MEMBRANOUS GLOMERULONEPHRITIS ASSOCIATED WITH HEPATITIS C VIRUS INFECTION IN RENAL TRANSPLANT PATIENTS¹,²

Abstract

Background. 

Hepatitis C virus (HCV) infection has been described in association with various types of glomerular diseases, usually type I membranoproliferative glomerulonephritis and rarely membranous glomerulonephritis (MGN). In this article, we describe the first series of MGN exhibited in renal transplant patients and associated with HCV infection.

Methods. 

From January 1980 to December 1994, 2045 kidney transplantations were performed in our renal transplant units. A retrospective analysis demonstrated an overall 20% prevalence of HCV virus-positive patients; 409 transplanted patients were HCV positive (ELISA and RIBA).

Results. 

Fifteen patients developed an allograft MGN (3.66%) 24 months after renal transplantation. MGN appeared in the form of significant proteinuria (>1.5 g/24 h) with stable renal function. In all cases, graft biopsy demonstrated a thickening of the capillary wall, subepithelial electron-dense deposits, and IgG and C3 diffuse granular deposits along the basal membrane. Ten cases were considered de novo, two cases were considered recurrent MGN, and three cases were considered undetermined because the primary renal disease was chronic glomerulonephritis. All patients showed negative antinuclear antibodies and cryoglobulins, normal complement, and negative rheumatoid factors. During follow-up (an average of 2 years), 12 patients developed a progressive worsening of renal function, with increased serum creatinine and persistent proteinuria; 8 of the 12 patients returned to dialysis. Of the remaining three cases, two patients showed partial remission of nephrotic syndrome after high doses of steroids, and one patient persisted with stable renal function and proteinuria (<2 g/24 h.).

Conclusions. 

In summary, HCV is preferentially associated with MGN in renal transplant patients, rather than with membranoproliferative glomerulonephritis as in the normal adult population. MGN associated with HCV infection has a similar clinical picture and outcome to posttransplant idiopathic de novo MGN, with persistent massive proteinuria and progressive deterioration of renal function.

Liver disease is a frequent and important cause of mortality and morbidity after renal transplantation ^{(1, 2)}. Currently, it is well known that hepatitis C virus (HCV^*) represents the major cause of chronic liver disease in renal transplant patients ^(2-6). On the other hand, HCV has been described in association with different forms of renal disease, especially type II cryoglobulinemia, vasculitis, and glomerular diseases, usually cryoglobulinemic or noncryoglobulinemic type I membranoproliferative glomerulonephritis (MPGN), in a way similar to hepatitis B virus infection ^(7-9). Unlike in HBV-associated glomerulonephritis, identification of the HCV particles in the glomeruli have remained elusive until very recently. Okada et al. ⁽¹⁰⁾ have identified HCV core proteins in the basal membrane of the glomerulus by indirect immunofluorescence in two HCV-positive patients with membranous glomerulonephritis (MGN).

In transplant recipients, the development of MPGN in HCV-infected patients after liver transplantation ⁽¹¹⁾ or MGN after bone marrow transplantation has recently been reported ⁽¹²⁾. Roth et al. ⁽¹³⁾ reported that 5% of HCV-positive renal transplant patients may develop MPGN, often without cryoglobulinemia or rheumatoid factor. Gallay et al. ⁽¹⁴⁾ have also reported that HCV-positive recipients may develop chronic glomerulopathy with pathological features between MPGN and transplant glomerulopathy. In our renal transplant population with chronic HCV infection, we observed a series of 15 patients who developed an allograft MGN. Therefore, these findings prompted us to describe the clinical, immunological, and pathological characteristics of this glomerular lesion in chronic HCV-infected transplanted patients and to relate its development to HCV infection.

PATIENTS AND METHODS

Study Population

During the period between January 1980 and December 1994, 2045 kidney transplants were performed in our renal transplant units. A retrospective analysis in both units demonstrated an overall 20% prevalence of HCV-positive recipients, 409 being HCV-positive (ELISA and RIBA) transplanted patients. Fifteen of the 409 patients (3.66%) developed an allograft MGN and represent the study group.

