The Role And Mechanism Of LAPTM5-mediated Tubular Epithelial Cell Senescence In Kidney Fibrosis

Chronic kidney disease(CKD),considered as the persistent abnormalities in the structure or function of the kidney,is related to glomerular filtration rate(GFR)fell.Epidemiological surveys showed that the global prevalence rate of CKD was 9.1%in 2017,indicating that CKD has become a serious social health problem.However,chronic kidney disease causes irreversible kidney damage and progression to end stage renal disease(ESRD),undergoing dialysis or a kidney transplant necessary for survival,which causes a great disease burden and associated economic pressure to the patients.Therefore,elucidating the pathogenesis of CKD and seeking novel therapeutic targets,are of great scientific significance for the clinical diagnosis and treatment of CKD.The causes of chronic kidney disease are various,such as acute kidney injury and diabetes,kidney fibrosis is the common pathway during the progression of CKD to ESRD.Kidney fibrosis are charactered as glomerular sclerosis and renal tubulointerstitial fibrosis.Kidney tubulointerstitial fibrosis,accompanied by renal tubule atrophy,inflammatory cells infiltration,activation and aggregation of fibroblasts and extracellular matrix accumulation,is one of the major pathological features of CKD,which involves multiple cell types.Renal tubular epithelial cells(RTECs),as the main component of the kidney,are susceptible to a lot of injuries and play an important role in the progression of CKD.Mild tubular damage can be restored with limited repair ability.However,severe,repetitive or sustained insults,might cause damaged tubules entered into the stage of maladaptive repair,followed by loss of epithelial phenotype,disturbance of energy metabolism and cell-cycle arrest,finally leading to the kidney fibrosis.Recently,accumulating evidence indicated that renal tubular epithelial cell senescence plays an important role in the progression of kidney fibrosis.Despite growth arrest,senescent renal tubular epithelial cells metabolically active,secreting numerous cytokines to regulate the microenvironment of kidney.These cytokines are referred to as the senescence-associated secretory phenotype(SASP),such as IL-1β、IL-6、TGFβ.The secretion of SASP factors induce the activation and proliferation of fibroblasts via paracrine pathway,finally promoting the progression of kidney fibrosis.Lysosomal-associated protein transmembrane 5(LAPTM5)is a member of the LAPTM family.It is composed of 262 amino acids and the molecular weight is 29.9 kDa.It contains five transmembrane domains,three multipolyproline tyrosine(PY)motifs,and one ubiquitin interaction motif(UIM).Recent studies have shown that LAPTM5 is involved in the adjustment of multiple pathways of immune cells.LAPTM5 negatively regulates immune response by directly interacting with T and B cell receptors and mediating their downregulation.However,the function of LAPTM5 in the kidney remains unclear,especially in kidney fibrosis.Therefore,in this study,we aim to explore examined the role and underlying mechanisms of LAPTM5 in tubular senescence and kidney fibrosis in the study.Methods and Results1 The expression of LAPTM5 in fibrotic kidneys1.1 The expression of LAPTM5 was increased in mouse fibrotic kidneys(1)We performed global gene expression profiling in the cortex of kidney from aristolochic acid nephropathy(AAN),unilateral ureteral obstruction model(UUO)and double renal ischemia-reperfusion injury model(IRI),and found that LAPTM5 was increased in fibrotic kidneys.(2)By qRT-PCR,Western blot and In situ hybridization(ISH)staining,the upregulation of LAPTM5 was further confirmed in the cortex of kidney from 3 different mouse models induced by AAN,UUO and bilateral IRI,especially in renal tubules.1.2 The expression of LAPTM5 was increased in renal tubular epithelial cells with different stimulusLAPTM5 was significantly induced in normal rat renal tubular epithelial(NRK52E)cells with aristolochic acid(AA)or TGF-β1 treatment.1.3 The expression of LAPTM5 was increased in the kidney from patients with chronic kidney diseaseWe detected the expression of LAPTM5 in renal biopsies from patients with CKD.Compared with the normal kidney tissues from patients who underwent tumor nephrectomy without other renal disease,CKD samples showed significant interstitial fibrosis measured by Masson’s trichrome and Sirius Red staining.Our results further showed that LAPTM5 was significantly increased in the kidney from CKD patients,with upregulation of Vimentin.Importantly,linear regression analysis showed that the level of LAPTM5 in tubule was positively correlated with Vimentin staining,suggesting that LAPTM5 may play an important role in kidney fibrosis.2 The role of LAPTM5 in kidney fibrosis2.1 LAPTM5 deficiency attenuated kidney fibrosis in AAN(1)LAPTM5 knockout(Laptm5-/-)mice were established and confirmed by t genotyping.