This study aims to screen liquid–liquid phase separation (LLPS)-related genes modulating ubiquitination for diagnosing patients with diabetic nephropathy (DN). After performing differential gene expression analysis, ssGSEA, and immune infiltration analysis, LLPS-related genes modulating ubiquitination, key immune cells, and immune-related pathways were acquired. The correlations between LLPS-related genes and ubiquitination, key immune-related pathways were explored, respectively. The diagnostic genes were identified by four machine learning algorithms, followed by evaluation to predict the performance of the machine learning models on ubiquitination using receiver operating characteristic curves and a confusion matrix. In vitro experiments were applied to explore the function of signal transducer and activator of transcription 1 (STAT1) in DN. Totally, 513 differentially expressed genes were screened between the DN and control groups. The activity of the ubiquitin-mediated proteolysis pathway exhibited a significant difference between the DN and control samples. The fraction of a total of 10 immune cells exhibited a significant difference between the lower and higher ubiquitination groups. Totally, nine LLPS-related genes modulating ubiquitination were acquired, and these genes modulating ubiquitination were enriched in ubiquitin pathways and significantly positively correlated with the complement, IFN-α, and IFN-γ pathways. Two diagnostic genes were obtained, namely dual specificity tyrosine phosphorylation regulated kinase 2 (DYRK2) and STAT1. Notably, downregulation of STAT1 effectively alleviated IFN-γ, IFN-α, IL-1β, and TNF-α levels in D-glucose-treated HK-2 cells, as well as the protein level of FK2. LLPS-related STAT1 modulating ubiquitination accelerates the inflammatory response in DN.
Albasanz-PuigA, MurrayJ, NamekataM, et al.Opposing roles of STAT-1 and STAT-3 in regulating vascular endothelial growth factor expression in vascular smooth muscle cells. Biochem Biophys Res Commun, 2012; 428(1):179–184; doi: 10.1016/j.bbrc.2012.10.037
2.
BarbieDA, TamayoP, BoehmJS, et al.Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1. Nature, 2009; 462(7269):108–112; doi: 10.1038/nature08460
3.
Ben-NeriahY. Regulatory functions of ubiquitination in the immune system. Nat Immunol, 2002; 3(1):20–26; doi: 10.1038/ni0102-20
4.
ButturiniE, Carcereri de PratiA, MariottoS. Redox regulation of STAT1 and STAT3 signaling. Int J Mol Sci, 2020; 21(19):7034; doi: 10.3390/ijms21197034
5.
CaoM, ZhangX, WangX, et al.An overview of liquid-liquid phase separation and its mechanisms in sepsis. J Inflamm Res, 2025; 18:3969–3980; doi: 10.2147/jir.S513098
6.
ChenH, HuangS, QuanL, et al.Liquid–liquid phase separation: A potentially fundamental mechanism of sepsis. Cell Death Discov, 2025; 11(1):310; doi: 10.1038/s41420-025-02599-2
7.
ChenS, YeJ, ChenX, et al.Valproic acid attenuates traumatic spinal cord injury-induced inflammation via STAT1 and NF-κB pathway dependent of HDAC3. J Neuroinflammation, 2018; 15(1):150; doi: 10.1186/s12974-018-1193-6
8.
CockramPE, KistM, PrakashS, et al.Ubiquitination in the regulation of inflammatory cell death and cancer. Cell Death Differ, 2021; 28(2):591–605; doi: 10.1038/s41418-020-00708-5
9.
DaiL, GolembiewskaE, LindholmB, et al.End-stage renal disease, inflammation and cardiovascular outcomes. Contrib Nephrol, 2017; 191:32–43; doi: 10.1159/000479254
10.
DaoTP, CastañedaCA. Ubiquitin-modulated phase separation of shuttle proteins: Does condensate formation promote protein degradation? Bioessays, 2020; 42(11):e2000036; doi: 10.1002/bies.202000036
11.
