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Abstract: PO1097

New Method to Discriminate Function of A and B Type of Intercalated Cells in Split-Opened Collecting Ducts

Session Information

Category: Fluid, Electrolyte, and Acid-Base Disorders

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

Authors

  • Hassanzadeh Khayyat, Naghmeh, The University of Texas Health Science Center at Houston, Houston, Texas, United States
  • Tomilin, Victor N., The University of Texas Health Science Center at Houston, Houston, Texas, United States
  • Pyrshev, Kyrylo, The University of Texas Health Science Center at Houston, Houston, Texas, United States
  • Zaika, Oleg L., The University of Texas Health Science Center at Houston, Houston, Texas, United States
  • Pochynyuk, Oleh, The University of Texas Health Science Center at Houston, Houston, Texas, United States
Background

The collecting duct (CD) is a highly adaptive terminal part of the nephron, which is essential for maintaining systemic homeostasis. Electrically uncoupled principal and intercalated cells (PCs and ICs) perform different physiological tasks and exhibit rather distinctive morphology. However, acid-secreting A- and base secreting B-type of ICs cannot be easily separated in functional studies despite virtually mirrored localization of their transport acid-base systems. Thus, there is no consensus of whether and how systemic pH stimuli affect function and A/B cell type ratio in the CD. The technique of split-opening isolated CD allows unambiguous monitoring of alterations in function in many individual cells within the split-opened area. However, it is not possible to specifically change the driving force for Cl- at luminal or basolateral sides, which is used in perfused tubule studies to identify IC types of certain cells on periphery.

Methods

We used BCECF-sensitive intracellular pH (pHi) measurements in split-opened CDs followed by immunofluorescent (IF) detection of AQP2 and pendrin from WT and ClC-K2-/- mice to demonstrate that inhibition of this Cl- channel enables sorting out signals from A- and B- types of ICs.

Results

We show that ClC-K2 Cl- channel is exclusively expressed on the basolateral side of AQP2-negative ICs, where it likely participates in Cl--dependent H+/HCO3- transport. Indeed, ClC-K2 blocker, NPPB, had no effect on pHi in PCs, whereas it caused acidification or alkalization in different subpopulations of ICs in WT but not ClC-K2-/- mice. IF assay of the same CDs revealed that NPPB decreased pHi in pendrin-positive B-type and increased pHi in A-type of ICs. Induction of metabolic acidosis markedly increased A/B cell ratio from 74% to 145%. Furthermore, dietary acidification also resulted in significantly augmented H+ secretion (assessed as recovery after acidification) in A-type and decreased pH transport in B-type of ICs.

Conclusion

We show that inhibition of ClC-K2 can be employed to discriminate between A- and B-type of ICs in split-opened CD preparations. Using this method, we found that metabolic acidosis leads to augmented transport rate and increased total population of A-type in the CD.