Animals, surgery, and study design

The experiments were performed in accordance with the policies of the National Institutes of Health Guide for the Care and Use of Laboratory Animals for Scientific Purposes. Experiments were approved by the Institutional Animal Care and Use Committee at Indiana University. Male Sprague Dawley rats (250—300 g) were purchased from Envigo (Indianapolis, IN). Rats were housed in pairs in the Indiana University Laboratory Animal Resource center with 12 h light cycle with food and water available ad libitum. All surgeries were performed between 0900 and 1300. As per IACUC guidelines, rats were monitored daily after surgery and euthanasia is indicated for rats based on a scoring system to evaluate excessive distress including the presence of excessive weight loss, lethargy, and loss of thermoregulation.

Rats were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (5 mg/kg) and then placed on a heated pad to facilitate maintenance of body temperature. Anesthetized rats were subjected to bilateral renal ischemia–reperfusion I/R injury by clamping both left and right renal pedicle for 40 min to induce AKI, as described previously [15]. Sham-operated control rats were subjected to anesthesia and midline incision, but the kidneys were not touched. Rats were provided buprenorphine-SR (1 mg/kg) as analgesia.

Blood (~ 250 µl) was collected from the tail into heparinized-Eppendorf tubes and centrifuged at 3000 × g for 10 min. Plasma creatinine was quantified daily using a Point Scientific QT 180 Analyzer (Point Scientific Inc, Canton, MI, USA) and Angiopoietin-2 was evaluated on day 5 post IR using Angiopoietin-2 Quantikine ELISA Kit (Catalog #: MANG20, R&D Systems, Inc. MN). Rat plasma KIM-1 was measured using an assay from Mesoscale Discovery (Rockville, MD; R-PLEX Rat HAVCR1/KIM-1, catalog K1534UR-2).

At 24 h post-surgery, rats were randomized into two groups based on creatinine values such that the vehicle-control and treatment groups had similar levels of renal injury at the initiation of treatment (Fig. 1). Of 31 rats subjected to IRI, one had an extremely high 24-h creatinine value of 5.7 mg/dl and was excluded from the study, as rats with this degree of injury are not likely to survive for the duration of the experiment. All other rats were randomized into groups receiving subcutaneous injection of either ASC-CS (2 mg/kg; 1 ml) or saline-vehicle (1 ml). No rats in either group died or required euthanasia after randomization.

Experimental schema and timeline. Rats were anesthetized and subjected to bilateral renal ischemia–reperfusion (I/R) injury or sham surgery. At 24 h post-surgery, rats were randomized into two groups based on creatinine values such that the vehicle-control and treatment groups had similar levels of renal injury at the initiation of treatment. Rats were randomized into groups receiving subcutaneous injection of either ASC-CS (2 mg/kg; 1 ml) or saline-vehicle (1 ml) at 24 h and 72 h post I/R injury. After 5 days (120 h) recovery post-surgery, kidneys were harvested for histological evaluation and flow cytometry analysis of inflammatory cells

After 5 days recovery post-surgery (4 days post-treatment), animals were euthanized following deep isoflurane anesthesia and subsequent pneumo-thoracotomy and exsanguination. Kidneys were rapidly harvested for histological evaluation and flow cytometry analysis of inflammatory cells. For histology, portions of the kidney were fixed in 10% buffered formalin. A subset of 6 post-IRI rats (3 per group) were not euthanized on Day 5 and therefore were not included in analysis of tissue.