Original article 65
Hepatitis B virus-associated glomerulonephritis in HBsAg serological-negative patients Dan Li, Gao Gao, Hong Jiang, Zhenghao Tang, Yongsheng Yu and Guoqing Zang Objective The main objective of this study is to report the clinical and pathological characteristics of hepatitis B virus (HBV)-associated glomerulonephritis (HBV-GN) in serum hepatitis B surface antigen (HBsAg)-negative patients in China. HBV-GN is caused by the HBV’s attack on the kidney tissues, but definitive diagnostic criteria are still lacking. The diagnostic criteria used in China require HBsAg positivity in the serum, but research on occult HBV infection has shown that HBV infection is also found in serum HBsAg-negative patients. Clinical and pathological characterization of HBVGN in serum HBsAg-negative patients is required. Materials and methods Serologic and clinical findings and pathological characteristics of renal tissues in 18 HBVGN patients (11 men and seven women) with serum HBsAg negativity were analyzed retrospectively. Thirty-three HBVGN patients with serum HBsAg positivity and 59 patients with membranous nephropathy (MN) without any HBV infection markers in serum and renal tissues during the same period were included as controls. Results Among the 18 HBsAg-negative patients with GN, 12 had HBsAb positivity in their sera. None of the patients was positive for serum HBeAg. Proteinuria was the major clinical manifestation and the renal histopathology was
Introduction Since the first report of the association between chronic hepatitis B virus (HBV) infection and glomerular diseases in 1971 [1], almost all morphological forms of renal diseases including membranous nephropathy (MN), membranoproliferative glomerulonephritis (GN), mesangial proliferative GN, minimal change disease, immunoglobulin A (IgA) nephropathy, and focal segmental glomerulosclerosis have been described [2–4]. The isolation of immune complexes in the kidney suggests that the pathogenesis of the disease may have an immune complex basis. The pathogenetic mechanisms by which certain individuals with chronic HBV infection develop GN are unknown and numerous in-vitro investigations have supported a role for hepatitis B surface antigen (HBsAg) by various mechanisms [5,6]. Occult hepatitis B is defined by the presence of HBV DNA in the serum or the liver in the absence of HBsAg in the serum [7,8]. The diagnosis depends on the sensitivity of HBV DNA assays and the prevalence of HBV infection in the population [9,10]. The serum HBV DNA 0954-691X © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
characterized as MN. Immune fluorescence deposits in renal tissues consisted mainly of HBsAg. The degree of renal injury and the decrease in the C3 level were less than those in HBsAg-positive patients and idiopathic membranous nephropathy patients. Conclusion We propose to use the HBV marker in renal tissues as a new diagnostic criterion for HBV-GN. If MN patients have HBV marker positivity in renal tissues, HBVGN can be diagnosed even without HBsAg in the serum. This would improve the diagnostic accuracy and potential treatment efficiency. Eur J Gastroenterol Hepatol 27:65–69 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. European Journal of Gastroenterology & Hepatology 2015, 27:65–69 Keywords: glomerulonephritis, hepatitis B surface antigen, hepatitis B virus infection Department of Infectious Disease, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, People’s Republic of China Correspondence to Guoqing Zang, PhD, 600 Yishan Road, Xujiahui District, Shanghai 200233, People’s Republic of China Tel: + 86 21 24058673; fax: + 86 21 24058659; e-mail: [email protected] Received 5 August 2014 Accepted 3 October 2014
level in these patients is generally lower than 104 copies/ ml [11]. To date, it remains unclear as to whether HBVGN occurs in patients with serum HBsAg negativity. China is an endemic area of HBV infection. It is reported that about 30–35% patients with chronic liver disease of unknown causes have HBsAg negativity in the serum and/or peripheral blood mononuclear cells [12,13]. Yet, little is known about HBV-GN with serum HBsAg negativity. This study attempts to shed some light on the prevalence and the clinical and pathological characteristics of HBV-GN with serum HBsAg negativity.
