ABSTRACT
Objective:To evaluate the role of histone demethylase (JMJD3) in drug-induced acute kidney injury (AKI) in mice.Methods:Twenty-four male C57BL/6 mice, aged 8-10 weeks, weighing 20-30 g, were divided into 4 groups ( n =6 each) using a random number table method: control group (C group), AKI group, a specific JMJD3 inhibitor GSKJ4+ control group (GSKJ4 group), and GSKJ4-AKI group.Folic acid 250 mg/kg was injected intraperitoneally to develop AKI model.GSKJ4 20 mg/kg was intraperitoneally injected at 1 h before developing AKI model in GSKJ4-AKI group and at the corresponding time point in GSKJ4 group.Blood samples were collected at 72 h after development of AKI model for determination of serum BUN and Cr concentrations.The animals were then sacrificed and renal tissues were collected for microscopic examination of histopathological morphology (using HE and PAS staining) and for determination of cell apoptosis (by TUNEL) and expression of JMJD3, Bax and cleaved caspase-3 (by Western blot), the number of JMJD3, myeloperoxidase (MPO), F4/80 and CD3 positive cells, expression of cleaved caspase-3 and Bax, and expression of IL-1β, IL-6, TNF-α and monocyte chemotactic protein 1 (MCP-1) mRNA (by reverse transcription polymerase chain reaction). The damage to the renal tubules was scored. Results:Compared with C group, the serum BUN and Cr concentrations and renal tubular damage score were significantly increased, the number of JMJD3, myeloperoxidase (MPO), F4/80 and CD3 + positive cells was increased, the number of apoptotic cells was increased, and the expression of Bax, cleaved caspase-3, JMJD3 and IL-1β, TNF-α, IL-6 and MCP-1 mRNA was up-regulated in AKI group ( P<0.05), and no significant change was found in the parameters mentioned above in GSKJ4 group ( P>0.05). Compared with AKI group, the serum BUN and Cr concentrations and renal tubular damage score were significantly decreased, the number of JMJD3, myeloperoxidase (MPO), F4/80 and CD3 positive cells was decreased, the number of apoptotic cells was decreased, and the expression of Bax, cleaved caspase-3, JMJD3, and IL-1β, TNF-α, IL-6 and MCP-1 mRNA was down-regulated in GSKJ4-AKI group ( P<0.05). Conclusions:The mechanism of drug-associated AKI may be related to up-regulation of JMJD3 expression and thus induces cell apoptosis and inflammatory responses in mice.
ABSTRACT
Objective:To evaluate the role of CXC chemokine receptor 6 (CXCR6)-mediated activation of natural killer T (NKT) cells in renal fibrosis following acute kidney injury (AKI) in mice.Methods:Eighteen male wild-type C57BL/6 mice and 18 CXCR6 knockout C57BL/6 mice, aged 8-10 weeks, weighing 20-30 g, were divided into 6 groups ( n=6 each) using a random number table method: wild-type mouse control group (group WT-CON), CXCR6 knockout mouse control group (group CXCR6 -/--CON), wild-type mouse with AKI group (group WT-AKI), CXCR6 knockout mouse with AKI group (group CXCR6 -/--AKI), wild-type mouse with AKI + NKT cell adoptive transfer group (group WT-AKI-NKT) and CXCR6 knockout mouse with AKI + NKT cell adoptive transfer group (group CXCR6 -/--AKI-NKT). Folic acid 250 mg/kg was intraperitoneally injected to establish the model of renal fibrosis in mice with AKI.NKT cellsuspension 250 μl(1×10 6 cells) was injected through the tail vein on the 4th and 9th days after folic acid injection in group WT-AKI-NKT and group CXCR6 -/--AKI-NKT, respectively.Blood samples were taken from orbital at day 14 after folic acid injection for determination of the concentrations of serum blood urea nitrogen (BUN) and creatinine (Cr). The animals were sacrificed, and renal tissues were obtained for observation of the area of renal fibrosis (by Sirius red staining) and renal injury (using H&E staining) which was scored and for determination of the proportion of CD1d Tetramer+ cells (by flow cytometry), the number of CD206 and α-smooth muscle actin (α-SMA) double positive (CD206 + -α-SMA + ) cells (by immunofluorescence) and expression of interleukin (IL)-4 and IL-13 mRNA (by real-time polymerase chain reaction). Results:Compared with group WT-CON, the BUN and Cr levels, renal injury scores, area of renal fibrosis, proportion of CD1d Tetramer + cells and CD206 + -α-SMA + cell count were significantly increased, and the expression of IL-4 and IL-13 mRNA was up-regulated in group WT-AKI and WT-AKI-NKT ( P<0.05). Compared with group WT-AKI, the BUN and Cr levels, renal injury scores, area of renal fibrosis, proportion of CD1d Tetramer + cells and CD206 + -α-SMA + cell count were significantly increased, and the expression of IL-4 and IL-13 mRNA was up-regulated in group WT-AKI-NKT ( P<0.05), and the BUN and Cr levels, renal injury scores, area of renal fibrosis, proportion of CD1d Tetramer + cells and CD206 + -α-SMA + cell count were significantly decreased, and the expression of IL-4 and IL-13 mRNA was down-regulated in group CXCR6 -/--AKI ( P<0.05). Compared with group CXCR6 -/--CON, the BUN and Cr levels, renal injury scores, area of renal fibrosis, proportion of CD1d Tetramer + cells and CD206 + -α-SMA + cell count were significantly increased in group CXCR6 -/--AKI and group CXCR6 -/--AKI-NKT ( P<0.05). Compared with group CXCR6 -/--AKI, the BUN and Cr levels, renal injury scores, area of renal fibrosis, proportion of CD1d Tetramer + cells and CD206 + -α-SMA + cell count were significantly increased, and the expression of IL-4 and IL-13 mRNA was up-regulated in group CXCR6 -/--AKI-NKT ( P<0.05). Conclusion:CXCR6-mediated activation of NKT cells is involved in renal fibrosis following AKI in mice, and the mechanism may be related to promoting Th2 cytokine-mediated M2 macrophage-myofibroblast transformation.