The Effects of Helicobacter pylori Eradication on Proteinuria in Patients with Primary Glomerulonephritis

Background. Membranous nephropathy (MN) is a common cause of nephrotic syndrome. In most cases it is idiopathic, while it may also be secondary to many diseases. In this study, prevalence of H. pylori infection and the effects of H. pylori eradication on proteinuria levels were investigated. Methods. Thirty five patients with MN (19 male), 12 patients with IgA nephropathy (4 male) and 12 patients with focal segmental glomerulosclerosis (FSGS) (8 male) were studied. The presence of H. pylori antigen was investigated in renal tissues obtained by biopsy, and the effects of H. pylori eradication on proteinuria levels were investigated. Results. Immunohistochemistry with H. pylori antigen revealed no positive staining in the glomeruli of all patients. 19 patients (54%) with MN, 10 (83%) with IgA nephropathy and 4 (33%) with FSGS were positive for H. pylori stool antigen test (P = 0.045). Patients with H. pylori infection were administered eradication therapy (lansoprazole, 30 mg twice daily, plus amoxicillin, 0.75 g twice daily, plus clarithromycin, 250 mg twice daily, for 14 days). Before the eradication therapy the mean proteinuria of patients with MN, IgA nephropathy and FSGS were 2.42 ± 3.24 g/day, 2.12 ± 1.63 g/day and 1.80 ± 1.32 g/day, respectively. Three months after eradication, baseline proteinuria levels of patients with MN significantly decreased to 1.26 ± 1.73 g/day (P = 0.031). In all three groups there were no significant differences with regard to serum creatinine, albumin and C-reactive protein levels before and after eradication therapy. Conclusions. The eradication of H. pylori infection may be effective to reduce proteinuria in patients with MN, while spontaneous remission of MN could not be excluded in this patient cohort. This trial is registered with NCT00983034.


Introduction
Membranous nephropathy (MN) is a common cause of nephrotic syndrome (NS) in adults. In most cases it is idiopathic, while it may also be secondary to many diseases (e.g., infections, drugs, neoplasms, and autoimmune diseases) [1][2][3][4][5]. Histologically, MN is characterized by an accumulation of immune deposits on the outer aspect of the glomerular basement membrane. The immune deposits consist of IgG, often IgG4, so far unidentified antigens, and the membrane attack complex of complement C5b-9 [1,6]. Functional impairment of the glomerulus causing proteinuria results from the formation of subepithelial immune deposits and complement activation [7,8]. The key to a specific hypothesisdriven therapy is the understanding of the development of immune deposits, which first requires identification of the pathogenic antigen(s).
In 2009 Beck et al. identified the M-type phospholipase A2 receptor (PLA2R) as an important antigenic target [9]. The authors showed that PLA2R is expressed on podocytes and that antibodies against native PLA2R, primarily of the IgG4 subclass, were present in the serum of approximately 70% of patients with idiopathic MN [9]. The other antigens responsible for human MN have eluded identification, hepatitis B, hepatitis C, and Helicobacter pylori antigens; tumor antigens and thyroglobulin have also been detected in the subepithelial 2 International Journal of Nephrology deposits, but there is no real proof that these antigens are pathogenic [1-5, 10, 11]. However, recent reports addressed the pathogenic role of H. pylori infection in MN [12,13]. In these reports, remission of proteinuria was achieved in some patients following eradication of H. pylori infection [12,13]. These reports might indicate that H. pylori infection might play an important role in the development of this disease. However, no controlled prospective study on this issue has been performed.
In this study, the prevalence of H. pylori infection and the effects of H. pylori eradication on the course of disease in patients with NS were investigated. This prospective study also compared the effects of H. pylori eradication on proteinuria levels in patients with MN versus other primary glomerulonephritides including IgA nephropathy and focal segmental glomerulosclerosis (FSGS). We also investigated the presence of H. pylori antigen in renal tissue from needle biopsy samples, using immunohistochemistry.