Materials and methods The clinical data
A total of 51 patients from 552 patients, who underwent renal biopsies and were diagnosed with HBV-GN from January 2008 to June 2011, were enrolled in this study. Eighteen patients (35.3%) were found to have HBsAg negativity in the serum whereas the other 33 patients (64.7%) were HBsAg positive. None of them had received antiviral treatment before the biopsy. Fifty-nine DOI: 10.1097/MEG.0000000000000236
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66 European Journal of Gastroenterology & Hepatology 2015, Vol 27 No 1
idiopathic membranous nephropathy (IMN) patients, diagnosed during the same period and without any HBV infection markers in serum or renal tissue, were included as negative controls. Pathological classification and diagnosis criteria
The lesions of the 51 patients with glomerular nephritis were classified according to the 1990 WHO Classification Criteria [14]. The diagnosis of nephropathy was confirmed by pathology. Sections from all biopsy specimens were also stained routinely with hematoxylin and eosin (H&E), periodic acid-sliver methenamine, Masson’s trichrome, and antibodies against IgA, IgG, IgM, C3, C1q, and fibrinogen (Fib). All antibodies were fluorescein isothiocyanate (FITC) labeled and purchased from Dako (Dako A/S, Glostrup, Denmark). HBsAg and hepatitis B core antigen (HBcAg) were detected using an immunohistochemical method with monoclonal antibody mouse anti-HBsAg, polyclonal rabbit anti-HBcAg, polyclonal rabbit anti-mouse Igs/FITC, and polyclonal swine anti-rabbit Igs/FITC from Dako. Renal function staging and urine and renal parameters
The renal function was staged as chronic kidney disease I–V according to the American standard stages of renal function [15]. Changes in routinely determined urine and renal parameters including urine protein, blood urea nitrogen, and serum creatinine levels were documented. The concentration of protein in the urine was detected quantitatively using the sulfosalicylic acid method. The serum HBV markers and pathological subtypes of patients with high blood urea nitrogen and serum creatinine levels were analyzed.
age 44 years (range 17–72 years). No significant difference was found in age between the three groups. Of the 18 HBsAg-negative patients, 12 patients showed HBsAb positivity in their sera. None was positive for serum HBeAg. Positive anti-HBe, HBcAb, and anti-HBs were found in one, one, and 12 patients, respectively. Serological markers of HBV of the participants are shown in Table 1. Serologic and clinical findings
Proteinuria was the major clinical manifestation among the 51 HBV-GN patients, with 7.51 ± 4.18 g/24 h in the HBsAg-positive group and 5.61 ± 2.01 g/24 h in the HBsAg-negative group. Microscopic hematuria was found in 37 patients, with 27 in the HBsAg-positive group and 10 in the HBsAg-negative group. Thirty-five patients showed clinical manifestation of nephrotic syndrome, 21 in the HBsAg-positive group and 14 in the HBsAg-negative group (Table 2). Thirteen patients showed mild liver dysfunction and six patients showed renal dysfunction. No correlation was found between the degree of liver dysfunction and renal dysfunction. There was no significant difference between the two groups in the levels of serum albumin, triglyceride and aminotransferase, and proteinuria, except C3 and creatinine (P = 0.001 and 0.004, respectively). Of the 18 HBsAgnegative patients, hypocomplementemia was more severe in the 12 HBVAb patients than in the six HBV marker-negative patients (P = 0.048).
Ethics approval was obtained from the ethical committee of The Sixth Hospital, Shanghai Jiao Tong University, China.
The proteinuria level was 5.87 ± 2.45 g/24 h in the 59 IMN patients. Clinical findings showed nephrotic syndrome in 51 cases, renal dysfunction in eight cases, and liver dysfunction none of the cases compared with the HBsAg-positive GN group, a significant difference was found in the serum levels of creatinine and complement (P = 0.018 and 0.008, respectively).
Statistical analysis
Pathological characteristics of renal tissues
The statistical analysis was carried out using SPSS, version 16.0 (SPSS Inc., Chicago, Illinois, USA). Quantitative variables were expressed as mean values and SEs and those with abnormal distributions as median values and ranges. The Mann–Whitney test was used to compare quantitative variables between groups. Yate’s correction of contingency was used for qualitative data when appropriate. Significance was accepted at 5%.
The renal histopathology was characterized as MN in all 18 HBsAg-negative GN patients and in 32 HBsAgpositive GN patients, except one, who had membranoproliferative GN. Detectable rates of HBsAg, HBcAg,
Results Clinical characteristics of the participants
Of 18 HBsAg-negative patients with GN, 11 were men and seven were women, average age 38 years (range 18–68 years). Of 33 HBsAg-positive patients with GN, 23 were men and 10 were women, average age 47 years (range 20–60 years). Fifty-nine patients diagnosed with IMN during the same period were included in the control group. In all, 33 were men and 26 were women, average
Table 1
Distribution of serum markers
HBV markers HBsAg (+ ) HBeAg (+ ) HBsAg, HBeAg HBsAg, HBeAb HBsAg, HBcAb HBsAb, HBeAb HBsAb (+ ) HBeAb (+ ) Negative HBV DNA (+ )
(+ ) (+ ) (+ ) (+ )
HBsAg positive
HBsAg negative
IMN
33 6 6 24 3 0 0 24 0 15
0 0 0 0 0 1 12 1 6 0
6 3 3 2 2 2 32 6 21 2
Ab, antibody; Ag, antigen; HBcAg, hepatitis B core antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; IMN, idiopathic membranous nephropathy.
