CLEC14A Protects Against Podocyte Injury In Mice With Adriamycin Nephropathy

Focal segmental glomerulosclerosis(FSGS)is a common glomerular disease in children and adult nephrotic syndrome(NS)and it is one of the main causes of end stage renal disease(ESRD),the pathological feature is proteinuria.Podocyte injury is the main pathological manifestation of FSGS.As a component of glomerular filtration barrier,podocytes maintain the renal filtration function.Therefore,finding a target with podocyte protection is beneficial for diagnosing and treating focal segmental glomerulosclerosis.CLEC14A is a single-pass transmembrane glycoprotein that belongs to the vascular expressed C-type lectin family.It is found that CLEC14A is specifically expressed in vascular endothelial cells during embryogenesis and is also implicated in tumor angiogenesis.However,current understanding of biological functions of CLEC14A in podocytes is very limited.In this study,we demonstrated that CLEC14A was expressed in podocytes and protected against podocyte injury in mice with Adriamycin(ADR)-induced FSGS.First,we found that CLEC14A was downregulated in mice with ADR nephropathy and renal biopsies from individuals with FSGS and other forms of podocytopathies.Moreover,CLEC14A deficiency exacerbated podocyte injury and proteinuria in mice with ADR nephropathy accompanied by enhanced inflammatory cell infiltration and inflammatory responses in FSGS.In vitro,overexpression of CLEC14A in podocytes had pleiotropic protective actions,including anti-inflammatory effects and anti-apoptosis effects.Mechanistically,CLEC14A could directly binds HMGB1 in podocytes,restraining early growth response protein 1(EGR1)signaling which could promote uPAR expression.Taken together,our findings provide new insights into the pivotal role of CLEC14A in maintaining podocyte function,indicating that CLEC14A may be an innovative therapeutic target in FSGS.Objective1.Detect the expression patterns of CLEC14A on podocyte injury in ADR nephropathy.2.Explore the role of CLEC14A on podocyte injury in ADR nephropathy.3.Investigate the mechanism by which CLEC14A protects against podocyte injury in ADR nephropathy.Methods1 The expression patterns of CLEC14A in the pathogenesis of focal segmental glomerulosclerosis1.1 The expression of CLEC14A in renal biopsy samples of proteinuria patientsThe levels of CLEC14A in renal biopsies were confirmed from FSGS,MCD,DKD and MN by immunohistochemistry(IHC)staining.1.2 Detected the expression patterns of CLEC14A in the kidney from ADR miceWe characterized the expression pattern of CLEC14A in adult murine heart,liver,spleen,lung,kidney,brain,stomach,and intestine by Western blot.In the renal glomeruli from ADR mice,the expression of CLEC14A was detected by RT-qPCR.In the renal cortex from ADR mice,the expression of CLEC14A was detected by Western blot and immunohistochemistiy.Moreover,the changes of CLEC14A in podocytes were analyzed by double immunofluorescence(IF)staining for CLEC14A and podocytes marker(Synaptopodin)in samples and observed by a confocal microscope.1.3 The expression of CLEC14A was detected in different renal parenchymal cells with ADR treatmentThe expression of CLEC14A in renal parenchyma cell was detected by Western blot.Human podocytes(HPC),glomerular endothelial cells(GENC)and mesangial cells(MC)were treated by ADR.The expression of CLEC14A was detected by Western blot.2 The role of CLEC14A in ADR nephropathy2.1 FSGS model of CLEC14A knockout mice was establishedCLEC14A knockout mice was identified by tail genotyping.In the renal cortex,the expression of CLEC14A was detected by RT-qPCR and immunofluorescent analysis.2.2 CLEC14A deficiency exacerbates renal injury in mice with ADR nephropathy2.2.1 CLEC14A deficiency exacerbates podocyte injury and proteinuriaThe urinary albumin/creatinine ratio,periodontal acid-schiff stain(PAS),transmission electron microscope(TEM),Western blot and IF staining of the podocyte specific marker nephrin and podocin,Western blot analysis of uPAR as well as IHC staining of ?3 integrin were used to detect glomeruli and podocyte injury.2.2.2 CLEC14A deficiency exacerbates renal inflammation in miceThe relative mRNA level of interleukin-6(IL-6),interleukin-1?(IL-1?),monocyte chemotactic protein 1(MCP-1)and tumor necrosis factor-?