Journal: JCI Insight

Article Title: NF- κ B–driven lymphangiogenesis affects kidney function via a VEGFR-3–mediated pathway

doi: 10.1172/jci.insight.198992

Figure Lengend Snippet: ( A ) WT mice were crossbred with VEGFR-3 Cre-expressing mice to generate Flt 4Cr exRel Aflox transgenic animals. Upon successful Cre-mediated recombination, exon 1 of the RelA gene, the neomycin resistance cassette, and promoter region were excised, resulting in subsequent GFP expression. The dotted box represents the components deleted upon recombination. Black and gray triangles denote loxP and frt sites, respectively. WT and its recombined form were adapted from Heise et al. . ( B ) Visualization of renal lymph nodes (white arrow) and lymphatic vessels (yellow arrow) with associated lumbar lymph nodes (green arrow) following 5% Evan’s blue dye injection via the footpad, prior to surgical resection. ( C ) Hepatic eGFP RNA expression analysis relative to that of WT mice, presented as fold change, and normalized to Gapdh confirming successful gene knockout efficiency in tamoxifen-treated knockout mice ( N = 3) compared with tamoxifen-treated WT controls ( N = 4). ( D ) Real-time quantitative PCR validation of target gene knockout using primers spanning from exon 1 to exon 3. The observed reduction in RNA expression indicates successful exon 1 excision following Cre-mediated recombination. RelA transcript levels were normalized to Gapdh expression and are presented as fold change relative to control WT mice (WT, N = 4; KO, N = 3). ( E ) Experimental timeline depicting sequential tamoxifen and cisplatin treatments, with tissue collection occurring 3 days after high-dose cisplatin (20 mg/kg) or saline administration. The washout period was 10 to 11 days. PGK, phosphoglycerate kinase promoter; Neo pA, neomycin resistance gene-polyadenylation; WT, RelA fl/fl ; KO, VEGFR-3 RelA–/– . * P < 0.0332. C is unpaired t test (2 tailed). D is 2-way ANOVA.

Article Snippet: After a series of methanol dehydration/rehydration, delipidation with organic solvents (e.g., dichloromethane), permeabilization, blocking, and washing, kidneys were immunolabeled with LYVE-1 (1:200; Novus Biologicals, NB-600-1008) and VEGFR-3 (1:200; R&D Systems, AF743) for 96 hours at 37°C.

Techniques: Expressing, Transgenic Assay, Injection, RNA Expression, Gene Knockout, Knock-Out, Real-time Polymerase Chain Reaction, Biomarker Discovery, Control, Saline

Journal: JCI Insight

Article Title: NF- κ B–driven lymphangiogenesis affects kidney function via a VEGFR-3–mediated pathway

doi: 10.1172/jci.insight.198992

Figure Lengend Snippet: ( A ) Glomerular filtration rate (GFR) in control and cisplatin-treated knockout and WT mice (WT control, N = 4; KO control, N = 7; WT cisplatin, N = 15; KO cisplatin, N = 17). ( B ) Serum creatinine measurements in control and cisplatin-treated knockout and WT mice. Kidney function analysis included data from at least 4 independent knockout cohorts and 3 WT control cohorts (WT control, N = 13; KO control, N = 12; WT cisplatin, N = 18; KO cisplatin, N = 21). ( C ) Representative images of PAS-stained kidney sections from the cortices in knockout and WT mice. Black arrows indicate loss of brush border; white arrows indicate cortical casts. Scale bar: 100 μm. ( D ) Tubular casts, tubular necrosis, and brush border loss were scored in a blinded fashion from 0 (low injury) to 4 (high injury) using an area-based system. Sample sizes (WT control, N = 4; KO control, N = 6; WT cisplatin, N = 11; KO cisplatin, N = 10). Statistical significance ( P < 0.05) was determined using 2-way ANOVA. ** P < 0.0021, *** P < 0.002, **** P < 0.0001. WT, RelA fl/fl ; KO, VEGFR-3 RelA–/– .

Article Snippet: After a series of methanol dehydration/rehydration, delipidation with organic solvents (e.g., dichloromethane), permeabilization, blocking, and washing, kidneys were immunolabeled with LYVE-1 (1:200; Novus Biologicals, NB-600-1008) and VEGFR-3 (1:200; R&D Systems, AF743) for 96 hours at 37°C.

Techniques: Filtration, Control, Knock-Out, Staining

Journal: JCI Insight

Article Title: NF- κ B–driven lymphangiogenesis affects kidney function via a VEGFR-3–mediated pathway

doi: 10.1172/jci.insight.198992

Figure Lengend Snippet: ( A ) Flow cytometry gating strategy for analyzing CD45 + leukocyte populations in kidneys from a cisplatin-treated knockout mouse (20 mg/kg), including neutrophils, kidney-resident macrophages (KRMs), infiltrating macrophages (IMs), and inflammatory Ly6c hi IMs. ( B ) Quantification of KRMs (CD11b int , F4/80 hi ) as a percentage of total CD45 + cells in RelA-knockout and WT control mice. ( C ) Quantification of neutrophils (Ly6G + ) as a percentage of total CD45 + cells. ( D ) Quantification of IMs (CD11b hi , F4/80 int ) as a percentage of total CD45 + cells. ( E ) Quantification of inflammatory Ly6c hi IM cells as a percentage of total IMs (CD11b hi , F4/80 int ) (WT control, N = 9; KO control, N = 9; WT cisplatin, N = 10; KO cisplatin, N = 7). Cisplatin-treated mice with serum creatinine levels at least twice those of saline controls were included in this analysis. Graphs include data from 4 independent cohorts. * P < 0.0332, ** P < 0.0021, **** P < 0.0001. WT, RelA fl/fl ; KO, VEGFR-3 RelA–/– .