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Hepatitis B infection and glomerulonephritis Li et al. 67
Table 2
Indicators of clinical and biochemical in different groups
Indicators Number of cases Proteinuria (g/24 h) Albumin (g/l) Creatinine Aminotransferase Triglyceride C3 complement
HBsAg-positive GN
HBsAg-negative GN
IMN
33 7.51 ± 4.18 22.1 ± 10.1 110.2 ± 42.6 33 ± 17 3.36 ± 3.49 0.84 ± 0.26b
18 5.61 ± 2.01 26.5 ± 8.8 78.3 ± 19.7 22 ± 14 3.11 ± 3.43 1.36 ± 0.42
59 5.87 ± 2.45 24.2 ± 7.6 86.2 ± 31.2a 22 ± 12 2.65 ± 2.46 1.16 ± 0.36a
Ag, antigen; GN, glomerulonephritis; HBsAg, hepatitis B surface antigen; IMN, idiopathic membranous nephropathy. a Versus the HBsAg-positive GN group, P < 0.05. b Versus the HBsAg-negative GN group, P < 0.05.
Table 3
Immune fluorescence distribution of renal tissues
Indicators IgA IgG IgM C1q C3 Fib HBsAg HBcAg HBsAg + HBcAg
HBsAg-positive GN
HBsAg-negative GN
HBV-GN
IMN
23 33 15 24 21 13 21 12 6
16a 18 8 16a 17 10a 16 6 5
39a 51 23 40a 38a 23a 37 18 11
27 59 23 22 42 13 0 0 0
Ag, antigen; Fib, fibrinogen; GN, glomerulonephritis; HBcAg, hepatitis B core antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; Ig, immunoglobulin; IMN, idiopathic membranous nephropathy. a Versus the IMN group, P < 0.05.
and HBsAg + HBcAg were 63.6, 33.4, and 18.2% in the HBsAg-positive GN group and 88.9, 33.3, and 27.8% in the HBsAg-negative GN group, respectively. The immune fluorescence distribution of renal tissues is shown in Table 3. No significant difference was found in the deposition rate of immune fluorescence and detectable rate of HBV markers in renal tissues between the HBsAg-positive and HBsAg-negative groups. Electron microscopy showed more extensive subendothelial electron dense deposits in the mesangium and subendothelial space in the HBsAg-positive group than in the HBsAg-negative group, although this was not significant. These glomerular deposits consisted mainly of IgG and C3. Seven cases in the HBsAg-positive group and three cases in the HBsAg-negative group were directly diagnosed as having HBV-GN on the basis of the combination of glomerular deposits and electron microscopy findings. The others were characterized as atypical MN (24 cases) or secondary MN (two cases). The deposition of immune complexes showed no statistically significant difference between the two groups. Of the 59 IMN patients, 25 had positive HBV markers in serum. None had positive HBV markers in the renal tissues. The deposits also consisted mainly of IgG and C3. Compared with the HBV-GN group, the deposits of IgA, C1q, C3, and Fib were fewer (P = 0.001, 0.001, 0.029, and 0.01, respectively). Compared with the HBsAg-negative group, the deposits of IgA, C1q, and Fib were fewer (P = 0.001, 0.003, and 0.005, respectively).
This indicated that HBV-GN showed higher intensity deposits with multiple kinds and sites than IMN.
