|Year : 2013 | Volume
| Issue : 4 | Page : 751-758
|Immuno-histochemistry analysis of Helicobacter pylori antigen in renal biopsy specimens from patients with glomerulonephritis
Qian Li1, Xi Lin2, Zhongbiao Wu2, Lihuan He1, Weijun Wang2, Qizhi Cao1, Jianzhong Zhang1
1 State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
2 The First People's Hospital of Wenling, the Affiliated Wenling Hospital of Wenzhou Medical College, Zhejiang, China
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|Date of Web Publication||24-Jun-2013|
| Abstract|| |
This study was conducted to investigate the relationship between Helicobacter pylori infection and three varieties of glomerulonephritis. Renal biopsy specimens from patients with Henoch Schonlein Purpura nephritis (HSPN; n = 10), membranous nephropathy (MN; n = 9) and lupus nephritis (LN; n = 27) were studied using immuno-histochemical labeling to clarify the etiological significance of H. pylori antigen in this disease. Immuno-histochemical labeling was performed using a mixture of anti-H. pylori-antibody-positive serum from nine volunteers; a mixture of anti-H. pylori-antibody-negative serum from nine volunteers was used as control. Staphylococci protein-A labeled by horseradish peroxidase was used as the second antibody in this study. A total of 34 of the 48 specimens revealed positive reaction with the anti-H. pylori-positive serum and five of the 48 specimens revealed positive reaction with the anti-H. pylori-negative serum. Positive reaction against anti-H. pylori-positive serum was seen in 10/10 patients with HSPN, six of nine patients with MN and 18/27 patients with LN. Statistical analysis showed that the difference of the positive reaction between anti-H. pylori-positive and negative sera was significant (χ 2 = 36.318, P = 0.000). Our study indicates that H. pylori infection may be associated with the development and/or progression of HSPN, MN and LN.
|How to cite this article:|
Li Q, Lin X, Wu Z, He L, Wang W, Cao Q, Zhang J. Immuno-histochemistry analysis of Helicobacter pylori antigen in renal biopsy specimens from patients with glomerulonephritis. Saudi J Kidney Dis Transpl 2013;24:751-8
|How to cite this URL:|
Li Q, Lin X, Wu Z, He L, Wang W, Cao Q, Zhang J. Immuno-histochemistry analysis of Helicobacter pylori antigen in renal biopsy specimens from patients with glomerulonephritis. Saudi J Kidney Dis Transpl [serial online] 2013 [cited 2020 Feb 26];24:751-8. Available from: http://www.sjkdt.org/text.asp?2013/24/4/751/113871
| Introduction|| |
Helicobacter pylori (H. pylori) is a causal agent of glomerulonephritis in humans. Recently, a few papers have addressed of active chronic gastritis and peptic ulcer in the causal relationship between H. pylori and extragastric diseases, such as cardiovascular, immunological, hematological and various other pathologies. ,,, Some case reports have described the association between H. pylori infection and Henoch Schonlein Purpura (HSP), ,,,,, and some researchers have found that treatment of the H. pylori infection resulted in prompt resolution of the HSP  or that the eradication of H. pylori infection prevented recurrence of HSP. ,, A similar phenomenon has also been observed in a patient from Japan with membranous nephropathy (MN), whose nephroticrange proteinuria was markedly reduced after the eradication of gastric H. pylori infection.  In this study, we concentrated on diseases such as HSP, MN and systemic lupus erythematosus (SLE), which are considered to be immune complex-mediated diseases, characterized by the presence of immune complexes predominantly in dermal, gastrointestinal and glomerular capillaries. ,,,
It has been shown that the presence of antigastric antibodies induced by H. pylori infection was due to a cross-mimicry between Lewis blood group antigens expressed both by the proton pump of gastric epithelium and the bacterium itself. , Moreover, of special interest is the demonstration that antibodies against H. pylori may also react with some extragastric tissues such as the glomerular capillary walls, ductal cells of the salivary glands and renal tubular cells.  Besides, it has been reported that there exists high-affinity binding of H. pylori antigen to laminin and type IV collagen, , which are major components of the glomerular basement membrane. It was also observed that the presence of a specific antigen in the glomeruli of patients with MN and H. pylori infection may be involved in the pathogenesis of MN. ,
Based on the above background, it is suggested that H. pylori infection may be associated with some kinds of nephritis. In the present study, we collected specimens from 48 patients with nephritis in China, including Henoch Schonlein Purpura nephritis (HSPN), MN and lupus nephritis (LN), to investigate the relationship between nephritis and H. pylori infection using the immunohistochemistry (IHC) method.
| Materials and Methods|| |
Case definition symptoms (ICD-10)
All the study patients were diagnosed according to the definition of the International Classification of Diseases (ICD-10). 
