Na2hpo4 nah2po4

Feline renal tissue was obtained with owner informed consent during post-mortem examinations, and FPTEC and CKD-FCF were isolated as previously described (Lawson et al., 2018a (link), Lawson et al., 2018b (link)). FPTEC were derived from the kidneys of cats without azotaemia, and CKD-FCF were derived from the kidneys of cats with azotaemic CKD. Experiments utilising FPTEC took place in serum-free medium (pH 7.4) supplemented with 10 ng/mL epidermal growth factor (Invitrogen), 36 ng/mL dexamethasone (Sigma-Aldrich), 2 ng/mL triiodothyronine (Sigma-Aldrich), 1% insulin-transferrin-selenium (Thermo Fisher Scientific) and 1% antimicrobial solution (Gibco Antibiotic-Antimycotic (100x); Thermo Fisher Scientific). Experiments utilising CKD-FCF were performed in reduced serum DMEM-F12 (pH 7.4) containing 3% foetal bovine serum (FBS; Thermo Fisher Scientific). All experiments were performed using cells at passages 1 or 2. Experiments were performed in quadruplicate, where each experimental repeat represented a separate isolation from an individual cat. Phosphate was supplemented, from a standard concentration of 0.95 mM, by the addition of a pH 7.4 stock solution of sodium phosphate monobasic/sodium phosphate dibasic (NaH₂PO₄/Na₂HPO₄; Sigma–Aldrich).

ApoMb was prepared from horse-heart myoglobin (Sigma-Aldrich) following the butanone method to extract the heme group (as performed in^{50 (link)}), adapting the method described in^{60 (link)}), and then refolded by dialysis in 20 mM NaH₂PO₄/Na₂HPO₄ (Sigma Life Science, >99.5% and Sigma-Aldrich, >99%) pH 7 buffer and distilled water. Before storage in the freezer, the apo-Mb solution was lyophilized. To replace the exchangeable protons by deuterium ions, the freeze-dried apo-Mb powder was dissolved in heavy water (99.9% ²H, Sigma-Aldrich), incubated for 1 day, and lyophilized again. The obtained powder was stored at −20 °C. In order to obtain the molten globule state of apoMb the powder was dissolved in ²H₂O and centrifuged to remove the large aggregates. In the supernate solution of concentration 2 mg/mL and pH 6, ²HCl 0.1 M (Sigma-Aldrich) was added until the pH-read out value was 3.6 (monitored by pH meter Methrom). This corresponds to a a pD value of 4. The buffer exchanged protein solution was centrifuged (Heracus Instruments) to the final concentrations (Vivaspin 3,000 MWCO concentration units, Sartorius, Göttingen, Germany).

The PUFA-plasmalogen (vinyl ether) derivative 1-(1Z-octadecenyl)-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (PL-DHA-PE) and the fluorescent lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (18:1 Liss Rhod PE) were purchased from Avanti Polar Lipids, Inc. (Alabama). The composition of the scallop-derived plasmalogen extract was characterized by the provider as a mixture of ethanolamine vinyl ether phospholipid (49.4%), choline vinyl ether phospholipid (24.9%), cholesterol (16.0%), and ceramide aminoethyl phosphonate (9.7%). This natural plasmalogen combination with 70% vinyl ether phospholipid content is referred as scPL70. Monoolein (MO, C18:1c9, powder, ≥99%), retinoic acid (RA), vitamin E (VitE), 2,6-di-tert-butyl-4-methylphenol (BHT), and D-α-tocopherol polyethylene glycol-1000 succinate (TPGS-PEG₁₀₀₀) were purchased from Sigma-Aldrich. Water of MilliQ quality (Millipore Corp., Molsheim, France) was used for preparation of a phosphate buffer solution (NaH₂PO₄/Na₂HPO₄, 1 × 10⁻² M, pH 7, p.a. grade, Merck).