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FTO‐mediated m 6 A modification of SOCS1 mRNA promotes the progression of diabetic kidney disease

2022; Springer Science+Business Media; Volume: 12; Issue: 6 Linguagem: Inglês

10.1002/ctm2.942

2001-1326

Qiang Sun, Houfa Geng, Meng Zhao, Yang Li, Xi Chen, Qian Sha, Peng Lai, Daoquan Tang, Dongzhi Yang, Jun Liang, Mengzhe Guo,

Cancer-related molecular mechanisms research

N6-methyladenosine (m6A) is the most prominent and frequent internal messenger RNA (mRNA) modification and plays diverse roles in regulating functions of modified transcripts.1 However, the role of m6A modification in kidney disease remains rarely understood, especially at the onset of diabetic kidney disease (DKD).2 Here, we delineate the biological role of FTO-mediated m6A modification in DKD through imaging mass cytometry (IMC), LC/MS, and RNASeq methods. The results show that the loss of m6A levels by overexpressing FTO recapitulated human DKD by increasing the expression of suppressors of cytokine signalling 1 (SOCS1) protein level to alleviate inflammation response and kidney injury. Thus, FTO maybe a potential therapeutic target for DKD patients. To quantify the m6A level at cellular and spatial levels, we designed an IMC panel specific to kidney histology and used this to analyse kidney biopsies (Figure 1A and Table S1). IMC integrates IHC using metal isotope-tagged antibodies with laser ablation and mass-spectrometry-based detection to produce high-dimensional images,3 which allow simultaneously quantified the m6A levels and its regulators. We identified 17 676 cells and quantified the levels of m6A, regulators, and spatial characteristics at single-cell level (Figures 1 and S1). We identified five dominant cell clusters of proximal tubules, distal convoluted tubule, glomerulus (Glom) endothelial, macrophage, and stromal cell populations.4 IMC and IHC data demonstrated that the m6A levels were significantly increased in several types of cells, and FTO expression was significantly reduced (Figure 2). The results suggest that FTO plays important roles in m6A dynamics in DKD. Further analysis revealed that serum m6A levels were significantly upregulated in T2D and DKD patients (Figures S2 and S3a). RNASeq data showed that FTO was significantly downregulated in T2D and DKD patients, whereas other regulators remained unchanged (Figures S3b and S4). Further data also showed that the m6A levels remained significantly upregulated in T2D and DKD patients, and FTO level was negatively correlated with m6A levels (Figure S3c–e). Furthermore, re-analyses of public datasets revealed that FTO was significantly decreased in DKD or uremina patients (Figure S3f–h). To further investigate the role of FTO in DKD pathogenesis, high-concentration glucose (HG) treatment was used to simulate the phenotypes in DKD.5 HG treatment significantly reduced FTO expression, whereas the m6A RNA level was significantly increased (Figure S5a–d). Moreover, other regulators remained unchanged after the HG treatment (Figure S6). FTO overexpression or knock-down significantly reduced or augmented the m6A levels, respectively (Figure S5e–h). HG often triggers glucose-response transcriptional factor ChREBP expression,6 further analysis revealed that the FTO promoter had several ChREBP-binding sites, indicating HG may suppress FTO expression via activating ChREBP (Figure S5i). Together, these data reveal that the m6A modification levels are increased due to FTO downregulation in DKD. To investigate the molecular mechanism by how dysregulated FTO is involved in DKD. We performed MeRIP-seq in HMC after FTO overexpression, and the results showed that m6A peaks were significantly enriched at the 3′-UTR region (Figure 3A,C) and were characterized by the RAACH motif (Figure 3B). Overall, 25 genes were affected at both RNA expression and m6A levels. Pathway enrichment analyses revealed that the genes affected are involved in inflammation (Figure 3D,E and Tables S6 and S7). More specifically, the m6A level of SOCS1, a key regulator of inflammation, was significantly reduced after FTO overexpression (Figure 3F), which was confirmed by re-analyses of public datasets (Figure S7a). MeRIP-qPCR results showed that SOCS1 m6A level was significantly reduced on FTO overexpression (Figure 3G). Further data revealed that SOCS1 protein level was significantly increased when FTO was overexpressed (Figure 3H). SOCS1 mRNA expression was also positively associated with FTO expression (Figure S7b). Moreover, SOCS1 expression was significantly suppressed after FTO knock-down (Figure S7c). The inhibition of FTO induced by HG treatment could also result in the reduced expression of SOCS1 (Figure S7d). Further results showed that demethylation-inactive mutant FTO (H231A and D233A) had no effects on both m6A and protein level of SOCS1 compared with wild-type FTO (Figures