Pretreatment with a Novel Toll-like Receptor 4 Agonist Phosphorylated Hexaacyl Disaccharide Attenuates Renal Ischemia-Reperfusion Injury

Objective: Acute kidney injury is common in surgical and critically ill patients. This study examined if pretreatment with a TLR4 agonist attenuated ischemia reperfusion-induced AKI (IRI-AKI). Design: A blinded, randomized controlled study in mice pretreated with PHAD, a synthetic TLR4 agonist. Two cohorts of male BALB/c mice received intraperitoneal vehicle or PHAD (10, 20, or 40 µg); or intravenous vehicle or PHAD (2, 20, or 200 µg) at 48 and 24 hours prior to unilateral renal pedicle clamping and simultaneous contralateral nephrectomy. A separate cohort of mice received vehicle or 200 µg of PHAD followed by bilateral IRI-AKI. Mice were monitored for 3 days post-reperfusion and euthanized for analysis. Methods and Results: Kidney function was assessed by BUN and serum creatinine measurements. Kidney tubular injury was assessed by semi-quantitative analysis of tubular morphology on PAS-stained kidney sections, and kidney mRNA quantification of injury (N-Gal, Kim-1 and HO-1) and inflammation (IL-6, IL-1b and TNF-α) using qRT-PCR technique. Immunohistochemistry was used to quantify Kim-1 and F4/80 protein to assess kidney injury and macrophages, respectively. PHAD pretreatment yielded dose-dependent kidney function preservation during unilateral IRI-AKI. Histological injury and N-Gal mRNA were lower, and IL-1b mRNA was higher in 200 µg 3D 6-Acyl PHAD treated mice. Similar pretreatment protection was noted at 200 mg PHAD after bilateral IRI-AKI, with significantly reduced Kim-1 immunostaining in the outer medulla of mice treated with PHAD after bilateral IRI-AKI. Conclusions: PHAD pretreatment leads to dose-dependent protection from renal injury after unilateral and bilateral IRI-AKI in mice.