Patients.
Fifty-nine patients (31 males and 28 females) with biopsy confirmed primary glomerulonephritis and persistent proteinuria, between ages of 18 and 70 [(mean age 44 ± 14 (range 19-69)], and being followed-up for more than six months were enrolled in this single center prospective study. Patients were grouped according to their diagnosis 1 month prior to the study. Thirty-five patients with MN (19 male, mean age: 46 ± 14 years), 12 patients with IgA nephropathy (4 male, mean age: 40 ± 13 years), and 12 patients with FSGS (8 male, mean age: 42 ± 15 years) were studied. All patients had been routinely screened for serum HBsAg, anti-HCV, antinuclear antibodies (ANA), anti-double-stranded DNA (anti-dsDNA), and complements. Inclusion criteria were biopsyproven idiopathic MN, IgA nephropathy or FSGS, glomerular filtration rate (GFR) > 30 mL/min per 1.73 m 2 , persistent urinary protein excretion rate > 0.5 g/24 h over at least 6 months of full dose angiotensin converting enzyme inhibitors (ACEi) or angiotensin II receptor blockers (ARB) therapy, and no previous complete remissions or treatment with steroids or immunosuppressive drugs during the past year. Exclusion criteria were low (<30 mL/min/1.73 m 2 ) glomerular filtration rate (GFR), diabetes mellitus, secondary glomerulonephritis, viral hepatitis, and history of gastric surgery. The patients who were on antibiotics, bismuth containing drugs, H2 receptor blockers, proton pump inhibitors, and antacids 3 months before the enrollment were excluded from the study.
Body mass index (BMI) was calculated by using the formula of weight/height 2 (kg/m 2 ). Blood pressure (BP) was measured in the sitting position after 5 min of rest with an ERKA sphygmomanometer (PMS Instruments Ltd., Berkshire, UK) with appropriately sized cuff on the right upper arm. Hypertension was defined as systolic BP > 140 mmHg or diastolic BP > 90 mmHg.
Fasting serum samples for biochemical studies were obtained between 8.00 and 8.30 in all cases. Laboratory values including complete blood cell count and serum levels of urea nitrogen (BUN), creatinine, total protein, albumin, total-, HDL-, LDL-, and VLDL-cholesterol, and triglycerides were measured by standard enzymatic procedures. High sensitive C-reactive protein (hs-CRP) levels were measured by nephelometric method (Dade Behring, GmbH, Marburg, Germany). GFR was predicted by using Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation [14]. Urinary protein-to-creatinine ratios (Up/c) were used to measure level of proteinuria.
Examinations of the patients confirmed to good medical and laboratory practices and the recommendations of the Declaration of Helsinki on Biomedical Research involving Human Subjects.
This study was approved by the Istanbul School of Medicine Clinical Studies Board.

Detection of H. pylori Infection.
Rapid HpSA test (Linear Chemicals, Barcelona, Spain) was performed in fresh stool according to the manufacturer's recommendations without knowledge of the H. pylori status by a single biologist. Rapid HpSA test provides a simple alternative to the evaluation of gastric biopsy specimens for H. pylori and the value of test for the diagnosis of H. pylori infection is well documented in the previous reports [15,16]. This test is a rapid 10-minute test, qualitative via a lateral flow chromatography technique. It is simple to perform and has possible advantages over other noninvasive tests, detecting actual antigen indicating current active infection [17]. The test was considered negative if only blue colored band (control line) appeared across the white central area of the reaction strip and positive when, in addition to the control line, a distinguishable pinkish red band (test line) also appeared across the central zone of the reaction strip. Any line or color appearing after 10 min has no diagnostic value. Tests were also considered invalid if the control band was absent.

H. pylori Eradication Therapy.
Proton pump inhibitorbased triple therapy was administered to patients whose Rapid HpSA test was found positive. The therapy protocol was as follows: lansoprazole, 30 mg twice daily, plus amoxicillin, 0.75 g twice daily, plus clarithromycin, 250 mg twice

Results
The baseline clinical and laboratory features of the patients are shown in Table 1. There were no differences regarding age, gender, smoking status, systolic/diastolic BP, hemoglobin, glucose, serum creatinine, total cholesterol, LDL cholesterol, triglyceride, total protein, albumin, hs-CRP, GFR, and Up/c levels among the three groups (Table 1). Immunohistochemistry for H. pylori antigen revealed no positive staining in the glomeruli from renal biopsy specimens in all patients. 19 patients (54%) with MN, 10 patients (83%) with IgA nephropathy, and 4 patients (33%) with FSGS were positive for H. pylori stool antigen test ( = 0.045). The baseline clinical and laboratory features of the patients who were positive for H. pylori stool antigen test are shown in Table 2. There were no differences regarding age, gender, smoking status, systolic/diastolic BP, hemoglobin, glucose, total cholesterol, LDL cholesterol, triglyceride, total protein, albumin, hs-CRP, Up/c, and GFR levels among the three groups ( Table 2). The mean serum creatinine level of patients with MN (0.98 ± 0.29 mg/dL) was significantly lower than the patients in the other groups ( = 0.011). Patients with H. pylori infection were administered eradication therapy (lansoprazole, 30 mg twice daily, plus amoxicillin, 0.75 g twice daily, plus clarithromycin, 250 mg twice daily, for 14 days). All patients were receiving angiotensin converting enzyme inhibitors or angiotensin II receptor blockers since 6 months before the study entry. All patients had received immunosuppressive therapy including steroid, cyclosporine A, mycophenolate mofetil, and azathioprine and there was no need to change the treatment during the study period.
Eradication was successful in all patients. No significant side effect was observed in any patient during the eradication therapy. Laboratory features of patients before and after H. pylori eradication therapy are shown in Table 2. Three months after eradication therapy, there were no differences regarding systolic/diastolic BP, hemoglobin, serum glucose, creatinine, total protein, albumin, total cholesterol, LDL cholesterol, triglyceride, hs-CRP, and Up/c levels among the three groups ( Table 2). Before the eradication therapy, the mean urinary protein excretion of patients with MN, IgA nephropathy, and FSGS was 2.42 ± 3.24 g/day, 2.12 ± 1.63 g/day, and 1.80 ± 1.32 g/day, respectively. Three months after eradication therapy, baseline proteinuria levels of patients with MN significantly decreased to 1.26 ± 1.73 g/day ( = 0.031). Decrease was also noted in patients with IgA nephropathy (1.58 ± 1.59 g/day; = 0.13), however without reaching statistical significance. In FSGS group baseline proteinuria levels (1.80±1.32 g/day) increased to 1.91 ± 1.21 g/day, but the difference was not statistically significant ( = 0.91). In all three groups after the eradication therapy there were no significant differences with regard to hemoglobin, serum creatinine, total protein, albumin, total cholesterol, LDL cholesterol and hs-CRP levels compared to baseline values. GFR values after the eradication therapy were also similar to values before the therapy.

