Cryoglobulinemia in Chronic Hemodialysis Patients

Abstract

Background: Renal failure patients submitted to chronic hemodialysis can present with cryoglobulinemia. There are few studies on cryoglobulins in chronic hemodialysis patients. The aim of the present study was to determine the prevalence and to identify the components of cryoglobulins in chronic hemodialysis patients. Methods: Fifty-four patients on chronic hemodialysis were evaluated for the presence of cryoglobulins, after inclusion and exclusion criteria. The components of the cryoprecipitate were analyzed. Results: Cryoglobulins were detected in 83% (45/54) of the patients on chronic hemodialysis. The cryoprecipitate was constituted by IgG, IgM, IgA, and complement fractions C3 and C4. Conclusion: We concluded that there was a high prevalence of cryoglobulins in chronic hemodialysis patients, and the cryoprecipitate was constituted by IgG, IgM, IgA, and complement fractions C3 and C4.

• hemodialysis
• cryoglobulins
• renal failure
• hepatitis C virus
• immunocomplexes

INTRODUCTION

There are few studies on the presence of cryoglobulins in end-stage renal disease (ESRD) patients on chronic hemodialysis.

Cryoglobulinemia is defined as the presence of immunocomplexes that in vitro precipitate with the exposure to cold and resolubilize when rewarmed and in vivo deposit on endothelial surfaces, causing systemic vasculitis.¹ The mechanism of the resulting vascular inflammation is not perfectly understood.²

The immunocomplexes are constituted by antigens, antibodies, and complement system components. The antigens more frequently observed are autoimmune antibodies and hepatitis C virus (HCV) RNA. The circulating immunocomplexes are formed when there is a relative increase in the number of antigens in relation to antibodies, determining a type III hypersensibility reaction. The pathogenesis of the disease is a consequence of the immunocomplexes deposition which acts as a local mechanical barrier and activates the complement system, resulting in tissue lysis.³

It is possible that a significant quantity of cryoglobulins may be present in patients with ESRD on chronic hemodialysis.

The aim of the present study was to determine the prevalence and to identify the components of cryoglobulins in ESRD patients on chronic hemodialysis and to verify the association with chronic HCV infection.

PATIENTS AND METHODS

Patients with ESRD submitted to chronic hemodialysis and follow-up in the Nephrology Clinic were studied, and the cryoglobulins were analyzed at the immunology laboratory of the institution.

The ethics committee of the institution approved the study protocol (no. 176/10), and informed consent was obtained from every participant.

Table 1. Cryoprecipitate components in peripheral blood of the patients submitted to chronic hemodialysis, with or without hepatitis C virus (HCV) infection.

	IgA (mg/dL)	IgM (mg/dL)	IgG (mg/dL)	C3 (mg/dL)	C4 (mg/dL)
HCV-positive patients (n = 27)	2.26 ± 2.29	1.73 ± 1.64	13.47 ± 11.30	0.74 ± 0.48	0.38 ± 0.42
HCV-negative patients (n = 27)	0.99 ± 0.40	0.97 ± 1.12	5.44 ± 5.06	0.40 ± 0.41	0.17 ± 0.17
p-Value	0.021	0.079	0.004	0.015	0.035

Note: *p < 0.05, Student’s t-test.

*p < 0.05, Student’s t-test.

Fifty-four adult patients were selected with ESRD on chronic hemodialysis: 27 infected by HCV for more than 6 months and 27 patients without chronic HCV infection. The inclusion and exclusion criteria were based on clinical history, physical exams, and laboratory tests. The inclusion criteria included patients on hemodialysis for more than 12 months, without previous renal transplantation; with negative rheumatoid factor; with antinuclear antibodies, anti-double-strand DNA antibodies, and antithyroidal antibodies; and without infection episodes for 3 months prior to tests. patients excluded were those treated with immunosuppressors, interferon, or ribavirin; those with neoplastic disease, liver disease, or known autoimmune diseases; patients undergoing transfusions or vaccines within 3 previous months; and those who received recent surgical procedures; patients with clinical signs/symptoms or positive laboratory exams for infection, except HCV infection. The demographic and biochemical data were analyzed, as well as the etiology of renal failure to characterize the population studied.

All patients were undergoing hemodialysis for 4 h, three times per week, utilizing polysulfone membrane and standard bicarbonate solution. All patients were tested for HCV antibodies by the second- and third-generation enzyme-linked immunoassay method and for HCV RNA by reverse transcription-polymerase chain reaction (RT-PCR), using an Amplicor kit (version 2.0, Roche Diagnosis System, Branchburg, NJ, USA).

