| Objective: The objective of this research is to investigate the association between MIP-1β and clinical parameters in individuals diagnosed with diabetic nephropathy(DKD),examine the potential causal link between MIP-1β and diabetic nephropathy,and analyze the molecular mechanisms underlying the downstream regulation of MIP-1β in diabetic nephropathy through a combination of in vivo and in vitro experiments.Materials and Methods: 1.Bidirectional Mendelian randomization analysis was used to establish a clear causal relationship between cytokines and DKD using publicly available Genome-Wide Association Studies(GWAS)summary databases(including3,283 DKD cases and 210,463 controls)and databases from three independent Finnish cohorts(totalling 8,293 individuals’ inflammatory factor instrumental variables,including the Young Finns Study,FINRISK 1997,and FINRISK 2002).Nine Mendelian randomization analysis methods were employed to explore the causal relationship between the instrumental variables of exposure factors and outcome variables,along with pleiotropy and heterogeneity tests.2.A total of 128 patients diagnosed with DKD through renal biopsy at the People’s Hospital of Xinjiang Uygur Autonomous Region from February 2015 to December 2022,along with 42 patients with Type 2 diabetes and 67 healthy controls,were included to assess the serum and renal tissue expression of 4cytokines.3.A high glucose-induced in vitro model was established using the human glomerular endothelial cell line HRGEC,divided into six groups: NG;HG;HG+vector;HG+MIP-1β;HG+si-control;HG+si-MIP-1β.After 48 hours,q PCR,Western Blot,ELISA,TUNEL staining,cell flow cytometry,and ROS flow cytometry were used to assess MIP-1β expression,oxidative stress markers,endothelial-to-mesenchymal transition markers,and apoptosis.4.Co-immunoprecipitation(COIP)and phosphoproteomics were utilized to screen effectors regulated downstream of MIP-1β,focusing on DDX41.Confocal immunofluorescence observed co-expression of MIP-1βand DDX41 under high glucose conditions.Further in vitro experiments explored the regulatory relationship between MIP-1β,DDX41,and phenotypic changes across four groups: HG+vector;HG+MIP-1β;HG+DDX41;HG+MIP-1+DDX41.5.Two diabetic nephropathy mouse models were constructed to overexpress or knock out MIP-1β,observing its regulatory effects on DDX41 expression and phenotype.The models were divided as follows: db/m;db/db;db/db+r AAV;db/db+r AAV-MIP-1β;db/db+r AAVDDX41;db/db+r AAV-MIP-1β+r AAV-DDX41.ELISA,HE staining,Masson’s trichrome staining,ROS flow cytometry,q PCR,and Western Blot were used to assess biochemical indicators,glomerular pathological changes,ROS levels,and expression of proteins related to endothelial-mesenchymal transition.6.Network pharmacology and machine learning identified berberine from Berberis vulgaris as a potential compound for delaying DKD progression.An STZ-DN animal model was used to compare the effects of berberine on urinary protein,renal pathology,and the expression of MIP-1β and endothelial-to-mesenchymal transition markers using Western Blot and q PCR.Results: Mendelian randomization analysis revealed that patients with elevated levels of CRP,IL-10,MIP-1β,and IFNγ might be related to the risk of developing diabetic nephropathy.Among 26 inflammatory cytokines,CRP,IFNγ,IL-10,and MIP-1βwere found to be highly expressed in both the serum and renal tissues of patients with diabetic nephropathy,with IFNγ and MIP-1β showing a positive correlation with the urine protein-to-creatinine ratio and 24-hour proteinuria levels.At the cellular level,it was observed that under hyperglycemic conditions,the level of MIP-1β was higher than that in the normal control group.Overexpression of MIP-1β induced oxidative stress in endothelial cells,endothelial-to-mesenchymal transition,and apoptosis.Integrating COIP mass spectrometry with phosphorylation mass spectrometry results,five proteins covarying with MIP-1β were identified,including DDX41,RPL17,RPS23,FTSJ3,and KRT68.GO and KEGG analysis of these proteins revealed significant enrichment in biological pathways related to cell apoptosis,proliferation,cytokine response,and cytokine production.Among these,DDX41 was the focus,and bidirectional COIP validation showed that MIP-1β could interact with DDX41 protein.Confocal immunofluorescence results indicated co-expression of MIP-1β and DDX41 in human glomerular endothelial cells under high glucose conditions,with DDX41 expression being lower than in the normal control group.MIP-1β was able to inversely regulate the expression of DDX41;overexpression of DDX41 could alleviate oxidative stress response,endothelial-to-mesenchymal transition,and apoptosis.Phenotypic recovery experiments revealed that the regulatory relationship of MIP-1β on phenotype depends on the regulation of DDX41 expression.At the animal level,diabetic nephropathy models exhibited increased expression of MIP-1β and relatively lower expression of DDX41,with both proteins co-expressed in glomeruli.Overexpression of MIP-1β in DKD mice increased UACR,BUN,and serum creatinine levels,decreased serum albumin levels,exacerbated renal pathology,and intensified glomerular oxidative stress response and endothelial-to-mesenchymal transition.Conversely,overexpression of DDX41 in DKD mice lowered UACR,BUN,and serum creatinine levels,increased serum albumin levels,and improved renal pathology,oxidative stress response,and endothelial-to-mesenchymal transition.Phenotypic recovery experiments showed that overexpression of DDX41 on the basis of overexpressing MIP-1β could improve biochemical indicators and renal pathology in mice,with the regulation of glomerular oxidative stress and endothelial-tomesenchymal transition by MIP-1β depending on DDX41.Network pharmacology and machine learning approaches identified berberine,an active component of Berberis integerrima,as a potential compound for treating diabetic nephropathy;preliminary findings indicated that berberine could reduce 24-hour proteinuria and glomerular MIP-1βdeposition,and improve glomerular pathology and endothelial-to-mesenchymal transition.Conclusion: MIP-1β may contribute to the development of DKD by promoting glomerular oxidative stress,endothelial-to-mesenchymal transition,and apoptosis through the inhibition of DDX41.Berberis integerrima could represent a novel therapeutic for diabetic nephropathy,lowering renal MIP-1β deposition,warranting further in-depth exploration. |
