Preliminary Study Of The Association Of Common Food Allergens With The Pathogenesis Of IgA Nephropathy And Membranous Nephropathy

BackgroundAs early as in 1905,British physician France Hare found that many diseases were food related.Celiac disease and rheumatoid arthritis are generally thought to be closely related with a gluten diet and Insulin-dependent diabetes mellitus is associated with the intake of milk.Food intolerance as well as the intake of a variety of food proteins was reported to be associated with Irritable Bowel Syndrome and inflammatory bowel disease.Some scholars had proposed that food antigens might be associated with some glomerular diseases.The potential pathogenic role of food antigen in glomerular diseases was emphasized in IgA nephropathy.Food specific antibody level in patients with IgA nephropathy was significantly higher than healthy controls.Restricting certain foods reduced proteinuria as well as the pathological injury of patients with IgA nephropathy.According to a Japanese epidemiological investigation,the risk of IgA nephropathy increased significantly in subjects who often consumed a diet containing raw egg and rice.Food allergy was also reported to be associated with hormone dependent or resistant nephrotic syndrome.A low antigen diet alone reduced proteinuria or even induced complete remission in partial patients who were glucocorticoids dependent or resistant.Cationic bovine serum albumin(BSA)has been generally accepted as the target antigen of early-childhood membranous nephropathy recent years.IgA nephropathy(IgAN)is now the most prevalent form of glomerulonephritis worldwide and is characterised by immunohistochemical finding of IgA-and IgG-containing immune complexes in the glomerular mesangium of the kidney.IgAN is a common cause of end-stage renal failure.30%-40%patients will develop end-stage renal disease.The prevalence of IgA nephropathy has increased in recent years.The etiology and pathogenesis of IgAN is not clear now.In recent years Kloster Smerud et al proposed that food sensitivity might be involved in the pathogenesis of IgAN in a subgroup of patients.Some scholars have found that up to 50%of IgAN patients had increased rectal mucosal response to soybean and milk and proposed that food sensitivity might be involved in the pathogenesis of IgAN in a subgroup of patients.Dietary structure and eating habit in china differs from those in western.However,the relationship between food antigens and IgAN has not been reported in China,where IgAN is very common.Membranous nephropathy(MN)is the most common cause of nephrotic syndrome in adults.MN is characterized by the subepithelial deposition of immune complexes at the glomerular basement membrane that results in a membrane-like thickening.The prevalence of MN has increased significantly in China in recent years.According to the results from a single center,the prevalence of MN was 6.48%during 1997-1999 while it increased to 22.79%during 2009-2011.The etiology of idiopathic membranous nephropathy is still unknown.40%patients will progress to end-stage renal disease in 10 years.Treatment of MN is always controversial and challenging due to non-specific pathophysiology-driven therapy.It is of great importance to clarify the pathogenesis of idiopathic MN.It is generally considered that idiopathic membranous nephropathy(IMN)is an auto-immune disease and combination of circulating autoantibodies to glomerular antigens plays a central role in the pathogenesis of MN.It is of critical importance to identify pathogenic antigens.Recently,cationic bovine serum albumin,a modified food-derived antigen,has been considered to be related to childhood membranous nephropathy.We hypothesized that food antigen might also play a role in adult membranous nephropathy.To test whether food antigens have a potential role in the pathogenesis of IgA nephropathy and membranous nephropathy,we detected serum food specific antibodies of 6 kinds of common foods in patients with IgA and the glomerular deposition of food antigens which were major allergic protiens of egg,shrimp,fish,milk,hazelnuts,wheat,peanuts and soybeans.This project was supported by the Key Laboratory Construction Grant(No.SW201110010)from the Science,Industry,Trade and Information Technology Commission of Shenzhen Municipality and the Key Laboratory Project of Shenzhen(No.JC201005250073A).Objective1.To explore the relationship between the major allergens of 6 kinds of common foods and IgA nephropathy.2.To explore whether