Hepatitis C virus related cryoglobulinemic glomerulonephritis: Long-term remission after antiviral therapy

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1 Kidney International, Vol. 63 (2003), pp Hepatitis C virus related cryoglobulinemic glomerulonephritis: Long-term remission after antiviral therapy PAOLO ROSSI, TULLIO BERTANI, PIERO BAIO, ROBERTA CALDARA, PATRIZIA LULIRI, FRANCESCA TENGATTINI, PIERMARIO BELLAVITA, GIANNA MAZZUCCO, and ROCCO MISIANI U.O. di Medicina Interna I, U.O. di Nefrologia e Dialisi, U.O. di Immunoematologia e Centro Trasfusionale, Ospedali Riuniti, Bergamo, Italy; and U.O.A.D.U. Anatomia Patologica I, Azienda Ospedaliera S. Giovanni Battista, Università di Torino, Torino, Italy Hepatitis C virus related cryoglobulinemic glomerulonephri- branoproliferative glomerulonephritis with distinctive tis: Long-term remission after antiviral therapy. histologic characteristics that is usually associated with Background. Renal involvement in patients with hepatitis type II mixed cryoglobulinemia [1]. Although the patho- C virus (HCV) infection commonly manifests as cryoglobuligenesis of HCV-related CGN is still unclear, various nemic glomerulonephritis (CGN). The combination of interforms of antiviral therapy previously employed in chronic feron-alpha (IFN- ) and ribavirin, which is currently considered the standard antiviral therapy in chronic hepatitis C, could hepatitis C have been recently suggested by several aube difficult to carry out in cryoglobulinemic patients who are thors. A beneficial effect of treatment with interferon- frequently anemic, even in the absence of renal failure. Clinical and histologic long-term results of this therapeutic regimen (IFN- ) [2 5], ribavirin [6, 7], or both in combination [8, 9] have not been so far reported in patients with CGN. has been described, but a sustained response to these Methods. Three patients with HCV-related CGN and drugs has been rarely obtained. Moreover, evidence of slightly impaired kidney function were treated with IFN- and improvement in histologic features of CGN has been ribavirin for 12 months, and subsequently were followed up for 24 to 36 months. Two of these patients who were anemic reported in only isolated cases [10, 11]. We describe three were pretreated with erythropoietin (EPO). In each patient consecutive patients with HCV-associated CGN who renal biopsy was performed before starting therapy and re- achieved a long-term clinical and histologic remission peated 14 to 26 months after the end of treatment. following a combined antiviral treatment with IFN- and Results. In all three patients, antiviral therapy induced susribavirin. tained virologic response, which was followed by clear improvement in clinical, biochemical, immunologic, and histologic features. Clinical and biochemical improvement steadily progressed in all three patients, achieving normal or nearly normal results METHODS at the end of follow-up. In contrast, some immunologic fea- Patients tures, such as serum levels of C4 and rheumatoid factor activity, did not normalize in two and three patients, respectively. Posttory of leg purpura and arthralgia. In 1993 a diagnosis Patient 1 was a 58-year-old woman with a 10-year his- treatment renal biopsies showed mildly active histologic lesions. of HCV-related type II (IgG-IgM kappa) mixed cryoglo- Conclusion. Antiviral therapy with IFN- and ribavirin may bulinemia was established. At that time, no manifestation be considerably beneficial in patients with HCV-related CGN who obtain sustained virologic response. of renal disease was noted. She received two courses of IFN- therapy during a 2-year period, but no clear response could be observed. In 1996, she developed nephrotic syndrome and underwent renal biopsy that re- The most common expression of renal involvement in patients with hepatitis C virus (HCV) infection is cryoonly slight and temporary improvement in the manifesta- vealed the characteristic lesions of CGN. She obtained globulinemic glomerulonephritis (CGN). This is a memtions of renal disease after treatment with corticosteroids Key words: hepatitis C virus, cryoglobulinemia, glomerulonephritis, and cyclophosphamide. The patient was referred to our interferon-, ribavirin. unit in August Patient 2 was a 60-year-old man who reported a long- Received for publication July 29, 2002 and in revised form October 21, 2002, and January 5, 2003 lasting history of purpura. In 1996, he was admitted to Accepted for publication January 28, 2003 another hospital for nephrotic syndrome and slight elevation of alanine aminotransferase. He presented 2003 by the International Society of Nephrology positive 2236

