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Abstract: SA-PO1006

NFAT5 Regulates Endothelin Gene Expression: Possible Common Pathway in Skin and Kidney Responses to High Na+ Intake

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Lakshmipathi, Jayalakshmi, University of Utah Health Sciences Center, Salt Lake City, Utah, United States
  • Wheatley, Will, University of Utah Health Sciences Center, Salt Lake City, Utah, United States
  • Ramkumar, Nirupama, University of Utah, Salt Lake City, Utah, United States
  • Kohan, Donald E., University of Utah Health Sciences Center, Salt Lake City, Utah, United States
Background

High Na+ intake stimulates collecting duct endothelin-1 (ET-1) production which in turn promotes a natriuresis. High Na+ intake induced ET-1 involves several factors, however increasing extracellular osmolality, as occurs in the renal medulla during Na+loading, stimulates ET-1 by inner medullary collecting duct (IMCD3 cells) more potently than any other known stimulus. In addition, following a Na+ load, the skin stores Na+ leading to increased local osmolality; release of skin Na+ into the circulation (facilitating renal elimination) partly depends upon ET-1. The current studies were undertaken to determine how osmolality augments ET-1 in IMCD and skin.

Methods

Cultured IMCD3 and dermal microvascular endothelial cells (DMVEC) were studied. Osmolality was increased for 2-4 hr using 50 mOsm/L mannitol. ET-1/GAPDH mRNA were measured. ET-1 promoter/reporter constructs were transfected into cells and secreted luciferase/alkaline phosphatase assayed. NFAT content and subcellular distribution were analyzed by western analysis.

Results

Increasing osmolality increased IMCD3 ET-1 mRNA associated with an increase in nuclear fraction NFAT5 protein content. Osmolality-stimulated ET-1 was markedly reduced by NFAT5 siRNA. Transfection of 1, 2 or 3 kb (5’ to transcription start site) ET-1 promoter/reporter constructs revealed maximal osmolality induced activity in the 1 kb fragment. This region contains two NFAT5 consensus binding sites; mutation of both sites (TGGAAA to TCACGA) completely prevented hyperosmolality induced promoter activity. Increasing osmolality also increased DMVEC ET-1 mRNA content; initial studies suggest this is also NFAT5 dependent.

Conclusion

These studies identify for the first time that NFAT5 directly regulates ET-1 promoter activity. Both the skin and IMCD NFAT5/ET-1 may be involved in the Na+ homeostatic response to high Na+ intake. Thus, there may be a remarkably common system used by the skin and the kidney to regulate body Na+content.

Funding

  • Other NIH Support