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Characteristics of Glomerular Lesions in Hepatitis B Virus Infection
Characteristics of Glomerular Lesions in Hepatitis B Virus Infection Shin-ichi Takeda, MD, Hiroshi Kida, MD, Masahiro Katagiri, MD, Hitoshi Yokoyama, MD, Toshio Abe, MD, and Nobu Hattori, MD • In an attempt to clarify a participation of hepatitis B virus (HBY) in the development of hepatic glomerulopathy in adults, kidney specimens obtained from 151 patients with liver diseases were studied. Although mesangial proliferation was more severe in patients with chronic hepatitis or liver cirrhosis than in those with acute hepatitis, no significant difference was observed between 82 serum hepatitis B surface antigen (HBsAg)-posltlve (HBY-related group) and 69 negative patients (HBY-nonrelated group). However, double contours of the glomerular capillary walls were observed more often In the former group (18/82, P < .01), especially in the HBeAg-positlve subgroup (8/24, P < .001), than In the latter (3/69). In addition, glomerular capillary spike formation or a bubblelike appearance was observed in seven patients of the former group. Of these, all five patients examined were HBeAgpositive in their serum. By electron microscopic studies, subendothelial dense deposits and mesanglal Interpositions were observed more frequently in the HBY-related group, and subepithelial deposits were found only in the HBeAg-posltive subgroup. The immunofluorescence study revealed IgA-domlnant mesangial deposition in both HBY-related and nonrelated groups. As for the capillary wall deposits, however, IgG was dominant in 13 of the HBY-related group, but only one of the nonrelated group (P < .01). Furthermore, one patient in the HBY-related group showed capillary wail-dominant HBeAg combined with IgG deposition. These results suggest that HBYrelated glomerulopathy in adults may be characterized by capillary wall lesions, possibly attributed to deposition of immune complexes composed of HBY-related antigens, especially HBeAg and IgG class antibodies, and the glomerulopathy should be discriminated from so-called hepatic glomerulosclerosis. © 1988 by the National Kidney Foundation, Inc. INDEX WORDS: HBY-related glomerulopathy; double contour; spike formation; bubblelike appearance; HBeAg.
G
LOMERULAR LESIONS associated with chronic liver diseases have been termed hepatic or cirrhotic glomerulosclerosis and are characterized by light microscopic findings of mesangial proliferation and irregular thickening of the glomerular capillary walls as well as by immunofluorescence findings of IgA-dominant mesangial deposits.I- 3 Combes et al, 4 however, demonstrated granular deposits of both Australia antigen and IgG class antibodies along the glomerular capillary walls in an adult patient with acute hepatitis B complicated by membranous nephropathy, and thereby pointed out the possibility that hepatitis B virus (HBV) or its components might have been involved in the development of the glomerulopathy through formation and deposition of the immune complexes. Since then, membranous nephropathy (MN)S-S and membranoproliferative glomerulonephritis (MPGN)9.10 have been reported as common histologic features of HBV-related glomerulopathy, especially in children. In this study, we attempted to clarify pathologic characteristics of HBV-related glomerulopathy in adults, especially stressing glomerular capillary wall lesions, and to discriminate it from so-called hepatic or cirrhotic glomerulosclerosis.
PATIENTS AND METHODS
Patients Of the patients admitted with liver diseases to the Kanazawa University Hospital or its affiliated hospitals between April, 1972 and September, 1985, 82 serum hepatitis B surface antigen (HBsAg)-positive (HBV-related group) and 69 both HBsAg-negative and anti-HBs antibody (HBsAb)-negative patients (HBV-nonrelated group) were studied (Table 1). Thirty-nine of the former group had further examinations for serum HBeAg and HBeAb, which showed 24 patients positive to the antigen and 15 to the antibody. The latter group consisted of one patient with Wilson's disease, two with fatty liver, two with lupoid hepatitis, three with alcoholic liver cirrhosis, nine with primary biliary cirrhosis, and 52 with cryptogenic chronic liver diseases_ All patients included in this study were further divided into two disease categories: one with acute liver diseases including acute hepatitis (AH) and fulminant hepatitis (FH), and the other with chronic liver diseases such as chronic From the First Department of Internal Medicine, School of Medicine, Kanazawa University, Kanazawa, Japan_ Supported by a research grant (Progressive Renal Lesions) from the Intractable Disease Division, Public Health Bureau, Ministry of Health and Welfare, Japan. Address reprint requests to Shin-ichi Takeda, MD, First Department of Internal Medicine, School of Medicine, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa, 920, Japan. © 1988 by the National Kidney Foundation, Inc_ 0272-6386/88/1101-0011 $3.00/0
American Journal of Kidney Diseases, Vol XI, No 1 (January), 1988: pp 57-62
57
58
TAKEDA ET AL
Table 1.
Profile of Patients
Age (yr) (± SO) Sex (F/M) Liver diseases and source of kidney specimen (B/A) Acute liver diseases AH FH Chronic liver diseases CH LC LC+HCC
c:
HBV· Related (N = 82)
HBV· Nonrelated (N = 69)
51.7 ± 14.3 18/64
52.1 ± 13.3
o
.~
Severe
....
a:>
t
Moderate
a;
Mild
~
........................ ..... ....... .
