Original Report: Patient-Oriented, Translational Research

Nephrology

American

Journal of

Am J Nephrol 2012;35:134140 DOI: 10.1159/000335375

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

Membranoproliferative Glomerulonephritis and Mixed Cryoglobulinemia after Hepatitis C Virus Infection Secondary to Glomerular NS3 Viral Antigen Deposits
Stanislas Bataille a Gilles Kaplanski b Jos Boucraut c Philippe Halfon e Claire Camus e Laurent Daniel d Stphane Burtey a Yvon Berland a Bertrand Dussol a
Centre de Nphrologie et Transplantation Rnale, b Service de Mdecine Interne, c Laboratoire dImmunologie, Hpital de la Conception, et d Service dAnatomie Pathologique et de Neuropathologie, Hpital Timone, APHM, Aix-Marseille Universit, et e Laboratoire Alphabio, Marseille, France
a

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

Abstract Background: We report on 3 cases of membranoproliferative glomerulonephritis associated with mixed cryoglobulin in patients with hepatitis C virus (HCV) antibodies but a negative blood viral load. These cases explore the pathogenesis of the renal disease. Methods: We searched for occult HCV infection in peripheral blood mononuclear cells, cryoprecipitate, bone marrow cells, and glomeruli using ultrasensitive PCR assays and immunohistochemistry. We also looked for infraclinical B cell lymphoma by computed tomodensitometry, 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 antigen in glomeruli. HCV-associated lymphoma was excluded, but mild polyclonal B lymphocytosis was present in the

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 patients with hepatitis C-negative viral load, membranoproliferative glomerulonephritis could be induced by the persistence of HCV antigen in the kidney but not in hematopoietic 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 cause of membranoproliferative glomerulonephritis (MPGN). This condition is often associated with mixed cryoglobulinemia (MC) systemic vasculitis [13]. MC is reported in 5060% of HCV-infected patients [4]. MPGN can be secondary to HCV alone, HCV-associated MC, or MC alone [5]. HCV-infected patients with MC are more likely to develop MPGN [6].

2012 S. Karger AG, Basel 02508095/12/03520134$38.00/0 Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com Accessible online at: www.karger.com/ajn

Dr. Stanislas Bataille, MD Centre de Nphrologie et Transplantation Rnale, Hpital de la Conception 147 Boulevard Baille FR13005 Marseille (France) Tel. +33 49 138 3042, E-Mail stanislas.bataille@ap-hm.fr

Clinical features of MPGN are hematuria, proteinuria often in the nephrotic range, hypertension, and impaired renal function with progression to chronic kidney disease 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 [1013]. MC is also associated with occult HCV infection [14]. Finally, persistent cryoglobulinemic vasculitis has been reported despite successful HCV infection treatment [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 persistence 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 HCVRNA by routine RT-PCR, were seen at the Centre of Nephrology and Renal Transplantation of the University Hospital of Marseille. MPGN was diagnosed on kidney biopsy performed to explore 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 possible causes of MPGN. They underwent complete clinical examination, including the search for cirrhosis, MC systemic vasculitis, such as Raynauds syndrome, peripheral neuropathy or purpura, long-term fever, and peripheral adenopathy. Blood and urine samples were taken for biochemical and hematological studies including albuminemia, cryoglobulinemia research and typing, complement (CH50, C3 and C4), C3 nephritic factor (C3NeF),

creatininemia, 24-hour proteinuria and urine cytology, liver enzymes, 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 marrow 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/106 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 performed 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 manufacturers instructions using CTM 48 (Roche Diagnostics, Meylan, France). The sensitivity of our method was validated by detection of HCV-infected 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 described above. The mixture of PBMC and HCV genotype 1 sera was then quantified by the COBAS TaqMan assay. The detection limit was 15 IU/106 cells [16]. B lymphocyte monoclonal proliferation was investigated in blood and bone marrow by lymphocyte antigenic surface typing, protein electrophoresis and immunofluorescence, and by immunoglobulin (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 biopsies. 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 monoclonal 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 secondary antibody. This secondary antibody was incubated for 20 min at 37 C. The sections were then immersed in a diaminobenzidine tetrahydrochloride solution, kit K3468 (Dako, Carpinteria, Calif., USA) for 10 min. Afterwards, counterstaining was performed 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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Table 1. Clinical and biological features of HCV, MPGN, and cryoglobulin

