Myricetin induces M2 macrophage polarization to alleviate renal tubulointerstitial fibrosis in diabetic nephropathy via PI3K/Akt pathway

BACKGROUND Development of end-stage renal disease is predominantly attributed to diabetic nephropathy(DN).Previous studies have indicated that myricetin possesses the potential to mitigate the pathological alterations observed in renal tissue.Never-theless,the precise molecular mechanism through which myricetin influences the progression of DN remains uncertain.AIM To investigate the effects of myricetin on DN and explore its potential therapeutic mechanism.METHODS Db/db mice were administered myricetin intragastrically on a daily basis at doses of 50 mg/kg or 100 mg/kg for a duration of 12 wk.Subsequently,blood and urine indexes were assessed,along with examination of renal tissue pathology.Kidney morphology and fibrosis were evaluated using various staining techniques including hematoxylin and eosin,periodic acid–Schiff,Masson’s trichrome,and Sirius-red.Additionally,high-glucose culturing was conducted on the RAW 264.7 cell line,treated with 25 mM myricetin or co-administered with the PI3K/Akt inhibitor LY294002 for a period of 24 h.In both in vivo and in vitro settings,quantification of inflammation factor levels was conducted using western blotting,real-time qPCR and ELISA.RESULTS In db/db mice,administration of myricetin led to a mitigating effect on DN-induced renal dysfunction and fibrosis.Notably,we observed a significant reduction in expressions of the kidney injury markers kidney injury molecule-1 and neutrophil gelatinase associated lipocalin,along with a decrease in expressions of inflammatory cytokine-related factors.Furthermore,myricetin treatment effectively inhibited the up-regulation of tumor necrosis factor-alpha,interleukin-6,and interluekin-1βinduced by high glucose in RAW 264.7 cells.Additionally,myricetin modulated the M1-type polarization of the RAW 264.7 cells.Molecular docking and bioinformatic analyses revealed Akt as the target of myricetin.The protective effect of myricetin was nullified upon blocking the polarization of RAW 264.7 via inhibition of PI3K/Akt activation using LY294002.CONCLUSION This study demonstrated that myricetin effectively mitigates kidney injury in DN mice through the regulation of macrophage polarization via the PI3K/Akt signaling pathway.

Expression of Connective Tissue Growth Factor in Renal Tubulointerstitial Fibrosis in Rats and Its Pathogenic Role

Summary： In order to explore the role of connective tissue growth factor （CTGF） in the pathogenesis of renal tubulointerstitial fibrosis, 48 Wistar rats were randomly divided into sham-operated and unilateral ureteral obstruction （UUO） group. On the postoperative day 1, 3, 7 and 14, the rats were killed and the kidneys were removed. The renal tubulointerstitial injury index was evaluated according to the MASSON staining. The mRNA levels of CTGF, transforming growth factor β1 （TGF-β1）. collagen [ （col I ）, and plasminogen activator inhibitor-1 （PAI 1） were detected using rexerse transcriptional-polymerase chain reaction （RT PCR）. Immunohistochemistry was performed to evaluale the protein expression of the above factors, and the relations among them were analyzed. Quantitative expression of CTGF protein in the kidneys was also assessed using Western blot. The results showed that TGF-β1 mRNA level was increased at first day after UUO, followed by a marked elevation of CTGF mRNA level, which began to increase 3 days after UUO （P〈0.01）. With the progression of the disease, the mRNA expression of CTGF, col I and PAI-1 was increased progressively. Immunohistochemistry revealed that the CTGF protein expression was significantly increased in fibrotic areas and tubular epithelial cells 3 days after UUO. On the post-UUO day 7, the protein level of CTGF was positively related to the renal tubulointerstitial injury index （r =0.62, P〈0.01）, the expression of TGF-β1 （r=0.85, P〈0.01）, colI （r=0.78, P〈0.01）, and PAI-1（r=0.76, P〈0.01）. Upon Western blot analysis, CTGF protein expression began to increase 3 days after UUO, and appeared progressively throughout the time course （P〈0.01, as compared with sham-operated group）. It is concluded that CTGF can be induced by TGF-β and mediate various profibrotic actions of this cytokine, such as increasing extracellular matrix （ECM） synthesis and decreasing ECM degradation. The increased expression of CTGF may play a crucial role in the development and progression of tubulointerstitial fibrosis.

