Abstract: FR-PO583
Lymphatic Endothelial Cell Permeability Is Regulated by Sodium and Nkcc1
Session Information
- Fluid, Electrolyte, and Acid-Base Disorders: Basic
October 25, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Fluid, Electrolytes, and Acid-Base Disorders
- 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic
Authors
- Zhong, Jianyong, Vanderbilt University Medical Center, Nashville, Tennessee, United States
- Yang, Haichun, Vanderbilt University Medical Center, Nashville, Tennessee, United States
- Kon, Valentina, Vanderbilt University Medical Center, Nashville, Tennessee, United States
- Shelton, Elaine L., Vanderbilt University Medical Center, Nashville, Tennessee, United States
Background
Interstitial sodium (Na+) accumulation is a halmark of proteinuric kidney disease. Lymphatic endothelial cells (LECs) play an integral role in regulating interstitial fluid uptake and transport. Interstitial fluid drains into lymphatic capillaries composed of permeable LECs with button-like cell junctions. These capillaries transition into contractile collecting vessels lined with relatively impermeable LECs with continuous zipper-like junctions, which facilitate the transport of fluid without leakage. We have previously shown that increased Na+ diminished the pumping capacity of renal lymphatic collecting vessels and reduced the activity of the Na-K-2Cl cotransporter, Nkcc1. In addition, furosemide, a Nkcc1 inhibitor, blunted lymphatic vessel pumping dynamics. Of note, ethacrynic acid, another Nkcc1 inhibitor, had no effect on vessel contractility. In this study we evaluated the effects of increased Na+ and Nkcc1 inhibition on LEC barrier formation and integrity.
Methods
Mouse LECs were cultured in normal or high Na+ conditions with or without bumetanide, a Nkcc1 inhibitor. Bulk RNA sequencing and qRT-PCR identified differentially expressed genes. LECs were also used in Trans-Epithelial Electrical Resistance (TEER) assays to measure changes in monolayer formation and barrier integrity.
Results
RNA sequencing revealed high Na+ upregulated 456 genes and downregulated 499 genes. Addition of bumetanide reversed these effects for 136 genes and augmented these effects for 2 genes. TEER assays found LECs exposed to high Na+ could establish a monolayer, but that monolayer was more permeable when compared to cells cultured in normal Na+ conditions. qRT-PCR showed tight and adherens junction genes were decreased in high Na+ conditions. Monolayer integrity further decreased with addition of bumetanide, but not ethacrynic acid.
Conclusion
These results demonstrate that Na+ avid states promote LEC permeability and that bumetanide-induced inhibition of Nkcc1 can further augment this effect. While increasing permeability in lymphatic capillary LECs may be conducive for draining excess interstitial fluid, altering the permeability of LECs in collecting vessels may cause leakage and be detrimental to fluid clearance. In addition, ethacrynic acid may be superior to other loop diuretics due to the lack of off target effects on lymphatic vessel contractility and permeability.
Funding
- NIDDK Support