(TNF-?)in renal cortex of ADR mice were detected by RT-qPCR.In addition,CD68 and Ly6B in glomerulus of ADR mice were detected by immunofluorescence staining.2.3 Overexpression of CLEC14A ameliorates renal injury in vivo2.3.1 Construct mouse model of overexpression of CLEC14ACLEC14A lentivirus(pGLV3-Clec14a,1 × 105 IU/?l)or negative control(pGLV3-null)was injected into the kidney parenchyma of WT mice eight weeks old,tail intravenous injection ADR one day later to construct the model of FSGS.In the renal cortex,the expression of CLEC14A was detected by Western blot and immunofluorescent analysis.2.3.2 CLEC14A ameliorates podocyte damage,proteinuria as well as renal cortical inflammation in a mouse model of FSGSThe urinary albumin/creatinine ratio,serum creatinine,periodontal acid-schiff stain,transmission electron microscope as well as immunofluorescence staining of the podocyte specific marker nephrin and podocin were used to detect glomeruli and podocyte injury.The relative mRNA level of IL-1?,MCP-1 and TNF-? in renal cortex of ADR mice were detected by RT-qPCR.2.4 Overexpression of CLEC 14A ameliorates podocyte damage in ADR-treated podocytesCLEC14A adenovirus transfection was used to overexpress CLEC14A in podocytes.Proinflammation response was detected in podocytes under ADR.Podocyte apoptosis was detected by flow cytometry and Casepase3 apoptosis kit.F-actin immunofluorescence staining and confocal imaging was used to observe the rearrangement of podocyte cytoskeleton.The podocyte specific marker nephrin and podocin were detect by Western blot.The expression of podocyte damage factor uPAR was detected by RT-qPCR and Western blot.Immunofluorescence staining and confocal imaging was used to observe the activity of ?3 integrin.3 Mechanisms by which CLEC14A alleviates podocytes injury in ADR nephropathy 3.1 CLEC14A directly binds to HMGB1 and thereby inhibits HMGB1 releaseStudies have shown that CD 141,which belongs to C type lectin family 14,binds to HMGB1 and inhibit its downstream inflammatory response.The binding of CLEC14A to HMGB1 was also detected.The expression of HMGB1 in podocytes under ADR was detected by Western blot.Immunofluorescence staining and confocal imaging was used to observe the expression and distribution of HMGB1 in podocytes under ADR.The expression of HMGB1 in medium under ADR was detected by Western blot.The binding of CLEC14A to HMGB1 in podocytes under ADR was detected by Co-IP.3.2 CLEC14A suppresses HMGB1 downstream signaling in podocytes3.2.1 CLEC14A suppresses inflammatory response through HMGB1rHMGB 1 was used to treat podocytes,and CLEC14A adenovirus transfection was used to overexpress CLEC14A in podocytes.The activity of NF-?B p65 was detected by Western blot.The relative mRNA level of IL-1?,IL-6 and TNF-? in podocytes were detected by RTqPCR.3.2.2 Gene sequence to detect CLEC 14A downstream signaling pathwayBy RNA sequencing for global gene expression analysis,we observed the significant changes of genes that important for regulating cellular inflammation in ADR-treated podocytes compared with their controls.3.2.3 CLEC14A suppresses the expression of EGR1 through HMGB1CLEC14A adenovirus transfection was used to overexpress CLEC14A in podocytes,the expression of EGR1 under ADR was detected by Western blot.HMGB1 siRNA was used to silence HMGB1 in podocytes,the silence efficiency of HMGB1 was validated by Western blot.The expression of EGR1 under ADR was detected by Western blot.In order to clarify the regulatory effect of CLEC 14A on EGR1 expression though HMGB1,rHMGB 1 was used to treat podocytes,and CLEC14A adenovirus transfection was used to overexpress CLEC 14A in podocytes,the expression of EGR1 was detected by Western blot.3.3 CLEC14A regulates podocyte injury through EGR1 and its downstream uPAR signaling pathway3.3.1 Inhibition of EGR1 rescues the functional defect in CLEC14A-deficient podocytesEGR1 siRNA and CLEC14A siRNA were used to silence EGR1 and CLEC14A in podocytes,the silence efficiency of EGR1 and CLEC14A werevalidated by Western blot.The relative mRNA level of IL-6,IL-1?,MCP-1 and TNF-? in podocytes under ADR were detected by RT-qPCR.Podocyte apoptosis was detected by flow cytometry.F-actin immunofluorescence staining and confocal imaging were used to observe the rearrangement of podocyte cytoskeleton.The podocyte specific marker nephrin and podocin were detect by Western blot.The expression of podocyte damage factor uPAR was detected by RT-qPCR.3.3.2 CLEC14A deficiency activates EGR1 and uPAR signaling in mice with ADR nephropathyThe relative mRNA level of EGR1 and uPAR in renal cortex from ADR mice were detected by RT-qPCR.The expression of EGR1 and uPAR in the glomeruli from ADR mice were detected by immunohistochemistry.To explore the regulatory effect of CLEC14A on uPAR downstream molecule ?3 integrin in ADR nephropathy,the ?3 integrin activity was detected by immunohistochemistry.Podocyte death was