Study On The Inflammatory Osteoclastogenesis In CKD-MBD

Objective:Chronic kidney disease(CKD)is recognized as an important disease that seriously endangers health,with an estimated global prevalence of13.4% and a prevalence of 10.8% in Chinese adults.Chronic kidney disease-mineral and bone disorder(CKD-MBD)is a serious complication of CKD.With the progression of the disease,most patients develop low bone mineral density and osteoporosis.The risk of bone fracture is significantly elevated in CKD-MBD.CKD-MBD is one of the critical causes of adverse outcomes such as disability and death in patients with CKD.Previous studies have shown that CKD patients are in a state of systemic chronic inflammation.Inflammation can promote the formation of osteoclasts in patients,and the pro-inflammatory cytokine tumor necrosis factor alpha(TNFα)is significantly positively correlated with the probability of fractures in CKD patients.The molecular mechanism of how TNFα induces inflammatory osteoclasts and bone resorption remains to be explored.In this study,we integrate and analyze the clinical monocytes sequencing data of multi-center CKD patients,expecting to discover some novel signaling pathways that synergize with TNFα to activate osteoclast function in patients.We take advantage of cutting-edge multi-omics sequencing technologies such as high-throughput transcriptome and multiple epigenome sequencing at the same time combined with molecular experiments.We integrate information at different levels such as m RNA,chromatin and transcription factors,and explore the molecular mechanism of inflammatory osteoclastogenesis in CKD-MBD.This study can provide novel insights into the pathophysiological mechanism of bone damage in CKD-MBD,propose potential therapeutic targets and therapeutic strategies for clinical treatment,and ultimately achieve the purpose of improving the prognosis of CKD patients and alleviating the social medical burden.Method:1.The clinical sequencing data of monocytes from 19 CKD patients and 11 healthy people in multiple centers were identified from gene expression omnibus database(GEO).The multiple datasets were integrated to obtain the enrichment pathways and key genes of CKD patients.The relationship between different signaling pathways and osteoclast signaling pathway in CKD was analyzed.2.To establish an in vitro human osteoclast culture system,CD14+monocytes from human peripheral blood were purified.The experimental conditions were divided into: control group,transforming growth factor beta(TGFβ)group,TNFα group,and TGFβ+TNFα group.The regulatory effects of TGFβ and TNFα on osteoclastogenesis,mineral absorption function of osteoclasts,the phagocytosis of zymosan by macrophages and the expression of inflammatory cytokines were examined.3.Multi-omics sequencing research methods: RNA-seq,ATAC-seq,H3K4me3/H3K27me3/H3K27 ac CUT&RUN-seq were performed using the samples of TGFβ/TNFα induced osteoclasts.De novo motif analysis was performed to identify specific transcription factors.The functions of transcription factors in the process of TGFβ/TNFα-induced inflammatory osteoclasts and inflammation were examined by gene silencing or knockout using in vitro culture system.4.The expression of the identified transcription factors in the integrated transcriptome data of CKD patients was examined.Results:1.Compared with healthy people,the osteoclast signaling pathway is significantly active in CKD patients,and the TGFβ signaling pathway and TNFα signaling pathway are also significantly up-regulated.The activity of the TGFβ pathway is significantly positively correlated with the activity of the osteoclast pathway.TGFβ/TNFα synergistically induces the activity of inflammatory osteoclasts,which is an important cause of bone damage in CKD-MBD.2.TGFβ negatively regulates the expression of TNFα-induced interferon-stimulated genes and other inflammation-related genes by regulating chromatin accessibility and histone modification,and at the same time activates a large number of osteoclast-related genes.TGFβultimately inhibits TNFα-induced macrophage inflammatory response and significantly enhances the ability of TNFα to induce osteoclastogenesis.3.Three specific key transcription factors were identified in the process of TGFβ/TNFα-induced inflammatory osteoclastogenesis: MYB proto-oncogene like 2(MYBL2),interferon regulatory factor 1(IRF1)and interferon regulatory factor 8(IRF8).Among them,MYBL2 is a positive transcription factor,while IRF1 and IRF8 are negative transcription factors.The expression of MYBL2 was significantly increased in CKD patients,while the expressions of IRF1 and IRF8 were significantly decreased.Conclusions:Taken together,we found that TGFβ/TNFα synergistically drive the inflammatory osteoclastogenesis through epigenetic regulations such as chromatin accessibility and histone modification.This finding provides a further theoretical basis for understanding the pathogenesis of inflammatory bone damage in CKD-MBD patients,and also provides new therapeutic targets and strategies for clinical treatment of bone damage.