Graduation Date

Spring 5-5-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Cellular & Integrative Physiology

First Advisor

Steven C. Sansom

Abstract

Because of its cardio-protective benefits, diets that are low in Na+ and high in K+ are often warranted in conjunction with use of diuretics such as furosemide for treating hypertension. It is critical to understand how such diets influence the diuretic actions on renal K+ handling in order to choose the best drug for patients.

Furosemide is a commonly used K-wasting diuretic. It increases urinary K+ excretion by increasing distal Na+ delivery that stimulates K+ secretion. However, in wild-type mice (WT) adapted to an alkaline low Na+ high K+ diet (LNaHK), both 12-hour IP furosemide injection and 7-day furosemide water treatment decreased renal K+ clearance. We hypothesized that there is furosemide-sensitive net K+ secretion in WT adapted to LNaHK. Free-flow micropuncture experiments showed that furosemide increased [K+] in the early distal tubule (EDT [K+]) in WT on a regular diet (RD) but decreased EDT [K+] in WT on LNaHK. Furosemide did not affect EDT [K+] in ROMK knockout mice (ROMK KO) on LNaHK or WT on an alkaline normal Na+ high K+ diet (HK). Furosemide-sensitive Na+ excretion was significantly greater in mice on LNaHK than those on RD or HK. Patch clamp analysis of split-open thick ascending limbs (TAL) revealed that the 70-pS ROMK channel exhibited a higher open probability (Po) but similar density in WT on LNaHK, compared to WT on RD. These results indicate that mice adapted to LNaHK exhibit furosemide-sensitive net K+ secretion in the TAL that is dependent on increased NKCC2 activity and mediated by ROMK channels.

Furosemide acidifies the urine by increasing acid secretion via the Na+-H+ exchanger 3 (NHE3) in TAL and vacuolar H+-ATPase (V-ATPase) in the distal nephron. Because alkaline urine is required for BK-αβ4-mediated K+ secretion, we hypothesized that furosemide reduces BK-αβ4-mediated K+ secretion by acidifying the urine. Furosemide water treatment for 11 days led to decreased urine pH in both WT and BK-β4 knockout mice (BK-β4 KO) with increased V-ATPase expression and elevated plasma aldosterone levels. However, furosemide water decreased renal K+ clearance and elevated plasma [K+] in WT but not BK-β4 KO. Addition of acetazolamide in furosemide water restored urine pH along with renal K+ clearance and plasma [K+] to similar levels as control group. Additionally, western blotting and immunofluorescence staining showed that furosemide decreased cortical expression of BK-β4 and reduced apical localization of BK-α in connecting tubules (CNT); they were restored by adding acetazolamide to furosemide water. These results indicate that in mice adapted to HK, furosemide reduces BK-αβ4-mediated K+ secretion by acidifying the urine.

In conclusion, by inhibiting net K+ secretion in the TAL and BK-αβ4-mediated K+ secretion in CNT, furosemide acts as a K-sparing diuretic in mice adapted to LNaHK and HK.

Available for download on Saturday, April 27, 2019

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