Bilateral renal IRI and IPC mouse model
Briefly, experimental mice were anesthetized with isoflurane (1.6–1.8%). Under aseptic conditions, two dorsal flank incisions were performed and the renal pedicles of both sides were identified and clamped using a small non-traumatic clamp (4*0.75 mm/16 mm, RWD Life Science, San Diego, CA, USA). After visual confirmation of ischemic changes, the kidneys were returned to the retroperitoneal cavity and vessel occlusion was maintained for 15 minutes. The clamps were then released and blood flow restoration confirmed visually. Incisions were closed in two layers using 4 − 0 absorbable sutures and animals were volume resuscitated using normal saline (0.2 mL) injected subcutaneously. Subcutaneous buprenorphine (0.075 mg/kg) was given every 8–12 hours to ensure post-operative analgesia till 48 hours. 4 days later, the same procedure was performed again expect for the vessel occlusion maintaining for 30 minutes. Animals in sham-operated group were anesthetized and two flank incisions performed, renal pedicles dissected but not clamped. They were maintained for corresponding minutes mentioned above and the incisions were then closed in two layers.
To study acute phase of renal IRI, we allowed reperfusion for 2 days post-ischemia. To investigate post-ischemic progression to fibrosis, mice were followed for 42 days after injury. Following the reperfusion phase, animals were anesthetized, blood was obtained by mice tail at the time-point of 24 hours, 72 hours, and weekly from 1 week to 5 weeks after the surgery, and by cardiac puncture at the time-point of 6 weeks. Native kidneys were obtained from healthy anesthetized animals and processed immediately.
Mice were euthanized by high concentration of isoflurane. Kidneys were surgically removed under anesthesia after reperfusion for 20 mL of phosphate-buffered saline (PBS), and then sectioned after removal of the renal capsule and extrarenal structures. Each kidney was sectioned along the transverse axis into two pieces: upper (3- to 4-mm length) and lower (3- to 4-mm length). The upper pieces were fixed in 4% paraformaldehyde in 1X PBS for 24 hours at room temperature. Following fixation, tissue samples were routinely processed and embedded in paraffin wax (Fisher Scientific, Pittsburgh, PA, USA), and then for H&E staining, picrosirius red staining and immunofluorescence staining, etc. The medulla of the lower pieces were removed and the cortex were cut into small pieces and then flashed frozen in liquid nitrogen and then stored at -80 °C for further protein studies.
Detection of kidney injury related indicators
Western blot and immunofluorescence staining were used to detect the expression of KIM-1, cytokines including connective tissue growth factor (CTGF), alpha smooth muscle actin (α-SMA), transforming growth factor-β (TGF-β), etc. and collagen I and IV. They were all performed according to the protocols provided by the manufacturer (Sigma-Aldrich, St. Louis, MO, USA). The antibodies for western blot were listed as bellows. KIM-1: primary antibody, goat anti-mouse KIM-1 (1:800, R&D Systems, Minneapolis, MN, USA); secondary antibody, HRP-conjugated donkey anti-goat IgG antibody (1:5000, Jackson ImmunoResearch Laboratories, WestGrove, PA, USA). CTGF: primary antibody, rabbit anti-mouse CTGF (1:1000, GeneTex, Irvine, CA, USA); secondary antibody, HRP-linked goat anti-rabbit IgG antibody (1:2500, Cell Signaling Technology, Danvers, MA, USA). α-SMA: primary antibody, rabbit anti-α SMA antibody (1:500, abcam, Cambridge, MA, USA ); secondary antibody, goat anti-rabbit IgG H&L (HRP) (1:2500, abcam, Cambridge, MA, USA). TGF-β, anti-TGF β1 antibody produced in rabbit affinity isolated antibody (1:500, Sigma-Aldrich, St. Louis, MO, USA); secondary antibody, goat anti-rabbit IgG H&L (HRP) (1:2500, abcam, Cambridge, MA, USA). Those for immunofluorescence staining were as followings. KIM-1, goat anti-mouse KIM-1 (1:200, R&D Systems, Minneapolis, MN, USA) and Cy™3 AffiniPure Donkey Anti-Goat IgG (H + L) (1:1000, Jackson ImmunoResearch Laboratories, WestGrove, PA, USA). Collagen IV, rabbit anti-collagen IV antibody (1:100, abcam, Cambridge, MA, USA) and goat anti-rabbit IgG H&L (Cy3 ®) (1:1000, abcam, Cambridge, MA, USA). Collagen I, rabbit anti-collagen I antibody (1:500, abcam, Cambridge, MA, USA) and goat anti-rabbit IgG H&L (Cy3 ®) (1:1000, abcam, Cambridge, MA, USA).
Picrosirius red staining and H&E staining, as well as sCr and BUN measurement were also conducted according to the manufacturer’s protocols (abcam, Cambridge, MA, USA).