ABS 078: The Role of NF-kB in Kidney Lymphangiogenesis

Mays, Dana ¹ ; Ghajar-Rahimi, Gelare ¹ ; Melkonian, Arin ¹ ; George, Jim ² ; Anupam Agarwal ¹

¹ UAB Heersink School of Medicine, Department of Nephrology
² UAB Heersink School of Medicine, Department of Cardiothoracic Surgery

Van Wickle (2025) Volume 1, ABS078

Introduction: Acute kidney injury (AKI) is a common clinical problem with substantial mortality, especially in hospital settings. Lymphangiogenesis (LA), a process in which the lymphatic system expands within the kidneys, is important in AKI and subsequent recovery. Inhibiting LA worsens the severity of AKI and results in increased NF-kB expression. Inhibiting NF-kB globally within the kidney has been shown to mitigate injury. We hypothesize that NF-kB has cell-specific roles within in the kidney, and that in lymphatic endothelial cells, it is required for LA. The role of NF-kB within LECs was investigated using cell culture. In one experiment, human dermal lymphatic endothelial cells were exposed to hypoxia as an in vitro correlate to ischemic kidney damage. In another experiment, NF-kB was knocked down using siRNA, and lymphangiogenic migration was assessed using a scratch assay. Expression of lymphangiogenic factors and markers in both experiments was measured by quantitative PCR and western blot. Our experiments showed that hypoxia and NF-kB knockdown conditions influenced levels of lymphangiogenic factors, including PROX1, LYVE1, and VEGFD. In the scratch assay, NF-kB knockdown mitigated LEC migration potential and had variable influence on several downstream genes including TNF-SF15 and PDPN. siRNA knockdown of NF-kB was confirmed by RT-PCR. These data will enhance our current understanding of molecular pathways involved in LA and provide possible avenues for therapeutic interventions for AKI patients.

Methods: In our first experiment, in vitro human dermal LECs, or hdLECs, were exposed to hypoxic conditions for half an hour, 6 hrs, and 24 hrs respectively, then media and cells were collected. A 0 hr time point sample was used as a control. Real-time PCR and Western blot analyses were performed on all samples.

In the second experiment, in vitro hdLECs were transfected with negative siRNA and with NF-kB knockdown siRNA respectively. siRNA, or small interfering RNA, is commonly used to temporarily knock down certain genes. An untransfected sample was used as a control. Real time PCR was performed on all samples. We performed a scratch assay with a pipette tip on all the samples from the NF-kB knockdown experiment to measure LEC growth capacity in the absence of NF-kB.

Results: We have shown that hypoxia alters lymphatic endothelial cell (LEC) gene expression over time, with PROX1 and LYVE1 reaching their lowest levels at 24 hours, and HIF1a and NF-kB peaking at 30 minutes and 6 hours, respectively. NF-kB knockdown reduced LEC migration in scratch assays and altered gene expression, increasing LYVE1 and HIF1a while decreasing PROX1, suggesting NF-kB’s regulatory role. Western blot analysis confirmed hypoxia induction and showed peak NF-kB activation at 6 hours. Together, these data support a model where NF-kB modulates LEC gene expression and function in response to hypoxic conditions.

Discussion: This pilot study shows that NF-kB does play a role in LECs, and it is important for LA. NF-kB does play a role in LECs, and it is important for LA. Future studies including in vitro tube formation assays and in vivo models of conditional NF-kB knockout mice will further explore this phenomenon.