S7e and 3I).Moreover, FTO-RIP-qPCR assays showed the direct binding of FTO on SOCS1 mRNA (Figure 3J). Our findings indicate that FTO can increase the expression of SOCS1 via removing its m6A. Inflammation plays vital roles in DKD, and SOCS1 is considered an important inflammation regulator.8, 9 Network and GSEA analyses showed that inflammation-related pathways including the JAK-STAT pathway were significantly repressed after FTO overexpression (Figure S8a,b). Moreover, FTO overexpression significantly inhibited inflammation via inhibiting JAK2/STAT3 phosphorylation. In contrast, FTO knock-down and HG aggravated DKD by promoting JAK2/STAT3 phosphorylation (Figure S8c–e). The rescue assay showed that p-JAK2 and p-STAT3 levels were significantly inhibited after SOCS1 knock-down (Figure S8f). Collectively, these results suggest that the JAK-STAT signalling pathway is activated due to the decreased FTO expression. We generated the db/db mice that were most commonly used T2D/DKD model.10 Intriguingly, injecting fto-overexpressing lentivirus significantly alleviated kidney damage, indicating that fto might be a potential therapeutic target (Figure S9a–g). LC/MS results showed that m6A levels were higher in db/db mice, and m6A levels were decreased after injecting FTO-overexpressing lentivirus (Figures 4A and S9h). qRT-PCR and analyses of public microarray data showed that fto expression was significantly reduced in various mouse models (Figure S9i–m). WB and IHC results showed that both fto and socs1 were significantly reduced in db/db mice, which led to inflammation response via promoting jak2 and stat3 phosphorylation (Figures 4B,C and S10). Surprisingly, fto overexpression significantly increased socs1 expression which alleviated inflammation response as the IHC and WB data showed (Figures 4B,C and S10). The H&E, PAS, SR, and IHC data further indicated that fto overexpression attenuated kidney injuries and fibrosis. Thus, our data suggest that fto overexpression can alleviate kidney injury via suppression of inflammation. In conclusion, our findings reveal a protective role of FTO during DKD pathogenesis. Mechanistically, the FTO/SOCS1/JAK-STAT axis promotes DKD pathogenesis via promoting inflammation (Figure 4D). Moreover, FTO expression is significantly decreased in DKD, and overexpression of FTO can dramatically alleviate kidney inflammation. Therefore, we suggest that therapeutic targeting of FTO in combination with current therapeutic approaches might be a new avenue for DKD treatment. No potential conflicts of interest relevant to this article were reported. Supporting Information Figure S1 Quality evaluation of IMC analysis of human kidney tissues: (a) The dot bolt analysis of synthetic RNA fragments with or without m6A modification; (b) summary example images of all 10 markers from different patients of the analysed cohort; (c) t-SNE plot showing the batch effect and the expression of Vimentin, aSMA, Nephrin, CD68, Aquaporin II, Collagen IV, and E-cadherin in each type of cell. Figure S2 Detection of m6A level by using LC-MS/MS method: (a) The calibration curve of m6A (top panel) and A (bottom panel) detected by LC-MS/MS; (b) the chromatograms of A (red) and m6A (blue) in serum samples of T2D, DKD, and healthy volunteers detected by LC-MS/MS. Figure S3 Decreased FTO expression levels are correlated with increased m6A modification levels in DKD patients: (a) overall m6A levels in serum samples of T2D, DKD, and healthy volunteers by LC–MS/MS; (b) heat map shows m6A regulators expression pattern in 15 serum samples. Five replicates for each group; (c) overall m6A levels in serum samples of T2D, DKD, and healthy volunteers from another cohort by LC–MS/MS; (d) qPCR analyses of FTO expression in serum samples of T2D, DKD, and healthy volunteers from cohort 2; (e) correlation between m6A levels and FTO mRNA expression in serum samples of T2D, DKD, and healthy volunteers from cohort 2; (f) the expression of FTO mRNA levels in blood samples of healthy volunteers and uraemia patients from GSE37171 dataset; (g,h) the expression of FTO mRNA levels in glomeruli of healthy volunteers and DN patients from GSE96804 (g) and GSE30122 (h) datasets. Data are represented as mean ± s.e.m. Statistical analyses were performed by two-tailed unpaired student t-tests and corrected for multiple comparisons using the Holm-Sidak method. Figure S4 Expression levels of m6A modification regulators in T2D, DKD, and healthy volunteers: (a) PCA plot of RNA sequencing after regressing out batch, sex and age in controls, T2D, and DN serums (n = 5 for each group); (b and c) qPCR analyses of YTHDC2 (b) and HNRNPA2B1 (c) expression in serum samples of T2D, DKD, and healthy volunteers from cohort 2; (d) qPCR analyses of key m6A modification regulators expression in serum samples of T2D, DKD, and healthy volunteers from cohort 1. Data are represented as mean ± s.e.m. Statistical