Fibrinogen Correlates With Podocyte Injury And The Progression Of Chronic Kidney Disease

Background and aimsFibrinogen has been reported to participate in inflammation in various diseases such as vascular disorders,rheumatoid arthritis via Toll-like receptor 4(TLR4).Previous studies have reported that TLR4 are expressed in immune cells,inflammatory cells and podocytes.Fibrinogen may activate macrophages to secrete interleukin 6,interleukin 8,tumor necrosis factor and monocyte chemotactic protein 1.Podocyte injury is involved in the formation of proteinuria,which leads to glomerular sclerosis.Some scholars report that in vitro fibrinogen stimulates podocytes to secrete chemokines and cytokines via TLR4.Our recent study has demonstrated that urinary fibrinogen levels in focal segmental glomerular sclerosis(FSGS)were significantly elevated.However,whether fibrinogen might induce podocyte damage through TLR4 singnaling pathway and be related to podocytopathy in FSGS remains unknown.Based on previous data,we aim to study the relationship between fibrinogen and podocytes' damage.Materials and methodsFirst,we observed fibrinogen-induced alterations in the actin cytoskeleton and apoptosis in cultured human podocytes with or without TLR4 siRNA transfection.In vitro we added fibrinogen into cultured human podocytes(20 ?g/ml,200 ?g/ml,800?g/ml,respectively),podocytes were incubated for 12 h or 24 h respectively;then we detected apoptotic rate by flow cytometry and evaluated F-actin by immunofluorescence test.In vitro the siRNA transfection experiment,first TLR4 siRNA or control siRNA was transfected into podocytes for 12 h,and subsequently cells incubated with fibrinogen 800 ?g/ml for 24 h.Then we detected TLR4 mRNA level by the qRT-PCR to observe blocking effect,meanwhile we assessed apoptotic rate and F-actin.And we measured the protein levels of TLR4,total NF ?B p65,phospho-NF ?B p65,total p38 MAPK and phospho-p38 MAPK in podocytes by Western Blot.Next,we measured the urinary fibrinogen levels by using ELISA and urinary ACR(the ratio of albumin to creatinine)in adriamycin-treated model and saline-treated mice.The correlation between the urinary fibrinogen and ACR was analyzed.We carried out pathologic examination and double immunofluorescence(IF)staining for TLR4,fibrinogen and podocin.Statistical analysisAll of the cell-based experiments were repeated over three times.The values were expressed as the means±standard deviations(SD)for continuous variables or medians and interquartile ranges(IQRs)(25th and 75th percentiles).GraphPad Prism 5 software(La Jolla,CA)and the 2-tailed t test were used to analyse the differences between any two groups.Correlations between variables were analysed using Spearman's method.P<0.05 was considered significant.ResultsFirst,fibrinogen dose-dependently induced actin cytoskeleton damage and apoptosis significantly in cultured podocytes with a fibrinogen-induced increase in TLR4 expression in comparison with untreated podocytes,and TLR4 siRNA transfection prevented these effects.TLR4 knockdown in podocytes inhibited the expression of TLR4 and activation of p38 MAPK and NF?B p65 induced by fibrinogen compared to the control siRNA.Next,the adriamycin-treated mice developed substantial proteinuria compared with the saline-treated mice.Elevated urinary fibrinogen levels were positively correlated with albuminuria in adriamycin-treated mice(r=0.65,P=0.0005).Typical focal segmental glomerular sclerosis and tubular atrophy with intratubular casts,and obvious foot process effacement were observed in adriamycin-treated mice at the 6th week.Fibrinogen and TLR4 were colocalized and exhibited increased expression in the podocytes of adriamycin-treated mice in comparison with saline-treated mice(P=0.0001 and P<0.0001,respectively);however,podocin expression was markedly decreased compared with that in the saline-treated controls.ConclusionsFibrinogen induced actin cytoskeleton damage and apoptosis in podocytes via the TLR4-p38 MAPK-NF?B p65 signaling pathway,and might participate in the podocytopathy of FSGS.Background and aimsChronic kidney disease(CKD)has been a worldwide public health problem with severe burden and a high risk contributor to cardiovascular disease(CVD)and end-stage renal disease(ESRD).Urinary fibrinogen levels were significantly elevated in FSGS patients in our previous study.Fibrinogen has been reported to be involved in renal tubulointerstitial fibrosis in mice model and podocyte injury.However,whether fibrinogen might be related to disease activity in patients with focal segmental glomerulosclerosis(FSGS)remains unknown.The relationship between urinary fibrinogen and long-term renal survival has not been clearly clarified in patients with CKD.Based on these data,we aim to study the relationship between urinary fibrinogen and FSGS activity and explore the effect of urinary fibrinogen on long-term renal survival for CKD patients.Subjects and methodsFirst,in order to study the relationship between urinary fibrinogen levels and FSGS activity,50 FSGS patients and 50 healthy controls were enrolled.The levels of urinary fibrinogen were measured by ELISA method.And we analysed the correlations between the urinary fibrinogen and 24 h proteinuria,urinary fibrinogen and foot process width(FPW)respectively.Meanwhile the changes of urinary fibrinogen levels after prednisone treatment were analysed in patients with