Liproxstatin-1 Attenuates Renal Fibrosis In Obstructive Nephropathy By Inhibiting Renal Tubular Epithelial Cells Ferroptosis

Background:Obstructive nephropathy is a common urologic disease in which the structure and function of the kidneys are damaged due to obstruction of urine excretion,and its most prominent pathological feature is renal fibrosis.The causes of obstructive nephropathy include urinary stones,deformities,tumors,and benign prostatic hyperplasia,etc.Studies have found that obstructive nephropathy is a chronic progressive and irreversible disease.After the obstruction is removed,renal function and renal structure are still difficult to recover,and the progress of fibrosis continues.At present,there is no drug can reverse or stop the progression of renal fibrosis.How to reduce the renal damage caused by obstruction and promote the recovery of renal structure and function after the obstruction is relieved is a clinical puzzle that needs to be solved urgently.Studying the mechanism of occurrence and development of renal fibrosis in obstructive nephropathy and exploring new anti-renal fibrosis targets are the key points.Ferroptosis is a newly type of regulated cell death characterized by iron-dependent and excessive lipid peroxidation.Renal tubular epithelial cells(TECs)are the initial responders to renal injury,and their injury is considered to be the initial link of renal fibrosis.It has been confirmed that ferroptosis is closely related to the occurrence and development of various diseases,and ferroptosis is an important death method of TECs during acute kidney injury.However,the link between ferroptosis and chronic renal fibrosis is still unclear.Liproxstatin-1(Lip-1)is a potent ferroptosis inhibitor and does not interfere with other classical types of cell death,but the role of Lip-1 in renal fibrosis has not been reported.Objective:This study aim to:(1)collect renal tissue samples from patients with clinical obstructive nephropathy and construct unilateral ureteral obstruction(UUO)mouse model to verify whether TECs undergo ferroptosis in obstructive renal fibrosis;(2)investigate the effect of Lip-1on ferroptosis of TECs in the UUO model and its effect on the degree of renal fibrosis and its mechanism;(3)explore the cellular and molecular mechanism of Lip-1 on renal fibrosis in vitro.Methods and Results:1.Ferroptosis of TECs in obstructive renal fibrosis1.1 Fibrosis and ferroptosis in renal tissue of obstructive nephropathyHuman obstructive nephropathy renal tissue and relatively normal renal tissue(far away from the tumor)were collected.HE staining and Masson staining were used to detect renal histopathological changes and collagen deposition.The results showed that the renal tissue of obstructive nephropathy had pathological structural damage,and collagen deposition was significantly increased.Perl’s staining and the level of glutathione peroxidase 4(GPX4)and E-cadherin(a marker of TECs)detected by immunohistochemistry in renal tissue reflected the ferroptosis of TECs in renal tissue.The results showed that compared with normal kidney tissue,iron deposition was increased in obstructive nephropathy renal tissue,the number of iron-positive cells was significantly increased,and the level of GPX4 in TECs was significantly decreased.1.2 Ferroptosis of TECs in the kidney tissue of UUO modelRenal fibrosis model was established by UUO method.Kidney tissues were collected 14 days after modeling.Perl’s staining,malondialdehyde(MDA)content and superoxide dismutase(SOD)activity,the levels of ferroptosis key molecules x CT and GPX4 detected by immunohistochemistry,the co-labeling of GPX4 and E-cadherin detected by immunofluorescence,and the co-labeling of TUNEL and E-cadherin detected by immunofluorescence,were used to reflecte the ferroptosis of TECs in the sham group and UUO group.The results showed that compared with the sham group,the number of iron-positive cells in the renal tissue of the UUO group was significantly increased,the content of MDA was significantly increased,the activity of SOD was significantly decreased,and the expressions of x CT and GPX4 were significantly decreased.The number of TUNEL~+cells in E-cadherin~+TECs increased significantly.In addition,by analyzing the high-throughput sequencing data in the GEO database,the expression changes of ferroptosis-related genes were reflected.The results showed that some important gene expression related to lipid metabolism pathway,iron metabolism pathway,glutathione pathway and iron autophagy pathway significant changed.These results indicated that ferroptosis occurred in TECs in UUO-induced renal fibrosis model.2.Lip-1 alleviates UUO-induced ferroptosis of TECs and renal fibrosis2.1 Lip-1 attenuates UUO-induced ferroptosis of TECsC57BL/6 mice were randomly divided into the following four groups:sham group,Lip-1 group,UUO group,and UUO+Lip-1 group.Kidney tissue and serum were collected 14 days after modeling.The kidney weight/body weight ratio,serum renal function and renal tissue structure in sham group and Lip-1 group were detected to reflect the effect of Lip-1 on normal kidney structure and renal function.The results showed that Lip-1 at 10 mg/kg/d had no significant effect on normal kidney tissue