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Ischemia-reperfusion injury is the commonest form of acute kidney injury (AKI). Tubular epithelial cell senescence contributes to incomplete recovery from AKI and predisposes to subsequent chronic kidney disease. In cultures of primary proximal renal tubular epithelial cells (RPTECs) subjected to anoxia or reoxygenation, we evaluated the role of indoleamine 2,3-dioxygenase 1 (IDO) in cellular senescence. Proteins of interest were assessed with Western blotting or enzyme-linked immunosorbent assay or histochemically. Under anoxia or reoxygenation, IDO expression and activity were increased. Moreover, the two IDO-derived pathways, the general control nonderepressible 2 kinase (GCN2K) pathway and the aryl-hydrocarbon receptor (AhR) pathway, were also activated. A DNA damage response (DDR) took place and led to increased levels of the cell-cycle inhibitors p21 and p16, and senescence-associated β-galactosidase (SA-β-Gal) activity. Cell proliferation was inhibited, and more IL-6 was produced. The IDO inhibitor 1-DL-methyl-tryptophan ameliorated the DDR, decreased p21, p16, and SA-β-Gal activity, restored cell proliferation, and decreased IL-6 production. The AhR inhibitor CH223191 did not affect the above parameters. In conclusion, anoxia and the subsequent reoxygenation upregulate IDO. IDO depletes tryptophan and activates GCN2K. The latter enhances the anoxia- or reoxygenation-induced DDR, resulting in increased p21 and p16 expression and eventually leading to RPTEC senescence. Since cellular senescence affects AKI outcome, the role of IDO in cellular senescence and the possible therapeutic role of IDO inhibitors deserve further investigation.

In antibody‑mediated rejection (ABMR), the graft endothelium is at the forefront of the kidney transplant against the assault from the recipient's humoral immune system, and is a target of the latter. The present study investigated the effect of antibodies against human leukocyte antigen (HLA) class I (anti‑HLAI) on the immunological properties of human glomerular endothelial cells. Additionally, the effect of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) inhibitor (everolimus), or the general control nonderepressible 2 kinase (GCN2K) activator (halofuginone) on anti‑HLAI antibody‑mediated alterations was assessed. Cell integrity was examined, an lactate dehydrogenase (LDH) release assay was performed and cleaved caspase‑3 levels were determined. Furthermore, cell proliferation was analyzed by performing a bromodeoxyuridine assay and the cellular proteins involved in signal transduction or immune effector mechanisms were assessed via western blotting. IL‑8, monocyte chemoattractive protein‑1 (MCP‑1), von Willebrand factor (vWF) and transforming growth factor‑beta 1 (TGF‑β1) were assayed via ELISA. The results revealed that anti‑HLAI triggered integrin signaling, activated mTOR and GCN2K, preserved cell integrity and promoted cell proliferation. Additionally, by increasing intercellular adhesion molecule 1 (ICAM‑1), HLA‑DR, IL‑8 and MCP‑1 levels, anti‑HLAI enhanced the ability of immune cells to interact with endothelial cells thus facilitating graft rejection. Contrarily, by upregulating CD46 and CD59, anti‑HLAI rendered the endothelium less vulnerable to complement‑mediated injury. Finally, by enhancing vWF and TGF‑β1, anti‑HLAI may render the endothelium prothrombotic and facilitate fibrosis and graft failure, respectively. According to our results, mTORC1 inhibition and GCN2K activation may prove useful pharmaceutical targets, as they prevent cell proliferation and downregulate ICAM‑1, IL‑8, MCP‑1 and TGF‑β1. mTORC1 inhibition also decreases vWF.

During the reperfusion phase of ischemia-reperfusion injury, reactive oxygen species (ROS) production aggravates the course of many diseases, including acute kidney injury. Among the various enzymes implicated in ROS production are the enzymes of the cytochromes P450 superfamily (CYPs). Since arylhydrocarbon receptor (AhR) controls the expression of certain CYPs, the involvement of this pathway was evaluated in reperfusion injury. Because AhR may interact with the nuclear factor erythroid 2-related factor 2 (Nrf2) and the hypoxia-inducible factor-1α (HIF-1α), whether such an interaction takes place and affects reperfusion injury was also assessed. Proximal renal proximal tubular epithelial cells were subjected to anoxia and subsequent reoxygenation. At the onset of reoxygenation, the AhR inhibitor {"type":"entrez-nucleotide","attrs":{"text":"CH223191","term_id":"44935898","term_text":"CH223191"}}CH223191, the HIF-1α activator roxadustat, or the ferroptosis inhibitor α-tocopherol were used. The activity of AhR, Nrf2, HIF-1α, and their transcriptional targets were assessed with western blotting. ROS production, lipid peroxidation and cell death were measured with colorimetric assays or cell imaging. Reoxygenation induced ROS production, lipid peroxidation and cell ferroptosis, whereas {"type":"entrez-nucleotide","attrs":{"text":"CH223191","term_id":"44935898","term_text":"CH223191"}}CH223191 prevented all. Roxadustat did not affect the above parameters. Reoxygenation activated AhR and increased CYP1A1, while {"type":"entrez-nucleotide","attrs":{"text":"CH223191","term_id":"44935898","term_text":"CH223191"}}CH223191 prevented both. Reoxygenation with or without {"type":"entrez-nucleotide","attrs":{"text":"CH223191","term_id":"44935898","term_text":"CH223191"}}CH223191 did not alter Nrf2 or HIF-1α activity. Thus, AhR is activated during reoxygenation and induces ROS production, lipid peroxidation and ferroptotic cell death. These detrimental effects may be mediated by AhR-induced CYP overexpression, while the Nrf2 or the HIF-1α pathways remain unaffected. Accordingly, the AhR pathway may represent a promising therapeutic target for the prevention of reperfusion injury.

