Pretreatment with a novel Toll-like receptor 4 agonist attenuates renal ischemia-reperfusion injury

Abstract

Acute kidney injury (AKI) is common in surgical and critically ill patients. This study examined whether pretreatment with a novel Toll-like receptor 4 agonist attenuated ischemia-reperfusion injury (IRI)-induced AKI (IRI-AKI). We performed a blinded, randomized-controlled study in mice pretreated with 3-deacyl 6-acyl phosphorylated hexaacyl disaccharide (PHAD), a synthetic Toll-like receptor 4 agonist. Two cohorts of male BALB/c mice received intravenous vehicle or PHAD (2, 20, or 200 µg) at 48 and 24 h before unilateral renal pedicle clamping and simultaneous contralateral nephrectomy. A separate cohort of mice received intravenous vehicle or 200 µg PHAD followed by bilateral IRI-AKI. Mice were monitored for evidence of kidney injury for 3 days postreperfusion. Kidney function was assessed by serum blood urea nitrogen and creatinine measurements. Kidney tubular injury was assessed by semiquantitative analysis of tubular morphology on periodic acid-Schiff (PAS)-stained kidney sections and by kidney mRNA quantification of injury [neutrophil gelatinase-associated lipocalin (Ngal), kidney injury molecule-1 (Kim-1), and heme oxygenase-1 (Ho-1)] and inflammation [interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (Tnf-α)] using quantitative RT-PCR. Immunohistochemistry was used to quantify proximal tubular cell injury and renal macrophages by quantifying the areas stained with Kim-1 and F4/80 antibodies, respectively, and TUNEL staining to detect the apoptotic nuclei. PHAD pretreatment yielded dose-dependent kidney function preservation after unilateral IRI-AKI. Histological injury, apoptosis, Kim-1 staining, and Ngal mRNA were lower in PHAD-treated mice and IL-1β mRNA was higher in PHAD-treated mice. Similar pretreatment protection was noted with 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. In conclusion, PHAD pretreatment leads to dose-dependent protection from renal injury after unilateral and bilateral IRI-AKI in mice.NEW & NOTEWORTHY Pretreatment with 3-deacyl 6-acyl phosphorylated hexaacyl disaccharide; a novel synthetic Toll-like receptor 4 agonist, preserves kidney function during ischemia-reperfusion injury-induced acute kidney injury.

Keywords: 3-deacyl 6-acyl phosphorylated hexaacyl disaccharide; Toll-like receptor 4 agonist; acute kidney injury; ischemia-reperfusion injury; phosphorylated hexaacyl disaccharide.

Graphical abstract

Figure 1.

Pretreatment with 3-deacyl 6-acyl phosphorylated hexaacyl disaccharide (PHAD) in mice that underwent unilateral ischemia-reperfusion injury-induced acute kidney injury. Mice were pretreated with intravenous (IV) PHAD at 2, 20, and 200 µg/mouse or vehicle control 48 and 24 h prior to undergoing right nephrectomy followed by clamping of the left renal pedicle for 28 min. A: blood was analyzed for blood urea nitrogen (BUN) and creatinine at baseline (day 0) and postinjury days 1 and 3. Results are expressed as means ± SE with n = 8. Two-way ANOVA was used to compare differences between PHAD- and vehicle-treated mice over time, with P values indicated. B: representative images of periodic acid-Schiff (PAS)-stained sections of the outer medulla day 3 after injury in sham, vehicle-treated, and PHAD-treated mice. Arrows point to casts within the collecting tubules. Scale bar = 100 µm. C: median tubular injury scores in the outer stripe of the outer medulla from PAS-stained sections day 3 after injury in sham, vehicle-treated, and PHAD-treated mice. D: 3-day survival curves. Group differences were compared by a log-rank test (P > 0.05). E: median quantitative RT-PCR for renal expression of neutrophil gelatinase-associated lipocalin (Ngal), kidney injury molecule-1 (Kim-1), interleukin-6 (IL-6), tumor necrosis factor-α (Tnf-α), and interleukin-1β (IL-1β) mRNAs. GAPDH was used as the internal control for quantitative RT-PCR, and change in threshold cycle (ΔC_t) values is graphed relative to the sham group. For C and D, individual data points and medians are shown and between-group differences were compared by one-way ANOVA using Dunnett’s post hoc correction for multiple between-group comparison Kruskal–Wallis test, with significant P values (<0.05) indicated. n = 6.

