Kidney Week

Abstract: SA-PO1024

Co-Localization of NKCC2 with AQP2 in the Collecting Duct Principal Cells Contributes to the Acute Diuretic Effects of Furosemide and Overall Water Handling by the Kidney

Session Information

• Fluid and Electrolytes: Basic - II
  October 27, 2018 | Location: Exhibit Hall, San Diego Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Fluid and Electrolytes

• 901 Fluid and Electrolytes: Basic

Authors

• Amlal, Hassane, University of Cincinnati, Cincinnati, Ohio, United States

Hassane Amlal,

• Amlal, Sihame, University of Cincinnati, Cincinnati, Ohio, United States

Sihame Amlal,

• Thakar, Charuhas V., University of Cincinnati, Cincinnati, Ohio, United States

Charuhas V. Thakar,

• Alam, Perwez, University of Cincinnati, Cincinnati, Ohio, United States

Perwez Alam,

• Ahmed, Rafeeq, University of Cincinnati, Cincinnati, Ohio, United States

Rafeeq Ahmed,

Background

The activity of Na⁺/K⁺/2Cl^- cotransporter (NKCC2) in the thick ascending limb (TAL) plays an important role in the process of urinary concentrating mechanism and the control of water balance by the kidney. Acute furosemide (NKCC2 inhibitor) administration to both human and animals is associated with a rapid (minutes) diuresis, natriuresis, diluted and acidified urine. This raises the question as to whether NKCC2 is co-localized with AQP2 in the collecting duct (CD) principal cells.

Methods

Male rats housed in metabolic cages were injected with furosemide (FURO, 5mg/kg BW) and urine was collected every hour for 2 hours and after 7 days of daily treatment. Urine of vehicle-injected rats (control) was collected over a 3-hour period and after 7 days. Urine volume, urine osmolality, Na⁺ excretion and urine pH were measured. Rat and human kidney tissues were used to examine the localization of NKCC2 and AQP2 and ENaC by immunofluorescence labeling technique.

Results

Compared to control group, furosemide-treated rats exhibited an increase in urine output and Na+ excretion by 9- and 33-fold, respectively, during the first hour of treatment. Urine osmolality decreased to 439 mosm/kg H2O during the first hour and remained low (450~ mosm/kg H2O) after 7 days vs. 1373 (3 hours) or 1635 (7 days) mosm/kg in control group. Urine pH decreased significantly (from 6.56 to 6.04, P<0.04) during the second hour of furosemide treatment. The later effect is inhibited in the presence of low dose of amiloride (ENaC inhibitor). Merged images of double immunofluorescence labeling of rat and human kidney tissues with AQP2 and NKCC2 or with AQP2 and ENaC showed NKCC2 expression in the apical membrane of both AQP-free (TAL), as well as AQP2 expressing cells; whereas ENaC is exclusively co-expressed with AQP2 in the CD principal cells of both rat and human kidneys.

Conclusion

We demobnstarte for the first time that NKCC2, ENaC and AQP2 are co-expressed in the apical membrane of principal cells in the cortical and outer medullary CD of both rat and human kidneys. The co-localization of NKCC2 with AQP2 contributes to the acute diuretic and natriuretic effects of furosemide, and the co-localization of NKCC2 with ENaC in the CD is responsible for furosemide-induced urinary acidification.

Funding

• NIDDK Support