The Role of NHE8 in the Regulation of Renal Proximal Tubule Calcium Reabsorption

• Author / Creator

  Wiebe, Shane A

• Abnormalities in calcium homeostasis can result in kidney stones, a painful and potentially life-threatening condition. Improved therapies for this disorder can only be achieved through increased fundamental knowledge about calcium handling. In the kidneys, filtered calcium (Ca2+) is reabsorbed along the nephron. To delineate the renal regulation of calcium reabsorption, we performed a mRNA micro-array on kidneys from mice treated with the calcium sensing receptor (CaSR) agonist cinacalcet. This revealed that the sodium/hydrogen exchanger isoform 8 (NHE8) expression was decreased by the CaSR agonist. These results were confirmed by quantitative PCR. Administration of vitamin D to mice also decreased NHE8 mRNA expression. In contrast, immunoblotting demonstrated increased renal NHE8 protein expression from the same mice treated with cinacalcet or vitamin D. We therefore hypothesized that NHE8, similar to NHE3, plays a role in calcium homeostasis by facilitating the reabsorption of filtered calcium. To assess this, we first validated a renal cell culture model, normal rat kidney (NRK) cells. To this end, we demonstrated apical expression of NHE8 in NRK cells using surface biotinylation and confocal immunofluorescence microscopy. Functional studies demonstrate 5-(N-Ethyl-N-isopropyl) amiloride (EIPA) inhibitable NHE activity at concentrations minimally attenuating NHE1 activity in AP-1 cells. To delineate the molecular role of NHE8 in calcium homeostasis, we measured transepithelial 45Ca2+ flux across confluent monolayers and 45Ca2+ uptake in the presence and absence of EIPA. There was no difference between groups suggesting NHE8 does not participate in cellular Ca2+ handling. However, ratiometric calcium imaging, a technique with greater temporal resolution, revealed enhanced Ca2+ uptake with EIPA treatment and after removal of extracellular sodium. Together, these results suggest NHE8 mediates Ca2+ efflux from NRK cells either directly or in collaboration with other transport mechanisms.

• Subjects / Keywords

  • Calcium Reabsorption
  • Renal Physiology
  • Kidney Stones
  • NHE8
  • Calcium Homeostasis

• Graduation date

  Fall 2016

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3RJ4932R

• 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.