20/49
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en
2 (2/0) 5 (0/5) 30 (24/6)
23 (7116) 22 (0/22)
1 (1/0) 8 (2/6)
10 (9/1) 31 (13/18) 19 (1118)
~
Absent
o
Pathological Examinations Kidney specimens in the HBV-related group were obtained by percutaneous biopsy in 33 patients and by autopsy in 49, and in the HBV-nonrelated group by biopsy in 26 patients and by autopsy in 43 (Thble I). These were observed under a light microscope after being fixed in a 10% formalin (pH 7.2), embedded in paraffin, cut at 4 p.m, and stained with hematoxylin and eosin, PAS reagent, Mallory-azan, and periodic acid silver methenamine (PAM). Mesangial proliferation was estimated as follows: absent, no mesangial proliferation; mild, mild mesangial proliferation; moderate, mesangial proliferation extended to the periphery of the tuft; severe, marked mesangial proliferation accompanied with luminal narrowing of glomerular capillaries. The grade of the capillary wall lesions, including double contours, spike formation (spike), and bubblelike appearance (bubble), were classified as follows: none, not observed; few, observed in a few capillary loops; scattered, observed in half of the tufts; diffuse, observed in almost all capillary loops. Fresh frozen specimens obtained from 59 HBV-related and 38 nonrelated patients were cut at 6 p.m and treated with fluorescein isothiocyanate-Iabeled anti-human IgG (-y-chain), IgA (a-chain), IgM (p.-chain), C3, and Clq rabbit sera (Behringwerke, Marburg, West Germany) for immunofluorescence studies. Other portions of 57 specimens were fixed with glutaraldehyde and osmium tetroxide, embedded in Epon (Epok) 812 (Oken Shoji Co, Tokyo, Japan), cut at 0.1 p.m, double-stained with uranyl acetate and lead citrate, and observed by Hitachi H600 electron microscopy (Hitachi Co, Tokyo, Japan). Urinary protein was measured using Kingsbury-Clark's method. Microscopic hematuria was diagnosed when there were more than 5 RBCs per high power visual field of spun urine. Serum HBsAg was determined by the reversed passive hemagglutination method, and HBsAb by the passive hemagglutination method. Both HBeAg and HBeAb were determined by radioimmunoassay. Statistical analyses were performed using Student's t test, Fisher's exact test and Wilcoxon's rank sum test.
C H
AHorFH
L C
LC+HCC
Fig 1. Mesangial proliferation in liver diseases. •, HBV-related group; 0, HBV-nonrelated group.
Abbreviation: BIA, biopsy/autopsy. hepatitis (CH), liver cirrhosis (LC), and liver cirrhosis with hepatocellular carcinoma (LC + HCC).
0000000
RESULTS
Light Microscopic Findings
Moderate-to-severe mesangial proliferation was observed only in patients with chronic liver diseases in both the HBV-related and nonrelated groups, but no difference was noted between the two groups (Fig 1). Double contours of the capillary walls, however, were observed more frequently in the HBV-related group (18/82,22.0%, P < .01; Fig 2), especially in the HBeAg-positive subgroup (8/24,33.3%, P < .001; Table 2), compared to the HBV-nonrelated group (3/69, 4.3%). All four patients who exhibited diffuse or, scattered double contours belonged to the HBV-related group. In addition, spikes or bubbles of the glomerular capilary wall, as shown in Fig 3, were observed in seven (8.5%) of the HBV-related group, but not in the HBV-nonrelated group. Of these, five patients who received examination were HBeAg-positive in their serum (Thble 2). Six of these seven patients were those with double contours, while the remaining patient with AH
::; o c: o
"
Diffuse
I
o Scattered
a:>
:c::J o
Cl
I.
tI",/ •••
Few
........................
None
I······ :::::: ..
~~~~~~~~:::
.....
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AHorFH
C H
L C
LC+HCC
Fig 2. Capillary wall lesions in liver diseases. •, HBV-related group; 0, HBV-nonrelated group; P.8tient with spike formation or bubblelike appearance; ., patient with membranous nephropathy.
e,
HBV-RELATED GLOMERULOPATHY Table 2.
59
Light Microscopic Findings in HBeAg-Positive and HBeAb-positive Subgroups Mesangial Proliferation
H BeAg-positive (N = 24) H BeAb-positive (N = 15) HBV-nonrelated (N = 69)
Minimal
Mild
Moderate
6
9
8
10
4
38
5
24
Double Contour Severe
None
Few
Scattered
Diffuse
16
6 (3)
1 (1)
1 (1)
0
12
3
0
0
2
66
3
0
0
Numbers in parentheses represent patients with spike formation and/or bubblelike appearance.
was diagnosed as MN based on the diffuse fine granular deposition of IgG, IgM, C3, and Clq along the glomerular capillary walls. Excluding the patient with AH and MN, spikes or bubbles were present in a "scattered" distribution in two patients and in a "few" distribution in the remaining four patients. Immunofluorescence Findings
In the mesangial area, IgA andlor IgG deposition was observed in 35 (59.3%) of the 59 patients in the HBV-related group and in 22 (57 .9%) of the 38 in the nonrelated group. The IgA dominant pattern was most frequently observed in both HBVrelated (15 159; 25.4 %) and nonrelated groups (121 38; 31.6%; Table 3). As for the capillary wall deposits, however, IgA andlor IgG was observed in 24 (40.7%) of the HBV-related and in nine (23.7%) of the nonrelated group. In 13 (22.0%) of the former, IgG was dominant, compared to one (2 .6 %) of the latter group (P < .01) . Similarly, these deposits were observed in 13 (61.9%) of the HBeAg-positive subgroup, nine of whom were
Fig 3. Light microscopic findings of a patient in the HBV-related group. Double contours, spike formation, and bubblelike appearance were observed (PAM original magnification x 780).
