49.73 1.83 ng/mg prot, = 5.33, = 0.0007). Conclusion: Platelets-derived P-selectin may be involved in the development of septic AKI through inducing neutrophil infiltration into kidney. for 5 min. (anti-Ly6G: 1.62 0.30 mg/dl vs. 2.68 0.27 mg/dl, = 5.76, = 0.0004; anti-P-selectin: 1.76 0.31 mg/dl vs. 2.68 0.27 mg/dl, = 4.92, = 0.0012; and platelet depletion: 1.93 0.29 mg/dl vs. 2.68 0.27 mg/dl, = 4.14, = 0.0032). Platelet amount significantly decreased compared to sham group (658.20 60.64 109/L vs. 822.00 48.60 109/L, = 4.71, = 0.0015) in septic mice, especially in platelet depletion group (240.80 44.98 109/L vs. 822.00 48.60 109/L, = 19.63, = Flopropione 0.0000). P-selectin activity was significantly improved in septic group compared to sham group (16.54 1.60% vs. 1.90 0.29%, = 15.64, = 0.0000) but decreased significantly in platelet depletion group compared to septic group (3.62 0.68% vs. 16.54 1.60%, = 12.89, = 0.0002). IHC analysis demonstrated that neutrophil infiltration improved in septic mice compared to sham group (36.67 3.79% vs. 9.17 1.61%, = 11.58, = 0.0003) and function-blocked organizations (anti-Ly6G: 36.67 3.79% vs. 15.33 1.53%, = 9.05, = 0.0008; anti-P-selectin: 36.67 3.79% vs. 21.33 1.53%, = 6.51, = 0.0029; and platelet depletion: 36.67 3.79% vs. 23.33 3.06%, = 4.75, = Flopropione 0.0090). MPO increased significantly in septic group compared to control (49.73 1.83 ng/mg prot vs. 13.04 2.16 ng/mg prot, = 19.03, = 0.0000) but decreased in function-blocked organizations compared to septic group (anti-Ly6G: 26.52 3.86 ng/mg prot vs. 49.73 Flopropione 1.83 ng/mg prot, = 9.59, = 0.0000; anti-P-selectin: 33.06 6.75 ng/mg prot vs. 49.73 1.83 ng/mg prot, = 4.85, = 0.0013; and platelet depletion: 33.37 2.25 ng/mg prot vs. 49.73 1.83 ng/mg prot, = 5.33, = 0.0007). Summary: Platelets-derived P-selectin may be involved in the development of septic AKI through inducing neutrophil infiltration into kidney. for 5 min. Platelet-rich plasma (PRP) was isolated and 5 l of PRP was added and fixed with 1% paraformaldehyde for 20 min. The CD62P (P-selectin)-FITC antibody (BD, USA) was added and incubated at space heat for 20 min before resuspension with 1 ml of phosphate-buffered saline (PBS) and tested by circulation cytometer. Renal function and cytokine analysis Blood sample’s serum creatinine and IL-1 concentration was measured with ELISA assay Kit (R&D Systems, USA) according to the manufacturer’s protocol. Histomorphometric analysis The kidneys fixed with 10% of formalin were inlayed in paraffin. Those cells were sliced up into 4 m solid sections following by hematoxylin-eosin (H&E) staining. Histologic morphology Flopropione was obtained for the loss of brush borders (0C3), tubular vascularization (0C3), and cell infiltration (0C3) by a blinded investigator. Dedication of myeloperoxidase activity Snap freezing kidneys were homogenized (20 mmol/L KPO4 buffer [pH 7.4]) and centrifugated at 17,000 for 10 min at 4C. Afterward, the supernatant was discarded and resuspended with IMP4 antibody 20 mmol/L KPO4 buffer (pH 7.4) and incubated for 20 min at 4C. After another centrifugation, the supernatant was used to detect the activity of myeloperoxidase (MPO). The results were determined by bicinchoninic acid assay (Pierce, USA) and indicated as models of MPO per gram of supernatant protein. Immunohistochemistry analysis Four micrometers sections of paraffin inlayed kidneys were clogged with 0.5% BSA for 30 min and incubated with anti-rabbit LY-6G (orb322983, Biorbyt, USA) antibody. After incubation of main antibody, a goat anti-rabbit secondary antibody was added and incubated at 37C for 60 min. Sections were washed with PBS and 3,3-diaminobenzidine chromogenic solutions was added to each slice, followed by hematoxylin staining and microscopic analysis by an experienced pathologist. Statistical analysis All data were indicated as mean standard deviation (SD). Statistical analysis was performed by SPSS version 19.0 (SPSS Inc., Palo Alto, California, USA). Unpaired 0.05 was considered statistically significant. RESULTS Cecal ligation and puncture-induced acute kidney injury accompanied by swelling After 48 h of CLP, compared to the sham group, animals of the septic group showed increased neutrophils count (1.70 109/L vs. 1.21 109/L, = 3.61, = 0.0059) and IL-1 levels (324.92 35.14 pg/ml vs. 125.54 42.08 pg/ml, = 10.47, = 0.0000) in peripheral blood [Figure 1]. The serum creatinine levels in septic mice.