Clinical Data

Before transplantation.. Pretransplantation records of these 15 patients were reviewed for age, gender, etiology of chronic renal failure, duration and type of dialysis, serum alanine aminotransferase (ALT) levels, serological tests for human immunodeficiency virus, hepatitis B virus status (HBsAg and HBcAc), and liver histology. In all available cases, pretransplantation-stored sera were tested for anti-HCV (ELISA and RIBA), HCV-RNA, and HCV genotype.

After transplantation. All transplant patients of our units were followed up regularly by the staff of the nephrology department. After transplantation, every patient was followed up once a month during the first year, every 2 months in the second year, and every 3 months in the following years. The monitoring consisted of periodic conventional biochemical tests (including renal function [serum creatinine and proteinuria] and liver enzymes). The monitoring of HCV-positive patients included yearly tests for anti-HCV (ELISA) and HCV-RNA (polymerase chain reaction; PCR). HCV genotypes were determined in the most recent monitoring of the study group. Liver biopsy was indicated only in patients with elevated (>2.5 times normal range) pre- or posttransplantation ALT levels, with the patient's consent. Renal biopsy was only performed by clinical indication (proteinuria [>1.5 g/24 h[for more than 3 months and/or deterioration of renal function). Serum complement levels, immunoglobulin, cryoglobulin, rheumatoid factor, and antinuclear antibodies were determined in the group of patients with MGN at the time of graft biopsy. Posttransplant immunosuppressive therapy, incidence of acute graft rejection, treatment with OKT3 or ALG, and the date and cause of graft failure were also recorded.

Definitions

Glomerular disease. Nephrotic-range proteinuria was diagnosed when proteinuria was higher than 3.5 g/day. Nephrotic syndrome was diagnosed when edema, hypoalbuminemia, and hypoproteinemia were also present.

The diagnosis of MGN was based on several factors: light microscopy, which showed diffuse thickening of the glomerular capillary wall; electronic microscopy, which demonstrated evidence of subepithelial electron-dense deposits; and immunofluorescence study, which showed IgG and C3 deposits diffusely located along the glomerular capillary wall ⁽¹⁵⁾.

Liver Disease

The presence of liver disease was based on serum ALT levels (normal range less than 45 IU/L). Chronic liver disease was diagnosed when elevated ALT levels were more than 2.5 that of normal liver values and were maintained for more than 6 months. Mild abnormalities were diagnosed if elevated ALT levels were less than 2.5 that of normal liver values. Pathologically, the diagnosis of persistent chronic hepatitis was based on infiltration of the portal areas with mononuclear cells, but without erosion of the limiting plate (piecemeal necrosis) or extension of the inflammation into the liver lobule.

Laboratory Tests for HCV

Anti-HCV. Sera were tested for anti-HCV using a second generation ELISA (Ortho HCV ELISA 2.0 test system; Ortho Diagnostic Systems, Raritan, NJ) and a second generation strip immunoblot assay (Ribatm HCV test system, Chiron Corporation, Emeryville, CA). Both tests were carried out according to the manufacturers' instructions.

HCV RNA. Sera from anti-HCV-positive recipients were tested by PCR, using a nested PCR protocol (Ravaggi, PCR Meth App, 1992).

HCV genotype. Sera were tested by a commercial method of reverse hybridization (Inno-Lipa HCV, Innogenetics).

Immunological Studies

Complement studies. C3 and C4 levels were measured by nephelometry (Beckman Array 360; Beckman Instruments, Fullerton, CA). Normal values were c3=83-171 mg/dl and c4=14-38 mg/dl.

Immunoglobulin. IgG, IgA, and IgM (normal values: IgG, 644-1436 mg/dl; IgA, 65-348 mg/dl; IgM, 55-206 mg/dl) and rheumatoid factor (normal value: 0-0.30 IU/ml) were also measured by nephelometry.

Antinuclear antibodies. Antibodies were measured by indirect immunofluorescence. Normal values are 0-1/40.