Figure 2.

Pretreatment with 3-deacyl 6-acyl phosphorylated hexaacyl disaccharide (PHAD) in mice that underwent bilateral ischemia-reperfusion injury-induced acute kidney injury. Mice were pretreated with intravenous PHAD at 200 µg/mouse or vehicle control 48 and 24 h prior to undergoing bilateral renal pedicle clamping for 24 min. A: blood was analyzed for blood urea nitrogen (BUN) and creatinine at baseline (day 0) and postinjury days 1 and 3. Results are expressed as means ± SE with n = 10. Two-way ANOVA was used to evaluate between-group differences over time (P < 0.05 for both BUN and serum creatinine), with P values shown after Sidak’s correction for multiple post hoc between-group comparisons at each time point. B: tubular injury scores in the outer stripe of the outer medulla from periodic acid-Schiff (PAS)-stained sections day 3 after injury. C: apoptosis in the outer stripe of the outer medulla from TUNEL-stained sections day 3 after injury. D: representative images of PAS-stained sections of the outer medulla day 3 after injury in sham, vehicle-treated, and PHAD-treated mice. Black arrows point to severely injured tubules showing flattening of the surviving epithelium and the presence of cellular debris within the tubular lumen. Yellow arrows indicate PAS-positive brush borders of uninjured S3 segment proximal tubule epithelial cells. Scale bar = 100 µm. E: representative images of TUNEL-stained sections of the outer medulla day 3 after injury in vehicle- and PHAD-treated mice. Red arrowheads indicate examples of TUNEL-positive tubular nuclei. Yellow dotted lines indicate necrotic TUNEL-positive tubular casts excluded from the analysis. Scale bar = 20 µm. F: quantitative RT-PCR for renal expression of neutrophil gelatinase-associated lipocalin (Ngal), kidney injury molecule-1 (Kim-1), interleukin-6 (IL-6), tumor necrosis factor-α (Tnf-α), and interleukin-1β (IL-1β) mRNAs day 3 after injury. GAPDH was used as the internal control for quantitative RT-PCR, and change in threshold cycle (ΔC_t) values is graphed relative to the vehicle group. For B, C, and F, individual data points and medians are shown, and P values with a significant difference are shown from a Mann–Whitney U test used to compare groups. n = 6 or 7.

Figure 3.

Immunofluorescence staining for kidney injury molecule-1 (Kim-1) and F4/80 after bilateral ischemia-reperfusion injury-induced acute kidney injury in mice pretreated with 3-deacyl 6-acyl phosphorylated hexaacyl disaccharide (PHAD). Mice were pretreated with intravenous PHAD at 200 µg/mouse or vehicle control 48 and 24 h prior to undergoing bilateral renal pedicle clamping for 24 min. A: representative images stained for expression of Kim-1, expressed by injured proximal tubules (left), and F4/80, expressed by renal macrophages (right), in sequential sections colabeled with Lotus tetragonolobus lectin (LTL) to mark proximal tubular cells in vehicle-treated vs. PHAD-treated mice 3 days after injury. White dotted lines indicate the junction between the cortex and outer medulla, which is only seen in top images. Scale bar = 50 µm. B: quantification of Kim-1- and F4/80-positive areas in the cortex (right) and outer stripe of the outer medulla (left) in vehicle- and PHAD-treated mice at day 3 after injury. Individual data points and medians are shown, and P values are shown from a Mann–Whitney U test used to compare groups. n = 8.

Figure 4.

Pretreatment with intravenous 3-deacyl 6-acyl phosphorylated hexaacyl disaccharide (PHAD) in mice that underwent bilateral ischemia-reperfusion injury-induced acute kidney injury. Mice were pretreated with intravenous PHAD at 200 µg/mouse or vehicle control 48 and 24 h prior to undergoing bilateral renal pedicle clamping for 24 min. Quantitative RT-PCR for renal expression of heme oxygenase-1 (Ho-1; A), interferon regulatory factor-1 (Irf1; B), chemokine (C-C motif) ligand 2 (Ccl2; C), and chemokine (C-C motif) ligand 3 (Ccl3; D) day 3 after injury. GAPDH was used as the internal control for quantitative RT-PCR, and change in threshold cycle (ΔC_t) values is graphed relative to the vehicle-treated group. Individual data points and medians are shown, and P values are shown from a Mann-Whitney U test used to compare groups. n = 6 or 7.