FSGS.Second,in order to explore the effect of urinary fibrinogen on long-term renal survival for patients with CKD,402 cases with stage 1-4 including 101 diabetic nephropathy(DN),94 idiopathic membranous nephropathy(IMN),55 idiopathic focal segmental glomerulonephritis(FSGS)and 152 IgA nephropathy(IgAN)were enrolled.The inclusion criteria were:(1)proteinuria>0.3 g/24 h;(2)a diagnosis proven by renal biopsy;(3)eGFR ?15 ml/min/per 1.73 m2;and(4)aged 18-65 years.The exclusion criteria were:(1)secondary causes;(2)viral infections;and(3)a family history of kidney disease.The median follow-up period from biopsy was 35 months(IQR:24.33-78.00 months).Clinical and laboratory data including age,gender,blood pressure,24-h urinary protein,serum albumin were collected within 1 month of renal biopsy.eGFR was calculated by using the equation of chronic kidney disease epidemiology(CKD-EPI)formula.Urinary fibrinogen was quantified by ELISA.The urinary creatinine(cr)level was used for normalization.So urinary fibrinogen/cr was used to analyze.The renal endpoint in our study was defined by the progression to eGFR<15mL/min per 1.73m2 or the initiation of renal replacement therapy for more than 3 months during the follow-up time.Interstitial fibrosis and tubular atrophy(IFTA)were scored as follows:0,absent;19<25%;2,25-50%and 3,>50%of the total area.Global glomerulosclerosis was defined as sclerosis area>50%of a glomeruli,and the definition of segmental glomerulosclerosis was any amount of the tuft involved in sclerosis,but not involving the whole tuft or the presence of an adhesion.Urine samples were collected in sterile plastic tubes at the time of biopsy,which were then centrifuged at 800×g for 10 min at 4? to remove cell debris.The supernatants of the samples were stored at-80? until further analysis.Statistical analysisStatistical analysisWe analysed all data using SPSS 19.0 statistical software(Chicago,IL).The non-normal distribution parameters were expressed as medians and interquartile ranges(IQRs)(25th and 75th percentiles)or percentages for categorical variables.The 2-tailed Mann-Whitney test or Kruskal Wallis test were used to analyse the differences between two or more groups.Paired t tests were used to analyse the change of urinary Fibrinogen levels after steroid treatment.Correlations between variables were analysed using Spearman's correlation.Urinary fibrinogen/cr was log10 transformed for regression analysis.Binary logistic regression models were used to evaluate the association between the baseline variables and the renal endpoint.Analysis of renal survival was estimated with the Kaplan-Meier method.Cox regression analysis was used to assess the relationship between baseline parameters and ESRD over time.Area under the receiver operating curves(ROC)was calculated to determine the discrimination ability of the corresponding models.Likelihood ratio test(LR test),Akaike information criterion(AIC),Itegrated Discrimination Improvement(IDI)and Net Reclassification Improvement(NRI)were used to compare the different models to analyze the value of urinary fibrinogen.P<0.05 was considered significant.ResultsUrinary fibrinogen reflected the podocyte injury and disease activity of FSGS In FSGS patients,elevated urinary fibrinogen levels were positively correlated with 24 h proteinuria(r=0.55,P<0.0001)and FPW(r=0.73,P<0.0001)respectively.After prednisone therapy,the urinary fibrinogen levels were significantly decreased in patients with complete remission(P<0.0001)but not in patients without remission(P=0.48).Urinary fibrinogen predicted the progression of CKDThe CKD patients were divided into three groups:tertilel(lowest level),tertile2(middle level),and tertile3(highest level)stratified by urinary fibrinogen/cr.Urinary fibrinogen/cr levels in CKD patients were higher than those of normal controls(P<0.0001).Urinary fibrinogen was positively correlated with proteinuria(r=0.644;P<0.0001),and interstitial fibrosis and tubular atrophy(IFTA)(r =0.104;P=0.037)respectively,but negatively correlated with eGFR(r=-0.198;P<0.0001)across a broader range of renal disease type(FSGS,IMN,IgAN and DN)and stage 1-4.Sixty-eight of 402(16.9%)CKD patients developed end-stage renal disease(ESRD)during the follow-up period.Kaplan-Meier analysis showed that higher urinary fibrinogen concentration at baseline was a significant higher risk for incident ESRD in CKD patients.In univariate and multivariate logistic regression and Cox regression analysis,urinary fibrinogen was demonstrated to be an independent risk factor of CKD progression to ESRD,even after adjusting for traditional factors such as age,sex,hypertension,proteinuria,disease type,eGFR and IFTA.The addition of urinary fibrinogen to traditional risk factors improved the prediction power for new-onset ESRD.The addition of the urinary fibrinogen to eGFR,proteinuria and hypertension increased the AUC from 0.73 to 0.76(LR test,P=0.0033)and AIC decreased from 333.64 to 326.99.The addition of urinary fibrinogen to traditional risks exhibited a better role in predicting CKD progression to ESRD compared with the combination of only eGFR,proteinuria and hypertension,especially in DN patients.Conclusions First,urinary fibrinogen levels reflected FSGS activity in FSGS patients.Second,urinary fibrinogen predicted adverse progression and incident ESRD in CKD patients despite of causes and stages independent of traditional risk markers...