NF-κB–driven lymphangiogenesis affects kidney function via a VEGFR-3–mediated pathway
Arin L. Melkonian, Amie M. Traylor, Anna A. Zmijewska, Kyle H. Moore, Gelare Ghajar-Rahimi, Stephanie Esman, Yanlin Jiang, Hani Jang, Babak J. Mehrara, Timmy C. Lee, James F. George, Anupam Agarwal
Arin L. Melkonian, Amie M. Traylor, Anna A. Zmijewska, Kyle H. Moore, Gelare Ghajar-Rahimi, Stephanie Esman, Yanlin Jiang, Hani Jang, Babak J. Mehrara, Timmy C. Lee, James F. George, Anupam Agarwal
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Research Article Cell biology Nephrology

NF-κB–driven lymphangiogenesis affects kidney function via a VEGFR-3–mediated pathway

Abstract

The lymphatic system maintains fluid homeostasis and orchestrates immune cell trafficking throughout tissues. While extensively studied in cancer and lymphedema, its role in nonlymphoid organs, particularly the kidney, remains an emerging area of investigation. Previous research established molecular connections among NF-κB, VEGFR-3, and PROX-1 in regulating lymphatic growth during inflammation, and studies using global knockout mice revealed that the NF-κB1 subunit (p50) influences lymphatic vessel density. However, the role of RelA — a key component of the canonical NF-κB heterodimer — in regulating lymphatic growth and kidney function following acute kidney injury (AKI) remains unexplored. Using an inducible, predominantly lymphatic endothelial cell-specific RelA-knockout mouse model, we demonstrated that RelA expression in VEGFR-3+ cells is essential for VEGFR-3–driven lymphangiogenesis following AKI. Knockout mice exhibited substantially worse kidney function, altered histological features, impaired VEGFR-3–dependent lymphangiogenesis, and dysregulated immune cell trafficking compared with WT mice. Compensatory upregulation of PROX-1 and podoplanin occurred despite decreased VEGFR-3 and LYVE-1 total protein expression, suggesting complex regulatory mechanisms. Our findings suggest that RelA is a critical sensor for inflammation and regulator of protective lymphangiogenesis following kidney injury and provide insights into potential therapeutic targets for improved kidney injury outcomes.

Authors

Arin L. Melkonian, Amie M. Traylor, Anna A. Zmijewska, Kyle H. Moore, Gelare Ghajar-Rahimi, Stephanie Esman, Yanlin Jiang, Hani Jang, Babak J. Mehrara, Timmy C. Lee, James F. George, Anupam Agarwal

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Figure 1

Mouse model, characterization, and experimental design.

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Mouse model, characterization, and experimental design. (A) WT mice were...
(A) WT mice were crossbred with VEGFR-3 Cre-expressing mice to generate Flt4CrexRelAflox 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. (46). (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, RelAfl/fl; KO, VEGFR-3RelA–/–. *P < 0.0332. C is unpaired t test (2 tailed). D is 2-way ANOVA.