Expression Of SEH In Renal Tubular Epithelial Cells Mediates Macrophages Infiltration And Polarization In IgA Nephropathy

Background and ObjectiveChronic kidney diseases(CKD)are known to be associated with inflammatory reactions.In its early phase,inflammation can be protective.However,prolonged inflammation precipitates renal injury and eventually leads to renal fibrosis and end-stage renal disease(ESRD).At the cellular level,the recruitment and activation of inflammatory cells is of central importance.Among these recruiting inflammatory cells,macrophages play a prominent role in regulating kidney inflammation and fibrosis.Monocytes isolated from renal artery were found to differentiate into classically activated M1 macrophages or alternative M2 macrophages in distinctive pathways.In response to ongoing injury,M1 macrophages promote inflammation and development of fibrosis.Depending on the microenvironment,M2 macrophages can be recruited from the circulation or transformed from M1 macrophages.M2 macrophages secrete factors that are able to repair kidney injury by promoting cell proliferation,reducing apoptosis,and stimulating angiogenesis.Numerous recent clinical studies have demonstrated that in CKD patients,the severity of the outcome positively correlates with the degree of macrophage infiltration.Epoxyeicosatrienoic acids(EETs),a group of cytochrome P-450(CYP450)metabolites of arachidonic acid(AA),play an important role in many physiological phenomena,including vasodilation,angiogenesis,anti-inflammation,anti-oxidation,anti-platelet aggregation,and promotion of sodium excretion.Soluble epoxide hydrolase(sEH)converts EETs to less potent diols,which are termed dihydroxyeicosatrienoic acids(DHETs).The activity of sEH is thought to be the main determinant of EET bioavailability.Either the genetic deletion or pharmacological inhibition of sEH results in the elevation of EET levels and enhancement of protective biological effects of EETs.It has been shown that inhibition of sEH decreases blood pressure,suppresses inflammatory response,and prevents progression of renal interstitial fibrosis in diabetic nephropathy,hypertensive nephropathy,and unilateral ureteral obstruction(UUO)models.However,the role of sEH in the pathogenesis of renal interstitial inflammatory is not well defined,especially the mechanisms of the cross-talk between tubular epithelial cells,macrophage activation,and polarization in conditions of sEH up-regulation.IgA nephropathy(IgAN),one of the most common causes of CKD in China,is accompanied by inflammatory infiltration of various degrees into tubulointerstitial area.In this study,we determined the level of renal sEH expression in IgAN patients and compared it with patients’ clinical and pathological characteristics.Furthermore,we used in vitro experiments to examine the role of tubular sEH on macrophage polarization and activation pathways.Our investigation highlights a potential therapeutic value of sEH inhibition in sustain inflammation and fibrosis of kidney.Therefore,our investigation was divided two parts.Part I:The correlation analysis of renal sEH expression and clinical and pathological characteristics in IgA nephropathy.To investigate the relationship of renal sEH expression and clinical and pathological characteristics in IgA nephropathy,we collected renal biopsy specimens and done immunohistochemical staining for sEH.The results provided clinic evidence to further explore the effect and mechanism of tubular epithelial sEH regulate macrophage polarization.Part II:The effect and mechanism of tubular epithelial sEH regulate macrophage polarization.we used urinary protein and different regulated methods of sEH to examine the role of sEH in inflammatory factor production for macrophages recruitment.Furthermore,we also exposed macrophage cells to different epithelial cells culture media conditioned to demonstrate the effect and mechanism of tubular sEH on macrophage polarization and activation pathways.Part I:The correlation analysis of renal sEH expression and clinical and pathological characteristics in IgA nephropathy.Materials and MethodsWe collected renal biopsy specimens,urine samples,and clinical and pathological data from 71 IgAN inpatients.Immunohistochemical staining for sEH was performed on frozen slides of biopsy sections from IgAN patients.According to the percentage and degree of positive sEH staining in the tubules,all IgAN patients were divided four group,from + to ++++.1.Collection of clinical parameters:age,sex,mean arterial pressure(MAP),24-h total urine protein,and estimated glomerular filtration rate(eGFR).2.Collection of histological parameters:mesangial hypercellularity,endocapillary hypercellularity,segmental glomerulosclerosis,tubular atrophy or interstitial fibrosis and tubular interstitial inflammation index.3.The expression of sEH protein level in IgAN patients were measured by western blot.4.The expression of MCP-1、IL-6、CSF-1、TNF-α mRNA level in IgAN patients were measured by real-time