The Role Of Urinary Angiotensinogen In The Renal Interstitial Inflammation And Renal Prognosis

Background:Renal tubulointerstitial fibrosis is the final outcome of almost all progressive chronic kidney diseases,and it is also a reliable predictor of prognosis.Moreover,long-term renal outcome is determined by the severity of tubulointerstitial involvement in most renal diseases.Renal interstitial inflammation serves as a primer that triggers tissue fibrogenesis and it correlated with the extent of fibrosis.Nonresolving inflammation is a major driving force in the development of fibrotic disease while renal fibrosis can be effectively ameliorated by inhibition of renal inflammation by a variety of means.It's important to evaluate the renal interstitial inflammation.The gold standard for judging the degree of tubulointerstitial lesions is renal biopsy.However,because of its contraindications and invasiveness,it can not be used as a routine and dynamic detection method.Therefore,the discovery of noninvasive markers that are closely related to interstitial inflammatory cell infiltration is of great value.To date,some clinical studies have been conducted to examine the utility of the biomarkers,mostly Kidney Injury Molecule 1(KIM-1)and Neutrophil Gelatinase–Associated Lipocalin(NGAL).Studies have shown that urinary KIM-1(uKIM-1)level reflects tissue KIM-1,and it is significantly associated withfibrosis and inflammation in different human renal diseases.Moreover,uKIM-1 also has prognostic value in acute kidney injury(AKI),chronic kidney disease(CKD),and transplant recipients,indicating that it can be used as a non-invasive biomarker and predictor of kidney disease.Unfortunately,the prognostic value of uKIM-1 was not strong as other biomarkers.However,increased uKIM-1 could indicate either injury or the repair response to injury,and the uKIM-1 level may not be able to differentiate the injury process with recover of AKI in patients accurately.Similarly,NGAL has been validated as a specific,sensible,and early predictor of AKI after cardiac surgery,contrast administration,septic shock,and even renal transplantation.Urinary NGAL(uNGAL)could also reflects the severity of kidney disease and predicts progression in CKD patients.Studies showed that uNGAL is associated with tubular atrophy and interstitial fibrosis in CKD patients and higher uNGAL predicts faster progression to ESRD.Unfortunately,there is less study about the NGAL with interstitial inflammatory cell infiltration though neutrophils and macrophages may be present in the interstitium.Recently,urinary angiotensinogen(uAGT)has been described as a novel prognostic biomarker of AKI.AGT was produced locally by proximal tubule cells,and there is substantial evidence that AngII present in renal tissues is generated locally from AGT delivered to the kidney.AngII is the major effector of the RAS.Activation of the intrarenal renin-angiotensin system(RAS)has long been recognized as an important contributor to CKD.Experiments have shown that target organs injured by locally AngII through the induction of non-infectious inflammation.Lai et al.found that interstitial inflammatory cell infiltration in IgA nephropathy is secondary to activation of RAS by situ hybridization.Notably,inhibition of AngII could elicit renal protection from inhibition of renal inflammation.uAGT has been proposed as a marker of intrarenal RAS activity,and it is predictive of progression of CKD Therefore,based on these reports,uAGT may be a useful non-invasive biomarker of renal interstitial inflammatory cell infiltration and a strong predictor for renal prognosis in renal insufficiency patients.Based on this,we will investigate the expression of uAGT,uKIM-1,and uNGAL,and the relationship with interstitial inflammatory cell infiltration andprognostic value in renal insufficiency patients.Purpose:1.We will explore the expression of uAGT,uKIM-1 and uNAGL in renal insufficiency patients and healthy subjects.2.The correlation between urinary biomarkers and pathological lesions in renal insufficiency patients will be explored.3.We will investigate the sensitivity,specificity,and AUC of uAGT,uKIM-1 and uNAGL in diagnosing renal pathologic lesions.4.The risk factors of renal prognosis in renal insufficiency patients will be investigated.Methods:Chinese patients with various degrees of renal impairment in Nephrology Hospital of the First Affiliated Hospital of Zhengzhou University from December2014 to April 2016 were screened.The Inclusion criteria were as follows: Age?18 years old;Serum creatinine ?115 umol/L(Enzyme Method,the normal range is 20-115 umol/L)or eGFR<60ml/(min×1.73m2)(eGFR calculated by CKD-EPI formula);Patients received renal bopsy.The exclusion criteria were as follows: Renal insufficiency patients caused by prerenal and postrenal renal diseases;Patients in CKD 5 stage(according to KDIGO2012 Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease);Renal biopsy specimens were less than 10 glomeruli;Patients received ACE inhibitor or Ang II type 1 receptor blocker.Renal pathology showed subacute tubulointerstitial nephropathy or was accompanied by subacute tubulointerstitial nephropathy.65 patients with renal insufficiency were enrolled and performed renal biopsy.12 healthy people were selected as healthy subjects.Blood samples were taken in the fasting state in the morning,and the first urine specimen of the day was also collected.The biochemical parameters,includingcreatinine,albumin,hemoglobin and proteinuria were measured at baseline in all