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
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Abstract |
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Methods. In this work, 20 patients with HCV-associated 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 anti-viral 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, P=0.001), serum albumin increased (from median 2.50 to 3.55 g/dl, P=0.012), lower viral titres (from median 1.15 to 0.53 mega-Eq/ml, P=0.049), 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
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Introduction |
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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 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 (n=20) diagnosed with HCV-related glomerulopathy.
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Patients and methods |
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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 5' 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 RTPCR with specific primers for HCV core region 5' NCR. Products were sequenced with AB1 prism DNA sequencing kit (PE Applied Biosciences, Warrington, UK).
Anti-viral treatment protocol
Interferon-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 mg/kg/day, 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. MannWhitney (non-parametric rank sum test), Fischer exact probability test, and chi-squared test were applied as appropriate. A P-value of <0.05 was considered significant.
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Results |
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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
RTPCR for HCV was positive in the cryoprecipitate of 14 cases, in four of seven formalin-fixed paraffin-embedded 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.
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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 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.
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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.
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Discussion |
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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 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 interferonribavirin 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 HCV-associated 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 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- 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- 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.
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Acknowledgments |
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Notes |
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References |
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