The Role And Regulation Of Glycometabolism In Mesangial Cell Proliferation In IgA Nephropathy

Background: IgA nephropathy(IgAN)is the most common primary glomerulonephritis,characterized by IgA immune complex deposition in the mesangial region along with mesangial cell proliferation,and is one of the main causes of end-stage renal disease(ESRD).In clinic,supportive treatment is the main treatment for IgAN,and there is a lack of specific therapy.Based on bioinformatics data,the exploration of molecular regulatory mechanisms of the early renal pathological manifestations-mesangial cell proliferation,can provide certain theoretical basis,new ideas and targets for drug discovery of IgAN.The kidney is an important site for glucose metabolism,recent studies have shown that glycolysis was the main energy source for renal cell selfrenewal and cell proliferation.PPAR-γ coactivator 1 alpha(PGC1α),as a transcription co-activator,plays a key role in regulating the expression of glucose metabolism genes in the kidney.Artemisinin analogs have been reported to significantly increase PGC1α level,and regulate the expression of glucose metabolizing enzymes and mitochondrial oxidative respiration function,thereby controlling cell energy and effectively treating autoimmune diseases.Its efficacy against IgAN is unknown.The purpose of this study was to explore the mesangial cell proliferation-related changes in glucose metabolism,and to clarify the regulatory mechanism of Dihydroartemisinin(DHA)on glucose metabolism and its therapeutic effect on IgAN.Methods:(1)The glomerular gene expression data sets of IgAN patients and healthy controls were obtained from GEO database,and Gene set enrichment analysis was performed using R software to investigate the difference in glucose metabolism gene expression and its correlation with clinical data were analyzed.Renal biopsy specimens diagnosed as minimal change disease,IgAN,and other immune-related nephropathy,membranous nephropathy,lupus nephritis,Henoch-Schonlein purpura nephritis were selected.Immunofluorescence staining was used to verify the differences in the expression of glucose metabolizing enzymes,and the relationship between enzyme and clinicopathology in IgAN patients was analyzed.(2)Aggregated IgA1(a IgA1)was used to stimulate human glomerular mesangial cells,the effects of PGC1α agonist/PGC1α inhibitor on mesangial cell proliferation were determined by CCK8 method.The cell cycle,apoptosis,mitochondrial membrane potential,and reactive oxygen were detected by flow cytometry.Western Blot was used to detect the expression of glucose metabolizing enzymes,and glycolysis/oxidative phosphorylation kit was used to evaluate the glycolysis energy and energy metabolic pathway.(3)The IgAN mouse model was established by oral mucosal immunization.After intragastric administration of DHA,the levels of serum creatinine,urine protein and urine creatinine were detected.The effects of DHA on renal pathology were observed by HE/PAS staining and IgA immunofluorescence staining.The expression levels of glucose metabolizing enzymes in glomeruli of each group were detected by immunohistochemical staining.(4)Mesangial cells were interfered with a IgA1 and DHA,and cell proliferation was detected by CCK8 and MTT method.Differential enrichment pathways were screened by high-throughput sequencing,and the transcription of PGC1α and glycometabolic enzymes expressions were analyzed.The metabolites of glucose metabolism were detected by energy metabolomics.Glycolytic energy and energy metabolic pathway were evaluated using glycolysis/oxidative phosphorylation kit,and the expression of glucose metabolizing enzyme was detected by q RT-PCR and western blot.Results:(1)Compared with healthy controls,IgAN glomerular glucose metabolism was significantly changed among immunological kidney disease,the expression of glycolytic enzyme HK1 was increased,and the expression of gluconeogenic enzymes PCK1 and FBP1 were decreased.Glycolytic enzyme HK1 transcription level was negatively correlated with estimated glomerular filtration rate(e GFR)and positively correlated with 24 h urinary protein level.The transcription levels of gluconeogenic enzymes G6 PC and FBP1 were positively correlated with e GFR,PCK1 and FBP1 expressions were negatively correlated with serum creatinine.The expression of PGC1α,a transcriptional regulator of glucose metabolism,was significantly correlated with the expression of the above enzymes,and was negatively correlated with urine erythrocyte count.The expression levels of HK1,PCK1,FBP1 and PGC1α were correlated with the degree of mesangial proliferation.(2)Compared with a IgA1 stimulation,the addition of PGC1αactivator ZLN005 inhibited mesangial cell proliferation within 24 h in a concentration-dependent manner.ZLN005 kept the cells in the G0/G1 cell cycle,reduced the proportion of S phase and G2/M phase cells,meanwhile promoted apoptosis.PGC1α inhibitor SR-18292 has a synergistic effect on a IgA1-induced mesangial cell proliferation.The mitochondrial membrane potential of mesangial cells decreased after a IgA1 stimulation,and tended to change after the intervention of SR-18292 or ZLN005.ZLN005 significantly down-regulated the a IgA1-induced HK1 expression and restored PGC1α expression.a IgA1 stimulation of mesangial cells promoted glycolysis,and metabolism transfer to oxidative phosphorylation after addition of ZLN005.(3)Compared with control group,serum creatinine was significantly increased in IgAN group.DHA administration significantly reduced urinary protein/creatinine,IgA deposition in the kidney,and mesangial cell proliferation in IgAN mice.The expression of HK1 was increased,and the expression of PCK1,FBP1,G6 PC and PGC1α were decreased in the glomerulus of IgAN group,while the expression of these glucose metabolism enzymes could be reversed by DHA therapy.(4)DHA intervention in mesangial cells for 24 h had a strong inhibitory effect on a IgA1-induced cell proliferation in a concentrationdependent manner.Full transcriptome sequencing enrichment analysis showed that DHA intervention significantly affected PPAR signaling pathway,increasing PGC1α and decreasing glycolytic enzyme(HK1,PKM,PFKP,PFKL)transcription.Metabolomic analysis showed that DHA significantly reduced the levels of phosphoenolpyruvate and lactic acid,which are the key components of glycolysis,and reduced ATP production in mesangial cells.Compared with a IgA1 stimulation,a IgA1+DHA stimulation caused the transformation of cellular metabolic pathway from glycolysis to oxidative phosphorylation.DHA significantly restored the increase of HK1 and the decrease of PGC1α expression induced by a IgA1.Conclusion: Glycometabolism was altered in IgAN glomerulus,and the expression of glycolytic enzymes such as HK1 was upregulated,while the expression of gluconeogenesis enzymes such as PCK1,FBP1,and glycometabolism-related transcriptional activator PGC1α were downregulated.Up-regulation of PGC1α expression in mesangial cells inhibited glycolysis and cell proliferation.DHA treatment can restore the expression of PGC1α and glycometabolism enzymes,transfer the metabolic pathway of mesangial cells from glycolysis to oxidative phosphorylation,and inhibit cell proliferation.These results provide new strategies for renal protection,as well as theoretical basis and intervention targets for DHA therapy of IgAN.