(2)LAPTM5 knockout attenuated tubular injury and tubulointerstitial fibrosis in AAN based on the results of HE staining,Masson’s trichrome and Sirius Red staining.(3)Moreover,LAPTM5 deficiency significantly reduced the protein level of Vimentin,α-SMA,Collagen Ⅰ and Collagen Ⅳ.2.2 RTEC-specific loss of LAPTM5 attenuated kidney fibrosis under AAN(1)Conditional knockout of LAPTM5 in tubular epithelial cell(Cre+/Laptm5fl/fl)mice were established and confirmed by genotyping.(2)LAPTM5 deficiency in TECs significantly attenuated tubular injury according to the HE staining.Compared with control group,AA-treated mice exhibited obvious deposition of extracellular matrix(ECM),but tubule-specific deletion of LAPTM5 alleviated kidney tubulointerstitial fibrosis evidenced by Masson’s trichrome and Sirius Red staining.(3)We further confirmed the antifibrotic effect of LAPTM5 deficiency in AAN by the reduction of markers associated with fibrosis,such as Vimentin,α-SMA,Collagen I and Collagen IV.2.3 LAPTM5 deficiency attenuated tubular senescence in AAN(1)In the AAN mice,LAPTM5 deficiency significantly reduced the protein level of age-related molecules p53,p16 and transforming growth factor TGF-β1.(2)LAPTM5 knockout increased the expression of Klotho in the kidney from AAN mice.2.4 RTEC-specific loss of LAPTM5 attenuated tubular senescence under AAN(1)LAPTM5 deficiency in TECs significantly reduced the expression of p53,p21 and p16 in the kidney from AAN mice.(2)RTEC-specific loss of LAPTM5 increased the expression of Klotho in the kidney from AAN mice.(3)LAPTM5 deficiency in TECs significantly reduced SA-β-gal activity.(4)We assessed the levels of senescence-associated secretory phenotype(SASP)factors and found that tubule-specific knockout of LAPTM5 reduced TGF-β1,IL-1β,IL-6 and CTGF expression in the kidney from AAN.2.5 LAPTM5 knockdown alleviated the loss of epithelial phenotype in NRK-52E with AA or TGF-β1 treatment(1)Gene silence of LAPTM5 decreased the protein level of α-SMA,Vimentin and Collagen I in NRK-52E with AA.(2)In addition,TGF-β1 treatment induced the expression of α-SMA,Vimentin and Collagen I,which was reversed by LAPTM5 knockdown in NRK-52E.2.6 LAPTM5 knockdown attenuated tubular epithelial cell senescence in NRK52E with AA treatment(1)Gene silence of LAPTM5 significantly inhibited upregulation of p21 and p16 in NRK-52E treated by AA,but increased the protein level of Klotho.(2)SASP markers,such as TGF-β1,IL-1β,IL-6 and CTGF,were significantly reduced after LAPTM5 knockdown in NRK-52E with AA treatment.2.7 LAPTM5 knockdown in NRK-52E inhibited the proliferation and activation of fibroblastsWe further explore the role of LAPTM5-mediated senescence in regulation of fibroblasts activation.The supernatant collected from NRK-52E cells with gene silencing of LAPTM5 could reduce the protein level of PCNA and α-SMA in NRK49F cells,suggesting that LAPTM5 deficiency in tubular epithelial cells inhibited proliferation and collagen production of fibroblasts.2.8 LAPTM5 deficiency attenuated kidney fibrosis in UUO(1)LAPTM5 knockout attenuated tubular injury and tubulointerstitial fibrosis in UUO based on the results of HE staining,Masson’s trichrome and Sirius Red staining.(2)LAPTM5 deficiency significantly reduced the expression of Vimentin,α-SMA,Collagen Ⅰ,Collagen Ⅳ,Fibronectin and increased the expression of E-Cadherin.2.9 RTEC-specific loss of LAPTM5 attenuated kidney fibrosis under UUOTubule-specific deletion of LAPTM5 ameliorated tubular atrophy and tubulointerstitial fibrosis in UUO mice according to the results of HE staining,Masson’s trichrome and Sirius Red staining,which was further confirmed by reduction of Collagen Ⅰ,Collagen Ⅳ,Vimentin and α-SMA.3 The mechanism of LAPTM5 in kidney fibrosis3.1 LAPTM5 might promote kidney fibrosis by regulating Notchl expression(1)LAPTM5 overexpression specifically enhanced the Notchl expression in NRK-52E cells.(2)Gene silence of LAPTM5 decreased the protein level of Notchl in NRK-52E with AA treatment.(3)LAPTM5 deficiency in TECs significantly reduced the expression of Notchl in the kidney from AAN mice.Conclusions1.In this study,we first demonstrated that the expression of LAPTM5 was significantly increased in fibrotic kidneys,especially in renal tubule.Notably,the upregulation of LAPTM5 was positively correlated with Vimentin in the kidney from CKD patients.These results indicated that LAPTM5 was involved in the kidney fibrosis and related to the injury of renal tubule.2.By establishing LAPTM5 global knockout mice and tubule-specific knockout of LAPTM5 mice,we demonstrated that LAPTM5-mediated tubular epithelial cell senescence contributed to kidney fibrosis,suggesting that LAPTM5 might be a therapeutic target for kidney fibrosis.