Animal models, biochemical analyses and tissue collection

Kcnj1^−/− mice³⁷ and wild-type (Kcnj1^+/+) littermates were housed in a light- and temperature-controlled environment with ad libitum access to tap water and standard chow (Diet AO3, SAFE; 25/18 GR Mucedola Srl, Settimo Milanese, Italy). Urine samples were collected using individual metabolic cages (UNO Roestvastaal BV, Zevenaar, The Netherlands) after appropriate training. Blood was collected by venous puncture or cardiac puncture at time of sacrifice and centrifuged at 2000 g for 15 minutes at 4 °C in heparin-coated tubes (Sarstedt AG, Nürnbrecht, Germany) in order to separate plasma from cells. Creatinine, electrolytes and urea were measured on a Synchron CX5 analyser (Beckman Coulter, Fullerton, CA, USA), according to the manufacturer’s instructions. Urine and plasma osmolality were measured on a multi-sample osmometer (Advanced Instruments Model 2020, Norwood, MA, USA). Urinary concentration of uromodulin in mouse urine was assessed by means of a commercial ELISA kit (ab245726, Abcam, Cambridge, UK). Following the collection of urine and plasma samples, mice were sacrificed either by decapitation or by cervical dislocation following anaesthesia with isoflurane (Minrad International Inc., Orchard Park, NY, USA) for the collection of the kidneys. One kidney was immediately homogenized for protein or RNA extraction, while the other was further processed for histological analyses. Kcnj1^fl/fl mice were generated by Ozgene (Perth, Australia) as described elsewhere (Penton et al., MS under revision) and were used to obtain kidney tissue for primary mTAL cell culture. Moreover, kidney lysates, frozen tissues and urine samples were obtained from tetracycline-inducible kidney-specific Kcnj1 KO mice that had been generated by crossing the Kcnj1^fl/fl mice with Pax8-rtTA-cre mice²⁴. All of the experiments were performed in accordance with the ethical guidelines at University of Zurich (Zurich, Switzerland) and the legislation of animal care and experimentation of Canton Zurich, Switzerland. The experimental protocols were approved by the appropriate licensing committee (Kanton Zürich Gesundheitsdirektion Veterinäramt; protocol ZH049/17) at the University of Zurich.