food antigens play a role in the pathogenensis of adult membranous nephropathy.Methods1 Detection of serum food specific antibodies of 6 common foods in patients with IgA nephropathy1.1 PatientsSerum samples were obtained from 31 patients with histologically proven IgAN and from 80 age-and sex-matched healthy volunteers from 181 Hosptital of Chineses People's Liberation Army and The Third Afiliated Hospital of Southern Medical University during Sept,2011-Sept,2012.1.2 MethodsA sensitive sandwich enzyme-linked immunosorbent assay(ELISA)has been used to detect serum IgA1,IgG and IgE antibodies to the food allergens of six common foods(peanuts,milk,egg,soy,shrimp and fish).We also observed clinical characteristics of the patients whose levels of multiple antibodies of more than 4 kinds of foods were significantly elevated.All the patients were examined for a history of food allergy using a questionnaire.1.3 Statistical AnalysisEach experiment was repeated for three times and the results presented are the mean of the triplicate values.Data were analyzed with SPSS(version 13.0).The nonparametric method(Mann-Whitney U test)was used for the skewed distribution of values.A P value less than 0.05 was considered statistically significant.2 Glomerular deposition of food antigens in adult patients with membranous nephropathy2.1 Patients67 renal specimens were obtained from patients with biopsy-proven MN at The Third Affiliated Hospital of Southern Medical University(Guangzhou,China)and The No.181 Hospital of Chinese People's Liberation Army(Guilin,China),from Sept,2012-Sept,2013.Renal specimens from 39 patients with IgA nephropathy(IgAN),24 patients with minimal change disease(MCD),31 patients with other kidney diseases were also obtained as controls in our study.2.2 Methods2.2.1 Specimen collectionRenal biopsy specimens were cut into 3 sections immediately after biopsy.Samples for light microscopy were fixed in FAA,samples for immunofluorescence staining were wrapped in saline-moistened gauze and for electron microscopy were fixed in 2%glutaraldehyde.2.2.2 Frozen and paraffin sectionsFrozen sections:Adjusted the temperature of freezing microtome to-20?,embedded renal tissues with OCT,set the slice thickness as 3-4 mm,attached renal tissues to the slides and stored the slides in freezing chamber.Paraffin sections:Fixed the renal tissues in FAA for more than 45 minutes,attached the 2-3um thick paraffin sections to slides which were pre-treated with poly-L-lysine solution after conventional dehydration,leaching with sodium and polyester wax embedding.2.2.3 Detection of food antigens in renal tissue.Frozen sections:The frozen sections were dried at room temperature for 30 minutes/with the cold wind for 4-5 minutes.The sections were fixed with 4?acetone for 10 minutes.Monoclonal antibodies against the eight kinds of food allergens were added after the specimens were immersed for 10 min in 5%normal goat serum.The specimens were incubated for 1hour at 37? or overnight at 4?.Then FITC-labeled goat-anti-mouse IgG was added and the stainning was observed under fluorescence microscope immediately or the specimens were kept in dark at 4? after adding anti-fading agent.Paraffin sections:Paraffin sections were baked at 60? for 2 hours,dewaxed by xylene,rehydrated by gradient alcohol and retrieved with trypsin K.The remaining steps were the same as the staining on frozen sections.2.2.4 Detection of M-type phospholipase A2 receptor 1 in renal tissueStaining steps of M-type phospholipase A2 receptor 1were similar as the stainning of food antigens.The difference was the replacement of the monoclonal antibodies against the eight kinds of food allergens with rabbit polyclonal antibody against PLA2R1 and replacement of the secondary antibody with FITC-labeled goat-anti-rabbit IgG2.2.5 Double immunofluorescence stainingWe detected IgG at the same time of the detection of food antigens and PLA2R1.The specimens were washed with PBS after adding anti-mouse/anti-rabbit secondary antibody and adding dylight 405-labeled goat-anti-human IgG(blue).2.2.6 Clinical data collection24 hours urine protein,serum creatinine,blood urea nitrogen and eGFR of the patients were recorded or calculated.Inquired and recorded Aallergic symptoms of the patients after eating suspicious foods was inquired and recorded.2.2.7 Statistical AnalysisData were analyzed with SPSS(version 13.0).Continuous variants were presented as mean ± standard deviation if they had normal distribution.Median and inter-quartile range