Discussion
In the present study, the eradication of H. pylori infection successfully reduced proteinuria in patients with MN. To our knowledge, this is the first study to show that the eradication of H. pylori infection has an ameliorating effect on urinary protein excretion.
Since the first report in 1997 in which Nagashima et al. have reported the presence of H. pylori antigens in the glomeruli of MN patients [11], few reports have been published on the association between H. pylori and MN [10][11][12][13]. In particular, a higher prevalence of H. pylori infection in patients affected by MN has been described and there is growing evidence for an association between H. pylori and MN. A recent study showed a significantly higher H. pylori infection rate (66%) in MN patients than in their control group (44%) consisting of normal healthy subjects [12]. In the present study, the prevalence of H. pylori infection was lower in patients with MN (54%) than in patients with IgA nephropathy (83%). Additionally, the prevalence of H. pylori infection was similar between patients with MN and FSGS. Barratt et al. also reported a high incidence of H. pylori seropositivity in patients with IgA nephropathy and suggested a possible explanation for this high incidence of H. pylori seropositivity [18]. As suggested by this study, in IgA nephropathy, there is an increase in gastric permeability resulting in excessive H. pylori antigenemia and systemic immune activation which may cause high incidence of H. pylori seropositivity [18]. However, the explanation for this high seropositivity rate is still unclear. Although previous International Journal of Nephrology 5 studies may suggest that MN patients are prone to H. pylori infection, it is possible that hypoalbuminemia or hypoglobulinemia (in the case of NS) may predispose patients to becoming immunocompromised and may lead to H. pylori infection [12]. However, in the present study, the serum albumin levels of patients with MN and IgA nephropathy were similar.
Another interesting finding of our study is that, although previous studies detected H. pylori antigens in the glomerulus of renal biopsy specimens from patients with MN, the immunohistochemistry findings in the present study revealed no deposition of H. pylori. These renal biopsies performed at the early stage of the MN for the histological diagnosis; thus, there is a possibility that enough amounts of H. pylori antigens, which can be detected by immunohistochemistry might not have deposited in our patients' glomeruli. However, there were also biopsy specimens showing stage 2 or 3 MN; thus, this hypothesis does not seem likely. On the other hand, there is another possibility that instead of H. pylori antigen, anti-H. pylori antibodies might be reacting with podocyte antigens to form immune deposits in situ under the subepithelium, resulting in the development of MN. If this is true, all MNs in the present study are supposed to be secondary MN, which can be demonstrated by the negative staining of IgG4 and PLA2R1. Although no definitive data are available on the pathogenesis of these phenomena, previous studies showed that antibodies against H. pylori may react with extragastric tissues, such as glomerular capillary walls, ductal cells of the salivary gland, and renal tubular cells [11,19]. However, the mechanisms behind this phenomenon are still unclear.
Previous case reports reported that eradication of H. pylori markedly reduced urinary protein excretions in four of five MN patients who were also receiving corticosteroid therapy [12,13]. The effects of H. pylori eradication on proteinuria levels in patients with MN versus other primary glomerulonephritides including IgA nephropathy and FSGS were assessed for the first time, in this cohort study. Three months after the eradication therapy, significant decrease in proteinuria levels and increase in serum albumin levels were noted in patients with MN. Decrease in proteinuria levels was also noted in patients with IgA nephropathy, however without reaching statistical significance. Patients with IgA nephropathy, FSGS, and MN may response differently to the therapy in terms of alleviation of proteinuria. However, these results also suggest that eradication of H. pylori infection has an ameliorating effect on urinary protein excretion in the course of MN. In other words, H. pylori infection may be a secondary etiological factor in MN and the eradication of this infection might be an affirmative therapy for MN patients with H. pylori infection. However, spontaneous remission of MN could not be excluded in this patient cohort. Previous studies reported that 10% of nontreated patients with MN underwent spontaneous remission at 12 months, 16% at 24 months, and 22% at 36 months [20]. Therefore, the reduction of proteinuria in the present study might either be the consequence of eradication therapy or represent spontaneous remission of MN. In order to minimize the effect of spontaneous remission on the results, the patient cohort was enrolled in this study after a six-month period of followup.
In conclusion, the eradication of H. pylori infection successfully reduced proteinuria in patients with MN, while spontaneous remission of MN could not be excluded in this patient cohort. Further well designed in vitro, epidemiological, and controlled intervention studies are needed in order to identify whether and by which molecular mechanisms H. pylori may play role in MN pathogenesis.