Discussion Occult HBV infection is characterized by positive HBV DNA in HBsAg-negative patients with or without serological markers of a previous HBV infection [11]. HBV may also be reactivated in patients undergoing anticancer chemotherapy [16,17]. Occult HBV infection may occur after complete clinical recovery from acute self-limited hepatitis; thus, HBsAg seroclearance does not necessarily imply HBV eradication [18,19]. In endemic areas, the anti-HBc IgG and anti-HBs as well as HBsAg are not sufficient markers to exclude HBV DNA carriers [20]. Very low viral load of HBV DNA in the serum, mutation of HBsAg, subtype of HBV, and the existence of immunologic escape strains contribute toward the separation of the HBV marker in the serum and in the kidney tissue. Meanwhile, the titer of the HBV antigen in the serum shows wave dynamics, and the increase and decrease in HBV antigen are not synchronized [21–25]. In this study, 18 HBsAg-negative patients showed HBsAg/HBcAg positivity in the kidney tissue, atypical or typical MN in histopathology, and subendothelial electron dense deposits in the mesangium and subendothelial space, supporting the diagnosis of HBV-GN [26,27]. It is interesting to find that two cases of HBsAgnegative GN with HBsAb positivity were induced by HBV vaccination. Whether an HBV vaccine would initiate an autoimmune mechanism requires further study and more samples. In the present study, HBV-GN mainly occurred in men, although the male predominance was less reported. Of the 51 patients with HBV-GN, 35 patients (68.6%) showed clinical manifestation as nephrotic syndrome. Most of them presented with proteinuria and none with isolated hematuria. This was in agreement with the results reported in the literature. There was a segregation phenomenon of C3 levels in the serum and the renal tissues. The serum C3 level was lower in HBsAg-positive patients than in HBsAg-negative patients, whereas the renal tissue C3 level showed no major difference in the two patient groups. A larger sample size and further investigations are needed to assess the pathophysiological effect of the separation of C3 levels in the serum and the renal tissues. Meanwhile, among the HBsAg-negative patients, those with positive HBV markers had a lower serum C3 level than those with negative HBV markers. Investigations on the renal gene expression profile with microarrays in transgenic mice that expressed HBsAg and HBcAg in the cytoplasm of renal tubular epithelial cells, but without replication of the entire virus, have shown upregulation of complement and coagulation pathways and acute-phase response genes and reduced circulating C3 levels [28].
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68 European Journal of Gastroenterology & Hepatology 2015, Vol 27 No 1
The renal damage was more severe in HBsAg-positive patients than in HBsAg-negative patients. This fact, combined with the existence of HBcAg in renal tissues, indicates that the HBV itself might be involved in pathogenesis directly. This needs more proof such as the existence and the pathogenicity of HBV viral particles in renal tissues. The observation of HBV DNA within the glomeruli in patients with GN [29–31] and the identification of both free and integrated HBV DNA in the kidneys of chronic HBsAg carrier suggest that HBV might also infect resident glomerular cells, leading to immune complex formation. Wang and colleagues found that the positive rate of HBV DNA in renal tubular epithelial cells was higher in the renal function serious group than that in the relatively light group. The longer HBV DNA in the renal unit and interstitial cells, the more severe the renal damage. These studies hint at the probability of HBV itself directly attacking renal cells. Thus, doctors need to focus on the change in hepatic function as well as the renal function when evaluating the damage of HBV. The renal damage is related to virus replication and, more importantly, linked to the immune response [32]. In the present study, the detection of HBsAg and HBcAg along with the expression of Ig types and the presence of complement components in the glomerular deposits suggests that an immune complex mechanism leads to glomerular injury [33]. The degree of renal injury and the decreased C3 level were more marked in HBV-GN patients than in IMN patients, indicating that HBV may damage the renal system through the C3 pathway. HBV-GN histopathology is characterized as atypical MN. Besides diffuse irregular thickening of the basement membrane, mesangial cells, and matrix hyperplasia, the tubulointerstitial system was also slightly damaged. Immune complex was extensively deposited at multiple sites. Different from typical MN deposits of IgG and C3, HBV-GN deposits consist of IgG, IgM, IgA, C3, and C1q. This is in agreement with other reports [29,34,35]. This indicates that the pathogenesis of HBV-MN involves insitu immune complexes and circular immune complexes. To date, there are no specific drugs or therapeutic maneuvers for HBV-GN. Some reports support antiviral treatment combined with hormone and immune inhibitor treatment [36,37]. However, so far, there is no report on the treatment of seronegative HBV-GN patients by the same modality as the IMN or HBV-GN-seropositive group. In this study, unfortunately, the treatment records of HBV-GN in patients with serum HBsAg negativity and positivity could not be tracked. Thus, the prognosis of serum HBsAg-negative HBV-GN also requires investigation.
mainly of HBsAg. The detection of HBV markers in renal biopsy specimens irrespective of the HBsAg status in serum is a positive finding. In our opinion, if MN patients have positive HBV markers in renal tissues, HBV-GN may be diagnosed even without HBsAg in serum. The findings would improve the diagnostic accuracy and potential treatment efficiency in HBV-GN.
Acknowledgements The authors thank Dr Wanzhan Liu, Minnesota, USA, for proof reading the manuscript for English language. This research is supported by a grant from the National Natural Science Fund For Young Scholars (No. 81102279). Conflicts of interest
There are no conflicts of interest.
