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Original Report: Patient-Oriented, Translational Research

Am J Nephrol 2012;35:134–140 DOI: 10.1159/000335375

Membranoproliferative Glomerulonephritisand Mixed Cryoglobulinemia after Hepatitis CVirus Infection Secondary to Glomerular NS3Viral Antigen Deposits

Stanislas Bataille a Gilles Kaplanski b José Boucraut c Philippe Halfon e

Claire Camus e Laurent Daniel d Stéphane Burtey a Yvon Berland a

Bertrand Dussol a

a Centre de Néphrologie et Transplantation Rénale, b Service de Médecine Interne, c Laboratoire d’Immunologie, Hôpital de la Conception, et d Service d’Anatomie Pathologique et de Neuropathologie, Hôpital Timone, APHM, Aix-Marseille Université, et e Laboratoire Alphabio, Marseille , France

3 patients. Remission occurred spontaneously in 1 patient, and in another patient it occurred after rituximab treatment. The third patient was lost to follow-up. Conclusions: In pa-tients with hepatitis C-negative viral load, membranoprolif-erative glomerulonephritis could be induced by the persis-tence of HCV antigen in the kidney but not in hematopoi-etic cells. Nonlymphomatous B cell proliferation may also be induced by chronic viral stimulation.

Copyright © 2012 S. Karger AG, Basel

Introduction

Hepatitis C virus (HCV) infection is the main causeof membranoproliferative glomerulonephritis (MPGN). This condition is often associated with mixed cryoglobu-linemia (MC) systemic vasculitis [1–3] . MC is reported in 50–60% of HCV-infected patients [4] . MPGN can be sec-ondary to HCV alone, HCV-associated MC, or MC alone [5] . HCV-infected patients with MC are more likely to develop MPGN [6] .

Key Words

Membranoproliferative glomerulonephritis � Hepatitis C virus � Cryoglobulinemia � B lymphocyte � HCV-NS3 antigen

Abstract

Background: We report on 3 cases of membranoprolifera-tive glomerulonephritis associated with mixed cryoglobulin in patients with hepatitis C virus (HCV) antibodies but a neg-ative blood viral load. These cases explore the pathogenesis of the renal disease. Methods: We searched for occult HCV infection in peripheral blood mononuclear cells, cryoprecip-itate, bone marrow cells, and glomeruli using ultrasensitive PCR assays and immunohistochemistry. We also looked for infraclinical B cell lymphoma by computed tomodensitom-etry, bone marrow aspiration and biopsy, and lymphocyte typing. Results: By PCR assays, we did not evidence occult hepatitis C infection in peripheral blood mononuclear cells, bone marrow cells, or cryoprecipitates. In the only patient with available kidney specimen, we evidenced HCV-NS3 an-tigen in glomeruli. HCV-associated lymphoma was exclud-ed, but mild polyclonal B lymphocytosis was present in the

Received: October 27, 2011 Accepted: November 27, 2011 Published online: January 13, 2012

NephrologyAmerican Journal of

Dr. Stanislas Bataille, MD Centre de Néphrologie et Transplantation Rénale, Hôpital de la Conception 147 Boulevard Baille FR–13005 Marseille (France) Tel. +33 49 138 3042, E-Mail stanislas.bataille   @   ap-hm.fr

© 2012 S. Karger AG, Basel0250–8095/12/0352–0134$38.00/0

Accessible online at:www.karger.com/ajn

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Clinical features of MPGN are hematuria, proteinuria often in the nephrotic range, hypertension, and impaired renal function with progression to chronic kidney dis-ease in some cases [6] . MPGN is defined by glomerular mesangial proliferation and thickening of the glomerular basement membrane and by subendothelial deposits of IgG and C3, or isolated C3 deposits.

Cryoglobulinemia is defined by the presence of serum of an abnormal immunoglobulin (Ig) that precipitates at 4   °   C and solubilizes at 36   °   C. Being based on � -globulin electrophoresis, it is classified according to the Brouet classification [7] . MC is characterized by the association of a polyclonal component, usually IgG, and sometimes a monoclonal component, usually IgM. HCV infection is the main cause of MC [8] . Mixed cryoglobulin has also an anti-IgG activity (rheumatoid factor activity).