2 Rossi et al: Treatment of cryoglobulinemic glomerulonephritis 2237 HCV testing and a liver biopsy demonstrated features All three patients required antihypertensive medicaconsisting with the diagnosis of chronic hepatitis C. Uri- tions, including angiotensin-converting enzyme (ACE) nary protein excretion was 5.1 g/day and serum creati- inhibitors and diuretics during both the periods of antivinine was 115 m/l. Renal biopsy disclosed a membrano- ral treatment and posttreatment follow-up. proliferative glomerulonephritis. The presence in the serum of mixed cryoglobulins (IgG-IgM kappa) led to diagnosis Evaluation of therapeutic response of CGN. He was treated initially with corticosteroids The patients were seen as outpatients every 2 weeks (25 mg prednisone) and subsequently with IFN- 2b 5 mil- for 8 weeks, and then at 4-week intervals during the lion units 3 times a week for 6 months. IFN- therapy treatment period. Subsequently the patients were folproduced a transient normalization of alanine amino- lowed-up every 6 months for 24 to 36 months. At each transferase in the absence of virologic response, whereas visit, renal and extrarenal manifestations of mixed cryothe manifestations of renal disease remained essentially globulinemia, along with the side effects of therapy, were unchanged. The patient was referred to our unit in Octo- carefully evaluated. ber The changes of kidney disease were monitored by Patient 3 was a 35-year-old man who was admitted means of clinical signs (edema, hypertension) and laboto our hospital in May 1998 for evaluation of severe ratory tests (proteinuria, albuminemia, and creatinine hypertension first noted 2 months earlier and unrespon- clearance). In addition, the three patients underwent a sive to 100 mg atenolol. On admission, the patient pre- first renal biopsy before starting antiviral therapy and a sented ankle edema, hypertension (190/120 mm Hg), second one 14 to 26 months after the end of treatment moderate proteinuria (2.7 g/day), mild renal insufficiency period. (serum creatinine 134 m/l) and positive serum testing A variety of clinical symptoms and signs, such as arfor HCV antibodies and HCV RNA. Moreover, the presthralgia and purpura, blood counts, and biochemical tests ence of type II mixed cryoglobulins (IgG-IgM kappa) served as indicators of extrarenal involvement and adwas demonstrated in his serum. Renal biopsy findings verse drug reactions. were consistent with the diagnosis of CGN. The levels of cryocrit, C 4, and rheumatoid factor activity in the serum were used to measure the immunologic Laboratory tests response. Liver and kidney function tests were performed ac- To assess the virologic response, the concentration of cording standard methods. Rheumatoid factor and com- HCV RNA in the serum was determined at fixed interplement levels were measured by rate nephelometry. vals, namely, after 4 weeks of ribavirin monotherapy, Cryoglobulins isolated from the serum were measured after 4 weeks of combined treatment, at the end of the as cryocrit (i.e., the percentage of packed cryoglobulins). treatment period, and then every 6 months during the HCV antibodies were detected using a second-generaposttreatment follow-up. tion enzyme immunoassay (Abbott Laboratories, North Chicago, IL, USA). Serum HCV RNA was quantified with a commercial reverse transcriptase polymerase chain RESULTS reaction (RT-PCR) assay (Amplicor HCV monitor, Virologic response Roche Diagnostic System, Branchburg, PA, USA). HCV genotyping was performed by PCR, using primers spewere HCV RNA positive, two having genotype 2b and At the start of antiviral therapy, the three patients cific for the HCV core region, as described [12]. the third one having the 2a type. After 4 weeks of ribavirin Treatment monotherapy, the viremia decreased by 1.2, 1.2, and The three patients received ribavirin at the initial dose 0.2 Log/mL in the three patients, respectively. After 4 of 15 mg/kg/day, which was adjusted to maintain hemo- weeks of combination therapy with ribavirin and IFN-, globin values between 10 and 12 g/dl on the basis of HCV disappeared from the serum of all three patients. fortnightly measurements. After 4 weeks of ribavirin Afterwards, this result was confirmed at the end of treat- monotherapy, IFN- 2b was added at a dose of 3 million ment period and maintained during the posttreatment units three times a week, and then the combined treatment follow-up. was continued for 1 year. Patients 1 and 3 who presented anemia before starting antiviral therapy were Clinical and biochemical response pretreated with erythropoietin (EPO), 10,000 UI, subcuno Following 4 weeks of treatment with ribavirin alone, taneously once a week until an hemoglobin value of 12 appreciable change in clinical or biochemical features g/dl or greater was obtained. Subsequently, EPO was could be observed. continued in association with ribavirin and IFN- administered As shown in Table 1, a few months after the start of as above described. combination therapy, edema and purpura disappeared

