The role of N ⁶ -methyladenosine (m6A) modifications in renal diseases is largely unknown. Here, we characterized the role of N ⁶ -adenosine-methyltransferase-like 3 (METTL3), whose expression is elevated in renal tubules in different acute kidney injury (AKI) models as well as in human biopsies and cultured tubular epithelial cells (TECs). METTL3 silencing alleviated renal inflammation and programmed cell death in TECs in response to stimulation by tumor necrosis factor–α (TNF-α), cisplatin, and lipopolysaccharide (LPS), whereas METTL3 overexpression had the opposite effects. Conditional knockout of METTL3 from mouse kidneys attenuated cisplatin- and ischemic/reperfusion (I/R)–induced renal dysfunction, injury, and inflammation. Moreover, TAB3 [TGF-β–activated kinase 1 (MAP3K7) binding protein 3] was identified as a target of METTL3 by m6A methylated RNA immunoprecipitation sequencing and RNA sequencing. The stability of TAB3 was increased through binding of IGF2BP2 (insulin-like growth factor 2 binding protein 2) to its m6A-modified stop codon regions. The proinflammatory effects of TAB3 were then explored both in vitro and in vivo. Adeno-associated virus 9 (AAV9)–mediated METTL3 silencing attenuated renal injury and inflammation in cisplatin- and LPS-induced AKI mouse models. We further identified Cpd-564 as a METTL3 inhibitor that had better protective effects against cisplatin- and ischemia/reperfusion-induced renal injury and inflammation than S -adenosyl- l -homocysteine, a previously identified METTL3 inhibitor. Collectively, METTL3 promoted m6A modifications of TAB3 and enhanced its stability via IGF2BP2-dependent mechanisms. Both genetic and pharmacological inhibition of METTL3 attenuated renal injury and inflammation, suggesting that the METTL3/TAB3 axis is a potential target for treatment of AKI.

Epigenetic changes are present in many physiological and pathological processes. The N⁶-methyladenosine (m6A) modification is the most common modification in eukaryotic mRNA. However, the role of m6A modification in diabetic nephropathy (DN) remains elusive. Here, we found that m6A modification was significantly upregulated in the kidney of type 1 and type 2 diabetic mice, which was caused by elevated levels of METTL3. Moreover, METTL3 is increased in podocyte of renal biopsy from patients with DN, which is related to renal damage. METTL3 knockout significantly reduced the inflammation and apoptosis in high glucose (HG)-stimulated podocytes, while its overexpression significantly aggravated these responses in vitro. Podocyte-conditional knockout METTL3 significantly alleviated podocyte injury and albuminuria in streptozotocin (STZ)-induced diabetic mice. Therapeutically, silencing METTL3 with adeno-associated virus serotype-9 (AAV9)-shMETTL3 in vivo mitigated albuminuria and histopathological injury in STZ-induced diabetic mice and db/db mice. Mechanistically, METTL3 modulated Notch signaling via the m6A modification of TIMP2 in an insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2)-dependent manner and exerted pro-inflammatory and pro-apoptotic effects. In summary, this study suggested that METTL3-mediated m6A modification is an important mechanism of podocyte injury in DN. Targeting m6A through the writer enzyme METTL3 is a potential approach for the treatment of DN.

Diabetic kidney disease (DKD) is one of the microvascular complications of diabetes mellitus and a major cause of end-stage renal disease with limited treatment options. Wogonin is a flavonoid derived from the root of Scutellaria baicalensis Georgi, which has shown a potent renoprotective effect. But the mechanisms of action in DKD are not fully elucidated. In this study, we investigated the effects of wogonin on glomerular podocytes in DKD using mouse podocyte clone 5 (MPC5) cells and diabetic mice model. MPC5 cells were treated with high glucose (30 mM). We showed that wogonin (4, 8, 16 μM) dose-dependently alleviated high glucose (HG)-induced MPC5 cell damage, accompanied by increased expression of WT-1, nephrin, and podocin proteins, and decreased expression of TNF-α, MCP-1, IL-1β as well as phosphorylated p65. Furthermore, wogonin treatment significantly inhibited HG-induced apoptosis in MPC5 cells. Wogonin reversed HG-suppressed autophagy in MPC5 cells, evidenced by increased ATG7, LC3-II, and Beclin-1 protein, and decreased p62 protein. We demonstrated that wogonin directly bound to Bcl-2 in MPC5 cells. In HG-treated MPC5 cells, knockdown of Bcl-2 abolished the beneficial effects of wogonin, whereas overexpression of Bcl-2 mimicked the protective effects of wogonin. Interestingly, we found that the expression of Bcl-2 was significantly decreased in biopsy renal tissue of diabetic nephropathy patients. In vivo experiments were conducted in STZ-induced diabetic mice, which were administered wogonin (10, 20, 40 mg · kg⁻¹ · d⁻¹, i.g.) every other day for 12 weeks. We showed that wogonin administration significantly alleviated albuminuria, histopathological lesions, and p65 NF-κB-mediated renal inflammatory response. Wogonin administration dose-dependently inhibited podocyte apoptosis and promoted podocyte autophagy in STZ-induced diabetic mice. This study for the first time demonstrates a novel action of wogonin in mitigating glomerulopathy and podocytes injury by regulating Bcl-2-mediated crosstalk between autophagy and apoptosis. Wogonin may be a potential therapeutic drug against DKD.