In patients with HCV-induced MPGN, HCV-RNA is usually found in plasma and in cryoprecipitate by routine PCR [9] . Occult HCV infection, i.e. detectable HCV-RNA in the liver or peripheral blood mononuclear cells (PBMC) in the absence of serum HCV-RNA, has recently been described in patients with abnormal liver enzyme levels [10–13] . MC is also associated with occult HCV infection [14] . Finally, persistent cryoglobulinemic vasculitis has been reported despite successful HCV infection treat-ment [15] , as has cryoglobulinemic vasculitis relapse in the absence of any evidence of HCV replication [8] .

In 3 patients with MPGN and anti-HCV antibodies without serum detectable HCV-RNA, we investigated whether MPGN could be associated either with persis-tence of HVC antigen in the kidney or hematopoietic cells or with B cell proliferation.

Subjects and Methods

In 2007 and 2008, 3 patients with MPGN, associated with type II MC and anti-HCV antibodies but no detectable plasma HCV-RNA by routine RT-PCR, were seen at the Centre of Nephrology and Renal Transplantation of the University Hospital of Mar-seille. MPGN was diagnosed on kidney biopsy performed to ex-plore glomerular proteinuria in the 3 patients. All 3 patients were known to have a history of HCV infection before kidney biopsy was performed. Each patient gave his written informed consent to the study.

Patients were questioned about HCV infection and other pos-sible causes of MPGN. They underwent complete clinical exami-nation, including the search for cirrhosis, MC systemic vasculitis, such as Raynaud’s syndrome, peripheral neuropathy or purpura, long-term fever, and peripheral adenopathy. Blood and urine samples were taken for biochemical and hematological studies in-cluding albuminemia, cryoglobulinemia research and typing, complement (CH50, C3 and C4), C3 nephritic factor (C3NeF),

creatininemia, 24-hour proteinuria and urine cytology, liver en-zymes, hepatitis B and HIV serologies, and antinuclear antibody. Furthermore, bone marrow aspiration and bone marrow biopsies were performed.

Occult hepatitis C infection was investigated by ultrasensitive PCR (usPCR) in plasma, PBMC, cryoprecipitates, and bone mar-row aspirates. Samples were stored at –80   °   C before analysis. HCV RNA was detected using a modified COBAS TaqMan HCV assay with a detection limit of 15 IU/10 6 cells. Detection of HCV-RNA in PBMC was performed on 1 million PBMC prepared with a BD CPT tube (BD Diagnostics). The RNA extraction step was per-formed using silica beads (NucliSens; Organon Teknika S.A., Fresnes, France), and real-time PCR was done with the COBAS TaqMan HCV assay, according to the manufacturer’s instruc-tions using CTM 48 (Roche Diagnostics, Meylan, France). The sensitivity of our method was validated by detection of HCV-in-fected sera diluted in negative PBMC. HCV genotype 1 serum quantified at 1,500 IU/ml was diluted by a factor of 10, and 1 ml of diluted serum was added to 1 million PBMC prepared as de-scribed above. The mixture of PBMC and HCV genotype 1 sera was then quantified by the COBAS TaqMan assay. The detection limit was 15 IU/10 6 cells [16] .

B lymphocyte monoclonal proliferation was investigated in blood and bone marrow by lymphocyte antigenic surface typing, protein electrophoresis and immunofluorescence, and by immu-noglobulin (A, M, D) and free light chain dosing. Histology and immunohistochemistry, including cell surface expression of CD20, CD5, and CD138, were performed on the bone marrow bi-opsies. All patients underwent thoracic and abdominal CT scan to search for adenopathy. One patient also had a TEP-TDM. Liver biopsies were not performed due to ethical reasons. Liver fibrosis was assessed by fibroscan.