Renal biopsy specimens were obtained from patients with HSPN, MN and LN by needle biopsy at The First People's Hospital of Wenling between 2001 and 2007. The biopsy material was fixed in 95% ethanol and processed routinely soon after the biopsy to obtain conventional paraffin sections. Of the 48 patients involved, there were 10 patients with HSPN, nine patients with MN, 27 patients with LN and two patients with immune-mediated nephritis of uncertain classification. The diagnosis of each type of nephritis was established on the basis of clinical symptoms and the characteristic findings on light microscopy and IHC. There were 16 male and 32 female patients, with a mean age of 34.17 years (10-70 years). The characteristics of these patients are listed in [Table 1].
|Table 1: Patient characteristics on renal biopsy and immuno-histochemistry labeling of the specimens.|
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The primary antibody employed in the present study was a mixture of the serum of nine volunteers who were anti-H. pylori-antibody positive during the 13C urea breath test (13 C-UBT) in 2004 and confirmed by Western blot (WB) during the present study. A mixture of serum obtained from nine volunteers who tested negative for anti-H. pylori antibodies in 2004 and also confirmed by WB during the present study served as controls. During the study, Staphylococci protein-A (SPA) labeled by horseradish peroxidase was used as the secondary antibody.
Immuno-histochemical labeling was achieved using the avidin-biotin-peroxidase method as follows. All specimens were divided into two groups, one each for testing with anti-H. pylori antibody-positive and anti-H. pylori antibody-negative sera. The sections were de-waxed in xylene, rehydrated in a graded ethanol series, placed in a plastic coplin jar filled with denaturing solution (citric acid buffer, pH 6.0), kept in a microwave on low for 10 min and then immersed in methanol containing 0.3% hydrogen peroxide to block their endogenous peroxidase activity. Thereafter, after washing in PBS three times, the sections were incubated with each primary antibody (work dilution 1:200) in moist boxes at 4°C overnight. The next day, the sections were rinsed in PBT at least three times and further processed for IHC by incubating with the secondary antibody (work dilution 1:400) in moist boxes at 37°C for 1 h. Finally, the sections were stained with diaminobenzidine (DAB) primarily and then restained with Harris's hematoxylin. The sections were dehydrated by torrefied at 37°C for two days and then transparented with dimethylbenzene. Lastly, they were mounted with neutral balsam for light microscopic examination.
Determination of the result
The determination of the result references the standard of Friedrichs. 
| Data Analysis|| |
SPSS 16.0 was employed in the present study. The statistical test is χ 2 test and/or Fisher's exact test, and the test level is α = 0.05.
| Results|| |
Of the 48 specimens, 34 had a positive reaction with the anti-H. pylori-positive serum and five specimens revealed positive reaction with the anti-H. pylori -negative serum. The number of positive reactions seen with anti-H. pylori antibody in patients with HSPN, MN, LN and immune-mediated nephritis was 10/10, six of nine, 18/27 and zero of two, respectively. The reactivity was observed along the glomerulus, tubular epithelium, protein cast and parietal epithelium of the Bowman's capsule. The positive reaction was observed at a single site or at a combination of the sites on one section ([Figure 2] and [Figure 3], negative controls are shown in [Figure 1]). Statistical analysis showed that the difference of the positive reaction rate between anti-H. pylori-positive and -negative serum was significant (χ 2 = 36.318, P = 0.000), and the positive rate of any two groups against anti-H. pylori-positive serum, among patients with HSPN, MN and LN, was identical (χ 2 = 13.348, P = 0.001). Among the three groups investigated, the group with HSPN showed the highest rate of positive reaction.
|Figure 1: Negative immune reactivity with mixed anti-H. pylori antibody-positive and -negative serum in the glomerulus of patient 16 (×1000). (a) Anti-H. pylori-antibody-positive serum, (b) anti-H. pyloriantibody- negative serum.|
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|Figure 2: Positive immune reactivity with mixed anti-H. pylori-antibody-positive and -negative serum in the glomerulus of patient 13 (×1000). (a) Anti-H. pylori-antibody-positive serum, (b) anti-H. pyloriantibody- negative serum. Arrows indicate the positive reaction observed along the glomerular (arrow 1) and tubular epithelium (arrow 2).|
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|Figure 3: Positive immune reactivity with mixed anti-H. pylori-antibody-positive and -negative serum in patient 18 (×1000). (a) Anti-H. pylori-antibody-positive blood sera, (b) anti-H. pylori-antibody-negative blood sera. Arrow indicates the parietal epithelium of Bowman's capsule.|
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| Discussion|| |
H. pylori is classified into two types based on gene sequences, East Asian and Western type. The differences between these two types lie mainly on the distribution of area and people, and pathogenicity. In the present study, we collected specimens from patients with nephritis in Wenling, Zhejiang province, China, which has a typical East Asian and East Asian-type population. Thus, the results of the present study may represent the characteristics of the East Asian population. Another feature of the present study is that we employed primary and secondary antibodies in the experiment. In order to capture the suspected antigens maximally, we chose to use a mixture of human serum, whose 13 C-UBT was anti-H. pylori-antibody positive, which was further confirmed by WB. Given this situation, the human serum mixture was considered to be polyclonal antibody against H. pylori, which also cover strains more commonly seen in China. Simultaneously, we used another mixture of serum that was anti-H. pylori-antibody negative on the 13 C-UBT and WB test as controls in order to rule out false-positive results. Besides, the secondary antibody we employed in the experiment was SPA, which has a high affinity with the IgG of humans, murine, rabbits, monkeys, etc. This high affinity helped in the detection of larger numbers of antigens or other immune complexes containing IgG.
