Investigation Into The Correlation Between Urinary L-FABP And Tubulointerstitial Damage

Objective: The prevalence of chronic kidney disease(CKD)is increasing every year in the world and CKD has become an independent risk factor for cardiovascular disease.But there is no method that can be used to detect CKD in the early stage,thus it restricts the timely treatments,although there may be no treatment which can completely reverse the progression of chronic kidney disease to end stage renal disease(ESRD).Therefore it is critical to find some markers which can detect CKD during the early stage and immediately interfere with the disease to slow down the progression to ESRD.The extent of tubulointerstitial injury has been found to be correlated more with the prognosis of chronic kidney disease than the degree of glomerular damage.But so far,except the renal biopsy,there are no other methods that can be used to detect the tubulointerstitial damage accurately.So it is necessary to find out an easy and practical method to monitor the tubulointerstitial injury and thus may reflect the degree of CKD.In this study we selected some clinical patients who were enrolled in our hospital and diagnosed with CKD,and observed the degree of the tubular damage and interstitial fibrosis,checked the serum and urinary biochemistry to investigate the relationship between defferent clinical parameters in CKD,especially to make sure whether urinary L-FABP can be used as an indicator of the tubulointerstitial damage in CKD and whether it can reflect the degree of CKD.We also try to further clarify the possible pathophysiological mechanisms of tubulointerstitial damage and the role of L-FABP in it.Methods:Patients presented with proteinuria,hematuria,edema or hypertension,diagnosed with CKD and willing to accept the renal biopsy were recruited for the study.Ones with other severe systemic diseases or taking ACEI,ARB,glucocorticoids were excluded from the study.The basic conditions of the patients were recorded,such as gender,age,blood pressure,past history,and so on.On the very day of receiving renal biopsy,blood and urine were selected as samples.5mL of the blood got in the morning was enough for the serum samples.Then,ALT,AST,BUN,SCr,CHO,TG were measured in serum samples by enzymatic methods.After the centrifugation,the supernatans of the urine were used to detect the urinary biochemistry.Total protein,ALB,creatinine were detected in urinary samples.Extent of urinary L-FABP was quantified using a commercially available ELISA kit by a two-step sandwich procedure.The urinay total protein,ALB,and L-FABP were expressed as ratios to the urinary cretinine respectively. For light microscopic analysis,the kidney samples obtained from the renal biopsy were dehydrated and embeded in paraffin,and then sliced into 3μm serial sections.Then the paraffined sections were used for PAS and MASSON staining and immunohistochemical examination.Immunohistochemical examination was to detect the expression of L-FABP in kidneys,and to check the location and degree of the expression. L-FABP immunostaining in the kidneys of the patients'biopsy specimens was performed with a polyclonal antibody against human L-FABP,FABP-2.A negative control without the primary antibody showed no staining.PAS and MASSON staining were for light microscopy analysis at 200 magnification.MASSON staining is used to detect the ratio of the area of interstitial fibrosis to the whole cortical area,while PAS staining is to define the tubular damage(including the tubular atrophy,tubular dilation, tubular cast formation,or without brush borders)ratio to the entire cortical area.Then the patients were devided into 3 groups according to the degree of the tubular damage:mild group,<10% of the cortical area injured;moderate group:10%～50% of the cortical area injured;severe group:≧50% of the cortical area injured. All values are expressed as the mean±SE.Differences among the three groups were analyzed by the Mann-Whitney U test.The correlation between two logarithmic values of clinical parameters including urinary hL-FABP was assessed by Pearson's correlation.The correlation between urinary L-FABP and the area ratio of tubular damage and interstitial fibrosis was assessed by Spearman's rank correlation.Statistical significance was set at P<0.05. These statistical analyses were performed with a computer software program for Microsoft Windows(SPSS,17.0).Results: We collected 74 patients and devided them into three groups according to the degree of renal tubular damages:mild group n=15,moderate group n=39,severe group n=20.The level of serum CHO,Cre and BUN in the severe group was significantly higher than that in the mild group(P<0.01),while it was not significantly higher than the moderate group(P>0.05);the concentration of serum TG,AST and ALT did not differ significantly among the three groups(P>0.05,between any two groups). By observing the urinary parameters we found that,the concentration of urinary protein,urinary albumin and urinary L-FABP was significantly higher in the severe group than either in the mild group or in the moderate group.Urinary protein(0.37±0.21g/g Cr,2.91±2.66g/g Cr and 2.91±2.66g/g Cr in mild,moderate and severe groups,respectively;P<0.01 mild group versus severe group; P<0.01 moderate group versus severe group);urinary albumin showed the same pattern(0.03±0.02 mg/mg Cr, 0.23±0.13 mg/mg Cr and 0.84±0.69 mg/mg Cr in mild,moderate and severe groups,respectively;P<0.01 mild group versus severe group; P<0.01 moderate group versus severe group);urinary L-FABP also followed the same pattern(8.93±5.11μg/g Cr,76.66±48.56μg/g Cr,389.99±267.12μg/g Cr in mild,moderate and severe groups,respectively;P<0.01 mild group versus severe group; P<0.01 moderate group versus severe group). Excretion of urinary L-FABP was significantly correlated with the degree of renal tubular damage(r=0.933,P<0.01)and the extent of renal interstitial fibrosis(r=0.897,P<0.01).Correlation between urinary L-FABP and serum BUN,Scr,CHO and TG was not significant(r=0.25, r=0.22, r=0.16 and r=0.10,respectively. P>0.05 urinary L-FABP versus any serum parameter),but it was significant between urinary L-FABP and urinary protein and urinary albumin(r=0.795 and r=0.914,respectively,P<0.01 urinary L-FABP versus any other urinary parameter).Immunohistochemical staining anti-L-FABP showed that,L-FABP was diffusely expressed throughout the proximal tubules,mainly in the cytoplasma and nucleus of the injured tubular cells,and the positive area was larger as the tubular injury became severer.Conclusions:With the severity of the tubulointerstitial damage in CKD,the expression of L-FABP in the kidney was elevated,and then induced the excretion of L-FABP from kidney into urine.Therefore,urinary L-FABP can be used to reflect the degree of tubulointerstitial damage and also can be used as an indicator for the degree of CKD.L-FABP is mainly expressed in the proximal tubules.Cytoplastic L-FABP can bind FFA and engage in the intracellular transportation to mitochondria or peroxisome where FFAs are metabolismed byβ-oxidation.Moreover,L-FABP can also transport FFAs from the cytosol to the nucleus and L-FABP interacts with the nuclear protein peroxisome proliferator-activated receptors(PPAR), which is a nuclear target of FFAs and initiates the gene expression of enzymes involved in lipid metabolism.FFA metabolism is involved in the tubulointerstitial damage and PPAR is correlated to the FFA metabolism,therefore,the legand PPAR on the surface of nucleus may play the key role in the pathophysiological mechanisms.