Studies To The Tubulointerstitial Lesions And Non-invasive Biomarkers Of IgA Nephropathy

Background and Objective:IgA nephropathy(IgAN) is the most common glomerulonephritis worldwide,which develops progressively in most patients with IgAN and 15-40%of of which eventually develops into end stage renal disease(ESRD) in 10-20 years.Tubulointerstitial lesion(TIL) is one of the strongest predictors of IgAN outcome.Up to now,renal biopsy has been the important method for diagnosing IgAN and evaluating assessing TIL.Due to the facts that renal biopsy is an invasive procedure with inherent risks,and difficult to be performed repeatedly,many patients can neither be diagnosed and treated in time,nor be followed up effectively.Therefore,it is necessary to find non-invasive diagnostic methods and markers of IgAN and TIL.In this study, through multi-factor logistic regression analysis of clinical and pathological data of patients with IgAN,clinical independent affecting factors were screened out associated with TIL,and function models were established for evaluating the degree of TIL.The association between urine neutrophil gelatinase-associated lipocalin(NGAl) and clinicopathological parameters in IgAN were also analyzed to explore the significance of NGAl in assessing TIL of IgAN.Besides,the magnetic separation system combining matrix-assisted laser desorption ionization -time of flight mass spectrometry(MALDI-TOF MS) was applied to explore non-invasive diagnosis model and potential markers of IgAN.Subjects and Methods:1.Clinical and pathological data of 1493 IgAN cases were analyzed,respectively.Clinical independent affecting factors associated with TIL were screened out from the model group to set up functional model assessing TIL,which was verified in the verifying group.2.Urine NGAL level was detected by ELISA in 115 cases with IgAN,30 cases with non-IgAN glomeruionephritis, and 30 healthy control persons in order to analyze the association between urinary NGAL level and clinicopathophysiological parameters.3.The magnetic separation system combining MALDI-TOF-MS was used for analyzing urinary peptide spectra of 32 patients with IgAN,36 patients with non-IgAN glomerulonephritis,and 30 healthy controls.ClinProtools 2.0 software principal constituent analysis method and SVM algrithm were applied for establishing diagnostic model.And differential peptides were discriminated with nano-LC-Q-FT-ICR-MS.Results:1.(1) The incidence of TIL was 87.2%in 1493 IgAN patients,with percentages of slight/moderate/severe TIL were 47.6%,23.8%,and 16.4%. (2)The degrees of TIL were positively correlated with age,blood pressure,serum creatinine,blood uric acid,triglyceride,cholesterol,urine protein,and NAG enzyme.The degrees of TIL were negatively correlated with haemoglobin,blood albumin,urine osmotic pressure;positively correlated to glomerulosclerosis, segmental damage,crescent,mesangial proliferation,arterial wall thickening,and arterial hyaline change.(3) The first functional model:Through logistic regression analysis in the model group,clinical independent affecting factors were screened out associated with or without TIL,and function models were established for confirming TIL,(?)_TIL1=e^a/(1+e^a),a=-4.0585+0.9335BP-0.0169Hb+1.4382Scr+ 0.0026Ua+0.0841ALB+0.2972UP+0.034Age.AUC of the ROC was 0.771. Using the cutoff of 0.83,the sensitivity,specificity and the accuracy was 81.5%, 33.3%and 88.4%,respectively.(4) The second functional model:Through logistic regression analysis in the model group,clinical independent affecting factors were screened out associated with or without moderate-to-severe TIL,and function models were established for assessing TIL degrees,(?)_TIL2=e^b/(1+e^b),b= -2.0187+0.8482BP-0.0317Hb+1.9032Scr+0.0050Ua+0.2539UP.AUC of the ROC was 0.855.Using the cutoff of 0.43,the sensitivity,specificity,and the accuracy was 66%,91.1%,and 81%,respectively.The diagnostic efficacy in the estimation group was similar to that of verifying group.2.(1)The level of urine NGAL in IgAN or non-IgAN glomerulonephritis patients was higher than that in healthy controls(P＜0.001).(2) The level of urine NGAL in IgAN was significantly correlated with many clinical and pathology parameters. (3) The independent influencing factors on urine NGAL included blood albumin, urine protein,NAG enzyme,urine osmotic pressure,and tubulointerstitial lesions. (4) To evaluate whether or not TIL existed,ROC analysis of urine NGAL combining NAG enzyme showed that AUC was 0.850,while the AUC for serum creatinine was 0.675.3.(1)The whole system of MB-IMAC-Cu²⁺ combining MALDI-TOF MS was effective in isolation of urine peptides,and the CV was less than 10%.(2) There were significant differences in the urine peptides peaks among IgAN group, non-IgAN glomerulonephritis,and healthy controls.The most optimal classification with highest accuracy eventually was established for the discrimination of IgAN patients from healthy individuals and other glomerulonephritis.When it was evaluated by cross-validation,the sensitivity and specificity to distinguish IgAN versus healthy controls were both 100%,and sensitivity and specificity to distinguish IgAN versus other glomerular diseases were 85.7%and 76.5%.(3) The peptide with m/z 1913.14 was identified as fragments of uromodulin.ROC analysis for m/z 1913.14 to distinguish IgAN versus healthy controls showed that the AUC was 0.998.ROC analysis for m/z 1913.14 to distinguish IgAN versus other glomerular diseases showed that the AUC was 0.815.Conclusions:1.The incidence of TIL in IgAN was high.The degrees of TIL were obviously correlated with many clinical and pathology parameters.The functional models for assessment of TIL were established with good sensitive,accuracy and good reproducibility,which can help predicting the degree of TIL and providing intervention early.2.The levels of urine NGAL in IgAN were obviously correlated with many clinical and pathology parameters,and could reflect the degree of illness.Combination of urine NGAL and NAG enzyme was better in evaluating whether or not TIL existed than serum creatinine,urine NGAL,urine NAG,or osmotic pressure alone.3.Analysis of urine peptides patterns by MB and MALDI-TOF MS was a non-invasive diagnostic tool with good sensitivity and specificity.Urine peptide marker of m/z 1913.14 was identified as the fragment of uromodulin,which may be used as a non-invasive way for clinical diagnosis of IgAN.