Take a look at the Recent articles

Prevalence of positive ANA, ANCA antibodies and 25(OH) vitamin D levels in patients in hemodialysis

Deisy da Silva Fernandes Nascimento

Department of clinical immunology, professor of pharmacy course at Universidade do Sul de Santa Catarina, Brazil

E-mail : bhuvaneswari.bibleraaj@uhsm.nhs.uk

Amanda Fernandes Freitas

Department of pharmacy, Universidade do Sul de Santa Catarina, Brazil

Brunna D’avila Pereira

Department of pharmacy, Universidade do Sul de Santa Catarina, Brazil

Mariana Caetano da Luz

Department of pharmacy, Universidade do Sul de Santa Catarina, Brazil

Morgana Miranda Clarinda

Department of pharmacy, Universidade do Sul de Santa Catarina, Brazil

Thiago Mamôru Sakae

Department of anesthesiologist, Professor of epidemiology in medicine course at UNISUL, Brazil

DOI: 10.15761/NRD.1000160

Article
Article Info
Author Info
Figures & Data

Abstract

Objective: To describe the prevalence and factors associated with the positivity of FAN-Hep2, ANCA and 25 (OH) vitamin D deficiency in users of a hemodialysis clinic in Southern Brazil.

Methods: A cross-sectional study was carried out in 68 patients from a hemodialysis clinic in the Southern Santa Catarina (Southern Brazil) from August to November 2013. An interview was conducted, data collection in an electronic medical record, FAN and ANCA research by indirect immunofluorescence WAMA and dosage of 25 (OH) vitamin D by chemiluminescence amplified in heparinized plasma.

Results: The prevalence of ANCA in a sample titrated 1:10 was 24.4%, with 10.3% presenting c-ANCA pattern and 14.1% presenting a p-ANCA pattern, of the total. The prevalence of ANA was 4.4%, and the prevalence of 25 (OH) vitamin D deficiency was 35.8%. No associations with clinical and sociodemographic characteristics were observed with autoantibodies.

Conclusion: We found a high prevalence of ANCA positive and 25 (OH) vitamin D deficiency. We emphasize the need for further studies in this group of patients to define the contribution of these tests to the diagnosis and prognosis of renal disease and its complications.

Keywords

autoantibodies, ANCA, anti-nuclear antibodies, kidney failure, 25 (OH) vitamin D

Introduction

According to data from the World Health Organization, an estimated 500,000,000 people are chronically infected with hepatitis B virus (HBV) or hepatitis C virus (HCV) worldwide. Due to these infections and their complications, such as cirrhosis and hepatic carcinoma, approximately 1,000,000 people die annually [1]. An important route of contamination by HBV and HCV viruses is the parenteral route. Blood transfusion, surgical procedures and hemodialysis are recognized as risk factors for these infections [2,3].

Extra-hepatic manifestations have been related to the presence of chronic hepatitis, especially with the presence of HCV. Autoimmune diseases are included in these extrahepatic manifestations, due to the induction of apoptosis by T cells, by mechanisms that involve the recognition of autoantigens [4]. Renal function during liver disease has also been the focus of recent research. Decreased peripheral vascular resistance promoting renal ischemia, renal damage in the tubular portion by elevated levels of hepatic metabolites, especially in cholestatic diseases, and deposition of immunocomplexes in the glomerular portion are the main mechanisms that explain the pathophysiological relationship between the kidney and liver [5].

Antinuclear Antibody (ANA) screening by indirect immunofluorescence is a sensitive and useful assay for screening of autoimmune diseases. The application and interpretation of the test must be judicious and confirmed by the search for specific autoantibodies through other techniques, such as ELISA and Western blot [6]. Antibody search for neutrophils (ANCA), however, provides a useful tool in the investigation of vasculitis [7]. Renal involvement in vasculitis associated with ANCA positivity is a frequent finding in clinical practice, and the presence of this marker is a negative indicator of prognosis [8].

It is also known that patients with renal disease have high frequency of 25 (OH) vitamin D deficiency, as well as, difficulty in activating this vitamin [9-11]. However, 25 (OH) vitamin D deficiency causes damage not only to bone metabolism, but systemic, since it is a source of immune system modulation. Clinical evidence corroborates this fact, through the relationship between vitamin D 25 (OH) deficiency and increased frequency of various autoimmune diseases [12].

Considering that the metabolism of vitamin D depends on hepatic and renal health, and the complexity of the pathophysiological mechanisms that relate the triad "autoimmunity - renal disease - liver disease", there is a need for studies that provide support to improve diagnosis and understanding of the clinical evolution of these diseases. The goal of this study is to characterize a group of patients with renal insufficiency regarding clinical aspects and to investigate the prevalence of autoantibodies by FAN, ANCA and 25 (OH) vitamin D. The identification of factors associated with the presence of these markers may contribute to refining the application and interpretation of the same in clinical practice.

The aim of this study was to describe the prevalence and factors associated with the positivity of FAN-Hep2, ANCA and 25 (OH) vitamin D deficiency in users of a hemodialysis clinic in Southern Brazil and associated factors.

