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Abstract: PO1401

Flow Regulation of WNK1 in the Cortical Collecting Duct (CCD)

Session Information

Category: Fluid, Electrolyte, and Acid-Base Disorders

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

Authors

  • Carrisoza-Gaytan, Rolando, Department of Pediatrics, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Flores, Daniel, Department of Pediatrics, Icahn School of Medicine at Mount Sinai, New York, New York, United States
  • Subramanya, Arohan R., Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Ray, Evan C., Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Kleyman, Thomas R., Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, United States
  • Satlin, Lisa M., Department of Pediatrics, Icahn School of Medicine at Mount Sinai, New York, New York, United States
Background

A high K diet (HKD) x 10 d increases (i) luminal flow rate in the distal nephron and (ii) expression of apical immunodetectable L-WNK1 in CCD intercalated cells (IC), which we propose enhances apical BK channel activity measured as flow-induced K+ secretion (FIKS) (Webb et al, 2015). We previously demonstrated that fluid shear stress (FSS) x 30 min induces expression of ERK and p-38, both BK channel modulators, in a CCD principal cell (PC) model (Carrisoza-Gaytan et al, 2014). The objective of this study was to test the hypothesis that an increase in tubular fluid flow rate rapidly induces apical localization of L-WNK1 in the CCD.

Methods

CCDs isolated from NZW rabbits fed a HKD x 10 d were microperfused at slow (n=4) or fast (n=4) luminal flow rates x 1 hr, fixed on the rig, immunoperfused with antibodies (Abs) directed against L-WNK1 (+ A488-fluorescent 2o Abs) and rhodamine-conjugated peanut lectin (PNA) or Dolichus biflorus lectin (DBA), which bind to apical surfaces of IC and PC, respectively, and examined by confocal microscopy. MDCK cells were subject to no (static), low or high FSS x 1 hr (n=3 each condition) and fixed for immunodetection of L-WNK1 and BKα in situ or harvested for semi quantitative immunoblotting of isolated plasma membranes.

Results

Expression of IC apical L-WNK1 relative to that in the total cell was 40.0±1.1 % greater in CCDs perfused at fast flow compared to those perfused at slow flow rates (p<0.001). In plasma membrane preparations of MDCK cells subject to low and high FSS, L-WNK1 abundance was 5.4±0.3 and 10.6±1.7 fold greater, respectively, to that measured in the absence of flow (p<0.001). In static MDCK cells, BKα did not colocalize with L-WNK1 at the apical membrane; however, colocalization of the two proteins was detected in cells subject to low or high FSS.

Conclusion

In conclusion, (i) apical expression of L-WNK1 in ICs in microperfused CCDs is rapidly stimulated by increases in luminal flow rate, and (ii) FSS favors apical colocalization of L-WNK1 with BKα, responses that may facilitate BK channel-mediated FIKS in the CCD.

Funding

  • NIDDK Support