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ROLE OF HEPATITIS B IN GLOMERULONEPHRITIS

Deepak N Amarapurkar*, Ashok L Kirpalani**,Anjali D Amarapurkar***
*Dept. of Gastroenterology-Hepatology, **Dept. of Nephrology, Bombay Hospital and Medical Research Centre and ***Dept. of Pathology BYL Nair Charitable Hospital and T N Medical College, Mumbai, India.

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Aim : To study relationship between hepatitis B infection and glomerulonephritis (GN)

Methods : Total 23 patients of glomerulonephritis and 5 patients with post renal transplant suspected to have rejection were evaluated, presenting with heavy proteinuria having normal BUN and serum creatinine. These were divided into two groups. HBsAg positive with GN 10 cases and HBsAg negative GN 18 cases. All patients were assessed for liver function tests, HBV markers, renal profile, kidney biopsies and liver biopsy whenever possible. Kidney tissue was stained with routine H and E, PAS, Gomori and immunoperoxidase stain for hepatitis B surface and core antigen. Electron microscopy was performed whenever possible. None of the 10 patients had overt liver disease.

Result : Out of 10 cases of HBsAg positive GN, 4 had membranous GN, 6 proliferative GN on histology. Except 2 cases, all showed deposition of surface and core antigen on glomerular basement membrane and also in tubular epithelium. 3/10 patients with active viral replication with GN were treated with interferon for 4 months showed excellent results. From 18 patients of HBsAg negative glomerular disease, 13 were cases of GN and 5 renal allograft rejection patients. 3/5 post transplant patients and 5/13 patients with GN showed positive staining for hepatitis B surface and core antigen. Kidney tissue obtained from autopsy in 6 patients with HBsAg +ve (liver cirrhosis 3, CAH 2, chronic carrier 1) who had no clinical or biochemical evidence of renal disease were also studied. None of these patients showed any deposits in the kidney.

Conclusion : To establish the relation between HBV and glomerular disease one needs to consider serological tests for HBV infection and kidney biopsy staining for HBV markers. Those patients who have active viral replication and glomerular disease need to be treated with interferon therapy.

 

INTRODUCTION

Hepatitis B virus (HBV) infection has been implicated in several extrahepatic disorders, most of which are mediated by immune complexes. [1-5] Renal disease is one of the HBV infection manifestations well documented in the literature. [6-8] Glomerulonephritis (GN) associated with HBV infection was first described by Combes et al, who showed Australia antigen antibody complexes in the glomerular basement membrane of an adult with membranous GN after post transfusion hepatitis. [9] Since then, many studies and case reports have been published linking HBV infection with immune complex induced GN.10-19 Several types of glomerular lesions have been described, membranous glomerulonephritis (MGN) and membranoproliferative GN (MPGN) being the most common.11-16 All the three antigens of HBV (HBsAg, HbcAg, HbeAg) have been demonstrated to be associated with HBV-GN.1,12,20 HBV associated GN occurs mainly in children, predominantly in males in areas where HBV infection is endemic. [11-14] , [17] , [18] , [21] There are very few adult patients reported, with the similar association. [2], [18] , [19] The diagnosis of HBV associated GN is established by serologic evidence of HBV antigen or antibodies, presence of immune complex GN on kidney biopsy and by demonstration of one or more HBV related antigens by immunohistochemistry.2 The prognosis of HBV associated GN is favourable in children as compared to adults. Few may progress to renal failure. [2], [13] , [21] The specific aims of this study were to demonstrate the HBV virus antigen antibody complexes in the kidney tissue and to study the relationship of hepatitis B virus with GN.

MATERIAL AND METHODS

Total 23 patients presenting with nephrotic range proteinuria, and 5 patients post renal transplant suspected to have rejection having normal serum creatinine and blood urea nitrogen, were included in this study. None of them had any complaints related to liver disease. All the patients were assessed for serological HBV markers (HBsAg, HBeAg, Anti HBeAg, IgM AntiHBc, HBVDNA by PCR), liver function tests, renal profile, kidney biopsies and liver biopsy whenever possible. Patients were grouped into two categories. Group I - HBsAg positive with GN 10 cases. Group II- HBsAg negative with GN 13 and 5 post renal transplant cases. HBsAg positive chronic liver disease patients without GN (autopsy study) - 6 cases served as controls.

Kidney biopsies taken in each patient were studied by light, immunoperoxidase and electron microscopy. For the light microscopic examination, sections were stained with H and E, periodic acid schiff (PAS) and Gomori Silver methenamine stain to establish the primary diagnosis of GN. Immunoperoxidase staining was done on the paraffin embeded tissue for the detection of hepatitis B surface and core antigen. HBsAg and HbcAg were stained with polyclonal rabbit anti HBs and anti HBc antibodies with the dilution of 1 : 4000 and 1 : 2000 respectively (Dako corporation). The reaction sites were shown by 3-3 diaminobenzedine tetrahydrochloride at PH 7.6. Positive results gave brown colour deposits. For the control, anti HBs and anti HBc antibodies were replaced by phosphate buffer saline. All the slides were studied for the detection of antigen antibody complexes. The results were noted by pathologist who was unaware of the HBV markers.