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Conclusion
HBV-GN with serum HBsAg negativity occurs mainly in men with histopathology characterized as atypical MN, and immune fluorescence deposits in renal tissues consist
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Hepatitis B virus-associated glomerulonephritis in HBsAg serological-negative patients Dan Li, Gao Gao, Hong Jiang, Zhenghao Tang, Yongsheng Yu and Guoqing Zang Objective The main objective of this study is to report the clinical and pathological characteristics of hepatitis B virus (HBV)-associated glomerulonephritis (HBV-GN) in serum hepatitis B surface antigen (HBsAg)-negative patients in China. HBV-GN is caused by the HBV’s attack on the kidney tissues, but definitive diagnostic criteria are still lacking. The diagnostic criteria used in China require HBsAg positivity in the serum, but research on occult HBV infection has shown that HBV infection is also found in serum HBsAg-negative patients. Clinical and pathological characterization of HBVGN in serum HBsAg-negative patients is required. Materials and methods Serologic and clinical findings and pathological characteristics of renal tissues in 18 HBVGN patients (11 men and seven women) with serum HBsAg negativity were analyzed retrospectively. Thirty-three HBVGN patients with serum HBsAg positivity and 59 patients with membranous nephropathy (MN) without any HBV infection markers in serum and renal tissues during the same period were included as controls. Results Among the 18 HBsAg-negative patients with GN, 12 had HBsAb positivity in their sera. None of the patients was positive for serum HBeAg. Proteinuria was the major clinical manifestation and the renal histopathology was
Introduction Since the first report of the association between chronic hepatitis B virus (HBV) infection and glomerular diseases in 1971 [1], almost all morphological forms of renal diseases including membranous nephropathy (MN), membranoproliferative glomerulonephritis (GN), mesangial proliferative GN, minimal change disease, immunoglobulin A (IgA) nephropathy, and focal segmental glomerulosclerosis have been described [2–4]. The isolation of immune complexes in the kidney suggests that the pathogenesis of the disease may have an immune complex basis. The pathogenetic mechanisms by which certain individuals with chronic HBV infection develop GN are unknown and numerous in-vitro investigations have supported a role for hepatitis B surface antigen (HBsAg) by various mechanisms [5,6]. Occult hepatitis B is defined by the presence of HBV DNA in the serum or the liver in the absence of HBsAg in the serum [7,8]. The diagnosis depends on the sensitivity of HBV DNA assays and the prevalence of HBV infection in the population [9,10]. The serum HBV DNA 0954-691X © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
characterized as MN. Immune fluorescence deposits in renal tissues consisted mainly of HBsAg. The degree of renal injury and the decrease in the C3 level were less than those in HBsAg-positive patients and idiopathic membranous nephropathy patients. Conclusion We propose to use the HBV marker in renal tissues as a new diagnostic criterion for HBV-GN. If MN patients have HBV marker positivity in renal tissues, HBVGN can be diagnosed even without HBsAg in the serum. This would improve the diagnostic accuracy and potential treatment efficiency. Eur J Gastroenterol Hepatol 27:65–69 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. European Journal of Gastroenterology & Hepatology 2015, 27:65–69 Keywords: glomerulonephritis, hepatitis B surface antigen, hepatitis B virus infection Department of Infectious Disease, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, People’s Republic of China Correspondence to Guoqing Zang, PhD, 600 Yishan Road, Xujiahui District, Shanghai 200233, People’s Republic of China Tel: + 86 21 24058673; fax: + 86 21 24058659; e-mail: [email protected] Received 5 August 2014 Accepted 3 October 2014
level in these patients is generally lower than 104 copies/ ml [11]. To date, it remains unclear as to whether HBVGN occurs in patients with serum HBsAg negativity. China is an endemic area of HBV infection. It is reported that about 30–35% patients with chronic liver disease of unknown causes have HBsAg negativity in the serum and/or peripheral blood mononuclear cells [12,13]. Yet, little is known about HBV-GN with serum HBsAg negativity. This study attempts to shed some light on the prevalence and the clinical and pathological characteristics of HBV-GN with serum HBsAg negativity.
