The addition of IL-10 restored the decreased expression of AT2R in cisplatin-treated HK-2 cells (Figures 4(a) and 4(b)). effect of coculture on cisplatin-treated HK-2 cells. Finally, PD123319, an AT2R antagonist, also reversed the Rabbit Polyclonal to GCVK_HHV6Z effect of IL-10 and coculture on the cell viability, death, and the expression of KIM, calbindin, TIMP-1, and Bcl-2 of cisplatin-treated HK-2 cells. Conclusions DN T cells protected HK-2 cells from cisplatin-induced injury through IL-10/AT2R axis, which may act as a potential target for the treatment of cisplatin-induced AKI. 1. Introduction Cisplatin is now widely applied for the treatment of multiple cancers, but its major adverse reaction, acute kidney injury (AKI), limits its clinical use and has raised wide concerns [1, 2]. Approximately one-third of patient suffers from AKI after the treatment of cisplatin, and there is no strategy to effectively protect against such AZ505 ditrifluoroacetate nephrotoxicity at present [3]. Hence, elucidating the pathogenesis of AKI induced by cisplatin is significant for developing agents that can attenuate this AZ505 ditrifluoroacetate serious side effect given its widespread use as chemotherapy. Over the past decades, the common mechanisms of cisplatin-induced AKI have been investigated extensively [4]. Multiple mechanisms are involved in cisplatin-induced nephrotoxicity, including oxidative stress, vascular injury, necrosis, and apoptosis, as well as inflammation. It is widely accepted that cisplatin-induced nephrotoxicity is attributed to platinum accumulation in renal tissue. The accumulation of cisplatin caused the excess production of reactive oxygen species (ROS) and tumor necrosis factor alpha (TNF-and nuclear factor-and [18]. However, there is very little information on the mechanism underlying the immunoregulatory roles of DN T cells to protect from cisplatin-induced AKI. The aim of the present study was to perform a preliminary investigation into the effects of DN T cells on cisplatin-induced AKI and the underlying mechanism. Human Kidney 2 (HK-2) cell line was selected as model for receiving cisplatin stimulation in this study since it maintains biochemical properties and activity similar to proximal tubule cells [19]. 2. Materials and Methods 2.1. Chemical Reagents AZ505 ditrifluoroacetate and Antibodies Cisplatin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-CD4 (RM4-5) APC/FITC/Pacific blue and anti-TCR(H57-597) FITC/BV421, CD45 (30-F11) APC-Cy7/BUV395, anti-CD8(53 6.7) PerCP-Cy5.5/APC-fire750, were purchased from BD Biosciences. Biotin-conjugated anti-CD16/32 (2.4G2) was purchased from BioLegend. Anti-AGTR2, anti-TIMP-1, and anti-KIM-1 antibodies were purchased AZ505 ditrifluoroacetate from R&D systems (MN, USA). Anti-GAPDH and anti-Bcl-2 antibodies were from Santa Cruz Biotechnology, Inc. (CA, USA). Anti-calbindin antibody was purchased from Cell Signaling Technology, Inc. (MA, USA). 2.2. Cell Culture HK-2 cell line was purchased from the Global Bioresource Center and cultured in HyClone? DMEM/F12 supplemented with 5% FBS (Gibco, USA) at 37C in a cell culture incubator. HK-2 cells were starved in the culture medium containing 0.5% FBS for 12?h and then administrated with cisplatin of indicated concentrations (20 or 40? 0.05 indicated a significant difference. 3. Results 3.1. DN T Cells Attenuated Cisplatin-Induced Injury of HK-2 Cells To verify the role of DN T cells in cisplatin-induced AKI, CCK-8 assay was conducted in HK-2 cells which were pretreated with cisplatin. The results showed that the addition of DN T cells improved the cell viability of HK-2 cells with either 20 or AZ505 ditrifluoroacetate 40? 0.05, ?? 0.01, and ??? 0.001. To further confirm the protective role of DN T cells in cisplatin-induced cell death, the HK-2 cells were treated with DN T cells and 40?in DN T cells were detected after being subjected to cisplatin or/and HK-2 cell coculture. Statistical significance was defined as ? 0.05, ?? 0.01, and ??? 0.001. Moreover, IL-17A, INF-in DN T cells have not changed. Surprisingly, the.