IgG-dominant, in comparison with two (20.0%) of the HBeAb-positive subgroup (P < .05). Incidentally, capillary wall lesions including double contours, spikes, and bubbles were likely to be observed more frequently in patients with IgG-dominant (9/13, 69.2%) rather than IgAdominant capillary deposits (217 , 28 .6 %) in the HBV-related group. In addition , one patient in the HBV-related group who showed double contours and bubbles had further examinations detecting depositions of HBV-related antigens using the monoclonal antibodies to HBsAg (no. 0012 , lot B20100 , 360274A, Hybritec Inc, La Jolla, CA) and HBeAg (clone AHl ; Green Cross Co, Osaka, Japan).11 These disclosed capillary wall-dominant HBeAg with IgG deposition (Fig 4) and mesangium-dominant HBsAg deposition . Electron Microscopic Findings
In both HBV-related (22129 , 75.9%) and nonrelated groups (22128, 78.6%), electron-dense deposits were most frequently observed in the mesangium (Table 4). On the other hand , subendothelial deposits , as shown in Fig 5, were observed more frequently in the HBV-related group (16/29,55.2 %, P < .001), especially in the HBeAg-positive subgroup (10/16, 62.5%, P < .001), than in the nonrelated group (3/28, 10.7 %). All patients who showed subepithelial deposits were included in the HBeAg-positive subgroup, except one patient who had no examination for HBeAg in serum. In addition, mesangial interpositions, which were thought to correspond with double contours in the light microscopic examinations, were observed in 14 (48.3%) of the HBV-related group, compared to three (10.7%) in the nonrelated group (P < .01). Notably, in the HBeAg positive subgroup, nine (56.3%) of the 16 patients revealed the lesions . In addition, seven (35 .0%) of 20 biopsy specimens in the HBV-re-
60
TAKEDA ET AL Table 3.
Glomerular Immunoglobulin Deposition in Liver Diseases Negative'
Mesangial deposition HBV-related (N = 59) HBV-nonrelated (N = 38)
10 3
Capillary deposition HBV-related (N = 59) HBeAg-positive (N = 21) HBeAb-positive (N = 10) HBV-nonrelated (N = 38)
13 (9) 9 1 1 (1)
10 7 4 (2) 2 1 1 (0)
15 12 7 (2) 2 0 7 (1)
24 16 35 (1) 8 8 29 (0)
Numbers in parentheses represent patients with capillary lesions such as double contours, spike formation, or bubblelike appearance. 'Both IgG and IgA were negative.
lated group, three (21.4%) of 14 in the nonrelated group, and all autopsy specimens except one in the nonrelated group showed small lucent areas as described by Berger et al. I Clinical Features
Urine protein excretion exceeding 0.5 gld was observed in 19 patients (23.2%) in the HBV-related group and in 11 (15.9%) in the nonrelated group. Hematuria was observed in 11 (13.4%) and 17 (24.6%) patients in the respective groups, with no apparent difference in incidence (Table 5). However, higher incidence of proteinuria was found in patients with capillary wall lesions (lSI 22; 68 .2 %), compared to those without them (lSI 129; 11.6%; P < .001), whereas mesangial proliferation did not affect the incidence of proteinuria.
Fig 4. Immunofluorescence findings of a patient in the HBV-related group. Capillary wall-dominant HBeAg deposition was observed (original magnlflcaton x 650).
DISCUSSION
Mesangial proliferation was more notable in patients with chronic liver diseases, both in the HBVrelated and nonrelated groups, but no difference was observed between the two groups. In addition, both groups frequently showed IgA-dominant deposition in the mesangial area. These findings appear to be common in chronic liver diseases, regardless of the pathogenesis, and constitute the principal pathologic feature described as hepatic glomerulosclerosis. 1·3 By contrast, double contours of the glomerular capillary walls by light microscopic studies, as well as subendothelial electron-dense deposits and mesangial interposition by electron microscopic studies, were more frequently observed in the HBV-related group, especially in the HBeAg-positive subgroup; spikes or bubbles of the capillary walls and subepithelial deposits were exclusively noted in this subgroup, suggesting the possibility of the involvement of HBV-related antigens, especially HBeAg, in the formation of the capillary wall lesions. Furthermore, the capillary wall lesions were simultaneously observed to be an IgGdominant deposition along the capillary walls, and in one patient with capillary wall lesions, HBeAg with IgG deposition was observed along the capillary walls. In this respect, several authors reported that HBeAg was detected in the same distribution as IgG in HBV-related MN12'14 and MPGN.IO,IS These data strongly suggest that the capillary wall lesions observed in the HBV-related group are essential features of HBV-related glomerulopathy and are attributed to glomerular capillary wall deposition of immune complexes composed of HBV-related antigens, especially HBeAg and IgGclass antibodies.