Patient 1 Mode of contamination HCV genotype Treatment Delay from serum viral clearance to MPGN Serum albumin, g/l Proteinuria, g/day eGFR, ml/min/1.73 m2 HTA Microhematuria Isotype of renal deposits Isotype of the cryoglobulin monoclonal subset C3, g/l [NV 0.751.44] C4, g/l [NV 0.10.34] CH50, % [NV 70130] Factor B, mg/l [NV 100450] IV drug abuse unknown none unknown 19 17.1 65 no yes IgM IgM 0.4 0.14 42 227

Patient 2 IV drug abuse 1a INF/ribavirin 4 months 23 3.44 87 no yes IgM IgM 0.68 0.19 52 111

Patient 3 unknown 2a/c INF/ribavirin 15 months 25 6.7 43 no yes IgG IgG 0.12 0.12 40 NA

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

intravenous drug abuse. Viral genotype was not determined. HCV replication disappeared spontaneously before 2002 on routine PCR in serum. At presentation, clinical 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) 3848 g/l], and an eGFR of 65 ml/min/1.73 m2, according to the Modification of Diet in Renal Disease Study (MDRD) equation [17], normal serum liver enzymes and prothrombin time, and no autoantibodies. C3 was 0.4 g/l (NV 0.751.44 g/l), C4 was 0.14 g/l (NV 0.10.34 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 mesangial hypercellularity and diffuse thickening of the glomerular basement membrane. Immunofluorescence showed endomembranous IgM, C3, and light chain deposits. No other causes of MPGN, like autoimmune diseases, neoplasms, or infections, were found. Chest and abdomen computed tomodensitometry showed no abnormalities. HCV-RNA was not present in serum, PBMC, cryoprecipitate, and bone marrow. We did not perform liver biopsy, since all liver enzymes were in the normal range. We unsuccessfully attempted to detect HCV-RNA in kidney biopsies because the conservation liquid used for tis136
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sue sampling contained formaldehyde a fixative known to degrade RNA. However, we could find HCV-NS3 antigen using immunohistochemistry. Immunostaining with anti-NS3 antibody showed numerous granular deposits along the glomerular basement membrane consistent 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 table2. P1 had type II MC with IgM monoclonal subset and mild lymphocytosis in bone marrow aspirations consisting of morphologically normal lymphocytes. Serum / light chain ratio was 1.79 (NV 0.261.65). 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 presented for the first time in January 2008. His medical history was remarkable for idiopathic deep venous thrombosis in September 2007, smoking, and past intravenous drug abuse. He had contracted genotype 1a HCV infection before 2005 and received combined interferon/ribavirin 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,
Bataille /Kaplanski /Boucraut /Halfon / Camus /Daniel /Burtey /Berland /Dussol

clinical examination showed Raynauds 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 m2 (MDRD), and normal serum liver enzymes and prothrombin time. C3 was 0.68 g/l, C4 was 0.19 g/l, and factor B was 111 mg/l. C3NeF was not present (tables 1 and 2). Kidney biopsy showed typical MPGN with endomembranous IgM, , and C3 deposits (fig. 1). Other causes of MPGN were not found. Searches for HCV-RNA in serum, 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 0.342.1), B lymphocyte count in blood at 868 cells/mm3 (NV 100450 cells/mm3), and a mild lymphocytosis in bone marrow consisting of morphologically normal lymphocytes. No adenomegaly was present on computed tomodensitometry. The patient spontaneously recovered. Proteinuria and cryoglobulinemia disappeared after 4 months and complement 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 cirrhosis, and had a liver fibrosis score of A3F4 estimated using Fibrotest in 2006. At presentation, HCV-RNA routine PCR was negative. The time lag between viral negativation 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 m2 (MDRD), and microhematuria (1,215 red blood cells/HPF). He had increased liver enzyme values: GOT 82 IU/l (NV 653 IU/l), GPT 57 IU/l (NV 840 IU/l), GGT 213 IU/l (NV 530 IU/l), alkaline phosphatases 107 IU/l (NV 35104 IU/l). Bilirubin was 18 mol/l (NV 534 mol/l) and prothrombin time was 59% (NV 170%) with factor V 0.68 U/ml (NV 0.71.2 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).