Materials and methods The clinical data
A total of 51 patients from 552 patients, who underwent renal biopsies and were diagnosed with HBV-GN from January 2008 to June 2011, were enrolled in this study. Eighteen patients (35.3%) were found to have HBsAg negativity in the serum whereas the other 33 patients (64.7%) were HBsAg positive. None of them had received antiviral treatment before the biopsy. Fifty-nine DOI: 10.1097/MEG.0000000000000236
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66 European Journal of Gastroenterology & Hepatology 2015, Vol 27 No 1
idiopathic membranous nephropathy (IMN) patients, diagnosed during the same period and without any HBV infection markers in serum or renal tissue, were included as negative controls. Pathological classification and diagnosis criteria
The lesions of the 51 patients with glomerular nephritis were classified according to the 1990 WHO Classification Criteria [14]. The diagnosis of nephropathy was confirmed by pathology. Sections from all biopsy specimens were also stained routinely with hematoxylin and eosin (H&E), periodic acid-sliver methenamine, Masson’s trichrome, and antibodies against IgA, IgG, IgM, C3, C1q, and fibrinogen (Fib). All antibodies were fluorescein isothiocyanate (FITC) labeled and purchased from Dako (Dako A/S, Glostrup, Denmark). HBsAg and hepatitis B core antigen (HBcAg) were detected using an immunohistochemical method with monoclonal antibody mouse anti-HBsAg, polyclonal rabbit anti-HBcAg, polyclonal rabbit anti-mouse Igs/FITC, and polyclonal swine anti-rabbit Igs/FITC from Dako. Renal function staging and urine and renal parameters
The renal function was staged as chronic kidney disease I–V according to the American standard stages of renal function [15]. Changes in routinely determined urine and renal parameters including urine protein, blood urea nitrogen, and serum creatinine levels were documented. The concentration of protein in the urine was detected quantitatively using the sulfosalicylic acid method. The serum HBV markers and pathological subtypes of patients with high blood urea nitrogen and serum creatinine levels were analyzed.
age 44 years (range 17–72 years). No significant difference was found in age between the three groups. Of the 18 HBsAg-negative patients, 12 patients showed HBsAb positivity in their sera. None was positive for serum HBeAg. Positive anti-HBe, HBcAb, and anti-HBs were found in one, one, and 12 patients, respectively. Serological markers of HBV of the participants are shown in Table 1. Serologic and clinical findings
Proteinuria was the major clinical manifestation among the 51 HBV-GN patients, with 7.51 ± 4.18 g/24 h in the HBsAg-positive group and 5.61 ± 2.01 g/24 h in the HBsAg-negative group. Microscopic hematuria was found in 37 patients, with 27 in the HBsAg-positive group and 10 in the HBsAg-negative group. Thirty-five patients showed clinical manifestation of nephrotic syndrome, 21 in the HBsAg-positive group and 14 in the HBsAg-negative group (Table 2). Thirteen patients showed mild liver dysfunction and six patients showed renal dysfunction. No correlation was found between the degree of liver dysfunction and renal dysfunction. There was no significant difference between the two groups in the levels of serum albumin, triglyceride and aminotransferase, and proteinuria, except C3 and creatinine (P = 0.001 and 0.004, respectively). Of the 18 HBsAgnegative patients, hypocomplementemia was more severe in the 12 HBVAb patients than in the six HBV marker-negative patients (P = 0.048).
Ethics approval was obtained from the ethical committee of The Sixth Hospital, Shanghai Jiao Tong University, China.
The proteinuria level was 5.87 ± 2.45 g/24 h in the 59 IMN patients. Clinical findings showed nephrotic syndrome in 51 cases, renal dysfunction in eight cases, and liver dysfunction none of the cases compared with the HBsAg-positive GN group, a significant difference was found in the serum levels of creatinine and complement (P = 0.018 and 0.008, respectively).
Statistical analysis
Pathological characteristics of renal tissues
The statistical analysis was carried out using SPSS, version 16.0 (SPSS Inc., Chicago, Illinois, USA). Quantitative variables were expressed as mean values and SEs and those with abnormal distributions as median values and ranges. The Mann–Whitney test was used to compare quantitative variables between groups. Yate’s correction of contingency was used for qualitative data when appropriate. Significance was accepted at 5%.
The renal histopathology was characterized as MN in all 18 HBsAg-negative GN patients and in 32 HBsAgpositive GN patients, except one, who had membranoproliferative GN. Detectable rates of HBsAg, HBcAg,
Results Clinical characteristics of the participants
Of 18 HBsAg-negative patients with GN, 11 were men and seven were women, average age 38 years (range 18–68 years). Of 33 HBsAg-positive patients with GN, 23 were men and 10 were women, average age 47 years (range 20–60 years). Fifty-nine patients diagnosed with IMN during the same period were included in the control group. In all, 33 were men and 26 were women, average
Table 1
Distribution of serum markers
HBV markers HBsAg (+ ) HBeAg (+ ) HBsAg, HBeAg HBsAg, HBeAb HBsAg, HBcAb HBsAb, HBeAb HBsAb (+ ) HBeAb (+ ) Negative HBV DNA (+ )
(+ ) (+ ) (+ ) (+ )
HBsAg positive
HBsAg negative
IMN
33 6 6 24 3 0 0 24 0 15
0 0 0 0 0 1 12 1 6 0
6 3 3 2 2 2 32 6 21 2
Ab, antibody; Ag, antigen; HBcAg, hepatitis B core antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; IMN, idiopathic membranous nephropathy.
