LPS-induced AKI protocol

The present study is divided into three protocols. All drugs administrations were intraperitoneal as we [7] and others [23] have reported and included six mice per group.

Fifty-four mice were randomized to prior-treatment or concurrent treatment groups to study anti-inflammatory and renoprotective effects of AT₂R activation against LPS-induced AKI 24-h post-LPS. Prior-treatment protocol: the AT₂R agonist C21 and antagonist PD123139 alone or together were administered on days 1 (first dose) and 2 (second dose). One hour after dose 2, LPS was administered. Various groups of mice were as follows: (i) sterile saline (control), (ii) LPS (5 mg/kg), (iii) AT₂R agonist C21 (0.3 mg/kg), (iv) C21+LPS, (v) AT₂R antagonist PD (5 mg/kg), (vi) PD+LPS, (vii) PD+C21+LPS. Concurrent treatment protocol: C21 alone or with PD123319 were given once 1 h prior to LPS administration. Treatment groups are as follows: (viii) C21+LPS (ix) PD+C21+LPS. All groups of mice 24-h post-LPS were killed.

Another 24 mice were randomly assigned to prior-treatment groups to study the immediate effects of AT₂R activation on LPS-induced inflammatory response 2-h post-LPS. In this protocol we used the following group of mice: (i) sterile saline (control), (ii) LPS (5 mg/kg), (iii) AT₂R agonist C21 (0.3 mg/kg) or (iv) C21+LPS. As aforementioned, the prior-treatment group included C21 administration of two doses on days 1 and 2. One hour after the second dose of C21, LPS was administered and then 2 h later, the mice were killed.

Another 12 mice were randomized to receive (i) sterile saline or (ii) AT₂R agonist C21 (0.3 mg/kg) on days 1 (first dose) and 2 (second dose) to determine the immediate effects of AT₂R activation on circulating and renal IL-10 at the time of LPS was given in above protocols. These mice were not subjected to LPS challenge. The mice were killed 1 h after the second dose of C21.

All mice were euthanized under isoflurane anesthesia. Spot urine was collected from the bladder. Biochemical analysis performed in spot/random urine sample was correlated with urine sample collected over 24 h from experimental animals [24,25] or from patients [26–28]. Blood for plasma was collected in Vacuette® K₃-EDTA-coated tubes (95057-227, VWR, Radnor, PA) through cardiac puncture and kidneys were harvested and stored at −80°C. Plasma was prepared by centrifugation (700×g, 20 min and 4°C) and stored at −80°C. In three mice from each group (protocol 1 only), the right kidney was clamped before whole-body fixation with sterile 4% sucrose in phosphate buffered saline (PBS) followed by formalin-free fixative (Milestone Medical). Kidneys were collected and kept in fixative for 24 h at 4°C and transferred to 30% sucrose in PBS for preparation of cryopreservation. Tissues were embedded in Optimal Cutting Temperature compound (OCT) and preserved at −80°C for microscopy.