Immunohistochemistry was carried out to detect HCV-NS3 on 4- � m frozen sections of renal biopsies using the mouse mono-clonal anti-human HCV-NS3 antibody (Millipore, Temecula,Calif., USA) as primary antibody. The sections were rehydrated in phosphate buffered saline (PBS) and then immersed into 0.3% hydrogen peroxide in PBS for 5 min at room temperature to avoid endogenous peroxidase activity. Then, they were incubated with 2% defatted milk in PBS at 37   °   C for 5 min to block non-specific staining. The primary antibody PBS was incubated overnight at 4   °   C at a dilution of 1/200. The sections were rinsed twice with PBS during 5 min. We used the detection system KP500 (Diagnostic Biosystems, Pleasanton, Calif., USA) as an indirect method with anti-mouse IgG conjugated with horseradish peroxidase as sec-ondary antibody. This secondary antibody was incubated for 20 min at 37   °   C. The sections were then immersed in a diaminoben-zidine tetrahydrochloride solution, kit K3468 (Dako, Carpinteria, Calif., USA) for 10 min. Afterwards, counterstaining was per-formed with hematoxylin.

Results

Patient 1 The first patient (P1) was a Caucasian 41-year-old male

examined in February 2008. His medical history included smoking and a 10-year history of HCV infection following

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intravenous drug abuse. Viral genotype was not deter-mined. HCV replication disappeared spontaneously be-fore 2002 on routine PCR in serum. At presentation, clin-ical examination was normal (no hypertension and no edema). Routine blood and urine tests showed nephrotic range proteinuria (17.1 g/day), microhematuria [302 red blood cells per high power field (HPF)], serum albumin of 19 g/l [normal values (NV) 38–48 g/l], and an eGFR of 65 ml/min/1.73 m 2 , according to the Modification of Diet in Renal Disease Study (MDRD) equation [17] , normal se-rum liver enzymes and prothrombin time, and no auto-antibodies. C3 was 0.4 g/l (NV 0.75–1.44 g/l), C4 was 0.14 g/l (NV 0.1–0.34 g/l), and factor B was 227 mg/l (NV 100–450 mg/l) ( tables 1 and 2 ). C3NeF was not present.

By light microscopy, kidney biopsy showed mild me-sangial hypercellularity and diffuse thickening of the glomerular basement membrane. Immunofluorescence showed endomembranous IgM, C3, and � light chain de-posits. No other causes of MPGN, like autoimmune dis-eases, neoplasms, or infections, were found. Chest and abdomen computed tomodensitometry showed no ab-normalities.

HCV-RNA was not present in serum, PBMC, cryopre-cipitate, and bone marrow. We did not perform liver bi-opsy, since all liver enzymes were in the normal range. We unsuccessfully attempted to detect HCV-RNA in kid-ney biopsies because the conservation liquid used for tis-

sue sampling contained formaldehyde – a fixative known to degrade RNA. However, we could find HCV-NS3 an-tigen using immunohistochemistry. Immunostaining with anti-NS3 antibody showed numerous granular de-posits along the glomerular basement membrane consis-tent with persistence of viral antigen in glomeruli ( fig. 2 ), whereas negative non-HCV-related MPGN control had a negative staining.

Immunological findings of the patients are shown in table 2 . P1 had type II MC with IgM � monoclonal subset and mild lymphocytosis in bone marrow aspirations con-sisting of morphologically normal lymphocytes. Serum � / � light chain ratio was 1.79 (NV 0.26–1.65). He was treated with angiotensin-converting enzyme inhibitor but was soon lost to follow-up.

