Screening and validation of key senescence-associated genes in diabetic nephropathy

YU Huiqing; WANG Xinling; PEI Yongchao; MA Jiaqing; LI Zhiguo; WU Xi'ai

doi:10.3969/j.issn.2096-9058.2025.04.007

Abstract：Objective To screen key senescence-associated genes closely associated with the development of diabet-ic nephropathy(DN)and to validate these genes.Methods Three DN-related human renal transcriptome datasets from the GEO database(GSE96804,GSE104948 and GSE30122)were standardized across platforms using the Shambhala algo-rithm.Differentially expressed genes(DEGs)were screened using the criteria of false discovery rate(FDR)adjusted P≤0.05 and fold change(FC)≥1.5.The DEGs were intersected with senescence-associated genes listed in the CellAge data-base to identify senescence-associated genes associated with DN.Functional enrichment analysis of these genes was per-formed using the clusterProfiler package(FDR-adjusted P≤0.05),and protein-protein interaction(PPI)networks were constructed using the STRING database to identify hub genes.Receiver operating characteristic(ROC)curve analysis was used to evaluate the discriminatory potential of the candidate genes for DN patients,and their expression was validated in an independent external dataset(GSE30528).BTBR ob/w heterozygous mice were bred,and at 5 weeks of age,mice were divided into BTBR w/w and BTBR ob/ob groups based on genotyping results.Fasting blood glucose,body weight,total cholesterol,kidney weight,24-hour urinary albumin,serum uric acid,serum creatinine,and blood urea nitrogen were monitored.Periodic acid-schiff(PAS)staining was used to assess renal pathological changes for assessing typical DN pa-thology.Quantitative PCR(qPCR)was conducted to validate the expression levels of key senescence-associated genes in mice renal tissues.Results A total of 918 DEGs associated with DN were identified,among which 90 were related to cel-lular senescence.Enrichment analysis indicated that these genes were primarily involved in biological processes such as"cellular response to hypoxia"and"cellular response to transforming growth factor beta(TGF-β)".PPI network analysis combined with validation in the GSE30528 dataset suggested that CDKN1B and MET may serve as key senescence-associat-ed genes in the development of DN.Compared to BTBR w/w mice,BTBR ob/ob mice exhibited higher levels of blood glu-cose,body weight,total cholesterol,kidney weight and 24-hour urinary albumin.Histological analysis revealed signifi-cant mesangial expansion and increased glomerulosclerosis index in BTBR ob/ob mice,indicating typical DN pathology.qPCR results showed that CDKN1B expression in kidney tissue of BTBR ob/ob mice(1.10±0.57)was lower than that in BTBR w/w mice(5.67±0.76),and the difference was statistically significant(P<0.05);however,there was no statisti-cally significant difference in MET expression between the two groups.Conclusions CDKN1B may be a core senes-cence-associated gene involved in the pathogenesis of diabetic nephropathy.Further research on this gene may provide new theoretical foundations and potential targets for the prevention and treatment of DN.