61
HBV-RELATED GLOMERULOPATHY Table 4.
Electron Microscopic Findings in Liver Disease Dense Deposits
HBV-related (N = 29) HBeAg-positive (N = 16) HBeAb-positive (N = 5) HBV-nonrelated (N = 28)
Mesangial
Subendothelial
Intramembranous
Subepithelial
22 (15) 11 4 22
16 (11) 10
6 (6) 6 0 3
5 (4) 4 0 0
3
Mesangial Interposition
14 (10) 9
3
Numbers in parentheses represent patients having examinations for HBeAg and HBeAb in serum.
Fig 5. Electron micrograph of a patient In the HBV-related group. Subendothelial electron-dense deposits as well as subepithelial and mesangial deposits were observed (original magnification x 2,480).
Table 5.
Incidence of Urinary Abnormalities Proteinuria
HBV-related (N = 82) HBV-nonrelated (N = 69) Patients with mesangial proliferation (N = 104) Patients without mesangial proliferation (N = 47) Patients with capillary lesions (N = 22) Patients without capillary lesions (N = 129)
Hematuria
<0.5
0.5-1.0
1.0-3.5
>3.5
(+)
(-)
63
13
4
2
11
71
58
4
6
17
52
80
16
5
3
20
84
41
2
4
0
8
39
7
11
2
2
5
17
114
6
8
23
106
62
TAKEDA ET AL
Although MN was considered to be a main histologic feature of HBV-related glomerulopathy in children, of the 82 patients with HBV-related liver diseases examined in this study, only one patient (1. 2 %) was diagnosed as having MN. In addition, our findings showed that double contours of the capillary walls occurred frequently in the HBV-related group, and that one third were associated with spikes or bubbles, indicating that adult HBVrelated glomerulopathy shows histologic features of MPGN rather than MN. In 1985 Yoshikawa et al 14 reported that children with HBV-related MN frequently showed mesangial and/or subendothelial deposits in comparison with those with
idiopathic MN. Furthermore, hypocomplementemia was noted in patients with HBV-related MN.6.16 These data suggest that HBV-related glomerulopathy shows various intermediate lesions ranging from MN to MPGN. In summary, HBV-related glomerulopathy in adults may be characterized by capillary wall lesions consisting of double contours, spikes, and bubbles, possibly attributed to deposition of immune complexes composed of HBV-related antigens, especially HBeAg and IgG class antibodies, and should be discriminated from so-called hepatic glomerulosclerosis.
REFERENCES 1. Berger J, Yaneva H, Nabarra B: Glomerular changes in patients with cirrhosis of the liver. Adv Nephrol 7:3-14,1977 2. Nakamoto Y, Iida H, Kobayashi K, et a1: Hepatic glomerulonephritis. Characteristics of hepatic IgA glomerulonephritis as the major part. Virchows Arch Pathol Anat 392:45-54, 1981 3. Endo Y, Matsushita H, Nozawa Y, et al: Glomerulonephritis associated with liver cirrhosis. Acta Pathol Jpn 33:333-346, 1983 4. Combes B, Stastny P, Shorey J, et al: Glomerulonephritis with deposition of Australia antigen-antibody complexes in glomerular basement membrane. Lancet 2:234-237, 1971 5. Takekoshi Y, Tanaka M. Shida N, et al: Strong association between membranous nephropathy and hepatitis-B surface antigenemia in Japanese children. Lancet 2:1065-1068, 1978 6. Wiggelinkhuizen J, Sinclair-Smith C, Stannard LM, et al: Hepatitis B virus associated membranous glomerulonephritis. Arch Dis Child 58:488-496, 1983 7. Hsu HC, Lin GH, Chang MH, et al: Association of hepatitis B surface (HBs) antigenemia and membranous nephropathy in children in Taiwan. Clin NephroI20:121-129, 1983 8. Seggie J, Nathoo K, Davies PG: Association of hepatitis B (HBs) antigenemia and membranous glomerulonephritis in Zimbabwean children. Nephron 38: 115-119, 1984 9. Brzosko WJ, Krawczynski K, Nazarewicz T, et al: Glomerulonephritis associated with hepatitis-B surface antigen immune complexes in children. Lancet 2:477-481, 1974