Cryoglobulin. The presence of cryoglobulin, proteins with the property of precipitating from cooled serum, was examined in serum at 4°C for 7 days.

RESULTS

Before Transplantation

Table 1 shows the demographic and clinical characteristics before renal transplantation of the recipients who developed MGN. There were 15 patients (7 males and 8 females) with an average age of 47.6±13.3 years, who had been on dialysis therapy for an average period of 51±30 months before transplantation. Primary renal disease was defined by renal biopsy in 14 of the 15 patients with allograft MGN (Table 1). None of the 15 patients had a positive HBsAg, nor human immunodeficiency virus or history of drug abuse. Six patients exhibited HLA DR5, two exhibited DR3, and none exhibited B18. Average HLA DR matching was 1.27±0.8.

Table 2 shows the characteristics of HCV before transplantation. Seven patients had normal liver values and the remaining eight showed mild liver abnormalities. HCV RNA was present in all of the 14 patients tested and the most frequent HCV genotype was 1b. Liver biopsy in one patient with mild liver abnormalities, HCV-RNA positive results, and 1b genotype demonstrated the presence of persistent chronic hepatitis. Information on HCV infection before dialysis was not available.

After Transplantation

Fifteen HCV-positive patients developed MGN after renal transplantation (3.66%), whereas only 6 of the 1636 (0.36%) anti-HCV-negative patients (P<0.001) presented this glomerular complication. Both groups of patients, HCV positive and HCV negative, were monitored regularly in our renal transplant units as mentioned before, and renal biopsy was only performed after clinical indication.

Twelve of the 15 patients with an MGN were treated with cyclosporine + prednisone, 2 patients were treated with azathioprine + prednisone, and 1 patient was treated with cyclosporine monotherapy. No changes in the immunosuppressive therapy were made during the follow-up. Acute graft rejection episodes were treated with intravenous methyl-prednisolone (1 g) for 3 consecutive days. Corticosteroid-resistant rejections were treated with muromonab-CD3 (OKT3; Ortho) (5 mg intravenously) for 10 days.

Glomerular Disease

Clinical and laboratory findings. All 15 patients presented significant proteinuria (>1.5 g/day) and microscopic hematuria, without purpuric lesions, in an average time of 18.2±14.5 months after renal transplantation (Table 3). In patients with recurrent MGN, there was a tendency to present proteinuria earlier than in patients with de novo glomerulonephritis (GN) (7±4.2 vs. 19.9±14.8 months, P<0.05). Graft biopsy was performed 24.2±15.8 months after renal transplantation. At the time of the biopsy, nine patients had a slight increase in serum creatinine (>1.5 mg/dl). Patient 6 also presented Fanconi syndrome (aminoaciduria, glucosuria, and phosphaturia). Allograft MGN was considered de novo in 10 of the 15 cases, recurrent in 2 cases, and undetermined in 3 cases with chronic glomerulonephritis as primary renal disease. Immunological studies performed in nine patients showed normal serum complement and rheumatoid factor levels and negative cryoglobulins. IgA and IgM were within the normal range and IgG was low in four patients, probably in relation to massive proteinuria. Antinuclear antibodies were also negative in all tested patients except in patient 3, who showed low positivity (Table 3).

Pathological analysis. In all cases except for the three patients with MGN grade I, pathological data demonstrated diffuse thickening of basement membranes and “spikes” on the silver stain. The cellularity was normal in all cases except in patient 6. Immunofluorescence microscopy revealed granular and diffuse deposits along the basal membrane containing C3 and IgG in all patients, and IgA in patient 3 and IgA-IgM in patient 14. Electron microscopy performed on 10 biopsy specimens (including patient 15 and the three patients with grade I MGN) showed subepithelial electrondence deposits (Fig. 1, patient 3) without mesangial cell interposition or radiolucent subepithelial spaces, except in patient 15. Renal biopsies of patients 5 and 14 had diffuse interstitial fibrosis with parietal thickening in the arteries and arterioles and tubular atrophy. Patient 4 exhibited stripped fibrosis with intimal proliferation and fibrosis in the arteries. Patients 8, 10, and 11 presented diffuse fibrosis associated with vascular sclerosis. The remaining nine patients showed no relevant tubular or interstitial lesions and patients 1, 2, and 15 showed only mild parietal thickening of the vessels with hyalinosis of the intima. Finally, patient 15 showed transplant glomerulopathy, confirmed by electron microscopy, with the presence of thickening and reduplication of the glomerular basement membrane and subendothelial spaces, associated with MGN.