Article Snippet: After a series of methanol dehydration/rehydration, delipidation with organic solvents (e.g., dichloromethane), permeabilization, blocking, and washing, kidneys were immunolabeled with LYVE-1 (1:200; Novus Biologicals, NB-600-1008) and VEGFR-3 (1:200; R&D Systems, AF743) for 96 hours at 37°C.

Techniques: Flow Cytometry, Knock-Out, Control, Saline

Journal: JCI Insight

Article Title: NF- κ B–driven lymphangiogenesis affects kidney function via a VEGFR-3–mediated pathway

doi: 10.1172/jci.insight.198992

Figure Lengend Snippet: ( A ) Lymphatic associated genes’ and ( B ) NF-κB and Vcam1 genes’ expression analyses from kidney lysate, presented as fold change relative to tamoxifen+saline WT controls. Analysis included data from 4 independent knockout cohorts and 3 WT control cohorts (WT control, N = 11; KO control, N = 7; WT cisplatin, N = 9; KO cisplatin, N = 15 for Flt4, Lyve-1, Ccl21a, Prox-1 , and Pdpn ; N = 12 for Vcam1 ; N = 14 for p50, Vegfc , and p52 ). All transcript levels were normalized to Gapdh . Statistical significance ( P < 0.05) was determined using 2-way ANOVA. * P < 0.0332, ** P < 0.0021, *** P < 0.0002, **** P < 0.0001. WT, RelA fl/fl ; KO, VEGFR-3 RelA–/– .

Article Snippet: After a series of methanol dehydration/rehydration, delipidation with organic solvents (e.g., dichloromethane), permeabilization, blocking, and washing, kidneys were immunolabeled with LYVE-1 (1:200; Novus Biologicals, NB-600-1008) and VEGFR-3 (1:200; R&D Systems, AF743) for 96 hours at 37°C.

Techniques: Expressing, Saline, Knock-Out, Control

Journal: JCI Insight

Article Title: NF- κ B–driven lymphangiogenesis affects kidney function via a VEGFR-3–mediated pathway

doi: 10.1172/jci.insight.198992

Figure Lengend Snippet: ( A ) Analysis of lymphatic marker protein expression in kidney lysate by Western blotting. Protein expression levels were quantified using densitometry and normalized to either β-actin or GAPDH as loading controls. Representative blots are shown with corresponding quantification. Densitometric analysis includes data from 3 independent knockout cohorts and 3 WT cohorts (WT control, N = 11; KO control, N = 8; WT cisplatin, N = 9; KO cisplatin, N = 15). Statistical significance ( P < 0.05) was determined using 2-way ANOVA. ** P < 0.0021, *** P < 0.0002, **** P < 0.0001. ( B ) Visualization of lymphatic structures in cleared kidney tissue using confocal microscopy. Images show quarter-sectioned, optically cleared kidneys from both a WT control and knockout mouse. Lymphatic vessels are labeled with two markers: LYVE-1 (red) and VEGFR-3 (green). Images shown are maximum intensity projections that have been processed with denoising and deconvolution algorithms using NIS-Elements. Original magnification, ×10. WT, RelA fl/fl ; KO, VEGFR-3 RelA–/– .

Article Snippet: After a series of methanol dehydration/rehydration, delipidation with organic solvents (e.g., dichloromethane), permeabilization, blocking, and washing, kidneys were immunolabeled with LYVE-1 (1:200; Novus Biologicals, NB-600-1008) and VEGFR-3 (1:200; R&D Systems, AF743) for 96 hours at 37°C.

Techniques: Marker, Expressing, Western Blot, Knock-Out, Control, Confocal Microscopy, Labeling

Journal: JCI Insight

Article Title: NF- κ B–driven lymphangiogenesis affects kidney function via a VEGFR-3–mediated pathway

doi: 10.1172/jci.insight.198992

Figure Lengend Snippet: ( A ) Representative confocal microscopy images (maximum intensity projections) of quarter kidney sections immunolabeled for VEGFR-3 (green, Alexa 647) and LYVE-1 (red, Alexa 594). Images compare VEGFR-3–specific RelA-deficient mice to floxed controls under baseline conditions and after cisplatin-induced AKI. 10× objective. ( B and C ) Quantitative analysis of lymphatic vessel morphology for ( B ) VEGFR-3 + and ( C ) LYVE-1 + vessels. Measured parameters include branch point density, total filament volume (μm 3 ), and total filament length (μm). Analysis was performed using Imaris Filaments module following image processing (denoising, deconvolution, thresholding). All measurements were normalized to imaging volume based on image field dimensions: x axis (2,038 pixels), y axis (2,038 pixels), and z axis (section thickness in μm), with a pixel size of 1.24 μm/pixel (WT control, N = 3; KO control, N = 3; WT cisplatin, N = 4; KO cisplatin, N = 5). WT, RelA fl/fl ; KO, VEGFR-3 RelA–/– .

Article Snippet: After a series of methanol dehydration/rehydration, delipidation with organic solvents (e.g., dichloromethane), permeabilization, blocking, and washing, kidneys were immunolabeled with LYVE-1 (1:200; Novus Biologicals, NB-600-1008) and VEGFR-3 (1:200; R&D Systems, AF743) for 96 hours at 37°C.

Techniques: Confocal Microscopy, Immunolabeling, Imaging, Control