TABLE 1 : GR I : HBs Ag +ve, with GN (10 cases)
Age	Sex	Kidney Histology	HBs Ag	HBe Ag	Anti HBe Ag	Liver histology	Immuno- peroxidase surface and core Ag
35	F	Membranous GN	+	—	+	Ground glass appearance	Positive
30	M	Proliferative GN	+	—	+	Chronic hepatitis	Negative
45	F	Membranous GN	+	—	+	Near normal	Positive
38	M	Proliferative GN	+	—	+	Ground glass appearance	Positive
27	M	Proliferative GN	+	+	—	Chronic hepatitis	Positive
42	M	Membranous GN	+	—	+	Ground glass appearance	Positive
30	F	Proliferative GN	+	+	—	Chronic hepatitis	Positive
23	M	Proliferative GN	+	+	—	Chronic hepatitis	Positive
40	M	Membranous GN + PAN	+	—	+	Chronic hepatitis	Negative
54	M	Proliferative GN	+	+	—	Chronic hepatitis	Positive

3/10 patients showing active viral replication were treated with antiviral therapy showed excellent results.

RESULTS

From Group I, out of 10 cases, 4 were membranous GN and 6 proliferative GN on histology (Table 1). PAS and Gomori stains revealed spikes along glomerular basement membrane and few showed wire loop appearance. Majority of them showed subendothelial or mesengial electrondense deposits on electronmicroscopy. Except two cases all showed deposition of surface and core antigen in the glomerular basement membrane or mesengium and also in the tubular epithelium (8 cases). Overall staining intensity for the core antigen was found to be stronger than surface antigen. Liver biopsies performed in all 10 cases showed chronic active hepatitis (6 cases) and chronic persistent hepatitis (4 cases). Three patients in this group showing active viral replication were treated with antiviral therapy. They were followed up for 18 months. Seroconversion occurred with disappearance of proteinuria.

From Group II, of 18 patients with glomerular disease, 13 were GN and 5 renal allograft rejection cases. In this group, 5 of 13 and 3 of 5 patients showed positive staining for surface and core antigen in the glomeruli or in the tubules (Table 2). In control group 6 patients with HBsAg positive without GN (autopsy data), liver histology was cirrhosis in 3, chronic active hepatitis 2, and chronic HBsAg carrier was seen in one case. These patients did not show any clinical or biochemical evidence of renal disease. None of them showed surface or core antigen deposits in the kidney (Table 3).

TABLE 2 GR : II : HB sAg negative, with GN (18 cases)
Kidney Histology	No of Cases	HBsAg	Immunoperoxidase surface and core antigen
Post transplant rejection (acute/chronic)	5	Negative	3 Positive
Membranous GN	4	Negative	Negative
Proliferative GN	5	Negative	3 Positive
Proliferative GN	2	Negative	Negative
Proliferative GN	2	Negative	2 Positive

TABLE 3 GR : III : HbsAg positive, no GN, (6 cases) (autopsy data)
Liver Histology	HB sAg(in serum)	Kidney Histology	Immunoper- oxidase surface and core Antigen
Cirrhosis	Positive	Normal	Negative
Cirrhosis	Positive	Normal	Negative
Cirrhosis	Positive	Normal	Negative
Chronic active Hepatitis	Positive	Normal	Negative
Chronic persistent Hepatitis	Positive	Normal	Negative
Chronic persistent Hepatitis	Positive	Normal	Negative

 

TABLE 4 Immunoperoxidase positivity in HB sAg negative GN (8 cases)
Age Years	Sex	Kidney Histology	Immuno-peroxidase surface and core Ag	Remarks
45	M	Post transplant chronic rejection	Positive	HBV +ve, HCV+ve with retrospect analysis
55	M	Post transplant chronic rejection	Positive	Anti HBc +ve, Anti HBs -ve exposure to B virus
40	M	Post transplant acute rejection	Positive	Cross reacting antigen
35	F	Proliferative GN	Positive	Probable false positivity
38	M	Proliferative GN	Weak Positive	C3+ Cross reacting antigen
40	F	Proliferative GN	Positive	IgM +C3+ Cross reacting antigen
29	F	Proliferative GN	Weak Positive	SLE -false positivity due to rheumatoid factor antigens
42	M	Proliferative GN	Positive	SLE -false positivity due to rheumatoid factor antigens

 

DISCUSSION

Since the discovery of Australia Antigen by Blumberg et al,22 at least 3 antigen antibody systems have been identified, which are responsiblefor immune mediated manifestations of hepatitis B virus infection. These extrahepatic manifestations of hepatitis B are due to deposition of circulating immune complexes. [2] , [9] , [16] , [20-26] Glomerulonephritis is being one of them in which immune complexes are deposited in the glomeruli. Several studies suggested the possible role of HBV in the development of glomerular diseases. [1-10] Glomerulopathies secondary to other infections than HBV, appear histologically similar to HBV associated GN. In order to support an immune complex pathogenesis, at least two types of evidences are essential. [2]
1) Serological evidence of HBV antigen or antibodies.
2) Demonstration of HBV related antigens in the kidney tissue by immunohistochemical techniques.