detected by immunofluorescence staining podocyte marker WT-1.3.3.3 Overexpression of CLEC14A down-regulates the expression of EGR1 in the glomeruli of mice with ADR nephropathyThe expression of EGR1 in the glomeruli from ADR mice was detected by immunohistochemistry.Results1 The expression patterns of CLEC14A in the pathogenesis of focal segmental glomerulosclerosis1.1 The expression of CLEC14A in renal biopsy samples of proteinuria patientsThe levels of CLEC14A in glomeruli were significantly reduced in renal biopsies from patients with FSGS by IHC staining.Downregulation of CLEC14A was further observed in renal biopsies from patients with other different forms of glomerular diseases such as DKD,MN and MCD.1.2 Detected the expression patterns of CLEC14A in the kidney from ADR miceIt was found that CLEC14A was present in all these tissues with a relative high expression in the lung and kidney.The levels of CLEC14A were markedly reduced in the kidney from ADR mice,by Western blot and IHC analyses.The mRNA levels of CLEC14A in the renal glomeruli were reduced detected by RT-qPCR.Immunofluorescent results showed a significant decrease of podocyte CLEC14A expression in the kidney from ADR mice.1.3 The expression of CLEC14A was detected in different renal parenchymal cells with ADR treatmentIn the kidney,CLEC14A was expressed in various types of renal parenchymal cells including human tubule epithelial cells cells,glomerular mesangial cells,glomerular endothelial cells,and podocytes.Western blot analysis showed that the levels of CLEC14A were reduced in HPC.However,other glomerular cells including GECs and RMCs exhibited no obvious changes in CLEC14A expression in response to ADR,indicating that the downregulation of CLEC 14A may selectively contribute to podocyte injury under ADR conditions.2 The role of CLEC14A in ADR nephropathy2.1 FSGS model of CLEC14A knockout mice was establishedTo elucidate the role of CLEC14A in podocytes in vivo,we generated CLEC14A knockout mice,which were identified by tail genotyping.In the renal cortex from Clec14a/-mice,the levels of CLEC14A were significantly reduced detected by RT-qPCR and immunofluorescent analysis.All mice were viable and fertile.2.2 CLEC14A deficiency exacerbates renal injury in mice with ADR nephropathy2.2.1 CLEC14A deficiency exacerbates podocyte injury and proteinuriaClec14a-/-mice were viable,phenotypically normal and had no significant defects in renal morphology and function.By using CLEC14A knockout mice,we generated ADRinduced FSGS model.FSGS Clec14a-/-mice exhibited a significant increase in urinary albumin excretion as compared with FSGS WT mice.Morphological examinations showed the glomerular and podocyte injuries as evidenced by glomerularbasement membrane(GBM)thickening,podocyte foot process broadening and effacement from FSGS WT mice,all of which were aggravated in FSGS Clec14-/-mice.The loss of key podocyte differentiation markers including Nephrin and Podocin was partially aggravated in FSGS Clec14-/-mice.The increase of podocyte injury factor uPAR was partially aggravated in FSGS Clec14a-/-mice.2.2.2 CLEC14A deficiency exacerbates renal inflammationCLEC14A deficiency enhanced inflammatory responses by increasing the levels of proinflammatory mediators,macrophage and neutrophil infiltration in the renal cortex and glomeruli from mice with ADR nephropathy.2.3 CLEC14A protects against podocyte injury in vivo2.3.1 Construct mouse model of overexpression of CLEC14ACLEC14A overexpression mice were constructed by CLEC14A-lentivirus transfection.In the renal cortex from Clec14a-/-mice,the expression of CLEC14A were significantly increased detected by Western blot and immunofluorescent analysis.2.3.2 Overexpression of CLEC14Aameliorates renal injury in vivoOverexpression of CLEC14A significantly ameliorated renal injury as evidenced by reduced albuminuria and Scr,decreased mesangial expansion,and ameliorated podocyte injury in Clec14a-/-mice with ADR nephropathy.2.4 Overexpression of CLEC14A ameliorates podocyte damage in ADR-treated podocytesOverexpression of CLEC14A significantly decreased the production of proinflammatory mediators,reduced Caspase3 activity and attenuated apoptosis in podocytes with ADR treatment.In addition,overexpression of CLEC14A also reduced the expression level of uPAR and the activity of ?3 integrin,and recovered the expression of nephrin and podocin,ameliorated actin cytoskeleton derangemen in podocytes with ADR treatment.3 Mechanisms by which CLEC14A alleviates podocytes injury in ADR nephropathy3.1 CLEC14A directly binds to HMGB1 and thereby inhibits HMGB1 releaseAlthough