Clinical course. The average follow-up after biopsy was 23±25 months. Five patients (2, 3, 6, 7, and 15) were treated with methylprednisolone (250 mg for 3 consecutive days and then prednisone 1 mg/kg/day, decreasing the dose after 6 weeks and returning to 10 mg/day); two of the five patients (2 and 3) showed partial remission of the proteinuria in an average time of 4 months. In nontreated patients, proteinuria persisted without significant changes. Eight patients (4, 5, 8, 9, 10, 11, 12, and 14) returned to dialysis, and graft loss was attributed to MGN and chronic rejection in all of them. Of the remaining seven, four showed chronic graft insufficiency, whereas the remaining three (two treated with steroids) have normal graft function. Finally, two patients on dialysis died (patients 11 and 12) during the follow-up, one due to ischemic cardiomyopathy and the other from an endometrial carcinoma.

Liver disease. Table 2 also exhibits the characteristics of HCV infection after transplantation with an average follow-up of 47.3±29.5 months. Eight patients presented normal liver values and seven presented mild liver abnormalities. All maintained anti-HCV + and HCV-RNA + after renal transplantation. Therefore, all patients presented ongoing HCV infection. Liver biopsies performed in three patients after renal transplantation (12, 14, and 26 months) showed persistent chronic hepatitis in two of them and minimal changes in the other. One patient (14) received a simultaneous hepatorenal transplant because of a severe portal hypertension associated with polycystic liver disease. Liver pathology was strictly normal except for hepatic cysts.

DISCUSSION

Glomerular abnormalities in patients with liver diseases occur primarily in patients with cirrhosis and viral hepatitis ⁽¹⁶⁾. The most common type of glomerular lesions in cirrhotic patients is a mesangial proliferation with IgA deposits ⁽¹⁶⁾. In patients with positive HBsAg with chronic liver disease the characteristic glomerular lesion is MPGN or MGN ⁽⁷⁾. Recently, chronic HCV infection has been associated with different varieties of glomerular diseases. Cryoglobulinemic or noncryoglobulinemic membranoproliferative type I glomerulonephritis represents the most frequent pathologic type, although other glomerular lesions such as MGN, mesangial proliferative GN associated with type III cryoglobulinemia, type III MPGN, endocapillary GN, and focal sclerosing forms have been occasionally described ^{(8, 9, 17, 18)}. Although MPGN usually represents the histological type associated with HCV infection, the report of Takishita et al. ⁽¹⁹⁾ that 8% of patients with MGN in Northern Japan had HCV antibodies and positive HCV-RNA as compared with less than 1% of patients with other GN (excluding MPGN), suggests the pathogenetic relevance of HCV in MGN. Moreover, the recent identification of virus structures (HCV core protein) in the glomeruli by indirect immunofluorescence in two cases of MGN, has reinforced the association between HCV and MGN ⁽¹⁰⁾.

Allograft MGN is usually a de novo disease with an accumulative incidence of approximately 1.5-2%, raising the prevalence with a follow-up higher than 8 years ⁽²⁰⁾. HLA-B35 and DR-3 have been associated with idiopathic MGN but not with de novo posttransplantation MGN. The HLA-DR5 antigen, associated with some autoimmune diseases, with a prevalence in the healthy European Caucasian population of around 19.5%, has been reported with increased frequency in de novo MGN ⁽²¹⁾. In our report, 5 of 15 patients were DR5 (40% vs. 19.5%, P<0.05). The clinical onset of proteinuria is delayed in de novo MGN, beginning at an average time of 19 months after transplantation compared with 10 months in recurrent MGN ⁽²²⁾. Many patients remained asymptomatic with a protein excretion rate in the nonnephrotic range for a long period of follow-up ^{(15, 20)}.