analyses were performed by two-tailed unpaired student t-tests and corrected for multiple comparisons using the Holm-Sidak method. Figure S5 High-concentration glucose triggers m6A via reducing FTO expression: (a) qPCR analyses of FTO mRNA expression in HMC or HK-2 cells after treatment with high-concentration glucose, normal-concentration glucose, or mannitol; (b) Western blot analyses of indicated proteins in HMC or HK-2 cells after treatment with high-concentration glucose, normal-concentration glucose, or mannitol; (c) overall m6A levels in HMC and HK-2 cells after treatment with high-concentration glucose, normal-concentration glucose, or mannitol by LC–MS/MS; (d) correlation between m6A levels and FTO protein expression in HK-2 and HMC cells; (e) Western blot analyses of indicated proteins in HMC or HK-2 cells after FTO overexpression; (f) overall m6A levels in HMC and HK-2 cells after FTO overexpression by LC–MS/MS; (g) Western blot analyses of indicated proteins in HMC or HK-2 cells after FTO knock-down; (h) overall m6A levels in HMC and HK-2 cells after FTO knock-down by LC-MS/MS; (i) the ChREBP binding motif presented by the JASPAR database and schematic illustration of the potential binding sites of ChREBP on the promoter of FTO. Data are represented as mean ± s.e.m. Statistical analyses were performed by two-tailed unpaired student t-tests and corrected for multiple comparisons using the Holm-Sidak method. Figure S6 Expression levels of m6A modification regulators in HMC and HK-2 cells after HG treatment: (a–d) qPCR analyses of METTL3 (a), YTHDC2 (b) and HNRNPA2B1 (c), and ALKBH5 (d) expression in HMC or HK-2 cells after treatment with high-concentration glucose, normal-concentration glucose, or mannitol. Data are represented as mean ± s.e.m. Statistical analyses were performed by two-tailed unpaired student t-tests and corrected for multiple comparisons using the Holm-Sidak method. Figure S7 SOCS1 is the potential target of FTO: (a) m6A abundances on SOCS1 mRNA transcripts in MONOMAC-6 cells as detected by MeRIP-seq from GSE76414 were plotted; (b) correlation between SOCS1 and FTO mRNA expression in serum samples of T2D, DKD, and healthy volunteers from cohort 2; (c) Western blot analyses of indicated proteins in HMC or HK-2 cells after FTO knock-down; (d) Western blot analyses of indicated proteins in HMC or HK-2 cells after treatment with high-concentration glucose, normal-concentration glucose, or mannitol; (e) Western blot analyses of indicated proteins in HMC cells after overexpressing empty vector, FTO wild-type or mutant plasmid. Data are represented as mean ± s.e.m. Statistical analyses were performed by two-tailed unpaired student t-tests and corrected for multiple comparisons using the Holm-Sidak method. Figure S8 Loss of SOCS1 promotes phosphorylation of JAK2 and STAT3: (a) Protein–protein interaction network of SOCS1 according to STRING analysis; (b) GSEA results of significantly altered pathways after FTO overexpression base on RNASeq data, NES, normalized enrichment score; (c) Western blot analyses of indicated proteins in HMC or HK-2 cells after FTO overexpression; (d) Western blot analyses of indicated proteins in HMC or HK-2 cells after FTO knock-down; (e) Western blot analyses of indicated proteins in HMC or HK-2 cells after treatment with high-concentration glucose, normal-concentration glucose, or mannitol; (f) Western blot analyses of indicated proteins in HMC after indicated treatments. Figure S9 In vivo experiments indicated the therapeutic value of fto: (a–c) Body weight (a), kidney weight (b), and kidney index (c) of db/m, db/db injected with control virus, and db/db with ftooverexpression lentivirus; (d–g) urinary protein (d), blood creatinine (e), blood glucose (f), and blood urea nitrogen (g) of db/m, db/db injected with control virus, and db/db with fto overexpression lentivirus; (h) relative m6A levels in the blood of db/m and db/db mice; (i–m) relative ftoexpression of kidney or glomeruli in different diabetic mouse models. Data are represented as mean ± s.e.m. Statistical analyses were performed by two-tailed unpaired student t-tests and corrected for multiple comparisons using the Holm-Sidak method. Figure S10 Quantification of m6A and indicated protein levels of db/m, db/db injected with control virus, and db/db with fto overexpression lentivirus: (a) Quantification of indicated protein in Figure 4 analysed by IHC results of db/m, db/db injected with control virus, and db/db with fto overexpression lentivirus; (b) Western blot analyses of indicated proteins of db/m, db/db injected with control virus, and db/db with fto overexpression lentivirus. Data are represented as mean ± s.e.m. Statistical analyses were performed by two-tailed unpaired student t-tests and corrected for multiple comparisons using the Holm-Sidak method. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.