Effect of combination therapy (ribavirin and interferon) in HCV-related glomerulopathy

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1 Nephrol Dial Transplant (2002) 17: Original Article Effect of combination therapy (ribavirin and interferon) in HCV-related glomerulopathy Alaa A. Sabry 1, Mohamed A. Sobh 1, Hussein A. Sheaashaa 1, Guara Kudesia 2, Graham Wild 2, Samantha Fox 2, Bart E. Wagner 2, William L. Irving 3, Anna Grabowska 3 and Abdel Meguid El-Nahas 3 1 Mansoura Urology and Nephrology Center, Mansoura University, Mansoura, Egypt, 2 Sheffield Kidney Institute, Northern General Hospital, Sheffield and 3 Queen s Medical Centre, Nottingham, UK Abstract Background. Hepatitis C virus (HCV) is a major cause of acute and chronic hepatitis throughout the world. Several extrahepatic manifestations, including glomerulonephritis, have been reported to be associated with this type of infection. Cryoglobulinaemic and non-cryoglobulinaemic membranoproliferative glomerulonephritis (MPGN) and membranous nephropathy (MN) are the commonest lesions associated with HCV. Results of treatment of these patients with interferon therapy have been disappointing, since relapse of the viraemia and subsequent relapse of the renal disease are major problems. Combination of interferon with ribavirin in patients with chronic liver disease has been shown to increase the rate of sustained response. Methods. In this work, 20 patients with HCVassociated glomerulopathy were subjected to an in-depth evaluation of their kidney lesions and HCV involvement. Laboratory, histopathological, immunohistochemical, and electron-microscopy techniques were used. The patients received interferon therapy for 12 months; in interferon-resistant subjects, interferon was combined with ribavirin. Results. MPGN was the commonest kidney lesion, being reported in 85% of these cases, followed by MN and mesangioproliferative glomerulonephritis (10 and 5% respectively). Mixed cryoglobulinaemia was encountered in 60% of the cases. Twelve months antiviral treatment resulted in aviraemia in 25% of cases, while liver enzymes were normalized in 75%, 24-h proteinuria significantly decreased (from median 4 g to 1.10 g, Ps0.001), serum albumin increased (from median 2.50 to 3.55 gudl, Ps0.012), lower viral titres Correspondence and offprint requests to: Dr A. A. Sabry, Mansoura Urology and Nephrology Center, Mansoura University, Mansoura, Egypt. asabry20@hotmail.com (from median 1.15 to 0.53 mega-equml, Ps0.049), and C3 and C4 concentrations returned to normal. Basal serum creatinine and viral titres were important determinants of response to treatment. Conclusion. This study supports the relationship between HCV and glomerulonephritis, especially MPGN, and the use of a combination of interferon and ribavirin in the treatment of selected cases of HCV-related glomerulopathy. Keywords: glomerulopathy; HCV nephropathy; interferon; membranoproliferative glomerulonephritis; ribavirin Introduction Glomerular disease frequently accompanies chronic liver disease, although it is clinically silent [1]. Accumulating epidemiological evidences suggest that there is an association between hepatitis C virus (HCV) infection and renal diseases, most commonly membranoproliferative glomerulonephritis (MPGN) and membranous nephropathy (MN) [2]. Focal segmental glomerulosclerosis (FSGS), mesangial proliferative glomerulonephritis with IgA deposition are especially common [3]. These patients may or may not have cryoglobulinaemia, and even if it is present they frequently lack other symptoms of essential mixed cryoglobulinaemia [4,5]. The results of interferon treatment for HCV-related MPGN have been disappointing [6]. In the great majority of these cases, interferon has been successful in inducing recovery of renal function with reduction of proteinuria, in association with clearance of HCV from the serum. Unfortunately, the disease tends to relapse shortly after therapy is stopped [7,8]. Ribavirin alone has recently been used in the treatment of patients with # 2002 European Renal Association European Dialysis and Transplant Association

