1 Sheffield Kidney Institute, 4 Department of Pathology, 5 Public Health Laboratories, Northern General Hospital, Sheffield, 3 Queens Medical Centre, Nottingham, UK and 2 Mansoura Urology and Nephrology Center, Mansoura, Egypt
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Abstract |
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Methods. Fifty patients were found to be HCV positive out of 303 who presented with a glomerulopathy between 1998 and 1999 at the Mansoura Urology and Nephrology Center, Egypt. Comprehensive investigations of these 50 patients were undertaken including an evaluation of their clinical, biochemical, histological, virological and immunological parameters. In addition, their kidney biopsy material was analysed by electron microscopy (EM) to detect viral particles, by immunohistochemistry to detect a viral core antigen and by RTPCR to detect RNA. This was compared with 50 HCV-negative controls.
Results. Positivity for HCV antibodies was higher among patients with GN (38%) compared with healthy blood donors (16%). Genotype 4 was sequenced in 70% of the HCV-positive samples examined. MPGN was the most common type of GN accounting for 54% of patients. Extrarenal manifestations were absent in the majority (80%) of patients even though 54% had cryoglobulinaemia. EM revealed virus-like particles in 50% of biopsies. Immunohistochemistry failed to reveal HCV-related antigens in kidney sections. HCV RNA was detected in the cryoprecipitates in 66% of patients and 22% of frozen renal sections. Control sections were negative.
Conclusion. Our findings suggest a causal link between HCV and GN based on the observation of virus-like particles as well as viral RNA within the kidney sections of patients with HCV-associated glomerulopathies.
Keywords: cryoglobulinaemia; glomerulopathy; hepatitis C virus; membranoproliferative glomerulonephritis
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Introduction |
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Several extrahepatic syndromes have been associated with chronic HCV infection including mixed essential cryoglobulinaemia (MEC) and a wide range of glomerulonephritides (GN) [5]. Amongst the different types of GN affecting patients infected with HCV, membranoproliferative GN (MPGN) is thought to be the most common [6]. An association has been described between this nephropathy and the presence of cryoglobulinaemia [7]. In spite of such associations, a direct link between the actual HCV infection and the nephropathy has been hard to prove in view of the difficulty in identifying the HCV within the kidneys [8]. While Johnson and collaborators failed to identify HCV antigens or RNA in the glomerular lesions [6], Yamabe and his colleagues [9] have localized the C22 antigen of HCV to the glomerular lesions in patients with MPGN. Similarly, Stokes and coworkers successfully detected HCV RNA in the serum of patients co-infected with HCV and HIV [10].
With the above data in mind, we have studied a population of HCV-infected individuals with nephropathy in an attempt to identify the viral particles, their core antigen and RNA within their kidneys.
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Subjects and methods |
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In the 50 HCV-positive patients other causes of GN were excluded by testing for diabetes mellitus (fasting and 2 h post-prandial blood glucose), autoimmune diseases (serum autoantibodies) and schistosomiasis (schistosomal circulating cathodic antigen by the double antibody sandwich ELISA technique). Renal biopsy was carried out in all 50 HCV-positive patients.
Genotyping
HCV genotyping was performed for 10 patients, serum samples were analysed by reverse transcription polymerase chain reaction (RTPCR) using primers specific for HCV core region and five NCR products were sequenced with the ABI prism DNA sequencing kit (PE Applied Biosciences, Warrington, UK).
Light microscopy
For light microscopy, renal specimens were fixed in formalin and embedded in paraffin before 4 µm sections were cut and stained with haematoxylin and eosin.
Electron microscopy
In eight patients (diagnosed by light microscopy to have MPGN in four, focal segmental glomerulosclerosis [FSGS] in two and mesangioproliferative GN in two), renal biopsy sections were examined by electron microscopy (EM). For EM, renal cores were preserved in 3% phosphate-buffered glutaraldehyde, diced into 1 mm cubes, rinsed in distilled water, transferred into 1% aqueous osmium tetraoxide and embedded in TAAB Emix resin. Sections of 0.5 µm were cut, mounted on glass slides, stained in 1% aqueous toludine blue in 1% sodium tetraborate for 15 s on a hot plate at 15 °C, cooled and examined by light microscopy for assessable glomeruli before being sectioned with a diamond knife in a Leica Ultracut E ultramicrotome, with interface colour gold of approximately 95 nm. Sections were stained by immersion for 7 min in 50% alcoholic saturated uranyl water, 3 min in Reynolds lead citrate and three washes in distilled water, and examined by a Philips 400 transmission electron microscope.
