The α7 nicotinic acetylcholine receptor agonist PNU-282987 ameliorates sepsis-induced acute kidney injury through CD4+CD25+ regulatory T cells in rats

Abstract

The ameliorative effects of α7 nicotinic acetylcholine receptor (α7nAChR) agonists have been demonstrated in acute kidney injury (AKI) caused by multiple stimulations. However, the ameliorative effect of α7nAChR on sepsis-induced acute kidney injury (SAKI) in the cecal ligation and puncture (CLP) model is unclear. Previous studies have demonstrated that α7nAChR is highly expressed on the surface of CD4+CD25+ regulatory T cells (Tregs). However, the role of Tregs in SAKI is unclear. We hypothesized that Tregs might play a role in the ameliorative effect of α7nAChR on SAKI. Hence, in this study, we determined the effects of PNU-282987 (a selective α7nAchR agonist) on SAKI and evaluated whether PNU-282987 would attenuate SAKI via regulating Tregs. Our study showed that immediate administration of PNU-282987 after CLP surgery in rats improved renal function, reduced levels of systemic inflammatory factors (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), etc.), inflammatory cell infiltration and tubular apoptosis in renal tissues, and increased forkhead/winged helix transcription factor p3 (Foxp3) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression indicating activated Tregs. Moreover, in in vitro experiments, isolated Tregs co-cultured with PNU-282987 also displayed enhanced expression of CTLA-4 and Foxp3. Furthermore, Tregs were co-cultured with PNU-282987 for 24 hours and then reinfused into rats through the tail vein immediately after CLP surgery, and a significant renal protective effect was observed 24 hours postoperatively. These results demonstrate that PNU-282987 exerts its renal protective effects on SAKI through activation of Tregs.

FIGURE 1

Establishment of sepsis-induced acute kidney injury model in rats. (A-C) The decremental urine output and increased serum creatinine and blood urea nitrogen were observed in the cecal ligation and puncture (CLP) group compared to the sham group; (D) representative H&E-stained sections (original magnification, ×400) of the rat kidney were obtained from the sham group 24 hours after surgery; and (E) Representative H&E-stained sections (original magnification, ×400) of the rat kidney were obtained from the CLP group 24 hours after surgery. The values are expressed as the mean ± SD (n = 8 in each group). ^##p < 0.01 versus the sham group.

FIGURE 2

Expression of a7 nicotinic acetylcholine receptor on the surface of rat CD4+CD25+ regulatory T Cells. (A) The purity of CD4+CD25+Tregs cells obtained after the single cells of normal rat spleen were sorted by magnetic beads was above 90%; (B) the a7nAChR band of Treg cells was clearly detected in Western blotting; and (C) After confocal fluorescence microscopy observation, Tregs cells emit green fluorescence under laser excitation.

FIGURE 3

PNU-282927 reduced the indicators of kidney injury 24 hours after cecal ligation and puncture (CLP) surgery in rats. (A-E) The levels of serum creatinine, blood urea nitrogen, urinary neutrophil gelatinase-associated lipocalin, kidney injury molecule 1, and urine output are shown in the four groups, respectively. The values are expressed as the mean ± SD (n = 8 in each group). ^##p < 0.01 versus the sham group; **p < 0.01 versus the CLP group.

FIGURE 4

PNU-282987 ameliorated renal histopathology and apoptosis 24 hours after cecal ligation and puncture (CLP) surgery in rats. (A) The representative images of H&E stained kidney sections (original magnification, ×200) in the sham, CLP, CLP + PNU-low, CLP + PNU-high groups; (B) the kidney histological damage scores from the four groups; (C) tubular apoptosis (TUNEL staining) and representative photomicrographs of rats (original magnification, ×200); and (D) apoptosis by counting the number of TUNEL positive cells per high power field using random sections and the mean apoptosis scores. The values are expressed as the mean ± SD (n = 8 in each group). ^##p < 0.01 versus the sham group; **p < 0.01 versus the CLP group.

FIGURE 5

PNU-282987 ameliorated systemic inflammation 24 hours after cecal ligation and puncture (CLP) surgery in rats. (A) Serum TNF-α levels are shown in the four groups and (B) IL-6 levels are shown in the four groups. The values are expressed as the mean ± SD (n = 8 in each group). ^##p <0.01 versus the sham group; **p < 0.01 versus the CLP group.

FIGURE 6

PNU-282987 increased expressions of Foxp3, CTLA-4, α7nAChR, and the production of TGF-β and IL-10 in Tregs of sepsis-induced acute kidney injury rats. (A) Representative flow cytometry images of Foxp3 on Tregs are shown in the four groups; (B) the MFI of Foxp3 on Tregs are shown in the four groups; (C) representative flow cytometry images of CTLA-4 on Tregs are shown in the four groups; (D) representative MFI of CTLA-4 on Tregs is shown in the four groups; (E) the a7nAChR band of Treg cells was clearly detected in Western Blotting; (F) the relative expression of α7nAChR was normalized to β-actin; and (G) the production of TGF-β1 was determined by ELISA. The values are expressed as the mean ± SD (n = 8 in each group). ^##p < 0.01 versus the sham group; **p < 0.01 versus the CLP group.

FIGURE 7

PNU-282987 increased expressions of Foxp3, CTLA-4 in Tregs in vitro, and the effects of PNU-282987 on production of TGF-β1 and IL-10 in Tregs in vitro. (A) Representative flow cytometry images of Foxp3 on Tregs are shown in the three groups; (B) the mean fluorescence intensity (MFI) of Foxp3 in Tregs is shown in the three groups; (C) representative flow cytometry images of CTLA-4 in Tregs are shown in the three groups; (D) representative MFI of CTLA-4 in Tregs is shown in the three groups; and (E) the production of TGF-β1 was determined by ELISA. The values are expressed as the mean ± SD (n=8 in each group). ^#p < 0.05 versus the Tregs group; ^##p < 0.01 versus the Tregs group.

FIGURE 8

Adoptive transfer of CD4⁺CD25⁺ regulatory T cells (Tregs) incubated in media containing PNU-282987 ameliorated kidney injury 24 hours after the cecal ligation and puncture (CLP) surgery in rats. (A-D) The levels of serum creatinine, blood urea nitrogen, urinary neutrophil gelatinase-associated lipocalin, and kidney injury molecule 1 are shown in the four groups; (E) representative images of the pathology (×200) are shown in the four groups; and (F) the kidney histological damage scores are shown in the four groups. The values are expressed as the mean ± SD (n = 8 in each group). ^#p < 0.05 versus the CLP + PBS group; ^##p < 0.01 versus the CLP + PBS group; **p < 0.01 versus the CLP + Tregs group.