Osmomat 030 osmometer

Urine volume was determined gravimetrically. Urinary osmolality (Uosm) was measured with the cryoscopic Osmomat 030 osmometer (Gonotec, Berlin, Germany). Urine sodium (UNa) and potassium (UK) concentration were measured by a flame photometer (BWB-XP, BWB Technologies Ltd, Newbury, UK). Urine flow (V), the excretion of total solutes (UosmV), sodium (UNaV) and potassium (UKV) were calculated using the standard formulas and standardized to g kidney weight (UXV/g KW). All measurements were performed in duplicates (technical replicates).

Human epithelial bladder transitional papilloma RT4 cells (ATCC, Manassas, VA) were cultured in McCoy's 5A modified medium (306 ± 12 mOsm, N = 3), supplemented with 10% heat inactivated fetal bovine serum. Once cells reached 90% confluency, they were sub‐cultured in a 1:10 dilution. Media were removed and replaced with fresh media every 2 days. Cells were incubated at 37°C with 5% CO₂. For regulatory experiments, cells were exposed to media containing additional NaCl, mannitol or urea for 48hr prior to RNA extraction or protein isolation. Osmolalities of experimental media were confirmed using an Osmomat 030 osmometer (Gonotec, Germany).

Human epithelial colorectal adenocarcinoma Caco‐2 cells (HTB‐37) were purchased (ATCC, Manassas, VA) and were cultured in Dulbecco's Modified Eagle Medium (DMEM) with a high d‐glucose content (4.5 g/L), containing l‐glutamine, and supplemented with 10% heat‐inactivated fetal bovine serum (Life Technologies, Dublin, Ireland). Once cells reached 90% confluency, they were subcultured in a 1:6 dilution every 5 days. Media were removed and replaced with fresh media every 2–3 days. Cells were incubated at 37°C with 5% CO₂. For regulatory experiments, cells which had reached 90% confluency were exposed to media containing additional NaCl, mannitol, or butyrate for 24 h prior to RNA extraction. Osmolalities of different experimental media were confirmed using an Osmomat 030 osmometer (Gonotec, Germany) and measurements (mean ± standard error) were as follows: “250 mOsm” = 246 ± 22 mOsm (N = 4); Control “350 mOsm” = 354 ± 13 mOsm (N = 5); “500 mOsm” = 502 ± 9 mOsm (N = 3); “600 mOsm” with NaCl = 595 ± 30 mOsm (N = 5); and “600 mOsm” with mannitol = 634 ± 15 mOsm (N = 4).

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-hydroxy-sn-glycero-3- phosphoglycerol (POPG), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn- glycero-3-phosphocholine (DMPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were purchased from Avanti Polar Lipids Inc. (Birmingham, AL). TMX, HTMX, and 5(6)-carboxyfluorescein (CF) (99% by HPLC) were from Sigma-Aldrich (Madrid, Spain). All the other reagents were of the highest purity available. Inorganic salts and buffers were of analytical grade. Purified water was deionized in a Milli-Q equipment from Millipore (Millipore, Bedford, MA), and had a resistivity of ca. 18 MΩ. Stock solutions of the various phospholipids and drugs were prepared in chloroform and stored at −80 °C. Phospholipid phosphorus was determined according to the method of Böttcher [26 (link)]. The buffer used through the work was 150 mM NaCl, 5 mM Hepes, pH 7.4, unless otherwise stated. Water, and all the buffer solutions used in this work, was filtered through 0.2 μm filters prior to use. The osmolarities of all the buffers and solutions were checked using a Osmomat 030 osmometer (Gonotec, Berlin, Germany).