Since the identification of the hepatitis C virus(HCV*) as the major cause of parentally transmitted non-A-non-B hepatitis in 1989, the prevalence of anti-HCV antibody among kidney recipients has been reported to be between 10 and 49% (reviewed in1). HCV infection has now been shown to be responsible for extra hepatic diseases in nonimmunocompromised individuals, especially glomerulonephritis (2). But little is known of its effect in renal transplantation, although some reports suggest that HCV-infected kidney recipients may develop recurrent or de novo membranoproliferative glomerulonephritis due to cryoglobulinemia (3), or the deposition of complexes containing viral antigen and anti-HCV antibodies(4). One recent study also suggested an association between chronic HCV infection and transplant glomerulopathy(5). The current study was therefore carried out to assess the relationship between pretransplantation anti-HCV antibodies and the occurrence of proteinuria and to determine the link between this disorder and short-term and long-term patient and graft survival.
All the patients admitted to our institution for kidney transplantation between July 1989 and December 1994 whose sera were routinely assayed for anti-HCV antibodies at the time of transplantation were included. Donor and recipient records were reviewed. The outcomes measured included patient survival, graft survival, and the occurrence of persistent proteinuria. Proteinuria (24 hr) was measured weekly during the first 2 months, monthly until month 12, and then every other month. Proteinuria was defined as urinary protein above 1 g/day at two consecutive assays. Persistent proteinuria was defined as protein remaining above 1 g/day over 3 months after onset. Transient proteinuria was not considered for the analysis.
Anti-HCV antibodies in donor and recipient specimens were assayed immediately before transplant by enzyme-linked immunosorbent assay (ELISA) 1(in 1989 and 1990) and by ELISA 2 (after January 1991). After-transplantation testing was done at 4 months and then every year. The specificity of the ELISA for anti-HCV-positive sera was checked with a second generation confirmation recombinant immunoblot assay (recombinant immunoblot assay 2, Ortho Diagnostics). Any HCV RNA in the sera of anti-HCV-positive patients was detected by polymerase chain reaction. Cryoglobulins were screened by immunobinding (Hydragel, Serbia) with antisera specific for heavy and light chains.
All the tissue specimens obtained by graft biopsy or nephrectomy from patients who developed proteinuria were evaluated blind by the same pathologist (J.C.) without knowledge of their clinical history. The only information given was that these were from renal transplants associated with significant proteinuria. When two biopsies were done, only the latest was taken into account. Tissue samples were examined by light and immunofluorescence microscopy. Electron microscopy studies were not routinely performed. Biopsies were classified as showing chronic rejection, transplant glomerulopathy, de novo glomerulopathy, or recurrent glomerulonephritis.
The product-limit method (Kaplan-Meier) was used to estimate the risk of each event, the occurrence of proteinuria until January 1997, return to dialysis, or death. Death was treated as a censoring date when the outcome was the occurrence of proteinuria or a return to dialysis. An association of clinical variables with each outcome was examined in univariate analysis using the log-rank test. In a conservative approach, covariates reaching a 0.2 type I significance level in univariate analysis were entered into a multivariate Cox regression analysis. Eligible risk factors were tested in stepwise forward and backward regression analyses. The significance level in multivariate analysis were set at alpha=0.05. All analyses were performed using BMDP software (6).
None of the 322 adult recipients (217 men and 105 women), mean age 43.9±12.5 years, included in the study was of African origin. Two patients who became HCV-positive after renal transplantation were excluded. Donors were men in 215 cases and women in 107. Mean age was 34.7±13.6 years. They were living-related in only two cases. All were HCV-negative, as the use of organs from HCV-positive donors is not permitted in France. Prophylactic treatment with polyclonal antilymphocyte serum (equine antilymphocyte globulin or rabbit antithymocyte globulin, Pasteur-Merieux, Lyon, France) was started on the day of transplantation and continued for 10-14 days. Oral cyclosporin (6 mg/kg/day) was started at the onset of adequate renal function (serum creatinine <2.5 mg/dl), with a target through-blood level of 150-200 ng/ml measured by monoclonal radioimmunoassay. Prednisone (1 mg/kg/day) was prescribed, with a reduction to a maintenance dose of 0.1 mg/kg/day at 6 months. Azathioprine, when given, was as an oral dose of 1.08±0.3 mg/kg/day. The baseline immunosuppressive regimen consisted of cyclosporin and prednisone in 118 patients and of triple therapy with azathioprine in 199 patients (5 patients never received these drugs because of death or early graft failure).