DaoTP, KolaitisRM, KimHJ, et al.Ubiquitin modulates liquid-liquid phase separation of ubqln2 via disruption of multivalent interactions. Mol Cell, 2018; 69(6):965–978.e966; doi: 10.1016/j.molcel.2018.02.004
12.
FanX, ZhangR, XuG, et al.Role of ubiquitination in the occurrence and development of osteoporosis (Review). Int J Mol Med, 2024; 54(2):68; doi: 10.3892/ijmm.2024.5392
13.
GallegoLD, SchneiderM, MittalC, et al.Phase separation directs ubiquitination of gene-body nucleosomes. Nature, 2020; 579(7800):592–597; doi: 10.1038/s41586-020-2097-z
14.
GaoC, HuangW, KanasakiK, et al.The role of ubiquitination and sumoylation in diabetic nephropathy. Biomed Res Int, 2014; 2014:160692; doi: 10.1155/2014/160692
15.
GaoL, ZhangW, ShiXH, et al.The mechanism of linear ubiquitination in regulating cell death and correlative diseases. Cell Death Dis, 2023; 14(10):659; doi: 10.1038/s41419-023-06183-3
16.
GerlachB, CordierSM, SchmukleAC, et al.Linear ubiquitination prevents inflammation and regulates immune signalling. Nature, 2011; 471(7340):591–596; doi: 10.1038/nature09816
17.
GraczykP, DachA, DyrkaK, et al.Pathophysiology and advances in the therapy of cardiomyopathy in patients with diabetes mellitus. Int J Mol Sci, 2024; 25(9):5027; doi: 10.3390/ijms25095027
18.
GraysonPC, EddyS, TaroniJN, et al.; Vasculitis Clinical Research Consortium, the European Renal cDNA Bank cohort, and the Nephrotic Syndrome Study Network. Metabolic pathways and immunometabolism in rare kidney diseases. Ann Rheum Dis, 2018; 77(8):1226–1233; doi: 10.1136/annrheumdis-2017-212935
19.
HarreiterJ, RodenM. [Diabetes mellitus: Definition, classification, diagnosis, screening and prevention (Update 2023)]. Wien Klin Wochenschr, 2023; 135(Suppl 1):7–17; doi: 10.1007/s00508-022-02122-y
20.
HespAC, SchaubJA, PrasadPV, et al.The role of renal hypoxia in the pathogenesis of diabetic kidney disease: A promising target for newer renoprotective agents including SGLT2 inhibitors? Kidney Int, 2020; 98(3):579–589; doi: 10.1016/j.kint.2020.02.041
21.
HiroiM, MoriK, SakaedaY, et al.STAT1 represses hypoxia-inducible factor-1-mediated transcription. Biochem Biophys Res Commun, 2009; 387(4):806–810; doi: 10.1016/j.bbrc.2009.07.138
22.
HuX, IvashkivLB. Cross-regulation of signaling pathways by interferon-gamma: Implications for immune responses and autoimmune diseases. Immunity, 2009; 31(4):539–550; doi: 10.1016/j.immuni.2009.09.002
23.
IvanovSV, SalnikowK, IvanovaAV, et al.Hypoxic repression of STAT1 and its downstream genes by a pVHL/HIF-1 target DEC1/STRA13. Oncogene, 2007; 26(6):802–812; doi: 10.1038/sj.onc.1209842
24.
JinQ, LiuT, QiaoY, et al.Oxidative stress and inflammation in diabetic nephropathy: Role of polyphenols. Front Immunol, 2023; 14:1185317; doi: 10.3389/fimmu.2023.1185317
25.
JohnsonWE, LiC, RabinovicA. Adjusting batch effects in microarray expression data using empirical Bayes methods. Biostatistics, 2007; 8(1):118–127; doi: 10.1093/biostatistics/kxj037
26.