Identification of genes related to tubulointerstitial injury in type 2 diabetic nephropathy based on bioinformatics and machine learning

Objective:To explore the genes related to renal tubulointerstitial injury in DN and to elucidate their underlying mechanism by using bioinformatics multi-chip joint analysis and machine learning technology,so as to provide new ideas for the diagnosis and treatment of DN.Methods:Four gene expression datasets of DN tubulointerstitial tissues were retrieved from the GEO database.GSE30122,GSE47185 and GSE99340 were used as the combined microarray datasets,and GSE104954 was used as the independent verification datasets.The differentially expressed genes(DEGs)were identified by R language,and Gene Ontology(GO)enrichment,KEGG pathway enrichment,Gene Set Enrichment Analysis(GSEA)and Immune Cell Infiltration Analysis were performed.Furthermore,LASSO regression,SVM-RFE and RF machine learning algorithm were used to screen core genes,while external validation and Receiver Operating Curve(ROC)analysis as well as the model of prediction nomogram were performed.Finally,the influence of the clinical characteristics of DN patients was explored by Nephroseq.Results:A total of 107 DEGs were obtained,enrichment analysis revealed that the tubulointerstitial injury in DN was mainly involved in adaptive immune response,lymphocyte mediated immunity,regulation of immune effector process and immune-inflammatory pathways such as staphylococcus aureus infection,complement and coagulation cascades,phagosomes,and Th1 and Th2 cell differentiation.In addition,cell adhesion molecule,cytokine-cytokine receptor interaction and ECM-receptor interaction pathways were also significantly enriched.Memory resting CD4 T cells,γδ毮T cells,resting mast cells and neutrophil cells were up-regulated,while CD8 T cells were down-regulated.Machine learning identified MARCKSL1,CX3CR1,FSTL1,AGR2,GADD45B as core genes with good diagnostic and predictive efficacy.Conclusion:The key pathological mechanism of tubulointerstitial injury in DN is immune disorder,inflammatory reaction,cytokine action and extracellular matrix deposition.Moreover,MARCKSL1,CX3CR1,FSTL1 may be the potential biomarkers for the diagnosis and prediction of DN.

Effect of Adhesion Molecule P-selectin and Dendritic Cells on Tubulointerstitial Lesions in IgA Nephropathy

To observe the expression of P-selectin and the localization of dendritic cells (DCs) in human kidney with IgA nephropathy, and to evaluate their function in human renal tubulointerstitial lesions and renal dysfunction, 45 biopsy specimens of patients with IgA nephropathy were divided into 3 groups according to the degree of renal tubulointerstitial lesions: i. e. mild group ( n =29), moderate group ( n =10), severe group ( n =6). Ten normal renal tissues severed as control. The expression of P-selectin was analysed by immunohistochemistry. CD1a +CD80 +DCs were investigated by double immunostaining method and the images were analyzed with Axioplan 2 microscopy. The experimental results showed that (1) P-selectin was not expressed in normal controls, but presented mainly in renal tubular epithelial cells, which was greater in severe group than mild and moderate groups. The expression of P-selectin was associated with the degree of renal tubulointerstitial lesions. (2) CD1a +CD80 +DCs were hardly observed in nomal renal tissues, but in renal tissues of patients with IgA nephropathy, CD1a +CD80 +DCs were mostly found in renal tubulointerstitium. Also the distribution area, number and density of CD1a +CD80 +DCs in severe group was much more than other groups, which was associated with the degree of renal tubulointerstitial lesions and the lever of serum creatine. The distribution of CD1a +CD80 +DCs was associated with the expression of P-selectin in patient′s renal tubulointerstitium. The present study demonstrated that P-selectin and DCs might play an important role in renal tubulointerstitial lesions of IgA nephropathy, and DCs recruited into the renal tissues with IgA nephropathy might be mediated by P-selectin.