Low salt diet

Polyclonal anti-KCC1 antibody was purchased from Alomone Labs, Israel. Bumetanide, amiloride, benzamil, barium, glibenclamide, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), CFTR(inh)-172, and forskolin were obtained from Sigma while GlyH 101 was from Calbiochem. All stock solutions were prepared in DMSO. Normal salt diet and low salt diet were purchased from Harlan Teklad. Ringer solution contains (in mM) 140.8 Na⁺, 5.2 K⁺, 123.5 Cl^-, 25 HCO₃^-, 1.25 Ca²⁺, 1.25 Mg²⁺ and 5 glucose, pH 7.4. Cl^--free Ringer solution contains HCO₃^- (25 mM) with no Cl^-. HCO₃^--free Ringer solution contains Cl^- with no HCO₃^-. Na⁺-free Ringer solution contains no Na⁺. In these solutions, isethionate was used in place of anion Cl^- and HCO₃^- and N-Methyl-D-glucamine (NMDG) was used as a substitute for Na⁺. HCO₃^--containing solutions were gassed with 95% O₂-5% CO₂; HCO₃^--free solution was gassed with 100% O₂.

Experiments were performed with non-fasting male adult Sprague-Dawley rats (5–10 week old with a body weight of 100–300 g) obtained from Charles River Laboratories. Before sacrifice, animals were subjected to one of the following three dietary treatments for a week: 1) normal salt diet (0.3% sodium and 0.5% chloride; Harlan Teklad, Madison, WI, USA; cat. no. T7012) plus tap water in drinking bottle, 2) low salt diet (0.1% sodium and 0.2% chloride; Harlan Teklad, Madison, WI, USA; cat. no. T7034) plus tap water in drinking bottle, 3) normal salt diet plus normal saline (0.9% NaCl) in drinking bottle to enhance daily salt intake. Animals were sacrificed with standard CO₂ inhalation, followed by cervical dislocation. A segment of distal colon of approximately 4–5 cm in length between the two landmark lymph nodes was excised and divided into early distal colon (the segment in proximity to the proximal lymph node), and late distal colon (the segment adjacent to the distal lymph node). The late distal colon was located approximately 1 cm from the anus. The use of animals, as well as the protocol for isolating colon tissues, was approved by the Institutional Animal Care and Use Committee (IACUC# 201507567) at the University of Florida.