10. Amemiya S, Ito H, Kato K, et al: A case of membranous proliferative glomerulonephritis type III (Burkholder) with the deposition of both HBeAg and HBsAg, Int J Pediatr Nephrol 4:267-273, 1983 11. Uemura Y, Fukuyama K, Nishida M, et al: Establishment of passive hemagglutination assay (PHA) system for antiHBc in plasma. Tohoku J Exp Med 149: 11-20, 1986 12. Takekoshi Y, Tanaka M, Miyakawa Y, et al: Free "small" and IgG associated "large" hepatitis B e antigen in the serum and glomerular capillary walls of two patients with membranous glomerulonephritis. N Engl J Med 300:814-819, 1979 13. Hirose H, Udo K, Kojima M, et al: Deposition of hepatitis B e antigen in membranous glomerulonephritis. Identification by F(ab '), fragments of monoclonal antibody. Kidney Int 26:338-341, 1984 14. Yoshikawa N, Ito H, Yamada Y, et a1: Membranous glomerulonephritis associated with hepatitis B antigen in children. A comparison with idiopathic membranous glomerulonephritis. Clin Nephrol 23:28-34, 1985 15. Collins AB, Bhan AK, Dienstag JL, et a1: Hepatitis B immune complex glomerulonephritis. Simultaneous glomerular deposition of hepatitis B surface and e antigens. Clin Immunol Immunopathol 26:137-153, 1983 16. Southwest Pediatric Nephrology Study Group: Hepatitis B surface antigenemia in North American children with membranous glomerulonephropathy. J Pediatr 106:571-578, 1985
G
LOMERULAR LESIONS associated with chronic liver diseases have been termed hepatic or cirrhotic glomerulosclerosis and are characterized by light microscopic findings of mesangial proliferation and irregular thickening of the glomerular capillary walls as well as by immunofluorescence findings of IgA-dominant mesangial deposits.I- 3 Combes et al, 4 however, demonstrated granular deposits of both Australia antigen and IgG class antibodies along the glomerular capillary walls in an adult patient with acute hepatitis B complicated by membranous nephropathy, and thereby pointed out the possibility that hepatitis B virus (HBV) or its components might have been involved in the development of the glomerulopathy through formation and deposition of the immune complexes. Since then, membranous nephropathy (MN)S-S and membranoproliferative glomerulonephritis (MPGN)9.10 have been reported as common histologic features of HBV-related glomerulopathy, especially in children. In this study, we attempted to clarify pathologic characteristics of HBV-related glomerulopathy in adults, especially stressing glomerular capillary wall lesions, and to discriminate it from so-called hepatic or cirrhotic glomerulosclerosis.
PATIENTS AND METHODS
Patients Of the patients admitted with liver diseases to the Kanazawa University Hospital or its affiliated hospitals between April, 1972 and September, 1985, 82 serum hepatitis B surface antigen (HBsAg)-positive (HBV-related group) and 69 both HBsAg-negative and anti-HBs antibody (HBsAb)-negative patients (HBV-nonrelated group) were studied (Table 1). Thirty-nine of the former group had further examinations for serum HBeAg and HBeAb, which showed 24 patients positive to the antigen and 15 to the antibody. The latter group consisted of one patient with Wilson's disease, two with fatty liver, two with lupoid hepatitis, three with alcoholic liver cirrhosis, nine with primary biliary cirrhosis, and 52 with cryptogenic chronic liver diseases_ All patients included in this study were further divided into two disease categories: one with acute liver diseases including acute hepatitis (AH) and fulminant hepatitis (FH), and the other with chronic liver diseases such as chronic From the First Department of Internal Medicine, School of Medicine, Kanazawa University, Kanazawa, Japan_ Supported by a research grant (Progressive Renal Lesions) from the Intractable Disease Division, Public Health Bureau, Ministry of Health and Welfare, Japan. Address reprint requests to Shin-ichi Takeda, MD, First Department of Internal Medicine, School of Medicine, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa, 920, Japan. © 1988 by the National Kidney Foundation, Inc_ 0272-6386/88/1101-0011 $3.00/0
American Journal of Kidney Diseases, Vol XI, No 1 (January), 1988: pp 57-62
57
58
TAKEDA ET AL
Table 1.
Profile of Patients
Age (yr) (± SO) Sex (F/M) Liver diseases and source of kidney specimen (B/A) Acute liver diseases AH FH Chronic liver diseases CH LC LC+HCC
c:
HBV· Related (N = 82)
HBV· Nonrelated (N = 69)
51.7 ± 14.3 18/64
52.1 ± 13.3
o
.~
Severe
....
a:>
t
Moderate
a;
Mild
~
........................ ..... ....... .