Patient 2 Patient 2 (P2) was a black 42-year-old male who pre-

sented for the first time in January 2008. His medical his-tory was remarkable for idiopathic deep venous throm-bosis in September 2007, smoking, and past intravenous drug abuse. He had contracted genotype 1a HCV infec-tion before 2005 and received combined interferon/riba-virin therapy for 70 weeks until viral clearance. In May 2005, he underwent liver biopsy with a fibrosis score of A2F2K11. The time lag between viral load negativation and renal impairment was 4 months. At presentation,

Table 1. Clinical and biological features of HCV, MPGN, and cryoglobulin

Patient 1 Patient 2 Patient 3

Mode of contamination IV drug abuse IV drug abuse unknownHCV genotype unknown 1a 2a/cTreatment none INF/ribavirin INF/ribavirin

Delay from serum viral clearance to MPGN unknown 4 months 15 monthsSerum albumin, g/l 19 23 25Proteinuria, g/day 17.1 3.44 6.7eGFR, ml/min/1.73 m2 65 87 43HTA no no noMicrohematuria yes yes yesIsotype of renal deposits IgM IgM IgG

Isotype of the cryoglobulin monoclonal subset IgM � IgM � IgG �C3, g/l [NV 0.75–1.44] 0.4 0.68 0.12C4, g/l [NV 0.1–0.34] 0.14 0.19 0.12CH50, % [NV 70–130] 42 52 40Factor B, mg/l [NV 100–450] 227 111 NA

I NF = Interferon; NA = not available; HTA = hypertension; NV = normal value.

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clinical examination showed Raynaud’s syndrome, and neither hypertension nor edema were found. Routine blood and urine tests showed nephrotic range proteinuria (3.44 g/day), microhematuria (1,215 red blood cells/HPF), serum albumin of 23 g/l, eGFR of 65 ml/min/1.73 m 2 (MDRD), and normal serum liver enzymes and pro-thrombin time. C3 was 0.68 g/l, C4 was 0.19 g/l, and fac-tor B was 111 mg/l. C3NeF was not present ( tables 1 and 2 ). Kidney biopsy showed typical MPGN with endomem-branous IgM, � , and C3 deposits (fig. 1). Other causes of MPGN were not found. Searches for HCV-RNA in se-rum, PBMC, cryoprecipitate, and bone marrow using usPCR were negative. Liver and kidney biopsies were not available for HCV-RNA PCR or immunohistochemistry. P2 had type II MC with IgM � monoclonal subset, plasma IgM dosing of 4.75 g/l (NV 0.34–2.1), B lymphocyte count in blood at 868 cells/mm 3 (NV 100–450 cells/mm 3 ), and a mild lymphocytosis in bone marrow consisting of mor-phologically normal lymphocytes. No adenomegaly was present on computed tomodensitometry.

The patient spontaneously recovered. Proteinuria and cryoglobulinemia disappeared after 4 months and com-plement levels normalized in the meantime. Six months later, his renal function was still normal.

Patient 3 Patient 3 (P3) was a Caucasian 51-year-old male who

presented for the first time in February 2008 with no medical history except for HCV infection from unknown origin before August 2006. Viral genotype was 2a/c. He received combined interferon/ribavirin therapy for 24 weeks. The patient was diagnosed with Child Pugh C cir-rhosis, and had a liver fibrosis score of A3F4 estimated using Fibrotest in 2006. At presentation, HCV-RNA rou-tine PCR was negative. The time lag between viral nega-tivation and renal impairment was 15 months. Clinical examination showed a sensitive peripheral neuropathy and skin lesions. He had neither hypertension nor edema. Routine blood and urine tests showed nephrotic range proteinuria (6.7 g/day), serum albumin of 25 g/l, eGFR of 43 ml/min/1.73 m 2 (MDRD), and microhematuria (1,215 red blood cells/HPF). He had increased liver enzyme val-ues: GOT 82 IU/l (NV 6–53 IU/l), GPT 57 IU/l (NV 8–40 IU/l), GGT 213 IU/l (NV 5–30 IU/l), alkaline phospha-tases 107 IU/l (NV 35–104 IU/l). Bilirubin was 18 � mol/l (NV 5–34 � mol/l) and prothrombin time was 59% (NV 1 70%) with factor V 0.68 U/ml (NV 0.7–1.2 U/ml). C3 was 0.12 g/l and C4 was 0.12 g/l. Factor B levels and C3NeF were not dosed ( tables 1 and 2 ).