Anti-HCV antibodies were detected in the pretransplant sera of 31 patients(9.6%). One of these was also Hbs-Ag-positive. After transplantation, two patients became anti-HCV and polymerase chain reaction-negative. Twenty-five patients (80.6%) with anti-HCV antibodies prior to transplantation tested positive for HCV RNA. Cryoglobulinemia was assayed in 24 of the 31 HCV-positive patients and was persistently or intermittently detected in 4 of them (16.7%).
Twenty patients died, but no death was attributed to liver failure. Forty-seven patients lost their graft during the study period. Proteinuria was higher than 1 g/day in 62 patients; it was transient in 18 patients and persistent in 44 (13.6%), in whom it occurred 1.3±1.4 years after transplantation. Of these 44, 11 developed nephrotic syndrome and 21 lost their graft. The impact of persistent proteinuria on graft survival is shown in Figure 1. Graft survival was significantly lower in recipients with proteinuria that in recipients without proteinuria(P<0.00001). One- and 5-year survival rates were 90.7% and 41.1% in proteinuric patients and 95.6% and 91.8% in nonproteinuric patients.Table 1 shows the characteristics of the patients with and without proteinuria at the time of renal transplantation. Comparison of the two groups by univariate analysis showed no difference in donor age, donor and recipient gender, causes of end-stage renal failure, cold ischemia time, number of transplantations, and panel-reactive antibodies. However, the proteinuric group had significantly more HLA-A,B mismatches and more anti-HCV-positive recipients than the nonproteinuric group. The patients with proteinuria were significantly younger than the patients without.
The results of the multivariate analysis using Cox regression modeling for time to proteinuria are shown in Table 2. The model identified three independent significant variables as predictors of proteinuria: the number of A-B mismatches, a second graft, and the presence of anti-HCV antibodies prior to transplantation. The duration of dialysis and age of the recipient seemed to be protective factors, with recipient age being close to statistical significance (P=0.07).
Figure 2 shows a significant difference in the cumulative probability of posttransplant proteinuria between recipients with and without anti-HCV antibodies (P<0.00001). At 1 year, the probability of proteinuria was 19.5% in the HCV-positive group and 7.5% in the HCV-negative group. At 3 years, it was 22.9% in the HCV-positive patients and 10.7% in the HCV-negative ones; at 5 years the figures were 45.1% and 13.1%, respectively.
There was no difference in the survivals of patients or grafts in the two groups. The 1- and 5-year patient survival rates were 100% and 82% in HCV-positive patients and 97.5% and 95.2% in the HCV-negative patients. The 1- and 5-year graft survival rates were 100% and 74.9% in the HCV-positive patients and 94.4% and 86.2% in the HCV-negative ones.
The proportions of patients experiencing episodes of acute graft rejection in the two groups were similar: 45.2% of HCV-positive and 45.8% of HCV-negative patients had at least one rejection episode, whereas 19.4% of the HCV-positive and 13.4% of the HCV-negative patients had more than one rejection episode.
The graft specimens taken for histopathological examination were from 35/44 patients after the onset of persistent proteinuria, but adequate specimens for examination by light microscopy and immunofluorescence were obtained from only 26 patients (7 HCV-positive and 19 HCV-negative). Chronic rejection was present in 3/7 HCV-positive and 11/19 HCV-negative; 1 HCV-positive and 4 HCV-negative patients experienced recurrent disease. The HCV-positive patients had more de novo glomerular lesions than the HCV-negative patients (3/7 vs. 4/19), but the difference was not statistically significant.
Proteinuria frequently occurs after renal transplantation. Proteinuria>1 g/day was found in 12.8-28.5% (7, 8). The transient proteinuria that is mostly due to acute rejection has no influence on 3- and 5-year graft survival, provided that adequate therapy is started and proteinuria is terminated within 3 months. But persistent proteinuria is strongly correlated with reduced graft survival (9). We find that 4.6% of our renal transplant recipients had a transient proteinuria and 13.3% had persistent proteinuria. A quarter of the latter had nephrotic syndrome, and nearly half lost their graft. Kaplan-Meier analysis confirmed that the recipients with persistent proteinuria had a worse graft outcome than non-proteinuric patients.