KratofilRM, KubesP, DenisetJF. Monocyte conversion during inflammation and injury. Arterioscler Thromb Vasc Biol, 2017; 37(1):35–42; doi: 10.1161/atvbaha.116.308198
27.
KumarK, Man-Un UngP, WangP, et al.Novel selective thiadiazine DYRK1A inhibitor lead scaffold with human pancreatic β-cell proliferation activity. Eur J Med Chem, 2018; 157:1005–1016; doi: 10.1016/j.ejmech.2018.08.007
28.
LeekJT, JohnsonWE, ParkerHS, et al.The sva package for removing batch effects and other unwanted variation in high-throughput experiments. Bioinformatics, 2012; 28(6):882–883; doi: 10.1093/bioinformatics/bts034
29.
LiB, YeS, FanY, et al.Identification of novel key genes and potential candidate small molecule drugs in diabetic kidney disease using comprehensive bioinformatics analysis. Front Genet, 2022; 13:934555; doi: 10.3389/fgene.2022.934555
30.
LiB, ZhaoX, XieW, et al.Integrative analyses of biomarkers and pathways for diabetic nephropathy. Front Genet, 2023a;14:1128136; doi: 10.3389/fgene.2023.1128136
31.
LiJ, ZhuK, GuA, et al.Feedback regulation of ubiquitination and phase separation of HECT E3 ligases. Proceedings of the National Academy of Sciences of the United States of America, 2023b;120(33):e2302478120; doi: 10.1073/pnas.2302478120
32.
LiuF, ChenJ, LiK, et al.Ubiquitination and deubiquitination in cancer: From mechanisms to novel therapeutic approaches. Mol Cancer, 2024; 23(1):148; doi: 10.1186/s12943-024-02046-3
33.
LiuW, WangZ, LiuS, et al.RNF138 inhibits late inflammatory gene transcription through degradation of SMARCC1 of the SWI/SNF complex. Cell Rep, 2023; 42(2):112097; doi: 10.1016/j.celrep.2023.112097
34.
LiuX, LinZ, YinX. Pellino2 accelerate inflammation and pyroptosis via the ubiquitination and activation of NLRP3 inflammation in model of pediatric pneumonia. Int Immunopharmacol, 2022; 110:108993; doi: 10.1016/j.intimp.2022.108993
35.
MaX, LiuR, XiX, et al.Global burden of chronic kidney disease due to diabetes mellitus, 1990–2021, and projections to 2050. Front Endocrinol (Lausanne), 2025; 16:1513008; doi: 10.3389/fendo.2025.1513008
36.
MikłoszA, ChabowskiA. Adipose-derived mesenchymal stem cells therapy as a new treatment option for diabetes mellitus. J Clin Endocrinol Metab, 2023; 108(8):1889–1897; doi: 10.1210/clinem/dgad142
37.
MłynarskaE, BuławskaD, CzarnikW, et al.Novel insights into diabetic kidney disease. Int J Mol Sci, 2024; 25(18):10222; doi: 10.3390/ijms251810222
38.
NakataS, WatanabeT, NakagawaK, et al.The dynamics of histone H2A ubiquitination in HeLa cells exposed to rapamycin, ethanol, hydroxyurea, ER stress, heat shock and DNA damage. Biochem Biophys Res Commun, 2016; 472(1):46–52; doi: 10.1016/j.bbrc.2016.02.057
39.
NihiraNT, YoshidaK. Engagement of DYRK2 in proper control for cell division. Cell Cycle, 2015; 14(6):802–807; doi: 10.1080/15384101.2015.1007751
40.
ParoliniF, TiraR, BarracchiaCG, et al.Ubiquitination of Alzheimer’s-related tau protein affects liquid-liquid phase separation in a site- and cofactor-dependent manner. Int J Biol Macromol, 2022; 201:173–181; doi: 10.1016/j.ijbiomac.2021.12.191
41.