patients,according to standard methods in the routine clinical laboratory.eGFR was assessed using the CKD-EPI formula.Urine samples were then stored at-80? until assayed.The expression of uAGT,urinary Kidney Injury Molecule 1(uKIM-1)and urinary Neutrophil Gelatinase-Associated Lipocalin(uNGAL)by Enzyme-linked Immunosorbent Assay(ELISA)were examined at baseline,and the pathology of kidney was evaluated at the same time.The specimens of renal tissue were processed for light microscopy,immunofluorescence,and electron microscopy.We assessed pathological injury by semiquantitative Katafuchi score.All these were done by at least two independent renal pathologists who were unware of the clinical status of each patient.The index of glomerular sclerosis was determined according to the percentage of obliterated glomeruli due to global sclerosis as follows: 0;no global sclerosis.1;global sclerosis in less than 10% of glomeruli.2;global sclerosis in more than 10% and less than 25% of glomeruli.3;global sclerosis in over 25% and less than 50% of glomeruli.4;global sclerosis in more than 50% of glomeruli.The severity of interstitial cell infiltration and interstitial fibrosis were semiquantitatively determined as follows: 0;none.1;occupying less than 25% of cortical area of biopsy specimen.2;occupying between 25% and 50% of cortical area.3;occupying over50% of cortical area.Index of tubular atrophy was graded as follows: 0;none.1;involving less than 25% of tubules.2;involving between 25% and 50% of tubules.3;involving over 50% of tubules.Glomerular,tubular and interstitial lesions were scored as 0,absent;1,mild;2,moderate;3 or 4,severe.After the baseline assessments,patients were followed prospectively until the end of the observation period or the progression endpoint was reached.The progression endpoint was defined as a decline of eGFR of?50% from baseline or ESRD or death.Statistical Analyses: SPSS 22.0 software was used for all analyses.Data were presented as mean±SD,median(interquartile range),or percentage frequency,as appropriate.Differences between groups were established by unpaired t test for normally distributed values and by Mann-Whitney U test for nonparametric values.Dichotomized values were compared using the x2 test.Pearson or Spearman correlation coefficients were used as appropriate to test correlations between eGFRand other variables.Before multiple linear regression was tested,all non-normally distributed values were log-transformed to better approximate normal distributions.To distinguish different pathologic lesions,receiver operating characteristics(ROC)curve analysis of urinary biomarkers was carried out to determine the area under the curve(AUC).ROC analysis was also used to calculate the AUC for uAGT,age and sBP and to find the best cut-off values for identifying the progression to renal endpoint.Kaplan-Meier curves were generated to assess renal survival.Adjusted risk estimates for progression endpoint were calculated using Cox proportional hazards regression model.All results were considered significant if P<0.05.Results:1.uAGT,uKIM-1,and uNGAL levels were significantly higher compared with those measured in healthy subjects(206.5[68.5,581.0] versus 0.14[0.04,2.79] ng/mg cr,P<0.0001),(1.9[1.0,3.6] versus 0.7[0.4,1.2] ng/mg cr,P<0.0001),and(81.1[10.5,362.0] versus 2.6[0.8,15.3]ng/mg cr,P<0.0001),respectively.2.On correlation analysis,the eGFR was directly correlated to hemoglobin(r=0.478,P<0.0001),T-CHO(r=0.321,P=0.012),and HDL(r=0.372,P=0.003),and an inverse correlation existed between eGFR and BUN(r=-0.54,P<0.0001),and uNGAL(r=-0.454,P<0.0001)(Table 2).Using eGFR as dependent variable in a multiple linear regression model,adjusted by age,hemoglobin,blood urea nitrogen(BUN),Ln uAGT,Ln uKIM-1,and Ln uNGAL,only the associations with hemoglobin(?=0.299,P=0.014),BUN(?=-0.402,P<0.0001),Ln uAGT(?=0.244,P=0.043),and Ln uNGAL(?=-0.379,P=0.007)are significant.3.A strongest correlation was showed between uAGT with interstitial inflammatory cell infiltration(r=0.366,P=0.003)and uAGT could reflect its severity(Table 4 and Table 5).ROC curve analysis showed that uAGT level was able to identify interstitial inflammatory cell infiltration with greater accuracy(AUC=0.664,P=0.023)than other pathologic lesions and urinary biomarkers(Figure 1).4.During the observational period(median follow-up of 22 months;range 0 to34),15 patients(23%)reached the renal endpoint.At baseline,patients reachedendpoints were significantly older and showed increased sBP and urinary AGT.67%of patients reached endpoints were chronic kidney disease patients.Kaplan-Meier survival curves in patients with age,sBP and uAGT level above the optimal cut-off experienced a significantly faster evolution to endpoint.These were also found in patients with glomerular sclerosis,moderate or severe interstitial inflammatory cell infiltration,interstitial fibrosis and tubular atrophy.Results from the multivariate Cox proportional hazards regression analysis showed that age(>51.5 years),chronic renalinsufficiency,uAGT(>166.8 ng/mg cr),and moderate or severe interstitial inflammatory cell infiltration predicted higher risk of progression endpoint independently in renal insufficiency patients.Conclusion:1.uAGT levels were enhanced in renal insufficiency patients compared with control subjects and can be a potential biomarker of severity of renal insufficiency.2.UAGT may be used as a non-invasive biomarker of interstitial inflammatory cell infiltration and is a strong predictor for renal prognosis in renal insufficiency patients.