20/49
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0000000000000000 000000000000 00
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en
2 (2/0) 5 (0/5) 30 (24/6)
23 (7116) 22 (0/22)
1 (1/0) 8 (2/6)
10 (9/1) 31 (13/18) 19 (1118)
~
Absent
o
Pathological Examinations Kidney specimens in the HBV-related group were obtained by percutaneous biopsy in 33 patients and by autopsy in 49, and in the HBV-nonrelated group by biopsy in 26 patients and by autopsy in 43 (Thble I). These were observed under a light microscope after being fixed in a 10% formalin (pH 7.2), embedded in paraffin, cut at 4 p.m, and stained with hematoxylin and eosin, PAS reagent, Mallory-azan, and periodic acid silver methenamine (PAM). Mesangial proliferation was estimated as follows: absent, no mesangial proliferation; mild, mild mesangial proliferation; moderate, mesangial proliferation extended to the periphery of the tuft; severe, marked mesangial proliferation accompanied with luminal narrowing of glomerular capillaries. The grade of the capillary wall lesions, including double contours, spike formation (spike), and bubblelike appearance (bubble), were classified as follows: none, not observed; few, observed in a few capillary loops; scattered, observed in half of the tufts; diffuse, observed in almost all capillary loops. Fresh frozen specimens obtained from 59 HBV-related and 38 nonrelated patients were cut at 6 p.m and treated with fluorescein isothiocyanate-Iabeled anti-human IgG (-y-chain), IgA (a-chain), IgM (p.-chain), C3, and Clq rabbit sera (Behringwerke, Marburg, West Germany) for immunofluorescence studies. Other portions of 57 specimens were fixed with glutaraldehyde and osmium tetroxide, embedded in Epon (Epok) 812 (Oken Shoji Co, Tokyo, Japan), cut at 0.1 p.m, double-stained with uranyl acetate and lead citrate, and observed by Hitachi H600 electron microscopy (Hitachi Co, Tokyo, Japan). Urinary protein was measured using Kingsbury-Clark's method. Microscopic hematuria was diagnosed when there were more than 5 RBCs per high power visual field of spun urine. Serum HBsAg was determined by the reversed passive hemagglutination method, and HBsAb by the passive hemagglutination method. Both HBeAg and HBeAb were determined by radioimmunoassay. Statistical analyses were performed using Student's t test, Fisher's exact test and Wilcoxon's rank sum test.
C H
AHorFH
L C
LC+HCC
Fig 1. Mesangial proliferation in liver diseases. •, HBV-related group; 0, HBV-nonrelated group.
Abbreviation: BIA, biopsy/autopsy. hepatitis (CH), liver cirrhosis (LC), and liver cirrhosis with hepatocellular carcinoma (LC + HCC).
0000000
RESULTS
Light Microscopic Findings
Moderate-to-severe mesangial proliferation was observed only in patients with chronic liver diseases in both the HBV-related and nonrelated groups, but no difference was noted between the two groups (Fig 1). Double contours of the capillary walls, however, were observed more frequently in the HBV-related group (18/82,22.0%, P < .01; Fig 2), especially in the HBeAg-positive subgroup (8/24,33.3%, P < .001; Table 2), compared to the HBV-nonrelated group (3/69, 4.3%). All four patients who exhibited diffuse or, scattered double contours belonged to the HBV-related group. In addition, spikes or bubbles of the glomerular capilary wall, as shown in Fig 3, were observed in seven (8.5%) of the HBV-related group, but not in the HBV-nonrelated group. Of these, five patients who received examination were HBeAg-positive in their serum (Thble 2). Six of these seven patients were those with double contours, while the remaining patient with AH
::; o c: o
"
Diffuse
I
o Scattered
a:>
:c::J o
Cl
I.
tI",/ •••
Few
........................
None
I······ :::::: ..
~~~~~~~~:::
.....
00000000°00000000000000000000000 00 0000000000000000
AHorFH
C H
L C
LC+HCC
Fig 2. Capillary wall lesions in liver diseases. •, HBV-related group; 0, HBV-nonrelated group; P.8tient with spike formation or bubblelike appearance; ., patient with membranous nephropathy.
e,
HBV-RELATED GLOMERULOPATHY Table 2.
59
Light Microscopic Findings in HBeAg-Positive and HBeAb-positive Subgroups Mesangial Proliferation
H BeAg-positive (N = 24) H BeAb-positive (N = 15) HBV-nonrelated (N = 69)
Minimal
Mild
Moderate
6
9
8
10
4
38
5
24
Double Contour Severe
None
Few
Scattered
Diffuse
16
6 (3)
1 (1)
1 (1)
0
12
3
0
0
2
66
3
0
0
Numbers in parentheses represent patients with spike formation and/or bubblelike appearance.
was diagnosed as MN based on the diffuse fine granular deposition of IgG, IgM, C3, and Clq along the glomerular capillary walls. Excluding the patient with AH and MN, spikes or bubbles were present in a "scattered" distribution in two patients and in a "few" distribution in the remaining four patients. Immunofluorescence Findings
In the mesangial area, IgA andlor IgG deposition was observed in 35 (59.3%) of the 59 patients in the HBV-related group and in 22 (57 .9%) of the 38 in the nonrelated group. The IgA dominant pattern was most frequently observed in both HBVrelated (15 159; 25.4 %) and nonrelated groups (121 38; 31.6%; Table 3). As for the capillary wall deposits, however, IgA andlor IgG was observed in 24 (40.7%) of the HBV-related and in nine (23.7%) of the nonrelated group. In 13 (22.0%) of the former, IgG was dominant, compared to one (2 .6 %) of the latter group (P < .01) . Similarly, these deposits were observed in 13 (61.9%) of the HBeAg-positive subgroup, nine of whom were
Fig 3. Light microscopic findings of a patient in the HBV-related group. Double contours, spike formation, and bubblelike appearance were observed (PAM original magnification x 780).