PhipsonB, LeeS, MajewskiIJ, et al.Robust hyperparameter estimation protects against hypervariable genes and improves power to detect differential expression. Ann Appl Stat, 2016; 10(2):946–963; doi: 10.1214/16-aoas920
42.
Rayego-MateosS, Rodrigues-DiezRR, Fernandez-FernandezB, et al.Targeting inflammation to treat diabetic kidney disease: The road to 2030. Kidney Int, 2023; 103(2):282–296; doi: 10.1016/j.kint.2022.10.030
43.
RitchieME, PhipsonB, WuD, et al.limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res, 2015; 43(7):e47; doi: 10.1093/nar/gkv007
44.
RussoMP, Grande-RattiMF, BurgosMA, et al.Prevalence of diabetes, epidemiological characteristics and vascular complications. Arch Cardiol Mex, 2023; 93(1):30–36; doi: 10.24875/acm.21000410
45.
ThipsawatS. Early detection of diabetic nephropathy in patient with type 2 diabetes mellitus: A review of the literature. Diab Vasc Dis Res, 2021; 18(6):14791641211058856; doi: 10.1177/14791641211058856
46.
WangH, ZhangY, XiaF, et al.Protective effect of silencing Stat1 on high glucose-induced podocytes injury via Forkhead transcription factor O1-regulated the oxidative stress response. BMC Mol Cell Biol, 2019; 20(1):27; doi: 10.1186/s12860-019-0209-0
47.
WangL, LiP. Arginine methylation-enabled FUS phase separation with SMN contributes to neuronal granule formation. Cell Rep, 2024; 43(8):114537; doi: 10.1016/j.celrep.2024.114537
48.
WangX, LiQ, SuiB, et al.Schisandrin A from Schisandra chinensis attenuates ferroptosis and NLRP3 inflammasome-mediated pyroptosis in diabetic nephropathy through mitochondrial damage by AdipoR1 ubiquitination. Oxid Med Cell Longev, 2022; 2022:5411462; doi: 10.1155/2022/5411462
WenJH, LiDY, LiangS, et al.Macrophage autophagy in macrophage polarization, chronic inflammation and organ fibrosis. Front Immunol, 2022; 13:946832; doi: 10.3389/fimmu.2022.946832
51.
WoronieckaKI, ParkAS, MohtatD, et al.Transcriptome analysis of human diabetic kidney disease. Diabetes, 2011; 60(9):2354–2369; doi: 10.2337/db10-1181
52.
WuL, LiuC, ChangDY, et al.Annexin A1 alleviates kidney injury by promoting the resolution of inflammation in diabetic nephropathy. Kidney Int, 2021; 100(1):107–121; doi: 10.1016/j.kint.2021.02.025
53.
WuY, ChenY, TianX, et al.Ubiquitination regulates autophagy in cancer: Simple modifications, promising targets. J Transl Med, 2024; 22(1):985; doi: 10.1186/s12967-024-05565-1
54.
XuY, LuJ, LiM, et al.Diabetes in China part 1: Epidemiology and risk factors. Lancet Public Health, 2024; 9(12):e1089–e1097; doi: 10.1016/s2468-2667(24)00250-0
55.
YamamotoK, GoyamaS, AsadaS, et al.A histone modifier, ASXL1, interacts with NONO and is involved in paraspeckle formation in hematopoietic cells. Cell Rep, 2021; 36(8):109576; doi: 10.1016/j.celrep.2021.109576
56.
YangD, YangC, HuangL, et al.Role of ubiquitination-driven metabolisms in oncogenesis and cancer therapy. Semin Cancer Biol, 2025; 110:17–35; doi: 10.1016/j.semcancer.2025.02.004
ZhangZ, ZhouF, LuM, et al.WTAP-mediated m(6)A modification of TRIM22 promotes diabetic nephropathy by inducing mitochondrial dysfunction via ubiquitination of OPA1. Redox Rep, 2024; 29(1):2404794; doi: 10.1080/13510002.2024.2404794
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