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Hepatitis B infection and glomerulonephritis Li et al. 67
Table 2
Indicators of clinical and biochemical in different groups
Indicators Number of cases Proteinuria (g/24 h) Albumin (g/l) Creatinine Aminotransferase Triglyceride C3 complement
HBsAg-positive GN
HBsAg-negative GN
IMN
33 7.51 ± 4.18 22.1 ± 10.1 110.2 ± 42.6 33 ± 17 3.36 ± 3.49 0.84 ± 0.26b
18 5.61 ± 2.01 26.5 ± 8.8 78.3 ± 19.7 22 ± 14 3.11 ± 3.43 1.36 ± 0.42
59 5.87 ± 2.45 24.2 ± 7.6 86.2 ± 31.2a 22 ± 12 2.65 ± 2.46 1.16 ± 0.36a
Ag, antigen; GN, glomerulonephritis; HBsAg, hepatitis B surface antigen; IMN, idiopathic membranous nephropathy. a Versus the HBsAg-positive GN group, P < 0.05. b Versus the HBsAg-negative GN group, P < 0.05.
Table 3
Immune fluorescence distribution of renal tissues
Indicators IgA IgG IgM C1q C3 Fib HBsAg HBcAg HBsAg + HBcAg
HBsAg-positive GN
HBsAg-negative GN
HBV-GN
IMN
23 33 15 24 21 13 21 12 6
16a 18 8 16a 17 10a 16 6 5
39a 51 23 40a 38a 23a 37 18 11
27 59 23 22 42 13 0 0 0
Ag, antigen; Fib, fibrinogen; GN, glomerulonephritis; HBcAg, hepatitis B core antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; Ig, immunoglobulin; IMN, idiopathic membranous nephropathy. a Versus the IMN group, P < 0.05.
and HBsAg + HBcAg were 63.6, 33.4, and 18.2% in the HBsAg-positive GN group and 88.9, 33.3, and 27.8% in the HBsAg-negative GN group, respectively. The immune fluorescence distribution of renal tissues is shown in Table 3. No significant difference was found in the deposition rate of immune fluorescence and detectable rate of HBV markers in renal tissues between the HBsAg-positive and HBsAg-negative groups. Electron microscopy showed more extensive subendothelial electron dense deposits in the mesangium and subendothelial space in the HBsAg-positive group than in the HBsAg-negative group, although this was not significant. These glomerular deposits consisted mainly of IgG and C3. Seven cases in the HBsAg-positive group and three cases in the HBsAg-negative group were directly diagnosed as having HBV-GN on the basis of the combination of glomerular deposits and electron microscopy findings. The others were characterized as atypical MN (24 cases) or secondary MN (two cases). The deposition of immune complexes showed no statistically significant difference between the two groups. Of the 59 IMN patients, 25 had positive HBV markers in serum. None had positive HBV markers in the renal tissues. The deposits also consisted mainly of IgG and C3. Compared with the HBV-GN group, the deposits of IgA, C1q, C3, and Fib were fewer (P = 0.001, 0.001, 0.029, and 0.01, respectively). Compared with the HBsAg-negative group, the deposits of IgA, C1q, and Fib were fewer (P = 0.001, 0.003, and 0.005, respectively).
This indicated that HBV-GN showed higher intensity deposits with multiple kinds and sites than IMN.