2 Interferon-a and ribavirin in HCV-related glomerulopathy HCV-associated mixed cryoglobulinaemia. However, complete and sustained virological response has never been reported [9]. Success of combination therapy in rapid clearance of HCV from serum, resolution of purpura, and marked improvement in renal function and proteinuria has recently been reported in a small group of patients [8]. Thus, we decided to study the effect of combination therapy (interferon plus ribavirin) in a group of patients (ns20) diagnosed with HCV-related glomerulopathy. Patients and methods This study included 20 HCV-infected patients with nephrotic syndrome. All patients were positive for HCV antibodies when screened by a third-generation ELISA technique (Abbott Diagnostics, USA). Confirmation of HCV infection was achieved by polymerase reaction (PCR) (Amplicor PCR Diagnostics, Roche Diagnostic Systems, USA). Other secondary causes of glomerulonephritis such as diabetes mellitus, schistosomiasis, and SLE were excluded. Laboratory investigations included urinalysis, serum electrolytes, creatinine, bilirubin, ALT, AST, and alkaline phosphatase test for rheumatoid factor and cryoglobulinaemia were carried out using standard methods. Additional virological studies included testing for HBsAg by ELISA (Abbott Diagnostics, USA) and HIV 1 and 2 (Murex HCV 1 and 2 immunoassay). Renal biopsies were carried out in all cases, but liver biopsies were obtained from only seven patients with elevated liver enzymes. Two patients with skin lesions were subjected to skin biopsy. Formalin-fixed and paraffin-embedded sections of kidney and liver tissues were stained and examined by light-microscopy. Reverse transcription (RT) polymerase chain reaction (nested PCR) Cryoprecipitates, paraffin-embedded liver and kidney sections, and frozen kidney tissues were tested for HCV- RNA by two round amplification PCR (nested PCR) using primers chosen from the highly conserved 59 region (Life Technologies Ltd, UK) [5,9]. Immunohistochemistry Formalin-fixed, paraffin-embedded liver and renal sections as well as frozen renal sections were subjected to immunohistochemistry using monoclonal antibody to HCV core AA1-120 (Biogenesis Ltd, UK) [5,10]. Electron microscopy Eight renal biopsy sections were randomly chosen and examined by electron microscopy (Philips 400 transmission EM). Kidney sections from three HCV-negative nephrotic patients were included in this test as controls. Viral genotyping HCV genotyping was performed on 10 patients using RT PCR with specific primers for HCV core region 59 NCR. Products were sequenced with AB1 prism DNA sequencing kit (PE Applied Biosciences, Warrington, UK). Anti-viral treatment protocol Interferon-a 2A was given subcutaneously in a dose of 3 MU three times weekly. The dose was adjusted according to patient tolerance. Patients were followed up weekly for 1 month and monthly thereafter for 12 months. Laboratory investigations at follow-up visits included urinalysis, serum creatinine, bilirubin, ALT, AST, alkaline phosphatase, CBC, and 24-h urinary protein excretion. At the 3-month follow-up qualitative HCV-RNA-PCR was carried out (Amplicor PCR diagnostics, Roche Diagnostic System, USA). Those with persistent HCV viraemia were given ribavirin in addition at a dose of 15 mgukguday, with dose modification when indicated. Treatment was continued to the complete 12 months. Quantification of HCV-RNA This was carried out for all patients at the beginning and at the end of anti-viral treatment using HCV-RNA 2.0 assay (bdna) (Chiron Corporation, Emeryville, CA, USA). Results from the Chiron luminometer are reported in relative luminescence units, a measurement of the amount of light emitted from each HCV capture well, which is proportional to the number of HCV-RNA mega-equivalent per millilitre present in the specimen [5]. Renal response to anti-viral treatment According to the renal response to anti-viral treatment, patients were divided into two groups: group I cases were those who showed favourable response (stable or decreased serum creatinine and proteinuria), and group II cases were those who showed a deterioration in their serum creatinine and proteinuria. The two groups were compared to identify factors affecting the renal response to anti-viral treatment. Statistical analysis Results are given as median and confidence intervals. Mann Whitney (non-parametric rank sum test), Fischer exact probability test, and chi-squared test were applied as appropriate. A P-value of was considered significant. Results 1925 General observations The clinical characteristics of the 20 patients with HCV-related glomerulopathy are given in Table 1. Males predominated, blood transfusion was reported in 42% of the cases, and a history of surgery was given in 60% of cases. Laboratory evaluation showed HCV antibodies and HCV-RNA in the serum of all cases, rheumatoid factor was positive in 15 cases while mixed cryoglobulinaemia was detected in 14. Of those with cryoglobulinaemia, extra-renal manifestations were reported in only eight patients, mainly in the form of purpura and arthralgia.