The present study shows 15 transplant patients with chronic HCV infection who developed proteinuria due to MGN after renal transplantation. Allograft MGN associated with HCV infection is clinically manifested by massive proteinuria, nephrotic syndrome, microscopic hematuria, and mild to severe progressive graft failure. Immunological laboratory data showed normal complement and rheumatoid factor levels with negative cryoglobulins and antinuclear antibodies. Serum immunoglobulin was also normal, except for low IgG levels in some patients because of massive proteinuria. It is also worth remarking that 14 of the 15 patients with MGN had HCV RNA in the serum, indicating active viral infection, although clinically the liver function was normal or exhibited only mild liver abnormalities. In other words, this glomerular complication could appear in chronically infected HCV patients independently of the liver disease. Pathological findings were basically similar to conventional MGN, except for the presence of vascular and interstitial features due to chronic allograft nephropathy. The clinical outcome of MGN associated with HCV infection was similar to de novo MGN with progressive renal failure, with almost 50% of the patients reaching end-stage renal disease 5 years after the diagnosis of MGN ^{(23, 24)}. In the present series, 8 of the 15 patients returned to dialysis therapy at an average time of 2 years after renal biopsy. Probably, chronic allograft lessions associated with MGN could contribute to the progression of renal insufficiency.

The pathogenesis of MGN in these patients may be related to the deposition of immune complexes containing HCV proteins in glomeruli ⁽²⁵⁾, like HBV infection. The role of HCV infection in the pathogenesis of allograft MGN seems unquestionable. The prevalence of MGN in the transplant population was significantly higher in HCV-positive than in HCV-negative patients. The presence of HCV RNA in the serum as an expression of “ongoing HCV infection,” the higher prevalence of MGN in HCV-positive than in HCV-negative patients, and the relationship between MGN and HCV infection reported in two bone marrow transplanted patients (detecting HCV RNA in renal biopsy specimens) ⁽¹²⁾, strongly suggests that MGN could also be associated with chronic HCV infection after renal transplantation. Also, the presence of two recurrent cases of MGN in the present series could represent another argument in the role of HCV infection. It is well known that recurrent MGN in renal transplant patients is a rare condition (3-5%); in fact, only 33 cases of recurrent MGN have as yet been reported ⁽²⁶⁾. Therefore, chronic HCV infection could facilitate the recurrence of MGN, although we do not know whether HCV infection was the cause of the primary MGN. It is well documented that after renal transplantation, HCV viremia increases and immunoglobulin synthesis decreases as a consequence of the immunosuppressive therapy ^(27-29). Therefore, all these changes could produce an imbalance in the antigen-antibody complex status and interfere with their clearance, predisposing the glomerular deposition of HCV structures.

Finally, we emphasize that renal transplant patients with chronic HCV infection with evidence of “ongoing infection” could develop urinary abnormalities, such as proteinuria, nephrotic syndrome, and microhematuria. Renal biopsy could demonstrate MGN in the absence of hypocomplementemia, rheumatoid factor, and cryoglobulinemia. Also, this pathological condition seems to be more frequent than in HCV-negative patients, and the clinical picture and outcome seems to be similar to the de novo MGN in renal transplant patients. Therefore, in renal transplant patients with proteinuria, serological tests for HCV infection and HCV-RNA, even with normal liver tests, should be performed as part of the differential diagnosis.

Acknowledgments. The authors thank Paloma Losada and Maria Jesus Domingo for HCV RNA and HCV genotype determination. The authors also thank Maria Antonia Rodriguez for her secretarial work.

Figure 1: Ultrastructure study revealing numerous electron-dense deposits in the epithelial side of glomerular basal membrane, partially surrounded by “spikes,” membrane basal-like. ME ×60.000 (patient 3).