IgG-dominant, in comparison with two (20.0%) of the HBeAb-positive subgroup (P < .05). Incidentally, capillary wall lesions including double contours, spikes, and bubbles were likely to be observed more frequently in patients with IgG-dominant (9/13, 69.2%) rather than IgAdominant capillary deposits (217 , 28 .6 %) in the HBV-related group. In addition , one patient in the HBV-related group who showed double contours and bubbles had further examinations detecting depositions of HBV-related antigens using the monoclonal antibodies to HBsAg (no. 0012 , lot B20100 , 360274A, Hybritec Inc, La Jolla, CA) and HBeAg (clone AHl ; Green Cross Co, Osaka, Japan).11 These disclosed capillary wall-dominant HBeAg with IgG deposition (Fig 4) and mesangium-dominant HBsAg deposition . Electron Microscopic Findings
In both HBV-related (22129 , 75.9%) and nonrelated groups (22128, 78.6%), electron-dense deposits were most frequently observed in the mesangium (Table 4). On the other hand , subendothelial deposits , as shown in Fig 5, were observed more frequently in the HBV-related group (16/29,55.2 %, P < .001), especially in the HBeAg-positive subgroup (10/16, 62.5%, P < .001), than in the nonrelated group (3/28, 10.7 %). All patients who showed subepithelial deposits were included in the HBeAg-positive subgroup, except one patient who had no examination for HBeAg in serum. In addition, mesangial interpositions, which were thought to correspond with double contours in the light microscopic examinations, were observed in 14 (48.3%) of the HBV-related group, compared to three (10.7%) in the nonrelated group (P < .01). Notably, in the HBeAg positive subgroup, nine (56.3%) of the 16 patients revealed the lesions . In addition, seven (35 .0%) of 20 biopsy specimens in the HBV-re-
60
TAKEDA ET AL Table 3.
Glomerular Immunoglobulin Deposition in Liver Diseases Negative'
Mesangial deposition HBV-related (N = 59) HBV-nonrelated (N = 38)
10 3
Capillary deposition HBV-related (N = 59) HBeAg-positive (N = 21) HBeAb-positive (N = 10) HBV-nonrelated (N = 38)
13 (9) 9 1 1 (1)
10 7 4 (2) 2 1 1 (0)
15 12 7 (2) 2 0 7 (1)
24 16 35 (1) 8 8 29 (0)
Numbers in parentheses represent patients with capillary lesions such as double contours, spike formation, or bubblelike appearance. 'Both IgG and IgA were negative.
lated group, three (21.4%) of 14 in the nonrelated group, and all autopsy specimens except one in the nonrelated group showed small lucent areas as described by Berger et al. I Clinical Features
Urine protein excretion exceeding 0.5 gld was observed in 19 patients (23.2%) in the HBV-related group and in 11 (15.9%) in the nonrelated group. Hematuria was observed in 11 (13.4%) and 17 (24.6%) patients in the respective groups, with no apparent difference in incidence (Table 5). However, higher incidence of proteinuria was found in patients with capillary wall lesions (lSI 22; 68 .2 %), compared to those without them (lSI 129; 11.6%; P < .001), whereas mesangial proliferation did not affect the incidence of proteinuria.
Fig 4. Immunofluorescence findings of a patient in the HBV-related group. Capillary wall-dominant HBeAg deposition was observed (original magnlflcaton x 650).
DISCUSSION
Mesangial proliferation was more notable in patients with chronic liver diseases, both in the HBVrelated and nonrelated groups, but no difference was observed between the two groups. In addition, both groups frequently showed IgA-dominant deposition in the mesangial area. These findings appear to be common in chronic liver diseases, regardless of the pathogenesis, and constitute the principal pathologic feature described as hepatic glomerulosclerosis. 1·3 By contrast, double contours of the glomerular capillary walls by light microscopic studies, as well as subendothelial electron-dense deposits and mesangial interposition by electron microscopic studies, were more frequently observed in the HBV-related group, especially in the HBeAg-positive subgroup; spikes or bubbles of the capillary walls and subepithelial deposits were exclusively noted in this subgroup, suggesting the possibility of the involvement of HBV-related antigens, especially HBeAg, in the formation of the capillary wall lesions. Furthermore, the capillary wall lesions were simultaneously observed to be an IgGdominant deposition along the capillary walls, and in one patient with capillary wall lesions, HBeAg with IgG deposition was observed along the capillary walls. In this respect, several authors reported that HBeAg was detected in the same distribution as IgG in HBV-related MN12'14 and MPGN.IO,IS These data strongly suggest that the capillary wall lesions observed in the HBV-related group are essential features of HBV-related glomerulopathy and are attributed to glomerular capillary wall deposition of immune complexes composed of HBV-related antigens, especially HBeAg and IgGclass antibodies.
61
HBV-RELATED GLOMERULOPATHY Table 4.
Electron Microscopic Findings in Liver Disease Dense Deposits
HBV-related (N = 29) HBeAg-positive (N = 16) HBeAb-positive (N = 5) HBV-nonrelated (N = 28)
Mesangial
Subendothelial
Intramembranous
Subepithelial
22 (15) 11 4 22
16 (11) 10
6 (6) 6 0 3
5 (4) 4 0 0
3
Mesangial Interposition
14 (10) 9
3
Numbers in parentheses represent patients having examinations for HBeAg and HBeAb in serum.