3 2238 Rossi et al: Treatment of cryoglobulinemic glomerulonephritis Table 1. Changes of clinical signs and laboratory values Patient 1 Patient 2 Patient 3 Parameters B 6m ET FU B 6m ET FU B 6m ET FU Edema Hypertension 170/90 140/80 130/85 135/85 165/90 135/80 140/80 135/80 190/ /80 110/80 120/80 Purpura Proteinuria g/day Albuminemia g/l Creatinine clearance ml/s Cryocrit % C 4 g/l Rheumatoid factor UI/ml Abbreviations are: B, baseline values; 6m, 6 months of treatment; ET, end of treatment; FU, end of follow-up;, absent or undetectable;, mild;, moderate;, marked. Normal values are proteinuria, 0.15 g/day; albuminemia, 3.5 g/l; creatinine clearance, 1.25 to 2.08 ml/s; cryocrit, undetectable; C 4, 0.15 to 0.40 g/l; rheumatoid factor, 20 UI/mL. Table 2. Renal biopsy findings Patient 1 Patient 2 Patient 3 Light microscopy FB SB FB SB FB SB Mesangial matrix Mesangial cells Monocyte infiltration GBM thickening and double contours Vasculitis Immunofluorescence Subendothelial deposits IgM IgM IgM IgM IgM IgM C 3 C 3 C 3 C 3 Mesangial deposits IgM IgM IgM IgM IgM IgM C 3 C 3 C 3 C 3 Electron microscopy Mesangial matrix Mesangial cells Monocyte infiltration Subendothelial deposits Mesangial deposits Abbreviations are: FB, first biopsy; SB, second biopsy; GBM, glomerular basement membrane;, undetectable;, occasionally seen;, mild;, moderate;, marked. patients. On light microscopy, we found moderate to severe increase in mesangial matrix, mesangial cells, and monocytes along with thickening of glomerular base- ment membrane (GBM) and formation of double con- tours (Fig. 1A). Only in one case, renal vasculitis was observed. Immunofluorescence study showed diffuse glomerular deposits of IgM apparently more abundant along the capillary walls than in mesangium Fig. 2A). In two patients, deposits of C3 with similar characteristics were demonstrated as well. On electron microscopy, the glomerular abnormalities found on light microscopy, such as increase in mesangial matrix, mesangial cells, and infiltrating monocytes, were confirmed. In addition, it was observed that the formation of double contours was mainly due to peripheral interposition of monocytes that were in close contact with subendothelial deposits consisting of amorphous or variously structured material. The second biopsy revealed obvious improvement in renal lesions. On light microscopy, the increase of mesangial matrix and mesangial cells was mild or occasionally in all three patients. At the same time, blood pressure normalized with reduced doses of antihypertensive drugs. Proteinuria decreased and creatinine clearance increased regularly throughout the observation period, returning into the normal range in two patients at the end of posttreatment follow-up. Cryoglobulins decreased to undetectable levels in the three patients before the completion of treatment. The serum concentration of C 4 became normal in one patient and increased to slightly subnormal levels in the others. Rheumatoid factor activ- ity clearly decreased in all patients, but in none of them achieved normal values. Hemoglobin concentration de- creased by 2.1 to 4.5 g/dl in the three patients, necessitat- ing a reduction in the dose of ribavirin in one of them. Other side effects, such as flu-like and gastrointestinal symptoms, albeit common, did not require modifications of therapy. Renal histologic changes Table 2 shows that, before starting antiviral therapy, renal histologic features were quite similar in the three

4 Rossi et al: Treatment of cryoglobulinemic glomerulonephritis 2239 Fig. 1. Patient 2. (A ) First biopsy. On light microscopy, the enlarged glomerulus shows marked endocapillary hypercellularity. Expansion of mesangial matrix and thickening of glomerular basement membrane with formation of double contours are also present (hematoxylin-eosin stain; magnification 250). (B ) Second biopsy. The glomerulus shows only mild increase of endocapillary cells and expansion of mesangial matrix (hematoxylin-eosin stain; magnification 250). seen (Fig. 1B). Monocyte infiltration and GBM thick- ening with double contours were rarely observed. Renal vasculitic lesions, previously demonstrated in patient 1, disappeared. On immunofluorescence, scanty deposits of IgM were still recognizable (Fig. 2B), while those of C3 were no longer detectable in both subendothelial and mesangial locations. The electron microscopy study confirmed that the mass of mesangial cells and mono- cytes that had been observed on the first biopsy was markedly reduced. Subendothelial deposits disappeared in two of three patients, whereas scanty deposits were still detectable in mesangium. units three times a week for 6 months) has been able to improve the renal function and/or reduce the proteinuria, possibly as a consequence of the clearance of HCV and the