*4.2. UUO Model*

UUO mouse model was performed as described earlier [7]. Briefly, NZO mice were anaesthetized by isoflurane (2.2%) supplied with air flow at approximately 350 mL/min. During the surgery mice were placed on a heating pad to prevent hypothermia. Preemptive analgesia with carprofen (5–10 mg/kg b.w) was subcutaneously used. Body temperature was maintained at 37.5 ◦C and monitored during surgery using a temperature controller with a heating pad (TCAT–2, Physitemp Instruments, Clifton, NJ, USA). In deep anesthesia, the anterior abdominal skin was shaved. Then, a midline laparotomy was conducted via an incision of the avascular linea alba, and the left ureter was exposed from left side. The ureter was then ligated twice close to the renal pelvis using a 5–0 polyglycolic acid (PGA) suture wire (Resorba®, Nürnberg, Germany). The linea alba and skin were closed separately. The wound was sanitized with a silver aluminium spray (Henry Schein®, Berlin, Germany), and 0.5 mL of warm (37 ◦C) isotonic sodium chloride solution was intraperitoneally injected. Subsequently, each mouse was placed in a cage in front of an infrared (IR) lamp and monitored until they recovered consciousness. For the following two days, mice received carprofen (2.5 mg/mL) in their drinking water (1:50) with a final concentration of 0.05 mg/mL. After surgery mice had free access to drinking water and chow. Seven days after UUO surgery, mice were sacrificed by overdose of isoflurane and cervical dislocation. The blood samples were collected for further analysis and left kidneys were removed immediately. The kidneys were divided into three portions. Upper part of the kidney tissue was frozen in isopthane. Middle part of kidney was immersed in 4% phosphate–buffered saline (PBS)-buffered formalin for histological assessment. The other left tissue was snap frozen in liquid nitrogen for RNA preparation.