Little is known about the causes of proteinuria. To our knowledge, only one study has used a multivariate analysis to test the influence of established prognostic factors on the occurrence of persistent proteinuria(10). This study did not find any correlation between proteinuria and donor or recipient factors. Our study shows that the presence of anti-HCV antibodies on the day of transplantation is a strong predictive factor of persistent proteinuria after renal transplantation that is independent of the rate of acute rejection episodes. Brunkhorst et al.(11) examined 996 patients with kidney grafts and found that 22.8% of their HCV-positive patients had proteinuria of more than 1 g/day, but they did not give the prevalence of proteinuria in the HCV-negative patients.
Histopathological studies on our patients with proteinuria showed that chronic rejection is the most frequent lesion leading to proteinuria in HCV-positive and HCV-negative recipients. None of our proteinuric HCV-positive recipients had membranoproliferative glomerulonephritis (MPGN), but nearly half of them had glomerular lesions, mostly transplant glomerulopathy. It was more frequent than in HCV-negative patients, but the difference was not statistically significant because of the small size of the population.
In the nontransplant setting, epidemiologic clinical and experimental data suggest that HCV infection may cause glomerular lesions. Type II cryoglobulinemic MPGN was first reported (12). However, HCV has also been implicated in the development of MPGN, membranous glomerulonephritis, mesangial proliferation, and focal sclerosis without cryoglobulinemia (13). MPGN due to HCV infection may be a recurrent disease (11) or de novo glomerulonephritis(1) after renal transplantation. Transplant glomerulopathy is considered to be a manifestation of subacute or chronic rejection (7). Gallay et al. (5) examined two cases of glomerulopathy in renal allografts of recipients with HCV infection and found features of both transplant glomerulopathy and MPGN. They speculated that some patients diagnosed as having transplant glomerulopathy may have had HCV-associated MPGN. There may be few or no immune complex deposits in immunocompromised patients with HCV-associated MPGN(13). It is therefore possible that they are not found in immunosuppressed patients, resulting in glomerular lesions that are indistinguishable from transplant glomerulopathy. HCV-positive patients were recently found to be more prone to chronic vascular rejection and acute transplant glomerulopathy during the first 6 months following renal transplantation (14). It was speculated that the association of HCV with transplant glomerulopathy may be because the virus has a direct effect on the kidney and/or because it causes pathogenic changes that affect mainly glomeruli and endothelial cells.
We have confirmed the influence of proteinuria on graft survival. However, despite the strong association between HCV infection and proteinuria, the presence of anti-HCV antibodies at the time of transplantation does not enhance the risk of acute rejection and does not adversely affect 1-and 5-year patient or graft survivals. This is in agreement with several studies(15) but in sharp contrast with others that have shown an increased risk of liver disease and death after renal transplantation in patients with HCV acquired before transplantation (16). Although it does not influence 5-year graft survival, HCV seropositivity may be a major cause of graft loss after a more extended follow-up.
We find that a second renal transplant is a significant risk factor of proteinuria. Kidney retransplantation is generally associated with a diminished overall graft survival (17). The higher risk of graft loss after a second transplant might reflect a greater immune responsiveness, leading to more chronic rejection.
The correlation between proteinuria and HLA mismatch showed a clear advantage for recipients with fewer HLA-A,B mismatches. HLA-DR mismatching was not a predictive factor of proteinuria in our study. Our results are concordant with those of a recent study showing that the HLA-A matching effect is a significant factor in first renal allograft survival in the 6 years after transplantation, unlike the strong short-lived effect of HLA-B and -DR matching, which can only be detected up to 2 years after transplantation(18).
Greater recipient age tended to be a protective factor (close to statistical significance) against proteinuria after renal transplantation in our patients. No data are available on the influence of recipient age on the occurrence of proteinuria. The importance of death with a functioning graft in analyzing late graft loss has been pointed out by some authors, who found that younger age was associated with poorer long-term survival. It is therefore possible that the greater prevalence of proteinuria in younger patients may be due to more frequent or more severe acute rejection leading to chronic rejection.
