The Mechanism Of IGFBP5 In The Progression Of Diabetic Kidney Disease

Background: Renal inflammation is a critical pathophysiological characteristic of diabetic kidney disease(DKD),and more and more studies show that inflammation plays an important role in the progression of DKD.However,the mechanism of the inflammatory response is complicated,and there are few effective treatments for renal inflammation that can be used clinically.Glomerular endothelial cells(GECs)are metabolic cells that are rich in a variety of organelles,require a large amount of energy supply and are sensitive to injury factors.The activation of ECs is a marker of diabetes mellitus,and GECs dysfunction plays a key role in the occurrence and progression of DKD.Endothelial energy metabolism depends on its own glycolysis.The critical modulator 6-phosphofructose-2-kinase/fructose-2,6-bisphosphatase 3(PFKFB3)regulates the rate of glycolysis.Previous studies have reported the important role of metabolic reprogramming in DKD,which is mainly manifested as enhanced aerobic glycolysis,increased glucose toxic metabolites,and mitochondrial damage,thus aggravating kidney damage.However,it is not clear whether GECs are associated with metabolic reprogramming during DKD development.Insulinlike growth factor-binding protein 5(IGFBP5)is an important secretory protein that is related to inflammation and fibrosis in several tissues.Single cell sequencing database analysis indicated that IGFBP5 was highly expressed in GECs.Studies have shown that the IGFBP5 level is significantly upregulated in DKD.However,the function of IGFBP5 and its mechanism in DKD remain unclear.Objective: To elucidate the role of IGFBP5 in DKD and its possible mechanism.Methods: The expression of IGFBP5 in kidney was analyzed by public database(“The Human Protein Atlas” and “Kidney Interactive Transcriptomics”).q PCR and Western Blot were used to evaluate the expression of IGFBP5 m RNA and protein in kidney tissues of db/db mice.RNA-seq was used to analyze the changes of endothelial cell signaling pathway induced by IGFBP5 overexpression.Endothelial cells were transfected with lentivirus/si RNA to achieve IGFBP5overexpression/knockdown,and the expression of inflammatory cytokines was detected by q PCR to observe the effects of IGFBP5 on endothelial inflammation.The diabetic model was established by streptozotocin(STZ)in IGFBP5 gene knockout mice.And the expression of inflammatory cytokines was detected by q PCR,and the renal pathology analysis was conducted to verify the possible promoting effect of IGFBP5 on DKD renal inflammation.Combined RNA-seq results and online analysis tools(jaspar.genereg.net and bioinfo.life.hust.edu.cn)showed that transcription factor EGR1 may bind to PFKFB3.The possibility of EGR1 binding to PFKFB3 was verified by double luciferase reporter gene and chromatin immunoprecipitation(Ch IP).Glycolysis metabolites were detected through IGFBP5 overexpressing or knock-down;Seahorse was used to elucidate the extracellular acidification rate.Using PFKFB3 mutant mice to establish diabetes model,analyze the effects of PFKFB3 on DKD renal inflammation.The effect of IGFBP5 on DKD kidney was observed by IGFBP5 lentivirus in diabetic mice.db/db mice were injected with 3-PO,a specific PFKFB3 inhibitor,and the effect on renal inflammation was detected.Results: Public database results showed that the expression of IGFBP5 was the highest in GECs in kidney,and increased in GECs of DKD patients.q PCR and Western Blot showed that IGFBP5 expression was increased in the kidney of db/db mice.RNA-seq suggested that IGFBP5 up-regulated the expression of endothelial inflammatory signaling pathways.Overexpression of IGFBP5 promoted endothelial inflammation,while inhibition of IGFBP5 weakened endothelial inflammation.Knockdown of IGFBP5 can reduce renal inflammation and proteinuria in DKD mice.RNA-seq suggested that IGFBP5 overexpression led to up-regulation of endothelial metabolism-related signaling pathways.q PCR and Western Blot results showed that IGFBP5 promoted the expression of EGR1 and PFKFB3.Ch IP and double luciferase reporter gene confirmed that PFKFB3 was the direct transcription target of EGR1.Overexpression of IGFBP5 enhanced glycolysis of endothelial cells and promoted the synthesis of glycolysis metabolites,such as lactate,pyruvate and F-2,6-BP.Knockdown of IGFBP5/PFKFB3 inhibited endothelial glycolysis.In addition,inhibition of PFKFB3 attenuated endothelial inflammatory response.PFKFB3 mutation could inhibit renal inflammation and reduce renal pathological changes in DKD mice.After IGFBP5 lentivirus intervention in wild diabetic mice,the renal pathological changes were aggravated.However,there was no renal pathology aggravation in PFKFB3 mutant diabetic mice after intervention.db/db mice intraperitoneally injected with 3-PO had similar effects with PFKFB3 mutation mice,reduced renal inflammatory response and proteinuria.Conclusion: High glucose stimulates the expression of IGFBP5 in endothelial cells,and IGFBP5 enhances PFKFB3-mediated glycolysis to promote endothelial inflammation and accelerate the progression of DKD by promoting the up-regulation of endothelial transcription factor EGR1.Our study elucidates the role and mechanism of IGFBP5 in DKD,suggesting that IGFBP5 may be a potential target for inhibiting DKD inflammatory response and delaying DKD progression.