Deciphering the Roles of 2 Intercalated Cell Proteins in Acid-Base and Ion Homeostasis: SLC26A7 and Claudin-4

• Author / Creator

  Ullah, AKM Shahid

• Few of the many crucial physiological functions are maintaining acid/base balance and electrolyte homeostasis. The last segment of the nephron, collecting duct, fine tunes these functions by secreting acids and reabsorbing bicarbonate through the specialized type-A intercalated cells (Type-A IC). These cells harbour acid secreting protein H+ATPase at the apical membrane and chloride-bicarbonate exchanger proteins, kidney anion exchanger 1 (kAE1) and SLC26A7 at the basolateral membrane. Mutations in either H+ATPase or kAE1 can cause distal renal tubular acidosis (dRTA), principally characterized by defective urine acidification. In the collecting duct, there are also the principal cells and the type-B intercalated cells which regulate the electrolyte homeostasis with the proteins localized in their apical and basolateral membranes. Tight junction (TJ) proteins occupy the gap between the cells on the apical side, facilitating paracellular ion transfer between lumen and interstitium.

  We characterized SLC26A7 protein’s expression and function in relation to kAE1 and dRTA mutant kAE1 R901X in normal growth condition and hyperosmotic growth medium mimicking medullary habitat of the protein. We confirmed that SLC26A7 protein acts as a chloride/bicarbonate exchanger in Madin Darby Canine Kidney (MDCK) cells. We also found that the protein is mostly intracellular in both polarized and non-polarized cells, and demonstrate a lower chloride/bicarbonate exchange activity than that of kAE1 protein. Co-expressing SLC26A7 and kAE1 proteins did not demonstrate additive chloride/bicarbonate exchange function compared to when they are expressed alone. Our experiments also showed that osmolarity does not affect the chloride bicarbonate activity in the cells expressing SLC26A7. Furthermore, we mimicked acidotic condition by decreasing pH of the growth medium and found reduced abundance of SLC26A7 protein. All these experiments suggest that SLC26A7 functions as a chloride/bicarbonate exchanger in MDCK cells independent of osmolarity and its cellular abundance is dependent on extracellular osmolarity and pH conditions.
  Our second set of study was on the tight junction protein claudin-4 (cldn-4), which acts as a chloride pore and sodium barrier in the collecting duct. Total knock out (KO) cldn-4 mice demonstrate lethal hydronephrosis, hypocalcemia and elevated fractional excretion of calcium and chloride ions. Principal cell (PC) specific cldn-4 KO show increased fractional excretion of chloride and sodium. We established IC specific cldn-4 KO mouse to obtain an overall picture of the role of cldn-4 in the collecting duct. At the steady state, IC cldn-4 KO mice do not demonstrate any gross physiological defect and are able to maintain normal acid-base and calcium homeostasis. To investigate if the mice are able to reabsorb sodium and chloride as efficiently as the WT, they were fed a NaCl depleted diet. In this condition, the mice displayed increased fractional excretion of sodium. At the basal state and salt depleted diet condition, the mice display increased expression of IC markers pendrin, kAE1 and sodium dependent chloride bicarbonate exchanger (NDCBE) without a change in the abundance of H+ATPase. When given 0.28M NH4Cl water, the mice are able to maintain pH homeostasis to the same degree as the WT mice. To investigate if the IC cldn-4 has any contribution to calcium homeostasis, the mice were fed a low calcium diet but displayed no perturbation in calcium homeostasis. Together all these investigations indicate that in the distal nephron, IC cldn-4 is not involved in pH and calcium homeostasis but is required for sodium reabsorption.

• Subjects / Keywords

  • Intercalated cells
  • SLC26A7
  • Claudin-4

• Graduation date

  Fall 2022

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-xe7j-ft38

• License

  This thesis is made available by the University of Alberta Library 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.