Hypertension is a primary modifiable risk factor for cardiovascular diseases, which often induces renal end-organ damage and complicates chronic kidney disease (CKD). In the present study, histological analysis of human kidney samples revealed that hypertension induced mtDNA leakage and promoted the expression of stimulator of interferon genes (STING) in renal epithelial cells. We used AngII- and 2K1C-treated mouse kidneys to elucidate the underlying mechanisms. Abnormal renal mtDNA packing caused by AngII promoted STING-dependent production of inflammatory cytokines, macrophage infiltration, and a fibrogenic response. STING knockout significantly decreased NF-κB activation and immune cell infiltration, attenuating tubule atrophy and extracellular matrix accumulation in vivo and in vitro. These effects delayed CKD progression. Immunoprecipitation assays and liquid chromatography-tandem mass spectrometry showed that STING and ACSL4 were directly combined at the D53 and K412 amino acids of ACSL4. Furthermore, STING induced renal inflammatory response and fibrosis through ACSL4-dependent ferroptosis. Lastly, inhibition of ACSL4 using siRNA, rosiglitazone, or Fer-1 downregulated AngII-induced mtDNA-STING-dependent renal inflammation. These results suggest that targeting the STING/ACSL4 axis might represent a potential strategy for treating hypertension-associated CKD.

Diabetes nephropathy (DN) is characterized by abnormal interactions between kidney‐infiltrating macrophages and glomerular mesangial cells. Recently, a novel cell‐cell communication mediated by exosomes has gained attention. Exosomes are membrane‐bound vesicles that contain a variety of molecules such as proteins, lipids, DNA, mRNA, and microRNAs. Exosomes play an important role in the pathogenesis of DN. In this study, we show that high glucose (HG) led to increased excretion of exosomes from macrophages. Mesangial cells took up exosomes in vitro , which resulted in the activation and proliferation of mesangial cells and the secretion of extracellular matrix and inflammatory cytokines. In addition, C57BL/6 mice injected with exosomes from HG‐treated macrophages showed morphologic and functional changes. We then showed that exosomes from HG‐treated TGF‐β1 knockdown macrophages induced less extracellular matrix and fewer inflammatory factors in mesangial cells compared with vector control. Our findings suggest that TGF‐β1 mRNA in exosomes serves a role between macrophages and mesangial cells by activating the TGF‐β1/ mothers against decapentaplegic homolog 3 pathway.—Zhu, Q.‐J., Zhu, M., Xu, M.‐X., Meng, X.‐M., Wu, Y.‐G. Exosomes from high glucose‐treated macrophages activate glomerular mesangial cells via TGF‐β1/Smad3 pathway in vivo and in vitro . FASEB J. 33, 9279–9290 (2019). www.fasebj.org

MLKL is a central mediator for necroptosis. Its knockout significantly relieves acute kidney injury (AKI). However, its upstream regulatory mechanism in AKI has not been fully elucidated. We recently reviewed how microRNAs (miRNAs), a type of well‐studied epigenetic regulator, play critical roles in AKI. Here, we evaluated miRNAs that potentially target MLKL and evaluated their function in human tubular epithelial cells in response to toxic and ischemic insults. TargetScan analysis showed that miR‐194‐5P, miR‐338‐3P, miR‐500a‐3P, and miR‐577 had MLKL binding sites. Although all 4 miRNAs are reduced in AKI, our data show that only hsa‐miR‐500a‐3P was significantly suppressed in cisplatin‐treated human tubular epithelial (HK2) cells. We further found that hsa‐miR‐500a‐3P alleviated cisplatin‐induced HK2 cell death, which was confirmed by transmission electron microscopy and flow cytometry. In addition, overexpression of hsa‐miR‐500a‐3P decreased kidney injury molecule‐1 mRNA and protein levels. Real‐time PCR, ELISA, and immunofluorescence data show that hsa‐miR‐500a‐3P protected against inflammatory response, evidenced by decreased monocyte chemotactic protein‐1 and proinflammatory cytokines TNF‐α and IL‐8. Further, hsa‐miR‐500a‐3P attenuated P65 NF‐κB phosphorylation and promoter activity. Mechanistically, luciferase reporter assay showed that hsa‐miR‐500a‐3P bound the 3′UTR of MLKL, thereby suppressing phosphorylation and membrane translocation of MLKL. In agreement with these findings, we identified that overexpression of hsa‐miR‐500a‐3P attenuated cell injury and the inflammatory response in response to sodium azide treatment in an in vitro model. Results show that circulating exosomes from patients with AKI down‐regulated miR‐500a‐3P, which suppressed cell injury and inflammation in HK2 cells. hsa‐miR‐500a‐3P alleviated toxic and ischemic insults that were triggered by cell necroptosis and the inflammatory response in human HK2 cells by targeting MLKL. This may serve as a novel therapeutic target for treatment of AKI.—Jiang, L., Liu, X.‐Q., Ma, Q., Yang, Q., Gao, L., Li, H.‐D., Wang, J.‐N., Wei, B., Wen, J., Li, J., Wu, Y.‐G., Meng, X.‐M. hsa‐miR‐500a‐3P alleviates kidney injury by targeting MLKL‐mediated necroptosis in renal epithelial cells. FASEB J. 33, 3523–3535 (2019). www.fasebj.org