The incidence of HBV GN ranges from 0.1 to 25%. [1] , [13] , [27] The children are commonly affected. There are very few studies of HBV GN affecting adult population.18,19 In our study the youngest patient was 15 years and eldest was 50 years of age, with the m:f ratio of 2 : 1. The most frequent presentation in our patients was nephrotic syndrome, not responding to the steroids. Small number of patients can also present with renal insufficiency. Frequent HBV infection is discovered by systemic investigations in-patients presenting with chronic GN. [1] , [2] The exact time relationship between the onset of HBV infection and the onset of GN is not known. Various types of glomerular lesions have been studied in association with HBV infection. [1] , [2] , [14], [15] . In our 10 cases of HBsAg positive GN, 4 were membranous GN and 6 proliferative GN seen histologically. From these, except 2 cases, all (80%) showed immune complex deposits of surface and core antigen in the glomerular basement membrane and tubules. Similar results were also reported in the literature. Brazoski et al14 were the first to suggest that HBV might be involved in the pathogenesis of high percentage of GN and found the presence of HBV in 18 of 52 patients, presenting with various types of glomerular disease. Subsequently, the high incidence of HBV infection was also stressed by Lagrue et al,28 Guardia et al,29 Nagy et al,30 who respectively demonstrated the presence of the virus in the serum in 12 of 217, 9 of 105 and 32 of 106 patients. In our study, core antigen showed strong positivity as compared to surface antigen. Fragments of core particle c and e are thought to be most important for the glomerular lesions. It was also interesting to note that in all positive cases, the deposits were seen in renal tubular epithelium. This probably could be due to cell mediated immune reaction. Similar type of observations were also made by Zhou S et al [31] and observed more positivity in the kidney tubules (21.54%) as compared to glomeruli (10.98%) Kleinknecht et al32 haveshown tubular basement membrane deposits of IgG and C3 in 11 out of 14 cases of HBsAg positive glomerular and non glomerular kidney diseases.

The liver histology in all our patients was chronic hepatitis (active or persistent). The histology can range from near normal to cirrhotic stage.1 A long interval may be there between the onset of glomerular disease and the discovery of HBV infection. High incidence (44%) of immune complex (surface and core) deposits were seen in HBsAg negative GN. These patients were 3 cases of post transplant rejection and 5 proliferative GN. Out of 5 cases of proliferative GN , 4 were found to have systemic lupus erythematosus. Such false positive staining can occur due to binding of antibodies with the rheumatoid factor activity previously deposited in the kidney. [2], [33] , [34] Out of 3 post transplant cases showing positivity for HBV, in 2 cases we could detect serological evidence of post exposure to B virus (Anti HBc positive) in the serum. In remaining two cases (1 post transplant, 1 proliferative GN) false positive result was thought to be due to other cross-reacting antigens. [33-35] Lack of specificity to anti serum used, insufficient sensitivity of technique and disappearance of HBsAg in serum are other factors for false positivity. [2], [33] , [36]

In our group of control patients with HBsAg positivity but without GN, none of them showed immune deposits (surface and core) in kidney tissue. The histopathology of the liver in these patients showed cirrhosis and or chronic hepatitis with normal histology of the kidney. Three patients of HBsAg GN showing active viral replication were treated with anti viral therapy. All of them showed seroconversion with the disappearance of proteinuria. The patients of HBV GN usually do not respond to steroid therapy. They may show relapses or lead to chronic renal failure. [37] Prognosis in the children is better as compared to the adults. [2] In adult patients the glomerular disease is slowly progressive. Interferon may play an important role in these cases. [2], [38] , [39]

In conclusion, to establish the relationship between HBV and glomerulonephritis, serological markers of HBV infection as well as kidney biopsy stained for HBV markers are essential. Investigations for the cause of glomerulonephritis should include a search for HBV markers, particularly in countries with a high prevalence of HBV carriers. Patients with active viral replication with GN show poor response to steroids. Antiviral therapy may be beneficial in such patients.

ACKNOWLEDGEMENT

We are thankful to Medical Research Society, Bombay Hospital for grant in aid for this published study.

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