Characterizing The Role of The Chloride/Bicarbonate Exchanger AE1 in Modulating Tight Junction Properties in Renal Collecting Duct Cells

• Author / Creator

  LASHHAB, RAWAD

• The human kidneys maintain acid/base balance and electrolyte homeostasis. In the collecting ducts, acid secretion and bicarbonate reabsorption are facilitated by type-A intercalated cells (type-A IC). Type-A IC cells express the acid-secreting vacuolar H+-ATPase pump at the apical membrane, and the kidney anion exchanger 1 (kAE1) at the basolateral membrane to reabsorb bicarbonate. Mutations in the gene encoding kAE1 protein lead to distal renal tubular acidosis (dRTA), a disease where patients suffer from hypokalemia, hyperchloremia, metabolic acidosis and a defective urine acidification among other symptoms. Type-A IC cells also express the tight junction (TJ) protein claudin-4 which facilitates paracellular Cl- reabsorption, and With-no-lysine kinases 1 and 4 (WNK1 & WNK4), two kinases that regulate claudin-4 phosphorylation.
  A membrane yeast two-hybrid assay performed by Dr. Reinhart Reithmeier (University of Toronto) revealed a physical interaction between kAE1 and claudin-4. We therefore hypothesized that this interaction contributes to acid/base balance and electrolyte homeostasis.
  First, we confirmed the physical interaction and colocalization between the two proteins in mouse inner medullary collecting duct (IMCD) cells and colocalization in mouse kidney sections. Upon inducibly expressing kAE1 protein in IMCDcells, the transepithelial electrical resistance (TEER) of the monolayer significantly decreased and thepermeability to Na+ and Cl-increased. These effects were dependent on kAE1 activity. When endogenous claudin-4 was knocked down, kAE1 expression had no further effect on TEER and TJ properties, indicating that kAE1 modulates TJ properties via claudin-4 protein.
  Next, we investigated the mechanism(s) by which kAE1 modulates claudin-4 function. Interestingly, kAE1 expression increased phoshorylation of claudin-4, WNK4 and SPAK (a downstream effector of WNK4). This effect was abolished using kAE1 E681Q inactive mutant, indicating that kAE1 activity is crucial for triggering these phosphorylation events. WNK463, a pharmacological inhibitor of WNKs including WNK4 resulted in a similar decrease in TEER as that of kAE1 expression. However, the combination of WNK inhibition and kAE1 expression resulted in an additive decrease in TEER rather than abolishing kAE1 effect upon WNK inhibition. Interestingly, claudin-3 abundance was reduced upon kAE1 expression and both claudin-3 and -4 decreased upon kAE1 expression and WNK inhibition. Therefore, although our findings indicate that kAE1 expression induces phosphorylation of claudin-4, WNK4 and SPAK, a definitive conclusion on the role of WNK4 in kAE1's impact on TJ properties could not be drawn.
  Finally, we assessed the effect of dRTA causing-mutants kAE1 S525F and R589H on TJ properties. Both mutants are targeted to the basolateral membrane and carry high mannose and complex oligosaccharide. However, they differ in their exchange activity rate. While kAE1 R589H mutant has a similar exchange rate as kAE1 WT, kAE1 S525F mutant is less active than kAE1 WT. Interestingly, assessing their effect on TEER, we found that functionally defective kAE1 S525F mutant had a lower impact on TEER, whereas fully active kAE1 R589H had a similar impact on TEER to kAE1 WT. These results indicated that kAE1 effect on TJ properties is proportional to its functional activity and is likely mediated by WNK4, SPAK and claudin-4 phosphorylation.

• Subjects / Keywords

  • PHYSIOLOGY

• Graduation date

  Fall 2021

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-s7b0-et70

• 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.