Fig 5. Electron micrograph of a patient In the HBV-related group. Subendothelial electron-dense deposits as well as subepithelial and mesangial deposits were observed (original magnification x 2,480).
Table 5.
Incidence of Urinary Abnormalities Proteinuria
HBV-related (N = 82) HBV-nonrelated (N = 69) Patients with mesangial proliferation (N = 104) Patients without mesangial proliferation (N = 47) Patients with capillary lesions (N = 22) Patients without capillary lesions (N = 129)
Hematuria
<0.5
0.5-1.0
1.0-3.5
>3.5
(+)
(-)
63
13
4
2
11
71
58
4
6
17
52
80
16
5
3
20
84
41
2
4
0
8
39
7
11
2
2
5
17
114
6
8
23
106
62
TAKEDA ET AL
Although MN was considered to be a main histologic feature of HBV-related glomerulopathy in children, of the 82 patients with HBV-related liver diseases examined in this study, only one patient (1. 2 %) was diagnosed as having MN. In addition, our findings showed that double contours of the capillary walls occurred frequently in the HBV-related group, and that one third were associated with spikes or bubbles, indicating that adult HBVrelated glomerulopathy shows histologic features of MPGN rather than MN. In 1985 Yoshikawa et al 14 reported that children with HBV-related MN frequently showed mesangial and/or subendothelial deposits in comparison with those with
idiopathic MN. Furthermore, hypocomplementemia was noted in patients with HBV-related MN.6.16 These data suggest that HBV-related glomerulopathy shows various intermediate lesions ranging from MN to MPGN. In summary, HBV-related glomerulopathy in adults may be characterized by capillary wall lesions consisting of double contours, spikes, and bubbles, possibly attributed to deposition of immune complexes composed of HBV-related antigens, especially HBeAg and IgG class antibodies, and should be discriminated from so-called hepatic glomerulosclerosis.
REFERENCES 1. Berger J, Yaneva H, Nabarra B: Glomerular changes in patients with cirrhosis of the liver. Adv Nephrol 7:3-14,1977 2. Nakamoto Y, Iida H, Kobayashi K, et a1: Hepatic glomerulonephritis. Characteristics of hepatic IgA glomerulonephritis as the major part. Virchows Arch Pathol Anat 392:45-54, 1981 3. Endo Y, Matsushita H, Nozawa Y, et al: Glomerulonephritis associated with liver cirrhosis. Acta Pathol Jpn 33:333-346, 1983 4. Combes B, Stastny P, Shorey J, et al: Glomerulonephritis with deposition of Australia antigen-antibody complexes in glomerular basement membrane. Lancet 2:234-237, 1971 5. Takekoshi Y, Tanaka M. Shida N, et al: Strong association between membranous nephropathy and hepatitis-B surface antigenemia in Japanese children. Lancet 2:1065-1068, 1978 6. Wiggelinkhuizen J, Sinclair-Smith C, Stannard LM, et al: Hepatitis B virus associated membranous glomerulonephritis. Arch Dis Child 58:488-496, 1983 7. Hsu HC, Lin GH, Chang MH, et al: Association of hepatitis B surface (HBs) antigenemia and membranous nephropathy in children in Taiwan. Clin NephroI20:121-129, 1983 8. Seggie J, Nathoo K, Davies PG: Association of hepatitis B (HBs) antigenemia and membranous glomerulonephritis in Zimbabwean children. Nephron 38: 115-119, 1984 9. Brzosko WJ, Krawczynski K, Nazarewicz T, et al: Glomerulonephritis associated with hepatitis-B surface antigen immune complexes in children. Lancet 2:477-481, 1974
10. Amemiya S, Ito H, Kato K, et al: A case of membranous proliferative glomerulonephritis type III (Burkholder) with the deposition of both HBeAg and HBsAg, Int J Pediatr Nephrol 4:267-273, 1983 11. Uemura Y, Fukuyama K, Nishida M, et al: Establishment of passive hemagglutination assay (PHA) system for antiHBc in plasma. Tohoku J Exp Med 149: 11-20, 1986 12. Takekoshi Y, Tanaka M, Miyakawa Y, et al: Free "small" and IgG associated "large" hepatitis B e antigen in the serum and glomerular capillary walls of two patients with membranous glomerulonephritis. N Engl J Med 300:814-819, 1979 13. Hirose H, Udo K, Kojima M, et al: Deposition of hepatitis B e antigen in membranous glomerulonephritis. Identification by F(ab '), fragments of monoclonal antibody. Kidney Int 26:338-341, 1984 14. Yoshikawa N, Ito H, Yamada Y, et a1: Membranous glomerulonephritis associated with hepatitis B antigen in children. A comparison with idiopathic membranous glomerulonephritis. Clin Nephrol 23:28-34, 1985 15. Collins AB, Bhan AK, Dienstag JL, et a1: Hepatitis B immune complex glomerulonephritis. Simultaneous glomerular deposition of hepatitis B surface and e antigens. Clin Immunol Immunopathol 26:137-153, 1983 16. Southwest Pediatric Nephrology Study Group: Hepatitis B surface antigenemia in North American children with membranous glomerulonephropathy. J Pediatr 106:571-578, 1985