Fig 1: Proposed model for this study.Inhibition of ALKBH5 protects I/R-induced kidney injury through increasing CCL28 level by m6A modification, and regulating downstream CCL28/Treg/inflammatory cells axis.
Fig 2: IOX1 and recombinant mouse CCL28 exerts protective effects in vivo during IRI.a Schematic illustrating experiment groups and treatment in 4 groups. b m6A dot blot assessed m6A mRNA methylation under IOX1 or CCL28 treatment in I/R-induced AKI mouse model. c, d Serum creatinine and BUN concentrations. e, f Tubular injury score and representative H&E staining image in different groups of renal tissues. g, h Representative flow cytometry dot plots and percentage of CD4+Foxp3+ cells among the CD4+ T cells in IRI mice with or without IOX1 and CCL28 antibody treatment. i Infiltration of Ly6G+ neutrophil and F4/80+ macrophages in different groups of renal tissues (f, i: Magnification: ×200, Scale bar: 100 µm). For c–e and i, n = 4, 5, 5, 5 for Sham, IRI, IRI + IOX1, IRI + CCL28 groups respectively. For h, n = 3, 4, 5, 5 for Sham, IRI, IRI + IOX1, IRI + CCL28 groups respectively. Each data point represents one animal, with unpaired two-tailed Student’s t-test performed. Data are shown as means ± SD.
Fig 3: ALKBH5 enhances Ccl28 mRNA stability in a m6A-dependent manner.a RNA level of Ccl28 in IRI-Alkbh5fl/flKspCre group and IRI-Alkbh5fl/fl group was examined by RT-qPCR. n = 3 biologically independent animals. b Protein level of CCL28 in IRI-Alkbh5fl/flKspCre group and IRI-Alkbh5fl/fl group was examined by western blot. c Representative IHC staining data of CCL28 in kidney biopsies from IRI mice. (Magnification: ×400, Scale bar: 40 µm). d Serum CCL28 level was examined by ELISA. n = 5 biologically independent animals. e m6A modification of Ccl28 mRNA was detected by MeRIP-qPCR analysis using anti-IgG and anti-m6A antibodies. n = 3 biologically independent animals. f Relative enrichment of Ccl28 mRNA associated with ALKBH5 protein was identified by RIP assays using anti-IgG and anti-FLAG antibodies. n = 4 biologically independent experiments. g Relative activity of the pGL3-empty, pGL3-CDS, pGL3-5’UTR, and pGL3-3’UTR luciferase reporter in Ad-Control and Ad-ALKBH5 groups. n = 3 biologically independent experiments. h Relative activity of the pGL3-empty, pGL3-WT, pGLMut1, pGLMut2, pGLMut3, and pGLMut4 luciferase reporter in Ad-Control and Ad-ALKBH5 groups. n = 3 biologically independent experiments. i ALKBH5-silenced and control cells were treated with actinomycin D and harvested at 0, 2, 4, and 6 h. RNA decay rate was determined to estimate the stability of Ccl28 mRNA (normalized to the expression at 0 h). n = 3 biologically independent experiments. j RT-qPCR analysis of Ccl28 in mRTECs after different insulin-like growth factor 2 binding proteins (IGF2BPs) knockdown compared to NC treatment. n = 3 biologically independent experiments. k Western blot analysis of CCL28 after IGF2BP2 knockdown in H/R-treated mRTECs. Three biological repeated immunoblots have been performed. l RT-qPCR analysis of RIP assays in H/R-treated mRTECs showing the direct binding between the IGF2BP2 protein and Ccl28 mRNA. n = 3 biologically independent experiments. m ALKBH5-silenced with/without IGF2BP2-silenced cells were treated with actinomycin D and harvested at 0, 2, 4, and 6 h. RNA decay rate was determined to estimate the stability of Ccl28 mRNA (normalized to the expression at 0 h). n = 3 biologically independent experiments. Data are shown as means ± SD. Unpaired two-tailed Student’s t-test for (a), (d–h), (j), (l).
Fig 4: CCL28/Treg/inflammatory cells axis is involved in mechanism of therapeutical effect of Alkbh5 deficiency on I/R-induced AKI.a Infiltration of Ly6G+ neutrophil and F4/80+ macrophages in different groups of renal tissues. b, c Serum creatinine and BUN concentrations. d, e Tubular injury score and representative H&E staining image in different groups of renal tissues. (a, e: Magnification: ×200, Scale bar: 100 µm). For b–d, Sham groups, n = 3; other groups, n = 5. Each data point represents one animal. Unpaired two-tailed Student’s t-test. Data are shown as means ± SD.
Fig 5: Alkbh5 deficiency increased the recruitment of Tregs through CCL28.a RNA level of Ccl28 in different times after I/R was examined by RT-qPCR. n = 3, 4, 4, 3, 3 biologically independent animals for Sham, 12 h, 24 h, 48 h, and 120 h groups, respectively. b The FACS analysis process of Tregs. c, d Representative flow cytometry dot plots and percentage of CD4+Foxp3+ cells among the CD4+ T cells at 0 h, 12 h, 24 h, 48 h, and 120 h after I/R. n = 3, 4, 4, 3, 3 biologically independent animals for Sham, 12 h, 24 h, 48 h, and 120 h groups, respectively. e The line chart showed the relationship of ALKBH5, CCL28, and Tregs. f, g Renal recruitment of CD4+Foxp3+ cells 24 h after IRI in 4 groups. n = 3 for Sham groups, n = 5 for IRI groups. Each data point represents one animal. h The CCL28 protein in serum from IRI-Alkbh5fl/flKspCre or IRI-Alkbh5fl/fl mice with or without CCL28 antibody treatment. i, j Representative flow cytometry dot plots and percentage of CD4+Foxp3+ cells among the CD4+ T cells in IRI-Alkbh5fl/flKspCre or IRI-Alkbh5fl/fl mice with or without CCL28 antibody treatment. n = 3 for IRI-Alkbh5fl/fl groups, n = 5 for Alkbh5fl/flKspCre groups. Each data point represents one animal. k CCL28 protein in supernatants from mRTECs cells incubated under hypoxic or oxic conditions with or without Ad-sh-ALKBH5 and CCL28 antibody treatment, as determined by ELISA. n = 3 biologically independent experiments. l, m Representative flow cytometry dot plots and percentage of recruited CD4+Foxp3+ cells among the CD4+ T cells in groups with or without hypoxic, Ad-sh-ALKBH5, and CCL28 antibody treatment. n = 3 biologically independent experiments. Data are shown as means ± SD. Unpaired two-tailed Student’s t-test for (a), (d), (e), (g), (h), (j), (k), and (m).
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