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Abstract: FR-PO520

Regulation of NKCC1, NKCC2, and NCC69 by TSC1/TSC2 Complex

Session Information

Category: Fluid, Electrolytes, and Acid-Base Disorders

  • 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic

Authors

  • Rodan, Aylin R., University of Utah Health, Salt Lake City, Utah, United States
  • Demaretz, Sylvie, Centre de Recherche des Cordeliers, Paris, France
  • Frachon, Nadia, Centre de Recherche des Cordeliers, Paris, France
  • Laghmani, Kamel, Centre de Recherche des Cordeliers, Paris, France
Background

Na-K-2Cl cotransporters NKCC1 and NKCC2 play important roles in numerous physiological processes and several human diseases such as salt losing tubulopathies, hypertension and cancer. In the kidney, NKCC1 plays a pivotal role in K+ secretion in the collecting duct while NKCC2 is the pacemaker of NaCl reabsorption in the thick ascending limb. Using the two-hybrid system, we identified tuberin (TSC2) as a binding partner of NKCC2. We therefore sought to characterize the mechanism by which TSC2, with or without hamartin (TSC1), could regulate NKCC2 and / or NKCC1.

Methods

NKCC1 and NKCC2 protein expression was monitored in HEK cells transiently or stably transfected with the cotransporters using immunoblot and confocal imaging. The stability of the cotransporters was assessed by cycloheximide chase assay. NCC69 function was evaluated in Drosophila Malpighian tubules overexpressing TSC1 and/or TSC2 in a control of Ncc69 mutant background.

Results

Co-immunoprecipitation experiments showed robust interaction between TSC2 and the complex-glycosalyed form of NKCC2 suggesting that the interaction takes a place at the post-Golgi level . TSC2 and/or TSC1 knock-down (KD) similarly increased total NKCC2 protein. Cycloheximide assays and leupeptin treatment (lysosome inhibitor) revealed that TSC1/2 KD upregulate NKCC2 by increasing its stability and maturation. Interestingly, similar to NKCC2, the KD of TSC1/TSC2 reproduced the same effects on NKCC1. To elucidate in vivo the role of TSC1/TSC2 in the regulation of NKCC, we took advantage of the expression of NCC69, the fly NKCC in the Drosophila melanogaster renal tubule. Importantly, while overexpression of either TSC1 or TSC2 alone had no effect, overexpression of TSC1 and TSC2 together resulted in decreased tubule K+ secretion in control tubules, but not in tubules lacking NCC69, strongly indicating that the two TSC function together to regulate NCC69 function.

Conclusion

We identified TSC1/TSC2 complex as a novel key player in the post-Golgi regulation of NKCCs. Our results are consistent with a model whereby TSC1 and TSC2 function together to decrease the expression of the cotransporters via the lysosome pathway. A better understanding of the regulatory pathways acting on NKCC1 and NKCC2 will ultimately help to identify new “druggable” targets to prevent and/or treat several disorders in which these two cotransporters are involved.

Funding

  • Government Support – Non-U.S.