In conclusion, we have described a significant relationship between persistent proteinuria and decreased renal allograft survival(19). The most compelling predictor of proteinuria was the presence of anti-HCV antibodies at the time of transplantation. Histopathological studies on the proteinuric patients showed a nonsignificantly higher frequency of de novo glomerular lesions in HCV-positive recipients. But the presence of anti-HCV antibodies does not impair 5-year patient or graft survival, despite the strong association between proteinuria and presence of these antibodies.
*Abbreviations: ELISA, enzyme-linked immunosorbent assay; HCV hepatitis C virus; MPGN membranoproliferative glomerulonephritis.
REFERENCES
1. Roth D. Hepatitis C virus infection and the renal allograft recipient. Nephron 1995; 71: 249.
2. Johnson R, Gretch D, Yamabe H, et al. Membranoproliferative glomerulonephritis associated with hepatitis C virus infection. N Engl J Med 1993; 328: 465.
3. Dussol B, Tsimaratos M, Lerda D, Casanova P, Brunet Ph, Berland Y, Hépatite virale C et glomérulonéphrite membranoproliférative chez une transplantée rénale. Néphrologie 1995; 16: 223.
4. Roth D, Cirocco R, Zucker K, et al. De novo membranoproliferative glomerulonephritis in hepatitis C virus-infected renal allograft recipients. Transplantation 1995; 59: 1676.
5. Gallay BJ, Alpers CE, Davis CL, Schultz MF, Johnson RJ. Glomerulonephritis in renal allografts associated with hepatitis C infection: a possible relationship with transplant glomerulopathy in two cases. Am J Kidney Dis 1995; 26: 662.
6. BMDP statistical software manual. Dixon WJ, ed. Los Angeles: University of California Press, 1990.
7. Vathsala A, Verani R, Schonberg L, et al. Proteinuria in cyclosporine-treated renal transplant recipients. Transplantation 1990; 49: 35.
8. Bear RA, Aprile M, Sweet J, Cole EH. Proteinuria in renal transplant recipients: incidence, cause, prognostic importance. Transplant Proc 1988; 20: 1235.
9. First MR, Vaidya PN, Maryniak RK, et al. Proteinuria following transplantation. Correlation with histopathology and outcome. Transplantation 1984; 38: 607.
10. Hohague H, Kleyer U, Bruckner D, August C, Zidek W, Spieker C. Influence of proteinuria on long-term transplant survival in kidney transplant recipients. Nephron 1997; 75: 160.
11. Brunkhorst R, Kliem V, Koch KM. Recurrence of membranoproliferative glomerulonephritis after renal transplantation in a patient with chronic hepatitis C. Nephron 1996; 72: 465.
12. D'Amico G, Fornasieri A. Cryoglobulinemic glomerulonephritis: a membranoproliferative glomerulonephritis induced by hepatitis C virus. Am J Kidney Dis 1995; 25: 361.
13. Johnson RJ, Willson R, Yamabe H, et al. Renal manifestations of hepatitis C virus infection. Kidney Int 1994; 46: 1255.
14. Cosio FG, Sedmak DD, Henry ML, et al. The high prevalence of severe early posttransplant renal allograft pathology in hepatitis C positive recipient. Transplantation 1996; 62: 1054.
15. Venkateswara Rao K, Ma J. Chronic viral hepatitis enhances the risk of infection but not acute rejection in renal transplant patients. Transplantation 1996; 62: 1765.
16. Bouthot BA, Murthy BVR, Schmid CH, Levey AS, Pereira BJG. Long-term follow-up of hepatitis C virus infection among organ transplant recipients. Transplantation 1997; 63: 849.
17. Abouljoud MS, Deierhoi MH, Hudson SL, Diethelm AG. Risk factor affecting second renal transplant outcome with special reference to primary allograft nephrectomy. Transplantation 1995; 60: 138.
18. Zantwoort FA, D'Amaro J, Persijn GG, et al. The impact of HLA-A matching on long-term survival of renal allografts. Transplantation 1996; 61: 841.
19. Pirsch JD, Ploeg RJ, Gange S, et al. Determinants of graft survival after renal transplantation. Transplantation 1996; 61: 1581.