3 1926 A. A. Sabry et al. Fig. 1. HCV antigen detected (arrow) in liver biopsy (avidin biotin peroxidase stain). Table 1. Demographic, clinical, and histological characteristics of 20 patients on interferon-a therapy Characteristic Number Demographic Maleufemale 13u7 Risk factors for HCV: History of hospital admission 10 Blood transfusion 8 Operation 12 History of abnormal liver enzymes 7 Clinical Generalized weakness 10 Peripheral oedema 17 Hepatomegaly 4 Palpable purpura 7 Arthralgias 4 Arterial hypertension 5 Laboratory HCV-RNA in cryoprecipitates 15 Mixed cryoglobulinaemia 14 Rheumatoid factor 15 HCV-RNA 20 HCV-Ab 20 Histological Renal pathology MPGN 7 MN 2 Mesangioproliferative glomerulonephritis 1 Liver biopsy CAH 3 CPH 4 Skin biopsy Acral necrolytic erythema 2 MPGN, membranoproliferative glomerulonephritis; MN, membranous nephropathy; CAH, chronic active hepatitis; CPH, chronic persistent hepatitis. Light microscopy of kidney sections showed mesangiocapillary glomerulonephritis (type 1) in 17 cases, membranous glomerulonephritis in two cases, and mesangioproliferative glomerulonephritis in one case. Light microscopy of liver sections obtained from seven patients showed chronic active hepatitis in three and chronic persistent hepatitis in four cases. Demonstration of HCV in cryoprecipitate, liver, and kidney tissues RT PCR for HCV was positive in the cryoprecipitate of 14 cases, in four of seven formalin-fixed paraffinembedded liver sections, in seven of 20 frozen renal biopsies, but in none of the formalin-fixed paraffin-embedded renal sections. Immunohistochemistry of liver and renal sections demonstrated HCV core AA antigen in two of seven paraffin-embedded liver sections (Figure 1), but not in frozen renal or liver tissue, nor in paraffin-embedded renal tissue. Electron microscopy of eight renal biopsy sections from HCV-positive cases showed immune complexes in the subendothelial and the paramesangial areas (Figure 2). In four of these cases virus-like particles were detected inside these immune complexes (Figures 3 and 4), while in all HCV-negative cases (controls) immune complexes were negative. Of 10 HCV-positive patients examined for viral genotype, sufficient amounts of HCV-RNA were extracted from serum of seven cases only. Genotyping of these cases demonstrated HCV type 4 in all of them. Outcome of anti-viral treatment At 3 months after initiation of interferon treatment, only four of the 20 treated patients showed negative HCV-RNA-PCR. The 16 non-responders were given

4 Interferon-a and ribavirin in HCV-related glomerulopathy 1927 Fig. 2. Electron microscopy for HCV-positive patients with subendothelial and paramesangial electron-dense deposits ( ). Fig. 3. Electron microscopy for HCV-positive patients with viral-like particles inside electron-dense deposits ( ). ribavirin. One of these showed response, while 15 showed persistent viraemia. Table 2 shows the effect of anti-viral treatment on laboratory and serological parameters of the 20 patients. HCV viral titres were significantly lower in treated patients. Also proteinuria, serum albumin, and complement components C3 and C4 were significantly improved after treatment. Of the 20 patients treated by interferon alone or in combination with ribavirin, five showed a progressive deterioration of kidney function and an increase in proteinuria. Table 3 shows, at the commencement of combination therapy, the baseline virological and biochemical parameters of patients who showed stabilization or improvement of kidney function, and their proteinuria on anti-viral treatment (group I), and those who showed deterioration of these parameters (group II). There was a tendency to higher basal viral titres and significantly higher basal serum creatinine concentrations in patients of group II. Table 4 shows adverse effects of anti-hcv treatment. Anaemia was clinically the most significant, and fever occurring early after treatment was the most frequent.

5 1928 A. A. Sabry et al. Fig. 4. Electron microscopy for HCV-positive patient showing virus-like particles inside electron-dense deposits (56 nm) with higher magnification. Table 2. Biochemical characteristics (median and range) of the 20 HCV-infected patients before and after anti-viral therapy Discussion Before After P-value Serum creatinine (mgudl) ( ) ( ) Proteinuria 24 h (g) ( ) ( ) Haemoglobin (gudl) ( ) ( ) Serum albumin (gudl) ( ) ( ) ALT (IUul) ( ) ( ) AST (IUul) ( ) ( ) C3 (mgudl) ( ) ( ) C4 (mgudl) ( ) ( ) HCV viral titre (MEquml) ( ) ( ) This work was conducted on 20 patients with nephrotic syndrome associated with HCV infection documented by both anti-hcv and HCV-RNA-PCR. Light-microscopy examination of kidney biopsies showed MPGN type l in 17 patients, while MN was observed in two and mesangial proliferative glomerulonephritis was documented in one case. The high prevalence of MPGN in our series is consistent with that reported in most of the literature. Schifferli and his colleagues [2] reported accumulating epidemiological evidence suggesting that there is Table 3. Parameters at start of anti-viral treatment (median and confidence intervals) of patients with favourable renal response (group I) and those with unfavourable response (group II) Group I Group II P-value Number Age (years) ( ) ( ) Serum creatinine (mgudl) ( ) ( ) Proteinuria 24 h (g) ( ) ( ) Serum albumin (gudl) ( ) ( ) HCV