For the cryoprecipitates analysis, peripheral blood samples were collected with their serums stored at 4°C for 15 days. After the cryoprecipitate formed, it was separated by centrifuging at 4°C and the serum was washed 10 times with saline solution. Parallel heating tests were performed on the cryoprecipitate, which dissolved at low temperatures and became soluble when heated. The cryoprecipitate components (IgG, IgM, IgA, C3, and C4) were evaluated by using simple radial immunodiffusion (NOR Partigen, Siemens Healthcare Diagnostics Marburg, Germany).⁴ The peripheral blood samples were collected just before the hemodialysis session.

Quantitative results expressed normal distribution and were analyzed using the Student’s t-test, and qualitative variables were compared by the χ² test. The considered significance level was 5% (p < 0.05).

RESULTS

Fifty-four patients submitted to chronic hemodialysis were studied and they presented with various etiologies of renal disease: chronic glomerulonephritis (18/54), hypertension and nephrosclerosis (17/54), chronic interstitial nephritis (3/54), urinary tract disorders (3/54), diabetic nephropathy (2/54), and unknown (11/54).

Among the patients, 27/54 had HCV infection, detected by RT-PCR, in serum 6 months before the study, while 27/54 did not present with HCV infection. The time on hemodialysis was 84.5 ± 44.7 months in the HCV-positive group and 77.1 ± 44.3 months in the HCV-negative group, without statistical significance (p = 0.6).

Among the 27 HCV-positive patients, 13 were male and 14 female, aged between 21 and 56 years (mean 43.8 ± 12.9 years); among the 27 HCV-negative patients, 11 were male and 16 female, aged between 25 and 50 years (mean 37.2 ± 12.1 years).

The positive and negative HCV patients showed, respectively, aspartate aminotransferase (AST; 21.9 ± 13.9 vs. 23.5 ± 17.3 U/L; p = 0.7) and alanine aminotransferase (ALT; 26.1 ± 28.2 vs. 24.0 ± 17.8 U/L; p = 0.7), and the C-reactive protein was at normal levels (0.48± 0.2 vs. 0.58 ± 0.3 mg/dL; p = 0.2), showing no difference between the groups. These data were used to identify the presence of acute hepatitis or other possible acute infections.

Cryoglobulins were detected in 83% (45/54) of all the patients studied. Among the 27 patients with chronic HCV infection, 93% (25/27) presented with cryoprecipitate, and among the patients without HCV, 74% (20/27) presented with cryoprecipitate. The difference in cryoglobulin prevalence in the two groups was not statistically significant (p = 0.7). None of the patients of the two groups manifested any clear clinical signs secondary to the presence of cryoglobulins.

Table 1 shows the constitution of the cryoprecipitate: IgG, IgM, IgA, and complement components C3 and C4. The results of the components IgA, IgM, IgG, C3, and C4 of the cryoprecipitate in the HCV-positive group showed significantly higher levels of IgG, IgA, C3, and C4, when compared to HCV-negative patients.

DISCUSSION

The present study detected cryoglobulins in the majority of the patients on chronic hemodialysis, with or without chronic HCV infection.

Patients with multiple blood transfusions, immunizations, neoplastic disease, liver or autoimmune diseases, and acute infections were excluded from the study because these could induce the formation of cryoglobulins.⁵

The clinical/laboratory examinations of the patients showed no clear symptoms of cryoglobulinemia, any other active inflammatory/infectious diseases, and normal values of C-reactive protein, AST, and ALT. The time on chronic hemodialysis was more than 5 years and similar between the groups with and without HCV infection.

The exclusion of fibrinogen by parallel heating of the cryoprecipitate and the identification and quantification of IgA, IgM, IgG, and complement components C3 and C4 confirm the presence of cryoglobulins.

The cryoprecipitate of the HCV-positive patients showed higher concentrations of IgG, IgA, C3, and C4, compared with that of the HCV-negative patients.