Intense reactivity with anti-H. pylori-antibody-positive serum was evident along the glomerulus in 34/48 specimens, which is consistent with the result of Nagashima's study.  Also, five of 48 specimens revealed positive reaction with anti-H pylori-antibody-negative blood sera; the difference was statistically significant (χ 2 = 36.318, P = 0.000).
Furthermore, we found that positive reactivity was mainly seen along the tubular epithelium, along with the glomerulus, protein cast and parietal epithelium of the Bowman's capsule in some specimens [Figure 2] and [Figure 3]. This finding is consistent with the study that showed that monoclonal antibodies against H. pylori cross-react with glomerular capillary walls and renal tubular cells.  Thus, we can suppose that the H. pylori strain may possess antigen components that the kidney expresses specifically; immune complexes may then form in situ or deposit from the circulation.
Among the three groups investigated, the HSPN group revealed the highest rate of positive reaction (10/10). The nature of occurrence of HSP is inflammation in capillaries mediated by polymeric IgA1-containing immune complexes predominantly deposited in the dermal, gastrointestinal and glomerular capillaries. ,, Considering the capillary lesion in HSPN, the components of basement membrane may be exposed to the antigen of H. pylori strains, and that makes it easier for the H. pylori antigen to bind to laminin or type IV collagen, and, finally, form immune complexes in situ. Another study found that the anti-CagA antibodies cross-react with antigens of both normal and atherosclerotic blood vessels,  which provided us a second mechanism, namely, mimicry between H. pylori antigens and vessels.
An epidemiological study performed in Japan in 2006 showed that a significantly higher H. pylori infection rate was found in patients with MN than in the control group. Moreover, eradication of H. pylori was achieved in four patients with MN and proteinuria who were receiving glucocorticoid therapy, three of whom experienced a reduction in proteinuria after eradication of H. pylori.  Besides, another case report described a patient with MN whose nephrotic-range proteinuria was markedly reduced after the eradication of gastric H. pylori infection.  A similar study was performed in China in 2005, which did not find any significantly higher H. pylori infection rate in patients with MN when compared with the control group. However, they found positive reactivity along tubular epithelium, protein casts and renal arterioles with polyclonal anti-H. pylori antibody by immuno-histochemical labeling. All the previous studies have suggested that H. pylori infection is associated with MN. The key point of the present study lies in the primary antibody employed in immuno-histochemical labeling. The primary antibody employed in the two previous studies was polyclonal antibody and monoclonal antibody against H. pylori, which was obtained from animal serum and purchased from a commercial company. Animal serum may react with human tissue as a result of species variation, and a false-positive result may be observed due to this mechanism. However, in the present study, human serum that showed positive with H. pylori antigen was employed as primary antibody, thereby eliminating false-positive results.
In murine studies on LN, anti-DNA antibodies have been found to be sequestered in the kidney, where they contribute to glomerulonephritis.  Several lines of evidence suggest that these antibodies deposit in the kidney by virtue of their cross-reactivity with renal antigens. Both anti-DNA antibodies and non-DNA-binding antibodies can deposit in the glomeruli, even when glomerular preparations have been treated with DNase.  Also, several renal protein antigens including fibronectin, laminin, α-actinin and heparan sulfate, are bound directly by monoclonal anti-DNA antibodies. ,, A recent study demonstrated that non-DNA-binding antibodies from a patient with lupus can bind both bacterial and renal antigens.  As H. pylori antigen is able to bind to laminin and type IV collagen with high-affinity, , H. pylori infection may contribute to the progression of LN.
In conclusion, the results of this study show some kind of association between H. pylori antigen and some immune-mediated nephritis. Identifying the exact H. pylori antigen (or cross-reacting antigen) in renal biopsy specimens from patients with immune-mediated nephritis is a crucial step toward a deeper understanding of the role of H. pylori infection in the mechanisms of the disease.
| Acknowledgments|| |
This work was supported by the Science and Technology Program of Zhejiang Province Public Technology Social Development Project (No. 2010C33035). The authors wish to thank Prof. Jingmin Zhao and his laboratory team from the 302 Hospital of PLA, Beijing, China, for the help on immunohistochemical analysis.
| Disclosure|| |
All the authors declared no competing interests.
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National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory for Infectious Disease Prevention and Control, Beijing
[Figure 1], [Figure 2], [Figure 3]
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