we found that ADR treatment didn't change the total level of HMGB1 in podocytes,IF results revealed that ADR treatment promoted HMGB1 translocation from nucleus to cytoplasm,which is essential for the release of HMGB1 into the extracellular space.We further found that ADR treatment increased the level of HMGB1 in supernatant of cultured podocytes and CLEC14A overexpression could inhibit the ADR-induced HMGB1 release.Moreover,Co-IP analysis revealed that the binding level of CLEC14A and HMGB1 was decreased after ADR treatment,which may be due to the reduction of CLEC14A.However,CLEC14A overexpression rescued the interaction with HMBG1 in the context of ADR as evidenced by the enhanced binding level of CLEC14A and HMGB1 in podocytes.Taken together,these results indicate that ADR treatment reduces the expression of CLEC14A and further decreases the binding level of CLEC14A and HMGB 1,thereby resulting in HMGB1 release.The extracellular HMGB1 acts as a danger signal to promote the podocyte injury.3.2 CLEC14A suppresses HMGB1 downstream signaling in podocytes3.2.1 CLEC14A suppresses inflammatory response through HMGB1CLEC 14A overexpression could inhibit the HMGB 1-induced NF-?B activation and inflammatory responses as documented by increased levels of NF-?B subunit phospho-p65(p-p65)and upregulated expression of proinflammatory mediators.3.2.2 Gene sequence to detect CLEC14A downstream signaling pathwayBy Agilent Whole Human Genome Oligo Microarray for global gene expression analysis,we observed the significant changes of genes important in regulating cellular inflammation in ADR-treated podocytes compared with their controls.Among them,CLEC 14A overexpression significantly reduced the levels of genes that govern inflammatory responses such as EGR1,p53 in podocytes with ADR treatment.3.2.3 CLEC14A suppresses the expression of EGR1 through HMGB1EGR1 is one target of HMGB 1 and plays a vital role in podocyte injury.CLE14A overexpression could inhibit rHMGB1-or ADR-induced EGR1 expression.Consistently,HMGB1 deficiency reduced EGR1 expression in podocytes with ADR treatment.Collectively,these results indicated that HMGB1/EGR1 may play a key role in mediating the function of CLEC 14A.3.3 CLEC14A regulates podocyte injury through EGR1 and its downstream uPAR signaling pathway3.3.1 Inhibition of EGR1 rescues the functional defect in CLEC14A-deficient podocytesSilencing of EGR1 reduced inflammatory responses and podocyte apoptosis.Moreover,gene silencing of EGR1 recovered the expression levels of nephrin and podocin and block actin cytoskeleton derangement in Clec14a-deficient podocytes.Finally,we found that EGR1 inhibition decreased the uPAR expression in Clec14a-deficient podocytes.Mechanistically,it was reported that EGR1 could activate ?-catenin expression,which could upregulate uPAR expression.These results indicating that EGR1 is one of critical components of a signal transduction pathway that links podocyte injury to CLEC14 deficiency in FSGS signaling.3.3.2 CLEC14A deficiency activates EGR1 and uPAR signaling in mice with ADR nephropathyEGR1 was markedly induced in the renal cortex from mice with ADR nephropathy,which was further enhanced by CLEC14A deficiency mice.Moreover,CLEC14A deficiency upregulated uPAR expression in mice with ADR nephropathy.It is known that uPAR is one of the important pathways capable of activating ?3 integrin and leading to podocyte loss.The activation of ?3 integrin could be detected by AP5 staining intensity.We found that Clec14a-/-dramatically enhanced AP5 staining in ADR nephropathy,suggesting that CLEC14A may be a regulator of uPAR/?3 integrin signaling.Finally,we found that CLEC14A deficiency exacerbated podocyte loss in mice with ADR nephropathy.3.3.3 Overexpression of CLEC14A down-regulates the expression of EGR1 in the glomeruli of mice with ADR nephropathyThe level of EGR1 was significantly decreased in glomeruli after transfection with Clec14a lentivirus in Clec14a-/-mice with ADR nephropathy.Conclusion and innovation1.Our study for the first time demonstrate that CLEC14A is significantly decreased in the renal biopsies from patients with FSGS and ADR-induced FSGS mice.2.CLEC14A difficiency significantly exacerbated the proteinuria and glomerularsclerosis in ADR nephropathy and overexpression of CLEC14A ameliorated podocyte injury.3.CLEC14A protects against podocyte injury by negatively regulating EGR1 signaling.This study helps to further elucidate the pathophysiological mechanism of kidney injury in FSGS and provides animal experimental basis and theoretical basis for the design and development of CLEC14A related drugs.