structure,kidney weight/body weight ratio and renal function.Iron content,MDA content,SOD activity,oxidized glutathione(GSSG)/reduced glutathione(r GSH)ratio,GPX4 protein level and cellular death situation reflected the effect of Lip-1 on ferroptosis of TECs.The results showed that Lip-1 could significantly inhibit the increase of iron content in UUO model renal tissue,inhibit the increase of MDA content and increase the activity of SOD,inhibit the increase of GSSG/r GSH ratio,significantly up-regulate the protein level of GPX4 in renal tubular epithelial cells,and inhibit TECs death.The above results showed that Lip-1 could reduce iron deposition,cell death,lipid peroxidation,and inhibited the downregulation of GPX4 expression induced by UUO,ultimately inhibiting ferroptosis in TECs.2.2 Lip-1 alleviates UUO-induced renal fibrosisTo explore the effects of Lip-1 on the degree of renal fibrosis.Kidney weight/body weight ratio,serum renal function,renal tissue morphological structure,interstitial collagen deposition,and collagen I level were detected in sham group,UUO group and UUO+Lip-1 group.The results showed that Lip-1 could reduce the UUO-induced increase of kidney weight/body weight ratio,serum creatinine and blood urea nitrogen in mice,improve the morphological and structural damage of kidney tissue,and significantly reduce renal interstitial collagen deposition and collagen I levels.By detecting the levels of the profibrotic factor transforming growth factorβ1(TGF-β1),connective tissue growth factor(CTGF)and platelet-derived growth factor(PDGF)in renal tissue,fibroblast proliferation indicators proliferating cell nuclear antigen(PCNA)and the level of fibroblast markerα-smooth muscle actin(α-SMA),we could investigate the mechanism of Lip-1 alleviating renal fibrosis.The results showed that Lip-1 could inhibit the expression of TGF-β1,CTGF and PDGF in UUO-induced renal fibrosis,and reduce the levels of PCNA andα-SMA.The above results indicate that Lip-1 can alleviate the degree of UUO-induced renal fibrosis,and its mechanism may be related to inhibiting the expression of profibrotic factors and inhibiting the proliferation and differentiation of fibroblasts.3.The cellular and molecular mechanism of Lip-1 alleviating UUO-induced renal fibrosis3.1 Lip-1 inhibits ferroptosis in HK2 cellsIn this study,we used RSL3 treatment or knockdown of GPX4 level(shGPX4)in HK-2 cells to induce ferroptosis.Detecting the changes of cell viability,iron content in cells and MDA content.The results showed that Lip-1 could significantly improve the decrease of cell viability induced by RSL3 or shGPX4,reduce the iron content in cells and inhibit the increase of MDA.This result indicated that Lip-1 could inhibit RSL3or shGPX4-induced ferroptosis in HK2 cells.3.2 Lip-1 inhibits the proliferation and differentiation of fibroblasts treated with HK2 cell culture mediumTo investigate the interactions between TECs and surrounding fibroblasts,we collected RSL3 and shGPX4-induced HK2-conditioned medium(CM)and co-cultured with fibroblasts.The proliferation of fibroblasts was detected by CCK8 assays,and the level ofα-SMA was detected by Western Blot to reflect the differentiation of fibroblasts.Our findings indicated that CM from RSL3 and shGPX4-treated HK2 cells significantly increased fibroblast cell proliferation and enhanced the expression ofα-SMA in fibroblasts,while the CM treated with Lip-1could inhibit this effect.The above results indicate that the substances released after ferroptosis of HK2 cells can promote the proliferation of fibroblasts and fibroblast-to-myofibroblast differentiation,and Lip-1 can effectively reduce the effect by inhibiting the ferroptosis of HK2 cells induced by RSL3 or shGPX4.3.3 Lip-1 reduces the secretion of profibrotic factors by inhibiting RSL3or shGPX4-induced ferroptosis in HK2 cellsTo further explore the mechanism of HK2 cells ferroptosis-mediated fibroblast proliferation and myofibroblast differentiation,we detected the contents of profibrotic factors TGF-β1,CTGF and PDGF in the CM of HK2 cells by ELISA.The results showed that the levels of TGF-β1,CTGF and PDGF in the HK2-CM were significantly increased after RSL3 or shGPX4-induced ferroptosis,and Lip-1 treatment significantly reduced the levels of profibrotic factors.The above results indicate that Lip-1 can reduce the proliferation and differentiation of fibroblasts by reducing the ferroptosis of HK2 cells and the secretion of profibrotic factors.Conclusion:1.Ferroptosis occurres in TECs in human obstructive nephropathy renal fibrosis tissue and UUO-induced renal fibrosis tissue.2.Lip-1 can alleviate UUO-induced ferroptosis and renal fibrosis in TECs,and its mechanism is related to inhibiting the level of profibrotic factors in renal tissue and inhibiting the proliferation and differentiation of fibroblasts.3.Lip-1 can alleviate RSL3-induced and GPX4 knockdown-induced ferroptosis as well as the secretion of profibrotic factors in HK2 cells.4.Lip-1 alleviates HK2 ferroptosis as well as the expression of profibrotic factors,thereby indirectly inhibiting the proliferation and activation of fibroblasts.This study is expected to provide insight into a new approach and new mechanism for the treatment of UUO-induced renal fibrosis.