viral titre (MEquml) ( ) ( ) an association between HCV infection and MPGN and MN. These findings were also reported by others [5,6]. Beside detection of anti-hcv antibodies and viral RNA in the serum of our nephrotic patients, we documented the presence of HCV viral particles in the cryoprecipitate, liver tissue, and kidney tissue specimens. Furthermore, the HCV specificity of these kidney lesions in our patients was supported by regression of proteinuria and normalization of C3 and C4 in response to interferon ribavirin treatment. It seems that the optimal treatment strategy for HCV-associated renal disease remains to be defined. Johnson et al. [7] reported that oral steroids with HCVassociated nephritis had no effect on renal function, although it may have improved the purpura. While pulse steroids were associated with improvement in renal function, these patients remain HCV-RNA

6 Interferon-a and ribavirin in HCV-related glomerulopathy Table 4. Adverse events of interferon and combined interferon ribavirin therapy in patients with HCV-related glomerulopathy Adverse events Interferon therapy Temporary discontinuation 5 1 of therapy Dose reduction Due to anaemia 4 7 Due to other adverse events Flu-like symptoms Headache 2 Fever 17 Gastrointestinal symptoms Anorexia 5 Vomiting 5 Nausea 5 Abdominal pain 1 Psychiatric symptoms Depression 1 1 Insomnia 1 1 Dermatological symptoms Alopecia 1 Combined therapy positive [7]. One problem with such therapy is the increase in HCV-RNA levels with its possible consequences on the underlying liver disease [10]. Cyclophosphamide was successfully used for treatment of the HCV-infected patients with cryoglobulinaemia and progressive renal insufficiency cause by MPGN. Unfortunately HCV-RNA levels increased in these patients [11]. Only a few studies, involving small number of patients, have examined the response of MPGN associated with chronic HCV infection to interferon-a treatment. Results of these studies have been inconsistent [4,7,12]. In our study, 12 months treatment with interferon alone (four patients) or combined with ribavirin (16 patients) resulted in a significant reduction of proteinuria, a rise in serum albumin, stabilization of serum creatinine, normalization of C3 and C4, and significant reduction in viral titres. Beneficial effects of interferon-a therapy on the clinical and biochemical manifestations of type II essential mixed cryoglobulinaemia in some uncontrolled studies [12,13] and in two controlled trials [14,15] had been reported even before the demonstration of the pathogenetic role of HCV infection in this disease. In our study, we observed that the introduction of ribavirin in addition to interferon resulted in little improved virological response (only one patient seroconverted out of 16 patients given the combined treatment); yet a satisfactory biochemical response was achieved in the form of a significant reduction of proteinuria and a rise in serum albumin. A similar observation was reported by Heagy et al. [16]. It was suggested that ribavirin acts on HCV-related glomerular or hepatic lesions through a mechanism other than a reduction in viral replication, Pham et al. [17] suggested that the non-specific effects of ribavirin might prove beneficial in non-viral immune glomerulonephritis. Five of our patients showed exacerbation of their proteinuria with progressive deterioration of renal function while on anti-viral treatment. Comparing these patients with those demonstrating a favourable renal response revealed a significantly higher serum creatinine before starting combination therapy; in addition, their initial viral titres tended to be higher. We have no explanation for this finding. Possibly these patients had a primary glomerular disease in which HCV had no pathogenic role, and this disease is exacerbated by a direct or indirect effect of interferon. Ohta et al. [18] reported exacerbation of glomerulonephritis in patients with chronic HCV infection after interferon therapy. The mechanism is not fully understood. In another study, anti-interferon IgG-antibody was found in the circulation and renal tissue of a patient who developed MPGN after interferon therapy for human immunodeficiency virus infection [19]. In conclusion, our study showed a documented link between cryoglobulinaemic and non-cryoglobulinaemic MPGN and chronic HCV infection. A possible role for HCV infection and other forms of GN requires further study. Detection of HCV-RNA in paraffin-embedded liver and frozen renal tissues is feasible. Biochemical response to interferon therapy in such cases improved when combined with ribavirin. Further studies using higher doses or pegylated forms of interferon on HCV-related MPGN are urgently needed. Acknowledgements. The authors wish to thank Dr Ibrahim Hefni for his generous grant, which covered the cost of all the laboratory work and that of the interferon and ribavirin used in this study. Also, the authors are grateful to the Northern General Hospital Trust Research Committee for its financial support, and acknowledge the secretarial work of Mrs Hend Sharabi. References Crawford DHJ, Endre ZH, Axelsen RA et al. Universal occurrence of glomerular abnormalities in patients receiving liver transplants. Am J Kidney Dis 1992; 19: Schifferli JA, French LE, Tissat JD. 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