Kidney biopsy showed MPGN, with IgG, C3, and � endomembranous deposits. No other cause of MPGN was found. As in P1 and P2, usPCR on serum, PBMC, cryoprecipitate, and bone marrow were negative but could not be performed in kidney or liver. No more bi-opsy material was available for immunohistochemistry.

The patient had type II MC with an IgG � monoclonal component. The plasma IgG and serum � / � free light chain ratio were 14.3 g/l (NV 6.9–14 g/l) and 1.78 (NV 0.26–1.65), respectively. He also had mild lymphocytosis in the bone marrow consisting of morphologically nor-mal lymphocytes. CT scan and PET-tomodensitometry showed no adenomegaly but splenomegaly with portal hypertension.

On the basis of severe renal and extra-renal manifesta-tions, the patient received 4 courses of rituximab 375 mg/m 2 /week followed by one injection of 375 mg/m 2 every 3

Table 2. Immunological findings

Patient 1 Patient 2 Patient 3

Serum immunoglobulins, g/lIgG [NV 6.9–14] 8.01 11.1 14.3IgA [NV 0.88–4.1] 1.7 3.51 3.48IgM [NV 0.34–2.1] 1.87 4.75 1.07

Kappa/lambda[NV = 0.26–1.65] 1.79 1.53 1.78

Blood lymphocyte typing, cells/mm3

CD3+ [NV 1,030–2,270] 1,400 1,601 1,154CD4+ [NV 640–1,450] 542 1,006 664CD8+ [NV 300–830] 783 504 456CD19+ [NV 100–450] 347 868 194CD19+/CD5+ [NV <120] 83 159 16

Bone marrow aspirationmild lymphocytosis 15% 15% 17%

Bone marrow lymphocyte typing, %/mm3

CD19+ 33% 48% NACD19+/CD5+ 5% 4% NACell surface �/� ratio

[NV 0.33–1.66] 1.6 0.5 NA

Bone marrow biopsyMorphology normal mild global

hyperplasianormal

ImmunohistochemistryCD 20, CD5, CD138

normal mild lympho-cytosis CD5+

normal

N A = Not available; NV = normal values. There are no normal values for bone marrow CD19+ and CD19+/CD5+ cell counts.

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months over a 6 months period. Nephrotic syndrome and ascites disappeared within the first month of treatment. At 20-month follow-up, proteinuria was negative and eGFR improved to 55 ml/min/1.73 m 2 . The patient had no adverse effect of rituximab.

Discussion

We report on 3 patients with MPGN associated with MC and a history of HCV infection. History of HCV in-fection was confirmed by the presence of anti-HCV anti-bodies in the serum of patients with a negative viral load. No evidence of occult HCV infection was found in PBMC and in the cryoprecipitate, and underlying B cell lympho-ma was not observed either, but HCV-NS3 antigen was present in the kidney biopsy in 1 patient.

Other forms of occult HCV infection were investigat-ed in our patients, but no HCV-RNA was detectable even using usPCR assays [10] . In the literature, occult liver HCV is defined as viral replication in liver tissue alone or additionally in PBMC besides constantly undetectable HCV-RNA in serum [18] . It is usually associated with ab-normal liver enzyme levels [10–12] . In our study, P1 and P2 had normal liver enzymes, and P3 had high liver en-zymes due to liver cirrhosis. Furthermore, in previous studies, 70% of patients with HCV occult infection in the liver also had occult infection in PBMC [10] . Because no liver biopsies were available, we cannot definitely exclude occult liver HCV infection.

To our knowledge, isolated occult kidney HCV infec-tion has never been described before. An HCV antigen, the HCV-NS3 protein, has been found in kidney biopsies of 2 patients out of 6 with anti-HCV antibodies, a negative routine viral load, and glomerulonephritis [19] . We could test only 1 patient for HCV-NS3 in kidney. He had granu-lar deposits along the glomerular basement membrane despite the 6-year time lag since viral blood negativation.

Because HCV-RNA was not investigated in the kid-neys, the significance of these deposits was unclear: are

Fig. 1. Endocapillary proliferative glomerulonephritis and tubu-lar injuries in P2. (Masson trichrome, ! 200).

