The Regulation of Na+/H+ Exchanger Isoform 1 in Kidney Cells

  • Author / Creator
    Aderibigbe, Ayodeji O.
  • The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitous plasma membrane protein that regulates intracellular pH by removing a proton in exchange for extracellular sodium. NHE1 is expressed in many tissues including the kidney where it has been demonstrated to play essential roles in pH and cell volume regulation. It has also been implicated in some pathological conditions of the kidney including ischemia and chronic kidney diseases. NHE1 has a large 315 amino-acid cytosolic regulatory domain that regulates the catalytic membrane domain. The cytosolic domain is mainly regulated by phosphorylation and protein interaction. This study examined how these regulatory mechanisms regulate NHE1 in kidney cells. The activation of NHE1 in myocardial cells by sustained intracellular acidosis (SIA) was shown to be mediated by ERK1/2 phosphorylation. In this study, we also demonstrate that SIA stimulates NHE1 activity in Mardin-Darby Canine Kidney (MDCK) cells. To characterize how SIA stimulates NHE1 in kidney cells, wild-type and mutant NHE1 cDNAs were stably expressed in MDCK cells and examined for activation and phosphorylation in response to SIA. All the cDNAs had a L163F/G174S mutation, which conferred a 100-fold resistance to EMD87580, an NHE1-specific inhibitor. This allowed us to assay exogenous NHE1 activity while inhibiting endogenous activity with EMD87580 and while inhibiting the NHE3 isoform of the Na⁺/H⁺ exchanger using the isoform-specific inhibitor S3226. We demonstrated that the amino acids Ser771, Ser776, Thr779, and Ser785 are important for NHE1 phosphorylation and activation after acute SIA. SIA also activated ERK-dependent pathways in MDCK cells, and this was blocked by treatment with the MEK inhibitor U0126. Treatment with U0126 also blocked activation of NHE1 by SIA. These results suggest that acute acidosis activates NHE1 in mammalian kidney cells and that in MDCK cells this activation occurs through phosphorylation of a distinct set of amino acids in the cytosolic regulatory tail of NHE1 by ERK1/2. We also examined how protein interaction regulates NHE1 activity. Using affinity chromatography with the C-terminus of NHE1, we determined the NHE1 binding proteins in the kidney which includes 14-3-3 protein, heat shock proteins (Hsp90 and Hsp70) and Na+/K+ ATPase. We also confirmed that 14-3-3 and heat shock proteins bind to or regulate NHE1 but could not confirm that Na+/K+ ATPase binds to the intact protein. The Hsp90 inhibitor 17-AAG decreased NHE1 activity and NHE1 phosphorylation in MDCK cells but did not decrease protein levels. Additionally, 17-AAG decreased phospho-AKT levels. Direct inhibition of AKT with MK2206 decreased NHE1 activity, though this effect was not additive with the effect of 17-AAG. These results are the first demonstration that in renal cells, NHE1 is associated with several regulatory proteins including Hsp90, and suggest that this interaction affect NHE1 function through altered phosphorylation of the protein via the AKT kinase.

  • Subjects / Keywords
  • Graduation date
  • Type of Item
  • Degree
    Doctor of Philosophy
  • DOI
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
  • Institution
    University of Alberta
  • Degree level
  • Department
    • Department of Biochemistry
  • Supervisor / co-supervisor and their department(s)
    • Fliegel, Larry (Biochemistry)
  • Examining committee members and their departments
    • Casey, Joseph (Biochemistry)
    • Holmes, Charles (Biochemistry)
    • Alexander, Todd (Pediatrics)
    • Stuart, David (Biochemistry)