PCR.5.Double immunofluorescence staining of sEH and CD68 was performed on frozen slides with sEH ++++ score samples from IgAN patients.6.sEH enzymatic activity was measured by urine 14,15-EET/14,15-DHET ELISA kit.Results1.According to the results of immunohistochemical staining and western blot for sEH and urine 14,15-EET/14,15-DHET ratio,the expression and enzymatic activity of sEH in IgAN patients was significantly higher compared to that in control group(P<0.05).2.Among the different four seh level group,the higher expression level of sEH was,the worse were the clinical parameters,including MAP,urine protein,and eGFR in IgAN patients(P<0.05).3.The level of renal tubular sEH expression was found to positively correlate with MAP(r = 0.408,P<0.05),the extent of proteinuria(r = 0.452,P<0.05),and tubular interstitial inflammation(r = 0.247,P<0.05).4.Using double immunofluorescence staining against sEH and the macrophage marker CD6 8,we discovered that sEH-positive tubules were surrounded by macrophages(CD68-positive cells)in IgAN tissues.5.Real-time PCR analysis revealed that expression levels of MCP-1,IL-6,CSF-1,and TNF-a mRNAs were significantly higher in both low(+/++)and high(+++/++++)sEH expression IgAN samples compared with their levels in control samples(P<0.05).Part Ⅱ:The effect and mechanism of tubular epithelial sEH regulate macrophage polarization.Materials and MethodsWe used immortalized human proximal tubular HK-2 cells and RAW264.7 mouse leukemic monocytes/macrophages to examine the role of sEH in macrophages activation and polarization.We treated HK-2 cells with urine protein,sEHI and sEH adenoviral vectors.we utilized RAW264.7 macrophage cells that were cultured with conditioned medium from different HK-2 cell culture media conditioned by the incubation with various substances affecting sEH amount.Control HK-2 cell culture media treatment with interferon y or interleukin-4 to induce M1 or M2 polarization for positive control.Results1.In HK-2 cells exposed to urinary proteins,the levels of MCP-1,IL-6,CSF-1,and TNF-α mRNAs were higher than control group and lower when sEH activity was inhibited by sEHI(P<0.05).sEHI did not affect the expression of sEH after exposure to urinary proteins,but dramatically inhibited sEH enzymatic activity.2.Supplementation of EET did not affect the expression of sEH after the incubation with urinary proteins.However,that treatment markedly reduced up-regulation of MCP-1,IL-6,CSF-1,and TNF-a mRNAs induced by the exposure to urinary proteins(P<0.05).3.Compared with control group,protein level of sEH and mRNA levels of MCP-1,IL-6,CSF-1,and TNF-a were significantly increased in sEH adenovirus infected HK-2 cell(P<0.05).4.Compared with control HK-2 cell conditioned medium,RAW264.7 cells treated with urinary protein and sEH adenovirus infected HK-2 cell conditioned medium and supplemented with IFN-y had significantly higher mRNA levels of iNOS and IL-6(P<0.05).Compared with urinary protein-treated HK-2 cell conditioned medium,RAW264.7 cells treated with urinary protein-and sEHI-treated,urinary protein-treated HK-2 cell conditioned medium supplemented with EET added and control HK-2 cell conditioned medium supplemented with IL-4 all had significantly higher mRNA levels of Arg-1 and IL-10(P<0.05).5.Compared with control HK-2 cell conditioned medium,RAW264.7 cells treated with urinary protein and sEH adenovirus infected HK-2 cell conditioned medium and supplemented with IFN-y had significantly higher protein levels of p-p65.Compared with urinary protein-treated HK-2 cell conditioned medium,RAW264.7 cells treated with urinary protein-and sEHI-treated,urinary protein-treated HK-2 cell conditioned medium supplemented with EET added and control HK-2 cell conditioned medium supplemented with IL-4 all had significantly higher protein levels of p-Akt.Conclusions1.The expression and enzymatic activity of sEH was significantly higher in IgAN patients.2.In IgAN,the expression of sEH in renal tubules were related to MAP,proteinuria and tubular interstitial inflammation.3.In IgAN,the expression of sEH in renal tubules promote the production of cytokines,which were associated with macrophage activation and polarization.4.We found that up-regulated sEH promoted M1 polarization associated cytokine production in HK-2 cells.sEHI administration and EET supplementation reversed the effect of sEH on cytokine production in HK-2 cells.5.sEH expression in tubular epithelial cells could promote M1 polarization and inhibit M2 polarization with production of cytokine and EET in a paracrine fashion.However,pharmacological inhibition of sEH and supplementation with EETs inhibited M1 polarization and stimulated M2 macrophage polarization.6.Activation of sEH promoted Ml polarization through NF-κB pathway and inhibit M2 macrophage polarization through PI3K pathway.through PI3K pathway.These data suggest that inhibition of sEH could be a new strategy to prevent the progression of inflammation and to attenuate renal tubulointerstitial fibrosis.