Fig. 2. HCV-NS3 immunohistochemistry in the kidney biopsy of P1. a Glomerulus showing a slight membranoproliferative feature with segmental deposits within the capillary walls ( ! 200). The deposits are stained by anti-HCV-NS3 antibody. b Few glomeru-li had a severe mesangial nodular pattern with numerous granu-lar deposits along the basement membrane also stained by anti-NS3 antibody. ( ! 400).

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they remnants from a past HCV infection or virus-in-duced direct kidney injuries? The latter is more likely, since it has previously been reported that patients with MPGN and an HCV-active infection showed a propor-tional presence of HCV-RNA and HCV core proteins in kidney glomeruli [20] . Moreover, Nepomnyashchikh et al. [21] reported a good correlation between HCV-NS3 liver deposits and the presence of HCV-RNA in tissue. In this report, the presence of HCV-NS3 on immunohisto-chemistry was correlated to the presence of HCV-RNA but not to the severity of the disease.

Two hypotheses can be made to explain negative se-rum viral load. A first hypothesis is that there remains viral replication of the virus only in kidney cells, i.e. oc-cult HCV infection. A second hypothesis is that serum viral load is fluctuating, leading to temporarily undetect-able levels. This last situation is well-known in acute hep-atitis C [18] .

A strong link between HCV infection and B cell mono-clonal lymphocytosis or B cell lymphoma has been re-ported [22–26] . No evidence of underlying B cell lym-phoma could be detected in our patients by conventional techniques such as lymphocyte count, B cell phenotyp-ing, serum � / � light chain ratio, and bone marrow ex-amination and phenotyping. Moreover, 1 patient recov-ered spontaneously and another one did not develop B cell lymphoma during the 20-month follow-up. Yet, indi-rect features of B cell monoclonal component were pres-ent in our 3 patients, including MC with a mildly in-creased � / � light chain ratio, and bone marrow lympho-cytosis. MC may be considered a prelymphomatous state, since the antigenic specificities of CDR3 in MC and B cell lymphoma are similar, suggesting that, in HCV-related B cell lymphoma, malignant cells could derive from rheu-matoid factor-producing clones [27, 28] . Our results are consistent with the persistence of rheumatoid factor-pro-ducing B cell clones.

A surprising result was the unusual absence of HCV-RNA in the cryoprecipitates [9, 29] , suggesting that the

cause of polyclonal immune activation was not HCV in-fection. It is possible that the rheumatoid factor was pre-cipitated with polyclonal IgG after an HCV-independent immune activation. Note that 1 patient was still using IV drugs and another one suffered from liver cirrhosis, two clinical settings favoring infection-related immune acti-vation [30, 31] . In support of this hypothesis was the com-plement consumption profile, which suggested activation of the alternative rather than the classical pathway. The classical pathway is usually activated by immune com-plexes, particularly MC [32, 33] . Finally, C3NeF was not present and cannot explain the low C3 complement lev-els. Another explanation might be that HCV-NS3 instead of HCV-RNA is the stimulating antigen in the cryopre-cipitate, but this hypothesis could not be tested.

Making a difference between an ongoing HCV infec-tion and a virus-induced B cell monoclonal proliferation is critical for treatment: in the former, antiviral therapy may be required, whereas in the latter, the therapy should target B cell proliferation using anti-B cell proliferative drugs such as rituximab.

In conclusion, we reported on 3 cases of MPGN and a history of HCV infection. Renal injury is likely to be vi-rus-induced because we found NS3 viral antigen in glo-meruli. All patients had a mild and reactive B cell mono-clonal proliferation. Residual viral antigen could be a per-sistent immunological stimulus explaining B lymphocyte stimulation, cryoglobulin formation, and renal injury.

Acknowledgements

We thank Dr. Noémie Jourde, MD, PhD, and Dr. Karin Mazodier, MD, for their help in recruiting the patients.

Disclosure Statement

The authors have no conflicts of interest to declare.

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