Immunohistochemistry
Immunohistochemistry was carried out on formalin-fixed and paraffin-embedded renal sections as well as on frozen renal cores (10 sections from patients with MPGN type I, four type II, three FSGS, two membranous nephropathy (MN), one mesangioproliferative GN). For paraffin sections, paraffin was removed by xylene, tissue rehydrated by immersion in decreasing grades of ethanol and endogenous peroxidase activity was inhibited by freshly prepared 3% hydrogen peroxide in absolute methanol. Sections were incubated with blocking serum (Vectastatin ABC kit, Vector Laboratories, Inc., Peterborough, UK). Overnight incubation (18 h) at 4 °C with the primary antibody (monoclonal antibody to HCV core AA 1120, Biogenesis Ltd, Poole, UK) was undertaken at different dilutions (1:50, 1:100, 1:250 and 1:300) in order to choose an optimal antibody concentration. ABC reagent was added (reagent A: avidin DH, reagent B: biotinylated horseradish peroxidase H, Vectastatin ABC Kit, Vector Laboratories, Inc.) and colour development was by the addition of aminoethylcarbazole (AEC reagent). The sections were counterstained with haematoxylin and eosin, and examined by light microscopy.
For frozen renal tissues, 5 µm cryosections were cut and stored at -80 °C before staining and fixation in cold acetone at 4 °C for 10 min before washing three times with phosphate-buffered saline (PBS) (5 min each). The rest of the steps were as described above for the paraffin-embedded blocks.
Different methods to enhance antigen retrieval were tried including heat (56 °C), incubation with proteinase K in PBS 20 µg/ml at 37 °C for 15 min and acid electroelution as described elsewhere [11].
Reverse transcriptionpolymerase chain reaction
RTPCR was performed on cryoprecipitates, when available, paraffin-embedded renal sections as well as frozen renal tissues. In brief, for formalin-fixed paraffin-embedded sections, RNA was extracted by tissue dewaxing in xylene followed by alcohol washes. In preliminary experiments two different methods for extraction were tried.
RNA was extracted by the proteinase digestion method (100 µg/ml) not by the guanidinum isothiocyanate method.
For cDNA synthesis 4 µl RNA was heated to 65 °C, chilled on ice, added to 3.5 Master Mix aliquots (First Strand cDNA Kit, Pharmacia) and incubated at 37 °C for 1 h. Amplification of 1 µm cDNA was performed in 24 µl reaction mixture containing 2.5 µl 10x PCR buffer, 0.25 µl dNTPs 10 µM, 0.15 µl Taq DNA polymerase (Boehringer Mannheim), 20 µl PCR water, 1 µl outer primer (sense), 1 µl outer primer (antisense). This was carried out for 35 cycles using the following programme: denaturation for 45 s at 94 °C, annealing for 45 s at 50 °C and extension for 2 min at 72 °C. A second PCR with nested inner primers was performed utilising 1 µl of the first PCR product as DNA template. The amplification cycles were identical to the first one [12].
Statistical analysis
Results are given as median and confidence intervals. MannWhitney (non-parametric rank sum) tests, Fisher exact probability tests and 2 tests were applied as appropriate. A P value of <0.05 was considered significant.
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Results |
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Despite the fact that 13 patients had elevated liver enzymes, clinical manifestations of hepatic disease were not frequent and only five had hepatomegaly. No other manifestations of liver disease were observed (Table 1).
At presentation, 27 patients tested positive for cryoglobulins; however, in 26 of them cryoprecipitates were typed and shown to contain both IgM and IgG. In 80% of the cryoglobulinaemic patients extrarenal manifestations of cryoglobulinaemia were absent. Arthralgias were present in 8%, purpura in 7% and peripheral neuropathy in 5% of the cryoglobulinaemia-positive patients.
Virological studies
In all patients, the diagnosis of HCV infection was confirmed by detection of HCV antibodies using third generation ELISA, and HCV RNA in serum by RTPCR as a confirmatory test. The testing was also undertaken on the cryoprecipitates from 25 patients, 17 of which were positive.
Genotyping was performed for 10 randomly chosen samples, in all of them RTPCR products were detected. However, only seven PCR products had sufficient quantities of cDNA for sequencing. All were classified as type 4 according to Simmonds and colleagues [13].
Detection of HCV antigen and RNA in tissue specimens
Immunohistochemistry
Immunohistochemistry was done on frozen and formalin-fixed paraffin-embedded renal sections from 20 patients who were HCV positive. Pre-treatment of the sections with heat or trypsinization did not affect the outcome as the samples remained negative.
Electron microscopy
Immune complexes were detected in the subendothelial space and paramesangial areas (Figure 1a). Virus-like particles were located inside the electron-dense deposits in 50% of cases (Figures 1b
and c
). In the three HCV-negative control patients with MPGN, neither electron-dense deposits nor virus-like particles were detected.
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RTPCR in renal sections
In frozen renal biopsies of 21 cryoglobulinaemic patients, four were positive for HCV RNA (Figure 2a). In non-cryoglobulinaemic patients, five out of 21 were positive (Figure 2b
). In each run positive and negative controls were included.
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Discussion |
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The seroprevalence of hepatitis C is thought to be greater in individuals with chronic GN compared with those suffering from other renal diseases [15]. The results of our study confirm this since we found a higher prevalence of HCV antibodies among patients presenting with glomerulopathy (38%).
