Lysyl Oxidase Promotes Renal Fibrosis Via Accelerating Collagen Crosslink Driving By AT1R/β-arrestin Pathway

Renal fibrosis is the hallmark and the common outcome of almost all progressive chronic kidney diseases(CKD)eventually progress to end-stage kidney failure(ESRD).At present,there is no effective treatment for renal fibrosis,and its emergcy for us to explore its mechanism to make effective treatment for clinical treatment.However,the pathogenesis of renal interstitial fibrosis is very complex,involving a variety of cells and signaling pathways,and lacks specificity.Therefore,we need to further study the pathogenesis of renal fibrosis,to provide new ideas and new therapeutic intervention targets for basic and clinical research of renal fibrosis.The pathogenesis of renal fibrosis is complex and the core event is an excessive,pathological accumulation of extracellular matrix(ECM).Accumulating evidences have shown that the formation of renal fibrosis can be induced not only pathological deposition of extracellular matrix,but also the over cross-linking of the ECM where the soluble monomers of collagen and elastin transformed into insoluble fibrous tissues and forming a dense network,leading to hard for degradation and renal interstitial fibrosis finally.To identify the factor responsible for the pathogenesis of renal fibrosis,unilateral ureteral obstruction-(UUO-),ischemia injury reperfusion-(IRI-)and folic acid-(FA-)induced mouse kidneys tissues performed by high-throughput RNA sequencing(RNA-seq).We found that lysyl oxidase(LOX)was significantly differentially expressed genes,which related to fibrosis overlapped UUO and FA models,suggesting that the expression of LOX was closely related to renal fibrosis.LOX is an extracellular enzyme responsible for initiating covalent cross-linking formation in collagen fibrils and elastin,which is essential for stabilization of collagen fibrils and elastin and prevent their degradation.Multiple studies have demonstrated that collagen cross-linking can play an important role in the occurrence and development of organ fibrosis by increasing the stiffness and non-compliance of ECM but independent of inflammatory pathways.Therefore,collagen crosslinking and crosslinking enzymes are new targets for anti-fibrosis therapy.Evidence have shown that LOX is associated with cancer and some fibrotic conditions.However,the role of LOX and its regulation in renal fibrosis is unclear.Therefore,we further elucidated its potential pathogenic role and underlying mechanism in renal fibrosis.As shown in our study,LOX was significantly upregulated in fibrotic kidney and mainly expressed in tubule epithelial cells.The cross-linking of ECM was also increased accompany with the upregulation of LOX which associated with the degree of renal fibrosis.Currently,the role of LOX has been studied in different tissues and cells,and the mechanisms for the regulation of LOX expression or activtity was complex.One study showed that ventricular remodeling induced by left atrial fibrillation was associated with upregulation the LOX,which induced by Angiotensin II(Ang II)treatment.Another study also showed that Ang II treatment lead to arterial hypertension,remodeling and sclerosis through increasing the activity of LOX.Growing evidence indicates that Ang II,a potent biologically active product of renin–angiotensin system(RAS),is a key regulator of renal fibrosis.Ang II type 1receptor(AT1R)mediates most functions of Ang II in RAS system and there are two signaling pathways downstream of Ang II-AT1R: G protein pathway and β-arrestin pathway.Studies have shown that β-arrestin signaling plays an important role in renal fibrosis development,but the specific downstream is still unclear.To explore the overexpression of LOX in renal fibrosis whether regulated by Ang II-AT1R-β-arrestin signaling,IRI mice and NRK-52 E cells treated with AT1R/β-arrestin specific activator SII([1-sar,4,8-ile]-angiotensin II)were used in vivo and in vitro.We found thatβ-arrestin-ERK pathway was active in IRI mice and treatment with SII in NRK-52 E cells increased the expression of β-arrestin1 and actived the β-arrestin-ERK pathway.Morover,the expression of LOX increased in NRK-52 E cells under SII treatment.To further explored the mechanism of LOX regulation,we next performed RNA-seq analysis of tissues collected from the IRI mouse kidneys.To identify key transcriptional programs involved in disease etiology,we performed target gene enrichment analysis using the transcription factor-target gene interaction database TRRUST(transcriptional regulatory relationships unraveled by sentence-based text mining).We identified that STAT3 mediated gene expression programs was significantly enriched which suggested that it might act as the downstream of theβ-arrestin-ERK pathway and involve in regulating the expression of LOX and the development of renal fibrosis.As shown in our study,the phosphorylation levels of STAT3 increased in IRI mice kidney tissues compared with sham kidney tissues.We also found that treatment with SII in NRK-52 E cells has a result in increasing ERK entered the nucleus and being phosphorylation,which promoted the STAT3 phosphorylation at the site of Tyr705 instead of Ser727.Moreover,the ERK/STAT3 signaling pathway was less activation when targeting β-arrestin with si RNA or ERK with its inhibitor under SII treatment.These findings suggested that the expression of LOX might depend onβ-arrestin/ERK/STAT3 signaling pathway.Moreover,we uncovered that STAT3 regulate LOX expression at the transcriptional level and this further supported by the direct binding of STAT3 on LOX promoter region demonstrated by Chromatin immunoprecipitation(Ch IP).Targeting of LOX with inhibitor BAPN effectively blocked LOX dependent ECM cross-linking,therefore suppressing the progressive of renal fibrosis in IRI mice.As above results shown that the upregulation of LOX invoved in renal fibrosis development and the expression of which was associated with the dgree of renal fibrosis in clinic renal biopsy tissues.As LOX is a cell secretory protein,thus we further investigate whether serum LOX can be act as a potential diagnostic biomarker for renal fibrosis.We found that the serum LOX level increased in patients with fibrosis kidneys compared to MCD kidneys and healthy kidneys.Moreover,the serum LOX level correlated with the severity of the renal fibrosis suggested that serum LOX is a potential diagnostic biomarker for kidney fibrosis.Collectively,these observations suggested that Ang II could upregulate LOX expression through AT1R-β-arrestin1-ERK-STAT3 pathway,increased the activatity of LOX,induced ECM over cross-linking and promoted renal interstitial fibrosis finally.IRI mouse model combining in vitro experiment used to clarify the regulating mechanism of Ang II-AT1R-β-arrestin1 pathway on regulation of LOX,reveal the important role of ECM over cross-linking in renal fibrosis.Thus,strategies targeting LOX could be a new avenue in developing therapeutics against renal fibrosis.