Higher rates of IgG in the cryoprecipitate are also found in chronic diseases, with a pronounced predominance of isotype IgG in the serum.^6,7

HCV is a positive-strand RNA virus with a 9.6 kb genome, which chronically infects over 170 million people worldwide.⁸ Patients with ESRD on hemodialysis present a greater risk of HCV infection due to the constant exposure to blood-derived products.⁹ Cryoglobulinemia is associated with an increased duration of the HCV infection. Patients with HCV and cryoglobulinemia have an apparent duration of the infection that is almost twice as long as that in HCV infection patients without cryoglobulinemia.¹⁰ Complex alterations in the immune system are observed in both patients on chronic hemodialysis for ESRD and chronic HCV infection.¹¹

The formation of immunocomplexes could be a result of the continuous presence of the antigen, inducing the production of antibodies. Among HCV-infected individuals, 74–86% of the patients developed persistent viremia.¹²

HCV infection frequently causes mixed cryoglobulinemia, characterized by immune complexes containing monoclonal and/or polyclonal immunoglobulins and complement, causing clinical manifestations of purpura, arthralgia, asthenia, B-cell lymphoproliferative disorder, and vasculitis in different organs, and it may also be involved in the pathogenesis of glomerulonephritis, mainly type I membranoproliferative glomerulonephritis.^2,13,14 In cryoglobulinemic glomerulonephritis, serum C4 levels are usually very low, as is the other early component C1q. This consumption of the early serum complement components, with the characteristic sparing of C3, might be attributed to the C4-binding protein that controls the mechanism of the classic pathway C3 convertase.¹⁵

The reported frequency of cryoglobulinemia among patients with chronic hepatitis C is variable, up to 60% in southern Europe, whereas the prevalence among HCV patients in the United States is lower (10–50%), and the prevalence of symptomatic cryoglobulinemia is much lower. It is unclear whether this difference is due to unidentified genetic factors.² Vigani et al.¹⁶ found cryoglobulinemia in 54% of the patients with HCV genotype 1 and in 71.4% of the patients with genotype 3.

In the literature, there are few studies which evaluated the presence of cryoglobulins in patients on chronic hemodialysis. Some studies have observed the association between HCV infection and cryoglobulinemia in patients with ESRD on hemodialysis.^17,18. Canavese et al.¹⁹ found cryoglobulinemia in 77% of the anti-HCV-positive versus 29% of the anti-HCV-negative patients on dialysis. Anis et al.¹⁸ found prevalence of cryoglobulins in patients either on hemodialysis or in postoperative renal transplantation to be higher (57.6%) among anti-HCV-positive patients, compared to the anti-HCV-negative patients (42.4%). Sens et al.²⁰ detected cryoprecipitate in 74.4% of the kidney transplant recipients, and 37.9% were HCV positive.

In the present study, a high prevalence of the cryoglobulins was observed in the peripheral blood samples of the patients with or without chronic HCV infection submitted to long-term hemodialysis treatment. Chronic kidney disease is a state of low-grade inflammation that contributes to the accelerated progression of chronic inflammatory disturbances. Platelet and leukocyte activation and interaction have an important role in this process.²¹ There is an anomalous response in uremia to specific antibodies and a decrease in cellular immunity.^22,23 Decreased CD4⁺ cell adhesion to the endothelium of healthy individuals in the presence of uremic patients’ serum was observed.²³ Menezes et al.²⁴ showed that there was a reduction in the ingestion phase of phagocytosis by polymorphonuclear neutrophil cells in renal transplant patients with cryoglobulins. The mononuclear and polymorphonuclear neutrophil phagocytic systems are important for the clearance of the immune complexes, and if their activity is compromised, there may be a longer persistence of circulating cryoglobulin immune complexes.

In patients on hemodialysis, leukocyte function is also disturbed because of bioincompatibility of some components of the hemodialysis membranes. Activation of cytokine and complement cascades occurs when blood comes into contact with hemodialysis membranes. These substances in turn alter inflammatory and immune responses of uremic patients.²⁵ Hemodialysis membranes with low biocompatibility, such as cuprophane, activate the complement system by alternate pathway and induce an inflammatory response.^25,26 In the present study, all ESRD patients with or without HCV infection were on chronic hemodialysis using polysulfone membranes. Synthetic polysulfone membranes trigger a lesser immediate immunologic reaction in comparison with cuprophane membranes, as they are more biocompatible.^26,27 However, the polysulfone membrane is capable of activating the complement system and accelerating the apoptosis of polymorphonuclear cells.^26,27

Thus, diverse possibilities may be considered for the patients studied to stimulate the formation and permanence of the circulating immunocomplexes; among them are the presence of HCV, the state of uremia, and even the hemodialysis membrane material.

In conclusion, in the group on chronic hemodialysis in this study there was a high prevalence of cryoglobulins, and the cryoprecipitate was constituted by IgG, IgM, IgA, and complement fractions C3 and C4, in patients with and without chronic HCV infection. We believe that cryoglobulins could be an aggravating factor in the disease. More studies are needed to clarify the role of these alterations and their clinical significance in uremic patients on chronic hemodialysis.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.