Hypoxia-inducible factor (HIF) prolyl-hydroxylase inhibitors are currently used for the treatment of renal anemia. Since HIF affects immune cells, we evaluated the effect of such a drug, the roxadustat, on adaptive immunity. Cell proliferation was assessed in a two-way mixed lymphocyte reaction (MLR) with BrdU assay. In CD4+ T cells isolated from the two-way MLRs, western blotting was performed to detect the impact of roxadustat on HIF-1α and HIF-2α, the apoptotic marker cleaved caspase-3, and the master transcription factors of CD4+ T cells differentiation towards Th1, Th2, Th17, Treg and Tfh subsets. The signature cytokines of the above CD4+ T-cell subsets IFN-γ, IL-4, IL-17, IL-10, and IL-21 were measured in the supernatants. For assessing humoral immunity, we developed a suitable antibody-mediated complement-dependent cytotoxicity assay. Roxadustat stabilized HIF-1α and HIF-2α, suppressed cell proliferation, inhibited CD4+ T-cell differentiation into Th1 and Th17 subsets, while it favored differentiation towards Th2, Treg and Tfh. Roxadustat suppressed humoral immunity too. These immunosuppressive properties of roxadustat indicate that the recently introduced HIF prolyl-hydroxylase inhibitors in medical therapeutics may render the patients vulnerable to infections. This possibility should be further evaluated in clinical trials.

BackgroundUrate in its crystal form is a known danger-associated molecular pattern, which after its internalization activates cells of the innate immune system. However, by inducing lipid raft sequestration and clustering of membrane-bound proteins with immunoreceptor tyrosine-based activation motifs, urate crystals can also activate cells of the innate immune system without previous internalization. Also, urate crystals trigger T-cell receptor signal transduction and induce T-cell proliferation. In this study, we evaluated whether urate crystals can also initiate B-cell receptor (BCR) signal transduction and promote B-cell proliferation.MethodsB cells were isolated from the blood of 10 individuals and cultured with or without urate at a concentration of 10 mg/dL, at which crystallization occurs. Phosphorylated Igα (CD79A) and c-Myc were assessed by Western blotting and B-cell proliferation with BrdU assay.ResultsUrate increased the level of phosphorylated Igα, a component of the BCR complex. Phosphorylation of Igα is the very proximal event in BCR signal transduction. Also, urate increased the expression of c-Myc, an essential transcription factor for BCR-induced B-cell proliferation. Finally, urate induces B-cell proliferation.ConclusionsUrate crystals trigger BCR signal transduction and induce B-cell proliferation. The clinical significance of urate-induced B-cell activation remains to be elucidated.

Comparative biology may reveal novel therapeutic strategies against human diseases. Ischemia‑reperfusion (IR) injury induces a number of diseases. It is known that hibernating mammals survive IR since during hibernation, prolonged periods of torpor with a marked decrease in blood flow and breathing rate are interrupted by short periods of arousal. In the present study, the differences in the character‑ istics of endoplasmic reticulum (ER) stress and the subsequent unfolded protein response, which are induced by IR and may cause cell death among humans, mice or the native hibernator Syrian hamster were examined in vitro using renal proximal tubular epithelial cells (RPTECs) derived from these three sources. RPTECs were subjected to anoxia or reoxygenation, both at 37˚C. Cell death was measured by LDH release assay. ER stress was assessed by determining the levels of phos‑ phorylated protein kinase RNA‑like ER kinase, ubiquitinated proteins and Bcl‑2‑associated X protein (Bax) by western blot analysis. For proteasomal activity, a specific assay was used. The results revealed that anoxia induced ER stress in all the evaluated RPTECs, from which only the hamster‑derived RPTECs recovered during reoxygenation. Anoxia and reoxy‑ genation increased protein ubiquitination in the human‑ and mouse‑derived RPTECs, whereas this was decreased in the hamster‑derived RPTECs. Anoxia enhanced proteasomal activity in all the evaluated RPTECs. In the human‑ and mouse‑derived RPTECs, reoxygenation reduced proteasomal activity, which remained high in the hamster‑derived RPTECs. Anoxia and reoxygenation increased Bax expression and induced cell death in the human‑ and mouse‑derived RPTECs, while neither Bax overexpression nor cell death occurred in the hamster‑derived RPTECs. Thus, on the whole, the find‑ ings of this study demonstrate that compared to human‑ or mouse‑derived RPTECs, those derived from the hamster recover more rapidly from ER stress following warm anoxia‑reoxygenation, possibly due to increased proteasomal function.