Discussion Occult HBV infection is characterized by positive HBV DNA in HBsAg-negative patients with or without serological markers of a previous HBV infection [11]. HBV may also be reactivated in patients undergoing anticancer chemotherapy [16,17]. Occult HBV infection may occur after complete clinical recovery from acute self-limited hepatitis; thus, HBsAg seroclearance does not necessarily imply HBV eradication [18,19]. In endemic areas, the anti-HBc IgG and anti-HBs as well as HBsAg are not sufficient markers to exclude HBV DNA carriers [20]. Very low viral load of HBV DNA in the serum, mutation of HBsAg, subtype of HBV, and the existence of immunologic escape strains contribute toward the separation of the HBV marker in the serum and in the kidney tissue. Meanwhile, the titer of the HBV antigen in the serum shows wave dynamics, and the increase and decrease in HBV antigen are not synchronized [21–25]. In this study, 18 HBsAg-negative patients showed HBsAg/HBcAg positivity in the kidney tissue, atypical or typical MN in histopathology, and subendothelial electron dense deposits in the mesangium and subendothelial space, supporting the diagnosis of HBV-GN [26,27]. It is interesting to find that two cases of HBsAgnegative GN with HBsAb positivity were induced by HBV vaccination. Whether an HBV vaccine would initiate an autoimmune mechanism requires further study and more samples. In the present study, HBV-GN mainly occurred in men, although the male predominance was less reported. Of the 51 patients with HBV-GN, 35 patients (68.6%) showed clinical manifestation as nephrotic syndrome. Most of them presented with proteinuria and none with isolated hematuria. This was in agreement with the results reported in the literature. There was a segregation phenomenon of C3 levels in the serum and the renal tissues. The serum C3 level was lower in HBsAg-positive patients than in HBsAg-negative patients, whereas the renal tissue C3 level showed no major difference in the two patient groups. A larger sample size and further investigations are needed to assess the pathophysiological effect of the separation of C3 levels in the serum and the renal tissues. Meanwhile, among the HBsAg-negative patients, those with positive HBV markers had a lower serum C3 level than those with negative HBV markers. Investigations on the renal gene expression profile with microarrays in transgenic mice that expressed HBsAg and HBcAg in the cytoplasm of renal tubular epithelial cells, but without replication of the entire virus, have shown upregulation of complement and coagulation pathways and acute-phase response genes and reduced circulating C3 levels [28].
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68 European Journal of Gastroenterology & Hepatology 2015, Vol 27 No 1
The renal damage was more severe in HBsAg-positive patients than in HBsAg-negative patients. This fact, combined with the existence of HBcAg in renal tissues, indicates that the HBV itself might be involved in pathogenesis directly. This needs more proof such as the existence and the pathogenicity of HBV viral particles in renal tissues. The observation of HBV DNA within the glomeruli in patients with GN [29–31] and the identification of both free and integrated HBV DNA in the kidneys of chronic HBsAg carrier suggest that HBV might also infect resident glomerular cells, leading to immune complex formation. Wang and colleagues found that the positive rate of HBV DNA in renal tubular epithelial cells was higher in the renal function serious group than that in the relatively light group. The longer HBV DNA in the renal unit and interstitial cells, the more severe the renal damage. These studies hint at the probability of HBV itself directly attacking renal cells. Thus, doctors need to focus on the change in hepatic function as well as the renal function when evaluating the damage of HBV. The renal damage is related to virus replication and, more importantly, linked to the immune response [32]. In the present study, the detection of HBsAg and HBcAg along with the expression of Ig types and the presence of complement components in the glomerular deposits suggests that an immune complex mechanism leads to glomerular injury [33]. The degree of renal injury and the decreased C3 level were more marked in HBV-GN patients than in IMN patients, indicating that HBV may damage the renal system through the C3 pathway. HBV-GN histopathology is characterized as atypical MN. Besides diffuse irregular thickening of the basement membrane, mesangial cells, and matrix hyperplasia, the tubulointerstitial system was also slightly damaged. Immune complex was extensively deposited at multiple sites. Different from typical MN deposits of IgG and C3, HBV-GN deposits consist of IgG, IgM, IgA, C3, and C1q. This is in agreement with other reports [29,34,35]. This indicates that the pathogenesis of HBV-MN involves insitu immune complexes and circular immune complexes. To date, there are no specific drugs or therapeutic maneuvers for HBV-GN. Some reports support antiviral treatment combined with hormone and immune inhibitor treatment [36,37]. However, so far, there is no report on the treatment of seronegative HBV-GN patients by the same modality as the IMN or HBV-GN-seropositive group. In this study, unfortunately, the treatment records of HBV-GN in patients with serum HBsAg negativity and positivity could not be tracked. Thus, the prognosis of serum HBsAg-negative HBV-GN also requires investigation.
mainly of HBsAg. The detection of HBV markers in renal biopsy specimens irrespective of the HBsAg status in serum is a positive finding. In our opinion, if MN patients have positive HBV markers in renal tissues, HBV-GN may be diagnosed even without HBsAg in serum. The findings would improve the diagnostic accuracy and potential treatment efficiency in HBV-GN.
Acknowledgements The authors thank Dr Wanzhan Liu, Minnesota, USA, for proof reading the manuscript for English language. This research is supported by a grant from the National Natural Science Fund For Young Scholars (No. 81102279). Conflicts of interest
There are no conflicts of interest.
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Conclusion
HBV-GN with serum HBsAg negativity occurs mainly in men with histopathology characterized as atypical MN, and immune fluorescence deposits in renal tissues consist
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