disappearance of cryoglobulins from the serum [2, 3]. Unfortunately, although more favorable results have been achieved in some patients treated with higher doses of IFN- [4, 10], in most cases the disease tends to recur upon discontinuation of the drug. The combination of IFN- with the nucleoside analog ribavirin has recently emerged as the most effective form of antiviral therapy in chronic hepatitis C, even though the latter drug given alone seems unable to suppress the replica- tion of HCV [13, 14]. Similar results have been observed in patients with CGN in whom the combined treatment with IFN- and ribavirin was able to obtain a long-lasting virologic and clinical response [8, 9], whereas ribavirin monotherapy produced some transient clinical improvement, but not the clearance of HCV from the serum [6, 7]. An important limitation of ribavirin is the tendency to cause hemolytic anemia, which may require early dis- continuation of this drug or even preclude its use in patients with low values of hemoglobin concentration DISCUSSION The current knowledge of close association between HCV infection and mixed cryoglobulinemia, with or without renal disease, has prompted a growing use of antiviral therapy in this condition. In many patients with CGN, IFN- monotherapy at standard doses (3 millions Fig. 2. Patient 3. (A ) First biopsy. Immunofluorescence study reveals intense glomerular deposition of IgM mainly along the capillary walls (magnification 250). (B ) Second biopsy. The glomerular deposits of IgM appear scanty, faint, and undefined in location (magnification 250).

5 2240 Rossi et al: Treatment of cryoglobulinemic glomerulonephritis [13 15]. Unfortunately, up to 70% of patients with mixed the clearance of HCV from the serum, could be related cryoglobulinemia show normochromic normocytic ane- to the slow and incomplete regression of the abovemia at presentation, even in the absence of renal failure mentioned immunologic abnormalities. [16, 17]. Indeed, two of our patients had anemia with Although much encouraging, these results are not sufunrevealing laboratory tests and bone marrow examina- ficient evidence to propose this form of treatment in all tion. In both cases, pretreatment with EPO enabled us to cases of HCV-related CGN. We are aware that, in pastart antiviral therapy with standard or slightly reduced tients with interferon resistant HCV genotypes or those doses of ribavirin. with severe and/or rapidly progressive manifestations In the three patients reported herein we had the op- of disease the response to antiviral therapy could be portunity of evaluating the effects of different modalities unsatisfactory [1, 21]. Furthermore, the use of interferon of antiviral therapy. Two of these patients had been pre- has been associated with the development of various viously treated with IFN- alone at standard or high doses, forms of renal disease or the exacerbation of preexisting but no appreciable benefit could be obtained. Similarly, CGN [21, 22]. In all these conditions where antiviral 4 weeks of ribavirin monotherapy did not appear to im- drugs are ineffective or contraindicated, as well as in prove the clinical condition in none of the three patients, CGN unrelated to HCV infection, immunosuppressive even though a clear reduction of HCV RNA serum con- therapy with or without plasmapheresis still maintains centration could be observed in two of them. In contrast, an important role [1, 21, 23]. With regard to antiviral 4 weeks of combination therapy caused persistent disap- therapy, it is possible that, over the next few years, the pearance of HCV viremia followed by clinical and bio- combination of peginterferon-alpha plus ribavirin, may chemical response. It is well known that, in patients with prove more efficacious than the classic combination therchronic hepatititis C, the HCV genotype is one of the apy in HCV-related CGN, as reported in chronic hepatimost important factors conditioning the results of antiviral tis [24]. Therefore, further studies in larger and heterogetherapy [13, 18]. Thus, it is likely that the presence of neous groups of patients are needed to establish the genotype 2 in all our patients may have fostered a sustained optimal treatment for HCV-related CGN. virologic response. A rapid and marked improvement in clinical signs was observed in all patients. Although the disappearance of edema and the normalization of blood ACKNOWLEDGMENT pressure could be ascribed to the intensive use of diuretof the manuscript. The authors thank Paolo Misiani for assistance in the preparation ics and antihypertensive drugs, the clearance of purpura appeared closely related to the virologic response, as Reprint requests to Rocco Misiani M.D., U.O. Medicina Interna I, reported in other studies [2, 19, 20]. 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