were used to demonstrate skewed distributed continuous variants.The categorical variables were recorded as absolute and relative(%)values or proportion.Student's t-test or Wilcoxon rank-sum test was used for continuous variants.Nonparametric Kruskal-Wallis test was used for continuous variants which were not normally distributed.Chi-squared test or Fisher's exact test was used for analyzing categorical variants.A two-tailed p value<0.05 was considered significant.Results1 Detection of serum food specific antibodies of food allergens of 6 kinds of common foods in patients with IgA nephropathy1.1 Serum levels of IgAl against six kinds of food allergens in the common allergic foodsFood(peanut Ara h 1,shrimp tropomyosin,soybean Gly m Bd 30K protein,ovomucoid,fish parvalbumin)specific IgAl in the serum of IgAN patients were significantly higher than that of the healthy controls.1.2 Serum levels of IgG against six kinds of food allergens of the common foodsFood allergens(peanut Ara h 1,shrimp tropomyosin,soybean Gly m Bd 30K protein,ovomucoid,fish parvalbumin)specific IgG levels in the serum of IgAN patients were significantly higher than that of the healthy controls.1.3 Serum levels of IgE against six kinds of food allergens of the common allergic foodsThere was no detectable food specific IgE in the serum of both IgAN group and healthy controls.1.4 Histories of food allergyAll patients in the present study denied symptoms of allergic reactions related to eating curtain foods and none of the patients had a clear history of food allergy.1.5 Clinical indicatorsWe observed the clinical characteristics of the 9 patients who had significantly increased serum levels of food specific(more than four kinds of foods)IgG and found all the 9 patients had proteinuria,while 5 patients with low serum levels of IgG against the six kinds of food allergens in the study showed only hematuria.2 Glomerular deposition of food antigens in adult patients with membranous nephropathy2.1 Renal deposition of food antigensGlomerular deposition of food antigens was found in 47 out of 67(70.1%)patients with MN,while only in 2 out of 39(5.1%)patients with IgA nephropathy.No food antigens were found in patients with other kidney diseases.2.2 Distribution of food antigens in the renal tissue of patients with MNMultiple food antigens were detected simultaneously in glomerulus.Positive results of egg protein were obtained in 28 cases(96.5%),for fish protein in 26 cases(89.6%),for gluten in 34 cases(85%),for shrimp protein in 35cases(83.3%)with shrimp protein,for milk protein in 31 cases(75.7%),for peanut protein in 29 cases(72.6%),for hazelnut protein in 18 cases(66.7%)and for soy protein in 15 cases(55.6%).2.3 Colocalization of food antigens and IgGWe assessed the location of food antigens and IgG by double-labelling confocal studies in patients with MN.Food antigens co-located with IgG in a fine linear and granular pattern along the glomerular capillary walls.2.4 Expression of M-Type Phospholipase A2 Receptor 1(PLA2R1)and co-localization of PLA2R1and IgGPLA2R1 staining was performed in 41 patients with MN and 51 patients with non-MN.PLA2R1 was detected in 33/36 patients with membranous nephropathy and 0/51 patients with non-MN.Double-labeling con-focal studies showed that PLA2R1 co-located with IgG in a fine linear and granular pattern.PLA2R1 was not only positive in idiopathic MN but also membranous lupus nephritis(membranous lupus nephritis,MLN).2.5 Clinical parameters and deposition of food antigens in patients with MNNearly all patients denied atopic allergic symptom after eating the eight kinds of food in our study and none of our patients provided a history of food allergy.There were no significant differences in clinical parameters between food antigen positive and negative group.Conclusions1.Some foods especially the highly allergic/agglutinin-rich ones might participate in the pathogenesis and progression of IgA nephropathy.2.Many kinds of food antigens could be detected in the glomerulus of patients with membranous nephropathy.Many kinds of food antigens or certain components of the foods might be involved in the pathogenesis of membranous nephropathy.The potential role of some kinds of food antigens or curtain components of foods such as dietary lectins in the development of MN merits considering,both in exploring the pathogenesis of MN and giving a specific treatment to the patients with MN,especially those patients received conventional therapy and with poor treatment effect.