVAN showing expression of APOL1 (red fluorescence), Cytok (green fluorescence), and SYNAPT (purple) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze PECs expressing APOL1. The processed images are showing randomly denoted colors of the area occupied by pixels expressing Cytok and APOL1. E: More than 20 glomeruli from eight biopsy specimens from patients with HIVAN were analyzed, and data of the area occupied by pixels expressing Cytok, SYNAPT, and APOL1 were collected. A dot plot shows the number of pixels co-expressing Cytok, SYNAPT, and APOL1 between controls and patients with HIVAN. A dot plot shows a comparison of the number of pixels in the area occupied by PECs co-expressing Cytok, SYNAPT, and APOL1. n  = 6 controls and 8 patients with HIVAN. ∗∗∗ P ÂÂ

Article Snippet: Cells were incubated with the following primary antibodies: APOL1 (1G12D11; 1.57 μg/100 μL; Proteintech, Chicago, IL), rabbit (rb)-PAX2 (EP3251; 0.145 μg/100 μL, Abcam, Cambridge, UK), rb–claudin 1 (0.175 μg/100 μL, Abcam), rb–Wilms tumor 1 (WT1) (0.36 μg/100 μL, Abcam), rb-podocalyxin (0.53 μg/100 μL, catalog number PAI-46170; Life Technologies, Carlsbad, CA), synaptopodin (P-19, 0.36 μg/100 μL, Santa Cruz Biotechnology Inc., Santa Cruz, TX), CD2AP (B-4, 0.36 μg/100 μL, Santa Cruz Biotechnology), and mouse-α-actinin (H-2; 0.1 μg/100 μL, Santa Cruz Biotechnology) for overnight at 4°C.

Techniques: Expressing, Labeling, Fluorescence, Software

Journal: The American Journal of Pathology

Article Title: Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition

doi: 10.1016/j.ajpath.2018.07.025

Figure Lengend Snippet: The apolipoprotein (APO) L1 expression is associated with the expression of parietal epithelial cells (PECs) transition markers. A: To evaluate PEC markers in transited PECs, undifferentiated PECs were incubated in special media for 14 days at 37°C. Protein blots of control (undifferentiated, 0 days) and differentiated (transited) PECs (14 days) were probed for PAX2 and reprobed for claudin 1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Representative gels are displayed. B: Cumulative densitometric data from protein blots from A are displayed in a bar diagram. PEC transition is associated with down-regulation of PAX2 and claudin 1 expression. C: To assess transition markers, protein blots from cellular lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from A ) were probed for APOL1 and reprobed for synaptopodin (SYNPT) and GAPDH. Protein blots from the same lysates were probed for α-actinin and reprobed for Wilms' tumor 1 (WT1) and GAPDH. Protein blots were also probed for CD2AP and reprobed for podocalyxin (PDX) and GAPDH. Representative gels are displayed. D: Cumulative densitometric data of protein blots generated in C are shown in a bar diagram. PEC transition manifests in the form of enhanced expression of podocyte markers. E: To evaluate transcription of PEC markers in transited PECs, RNAs were extracted from the lysates of undifferentiated (0 days) and differentiated (14 days) PECs (from 3A). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Transited PECs displayed an attenuated transcription of PEC markers. F: To determine the transcription of transition markers in transited PECs, cDNAs from E were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . Transited PECs display an enhanced transcription of transition markers. G: To determine the time course effect on the transcription of PEC markers during the transition, PECs were incubated in media for variable periods (0, 4, 8, and 14 days) at 37°C. RNAs were extracted, and cDNAs were amplified with specific primers for PAX2 and claudin 1 . The transcription of PEC markers deceases during the transition in a time course manner. H: To evaluate the time course effect on the transcription of PECs transition markers, cDNAs obtained from the 2 G (RNAs) were amplified with specific primers for APOL1 , WT1 , PDX , and SYNPT . The transcription of transition markers increases during the transition in a time course manner. I: PECs grown on coverslips were fixed on 0 (undifferentiated) and 14 days (differentiated) and labeled for APOL1, SYNPT, α-actinin, PDX, and WT1. Subsequently, PECs were examined under a confocal microscope. Representative fluoromicrographs (APOL1, SYNPT, α-actinin, and PDX displayed green and WT1 exhibited red fluorescence) are displayed. n  = 4 ( A and C–F ). ∗ P ÂÂ