In our study, light microscopy examination revealed a high incidence of MPGN. The high prevalence of cryoglobulinaemic and non-cryoglobulinaemic MPGN observed in our patients has been previously reported by others [6,8]. An association between HCV infection and FSGS has also been reported [16]. In fact, the high prevalence of FSGS in our series (24%) may reflect a real association between HCV and this type of renal lesion or it may reflect a selection bias. Only 4% of our patients had MN, with nephrotic-range proteinuria, mild elevation of liver enzymes, normal complement level, negative for both cryoglobulins and rheumatoid factor, which is consistent with previous observations [17]. Other forms of renal disease that have been previously linked to chronic HCV infection include acute exudative and proliferative glomerulonephritis and IgA nephropathy [18]. Of interest, the distribution and types of GN in the HCV-negative patients was comparable to those with seropositivity.
The prevalence of cryoglobulinaemia in our series of HCV-positive patients was 54%. However, despite the presence of cryoglobulinaemia, extrarenal manifestations were absent in the majority of patients (80%). It has been suggested that HCV infection is responsible for all cases of type II MEC and that the idiopathic (essential) type no longer exists [8,19]. In the two large studies of patients with type II MEC and MPGN the prevalence of HCV RNA was 81 and 100% [8,19]. Our data agree with Johnson and his colleagues [6], who found the prevalence to reach 62% in their patients. The same authors described a prevalence of 59% in a series of patients with HCV-associated nephropathy at presentation. However, an additional 26% developed cryoglobulinaemia during the subsequent months [20]. The same findings were described by Lunel and coworkers, who found 54% of patients with HCV-associated nephropathy to have type II cryoglobulinaemia [21].
In this study, we were not able to locate any virus-related proteins in formalin-fixed, paraffin-embedded or frozen renal biopsies. The negative renal results were in spite of the application of a variety of techniques for antigen retrieval on both paraffin-embedded and frozen sections. This failure to locate viral antigens in the kidney is in agreement with most of the reported literature [6,10,20] with few exceptions [11,22]. Sansonno and coworkers [11], in 12 patients with MPGN and cryoglobulinaemia, found HCV-specific immunoreactive deposits in 66.7% of kidney biopsy specimens. In fact, these authors used a pannel of HCV monoclonal antibodies including anti-C22-3, anti-envelope glycoprotein2 (E2)/NS1 antibody, anti-C33 antibody and a mixture of anti-C100 and anti-NS5 antibodies [11]. This may explain their positive results compared with other studies, including our own, where only one antibody was tested. Although some investigators have been unsuccessful in identifying viral proteins in renal specimens [6,20], others reported the detection of such proteins in the renal tissues of patients with HCV-associated glomerulonephritis [10,22]. Clearly, the presence of virus-like particles within the glomeruli does not necessarily implicate it in the pathogenesis of renal disease; rather it may simply represent antigen-trapping within an area of tissue damage [6,10].
The most reliable method for the diagnosis of HCV infection is the detection of viral RNA sequences by RTPCR in serum and tissue samples [1]. We were unable to detect HCV RNA in formalin-fixed, paraffin-embedded renal specimens. This may be explained by the fact that HCV is an RNA virus and is therefore more labile than DNA viruses, making it susceptible to damage by the process of fixation and prolonged paraffin embedding [23].
Interestingly, HCV RNA was extracted and amplified successfully in frozen renal tissues from nine of our patients (22%), both cryoglobulinaemic and non-cryoglobulinaemic, representing most of the histological classes included in our study (four MPGN, two MN, two FSGS and one mesangioproliferative GN). Failure to detect HCV RNA in the remaining samples could be explained by the presence of only a small amount of RNA, which may be below the detection sensitivity of the method used, or simply by the absence of HCV RNA in renal tissues. Our results for HCV RNA yield in renal tissues is much lower than those described by Stockes and colleagues [10], who were able to extract and amplify HCV RNA in 58% of their patients. However, their patients were co-infected with HIV raising the possibility that such a co-infection may lead to the over-expression of HCV RNA. Also the signals obtained by RTPCR could be due to circulating HCV-infected leukocytes within the kidney [24].
Previous studies have shown that aggregates or single virus-like particles of about 45 nm in diameter were detectable in the endoplasmic cisternae and in the cytoplasm of hepatocytes of HCV-infected patients [25]. Similarly, virus-like particles were demonstrated in human T- and B-lymphocyte cell lines [26]. We have demonstrated virus-like particles in the paramesangial electron-dense deposits by EM in 50% of patients studied. These particles were comparable to those previously described in the paramesangial dense deposits in a patient with HCV-related proliferative glomerulonephritis [27]. Their characteristic features support their classification as HCV-related particles.
In conclusion, HCV infection is frequent among the Egyptian population affecting about 16% of the whole population. This prevalence is higher among patients with chronic GN. There is a documented link between cryoglobulinaemic and non-cryoglobulinaemic MPGN and chronic HCV infection. A possible role for HCV infection in other forms of GN requires further investigation.
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Acknowledgments |
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Notes |
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References |
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