Table 2. Immunological findings

Patient 1 Patient 2 Serum immunoglobulins, g/l IgG [NV 6.914] 8.01 IgA [NV 0.884.1] 1.7 IgM [NV 0.342.1] 1.87 Kappa/lambda [NV = 0.261.65] 1.79
3

Patient 3 14.3 3.48 1.07 1.78 1,154 664 456 194 16 17% NA NA NA

11.1 3.51 4.75 1.53 1,601 1,006 504 868 159 15%

Blood lymphocyte typing, cells/mm CD3+ [NV 1,0302,270] 1,400 CD4+ [NV 6401,450] 542 CD8+ [NV 300830] 783 CD19+ [NV 100450] 347 CD19+/CD5+ [NV <120] 83 Bone marrow aspiration mild lymphocytosis 15%

Bone marrow lymphocyte typing, %/mm3 CD19+ 33% 48% CD19+/CD5+ 5% 4% Cell surface / ratio 1.6 0.5 [NV 0.331.66] Bone marrow biopsy Morphology Immunohistochemistry CD 20, CD5, CD138 normal normal

mild global normal hyperplasia mild lympho- normal cytosis CD5+

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

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 biopsy 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 6.914 g/l) and 1.78 (NV 0.261.65), respectively. He also had mild lymphocytosis in the bone marrow consisting of morphologically normal lymphocytes. CT scan and PET-tomodensitometry showed no adenomegaly but splenomegaly with portal hypertension. On the basis of severe renal and extra-renal manifestations, the patient received 4 courses of rituximab 375 mg/ m2/week followed by one injection of 375 mg/m2 every 3
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Color version available online

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

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 m2. 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 infection was confirmed by the presence of anti-HCV antibodies 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 lymphoma was not observed either, but HCV-NS3 antigen was present in the kidney biopsy in 1 patient. Other forms of occult HCV infection were investigated 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 abnormal liver enzyme levels [1012]. In our study, P1 and P2 had normal liver enzymes, and P3 had high liver enzymes 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.
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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 glomeruli had a severe mesangial nodular pattern with numerous granular deposits along the basement membrane also stained by antiNS3 antibody. (!400).

To our knowledge, isolated occult kidney HCV infection 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 granular deposits along the glomerular basement membrane despite the 6-year time lag since viral blood negativation. Because HCV-RNA was not investigated in the kidneys, the significance of these deposits was unclear: are
Bataille /Kaplanski /Boucraut /Halfon / Camus /Daniel /Burtey /Berland /Dussol

Color version available online

they remnants from a past HCV infection or virus-induced 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 proportional 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 immunohistochemistry was correlated to the presence of HCV-RNA but not to the severity of the disease. Two hypotheses can be made to explain negative serum viral load. A first hypothesis is that there remains viral replication of the virus only in kidney cells, i.e. occult HCV infection. A second hypothesis is that serum viral load is fluctuating, leading to temporarily undetectable levels. This last situation is well-known in acute hepatitis C [18]. A strong link between HCV infection and B cell monoclonal lymphocytosis or B cell lymphoma has been reported [2226]. No evidence of underlying B cell lymphoma could be detected in our patients by conventional techniques such as lymphocyte count, B cell phenotyping, serum / light chain ratio, and bone marrow examination and phenotyping. Moreover, 1 patient recovered spontaneously and another one did not develop B cell lymphoma during the 20-month follow-up. Yet, indirect features of B cell monoclonal component were present in our 3 patients, including MC with a mildly increased / light chain ratio, and bone marrow lymphocytosis. 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 rheumatoid factor-producing clones [27, 28]. Our results are consistent with the persistence of rheumatoid factor-producing B cell clones. A surprising result was the unusual absence of HCVRNA in the cryoprecipitates [9, 29], suggesting that the

cause of polyclonal immune activation was not HCV infection. It is possible that the rheumatoid factor was precipitated 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 activation [30, 31]. In support of this hypothesis was the complement consumption profile, which suggested activation of the alternative rather than the classical pathway. The classical pathway is usually activated by immune complexes, particularly MC [32, 33]. Finally, C3NeF was not present and cannot explain the low C3 complement levels. Another explanation might be that HCV-NS3 instead of HCV-RNA is the stimulating antigen in the cryoprecipitate, but this hypothesis could not be tested. Making a difference between an ongoing HCV infection 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 virus-induced because we found NS3 viral antigen in glomeruli. All patients had a mild and reactive B cell monoclonal proliferation. Residual viral antigen could be a persistent immunological stimulus explaining B lymphocyte stimulation, cryoglobulin formation, and renal injury.

Acknowledgements
We thank Dr. Nomie 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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