Article Snippet: Cells were incubated with the following primary antibodies: APOL1 (1G12D11; 1.57 μg/100 μL; Proteintech, Chicago, IL), rabbit (rb)-PAX2 (EP3251; 0.145 μg/100 μL, Abcam, Cambridge, UK), rb–claudin 1 (0.175 μg/100 μL, Abcam), rb–Wilms tumor 1 (WT1) (0.36 μg/100 μL, Abcam), rb-podocalyxin (0.53 μg/100 μL, catalog number PAI-46170; Life Technologies, Carlsbad, CA), synaptopodin (P-19, 0.36 μg/100 μL, Santa Cruz Biotechnology Inc., Santa Cruz, TX), CD2AP (B-4, 0.36 μg/100 μL, Santa Cruz Biotechnology), and mouse-α-actinin (H-2; 0.1 μg/100 μL, Santa Cruz Biotechnology) for overnight at 4°C.

Techniques: Expressing, Incubation, Wilms Tumor Assay, Generated, Amplification, Labeling, Microscopy, Fluorescence

Journal: The American Journal of Pathology

Article Title: Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition

doi: 10.1016/j.ajpath.2018.07.025

Figure Lengend Snippet: Podocyte expression of apolipoprotein (APO) L1 in Tg26:APOL1G1 transgenic mice. A: Renal cortical sections of control (FVB/N) and HIV transgenic mice expressing APOL1 (Tg26:APOL1) were co-labeled for APOL1 (red fluorescence) and synaptopodin (SYNAPT) (green fluorescence). Representative microfluorographs are shown. Foci of parietal epithelial cells (PECs) display expression of SYNAPT ( arrows ). B: Representative original images of a renal cortical section of control (FVB/N) and TG26:APOL1 mice showing expression of APOL1 (red fluorescence) and SYNAPT (green fluorescence) were captured using SlideBook software version 6.0. Images were then processed using CellProfiler pipeline to analyze co-expression of APOL1 and SYNAPT. The processed images show randomly denoted colors of the area occupied by pixels expressing SYNAPT and APOL1. C: Randomly selected glomeruli from FVB/N and TG26:APOL1 mice were analyzed using CellProfiler. Data for the area occupied by pixels expressing SYNAPT and APOL1 were collected and analyzed using GraphPad Prism 7 software. A dot plot shows a comparison of the number of pixels co-expressing SYNAPT and APOL1 between FVB/N and Tg26:APOL1 mice. n  = 4. ∗∗∗ P ÂÂ

Article Snippet: Cells were incubated with the following primary antibodies: APOL1 (1G12D11; 1.57 μg/100 μL; Proteintech, Chicago, IL), rabbit (rb)-PAX2 (EP3251; 0.145 μg/100 μL, Abcam, Cambridge, UK), rb–claudin 1 (0.175 μg/100 μL, Abcam), rb–Wilms tumor 1 (WT1) (0.36 μg/100 μL, Abcam), rb-podocalyxin (0.53 μg/100 μL, catalog number PAI-46170; Life Technologies, Carlsbad, CA), synaptopodin (P-19, 0.36 μg/100 μL, Santa Cruz Biotechnology Inc., Santa Cruz, TX), CD2AP (B-4, 0.36 μg/100 μL, Santa Cruz Biotechnology), and mouse-α-actinin (H-2; 0.1 μg/100 μL, Santa Cruz Biotechnology) for overnight at 4°C.

Techniques: Expressing, Transgenic Assay, Mouse Assay, Labeling, Fluorescence, Software

Journal: The American Journal of Pathology

Article Title: Role of Apolipoprotein L1 in Human Parietal Epithelial Cell Transition

doi: 10.1016/j.ajpath.2018.07.025

Figure Lengend Snippet: HIV, interferon (IFN)-γ, and vitamin D receptor (VDR) agonists induce apolipoprotein (APO) L1 and transition markers in parietal epithelial cells (PECs). A: To examine the effect of APOL1 stimulators on APOL1 induction and expression of PEC transition markers, PECs were incubated in media that contained vehicle [control (C)], VDR agonists (EB1089, 100 nmol/L), or IFN-γ (10 ng/mL) for 48 hours. Protein blots were probed for APOL1, Wilms' tumor 1 (WT1), podocalyxin (PDX), and α-actinin and reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Gels from two different lysates are shown. B: To assess the effect of HIV on APOL1 induction and expression of PEC transition markers, PECs were transduced with vector (Vec) or HIV [NL4-3, 10 3 green fluorescent protein (GFP)–expressing units (GEU)/mL]. Protein blots were probed for APOL1, WT1, podocalyxin, and α-actinin and reprobed for GAPDH. Gels from two different lysates are shown. C: Cumulative densitometric data from the cells treated with VDA and IFN-γ are shown. D: Cumulative densitometric data from the cells transduced with Vec or HIV are displayed. IFN-γ–, VDA receptor–, and HIV-induced APOL1 expression is associated with the expression of transition markers in PECs. E: To evaluate the effect of APOL1 induction on the transcription of PEC markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–treated cells ( A and B ). cDNAs were amplified with specific primers for PAX2 and claudin 1 . Cumulative data are shown in a bar diagram. APOL1 induction in PECs attenuates the expression of PEC markers. F: To examine the effect of APOL1 induction on the transcription of PEC transition markers, RNAs were extracted from the lysates of HIV-, IFN-γ–, and VDR agonist–-treated cells ( A and B ). cDNA was amplified with specific primers for APOL1 , WT1 , α-actinin, PDX , and CD2AP . Cumulative data are shown in bar graphs. APOL1 induction in PECs results in enhanced transcription of PEC transition markers. G: To visualize the expression of PEC transition markers in response to APOL1 inducers, PECs grown on coverslips were treated under similar conditions (as in A ) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Expression of APOL1, α-actinin, and PDX is indicated by green fluorescence and of WT1 by red fluorescence. H: To visualize the expression of PEC transition markers in response to HIV, PECs grown on coverslips were transduced with VEC (GFP positive) or HIV (GFP positive) and labeled for PEC transition markers. Representative fluoromicrographs are displayed. Both Vec- and HIV-transduced cells are GFP positive (indicated by green fluorescence). HIV-transduced cells display an overt expression of APOL1, synaptopodin (SYNPT), α-actinin, and WT1 (red fluorescence). n  = 4 ( A and B ); n  = 3 ( E and F ). ∗ P ÂÂ

Article Snippet: Cells were incubated with the following primary antibodies: APOL1 (1G12D11; 1.57 μg/100 μL; Proteintech, Chicago, IL), rabbit (rb)-PAX2 (EP3251; 0.145 μg/100 μL, Abcam, Cambridge, UK), rb–claudin 1 (0.175 μg/100 μL, Abcam), rb–Wilms tumor 1 (WT1) (0.36 μg/100 μL, Abcam), rb-podocalyxin (0.53 μg/100 μL, catalog number PAI-46170; Life Technologies, Carlsbad, CA), synaptopodin (P-19, 0.36 μg/100 μL, Santa Cruz Biotechnology Inc., Santa Cruz, TX), CD2AP (B-4, 0.36 μg/100 μL, Santa Cruz Biotechnology), and mouse-α-actinin (H-2; 0.1 μg/100 μL, Santa Cruz Biotechnology) for overnight at 4°C.

Techniques: Expressing, Incubation, Wilms Tumor Assay, Transduction, Plasmid Preparation, Amplification, Labeling, Fluorescence