Methods

A cross-sectional study was conducted, which census of the population of the Kidney Disease Clinic of the city of Tubarão – Santa Catarina, Southern Brazil, from August to November 2013. Patients with renal insufficiency undergoing hemodialysis treatment were included, who underwent periodic exams in the Laboratory of Clinical Analysis of the University of Southern Santa Catarina (LAC-UNISUL), aged 18 years or older. Exclusion criteria were: patient refusal to study, patients with cognitive impairment, previously diagnosed autoimmune diseases (lupus, rheumatoid arthritis, thyroiditis, etc.), and patients absent from hemodialysis sessions on at least three previously scheduled visits. This work was approved by the Research Ethics Committee (CEP) of UNISUL, protocol number 338.734.

Data collection was initiated with invitation and semi-structured questionnaire application. Dosing of 25 (OH) vitamin D was performed by amplified chemiluminescence (Ortho Clinical Diagnostics Johnson and Johnson) in heparinized plasma samples of these patients. The FAN and ANCA research by indirect immunofluorescence (WAMA diagnosis) was performed in heparinized plasma obtained from the Laboratory of Clinical Analysis of UNISUL.

Statistical analysis was done with SPSS software version 20.0. The mean, median and standard deviation for the continuous variables and proportions for the categorical variables were calculated. For the test of association between the variables of interest, Pearson's chi-square test and Fisher's exact test were used, when appropriate. The pre-established level of significance was 95%.

Results

From the 127 patients treated by the Renal Disease Clinic, 101 patients had collected heparinized plasma for periodic examinations by the LAC-UNISUL at the time of the study and were previously included in the study. Twelve losses occurred: two refusals, three deaths, two discharges, one patient with cognitive impairment and four absences at hemodialysis sessions. Of the remaining 89 participants, ten patients were lost due to lack of exams and eleven autoimmune diseases were excluded from the study: lupus (1), rheumatoid arthritis (3), vasculitis and thyroiditis (4) (Table 1 and 2).

Table 1. Sociodemographic Characteristics

Sociodemographic Characteristics

N

%

Gender

Female

25

36.8

Male

43

63.2

Race

White

57

83.8

Black

11

16.2

Age (years)

21-40

8

11.9

41-50

15

22.0

51-70

36

52.9

71-80

7

10.3

>80

2

2.9

Age (Mean ± standard deviation in years)

Mean=55.94

SD=14.79

Scholarity (completed)

0-4

23

33.8

5-8

27

39.7

9-11

14

20.6

12-16

Scholarity (mean ± standard deviation in years)

4

mean=6.81

5.9

SD=3.84

Table 2. Clinical characteristics of the patients

Clinical characteristics

N

%

Comorbidity

Arterial hypertension (HAS)

48

70.6

Diabetes Mellitus (DM)

31

45.6

Urinary infection of repetition

9

13.2

Kidney stone

6

8.8

Dyslipidemia

28

41.2

Hepatitis C

3

4.4

Hepatitis B

0

0

Transfusion

Never received

36

52.9

Received until 3 times

28

41.1

Received from 4 to 6 times

1

1.5

Received from 7 to 10 times

1

1.5

Received more than 10 times

2

3.0

Hemodyalisis’ time (in months)

1 – 24 months

43

63.2

25-50 months

10

14.7

51-80 months

7

10.3

81-100 months

2

3.0

101-180 months

6

8.8

Mean and standard deviation (SD) Primary causes of kidney injury 

mean=35.96

SD=38.92

Routine tests or doesn´t know the fact that related to the diagnostic of the kidney injury

27

39.7

Symptoms (pain, edema, weakness)

12

17.6

Hypertension

9

13.2

Infection of urinary tract

2

2.9

Diabetes

7

10.3

Vascular accident (angina, heart attack, stroke)

4

5.8

Hereditary / congenital

2

2.9

Kidney stone

2

2.9

Drug toxicity

3

4.4

Creatinine (pre-dialysis)

mean=9.93

SD=2.64

Urea

mean=156.44

SD=37.12

Platelets

mean=191313.24

SD=22.44

From the 32 patients who reported receiving a blood transfusion, 14 (20.6%) reported having received at least one prior to 1993. From the 68 patients, only 3 (4.4%) reported having been drinking alcoholic beverages socially. No patient had Hepatitis B infection and three patients (4.4%) had Hepatitis C.

In relation to ANA, 4.4% of the sample were positive. For ANCA antibodies, 51 (75.0%) patients had non-reagent results, and 17 (24.4%) had reagents. About ANCA reagents, 6 (8.8%) presented c-ANCA and 11 (16.2%) presented p-ANCA pattern (Table 3).

Table 3. Prevalence of positivity of ANA, ANCA, and levels of 25 (OH) vitamin D

Variable

N

%

ANA

positive

3

4.4

negative

65

95.6

ANCA

negative

51

75.0

mean cytoplasmic (c-ANCA)

6

8.8

Mean perinuclear (p-ANCA)

11

16.2

25 (OH) Vitamin D (25 OH cholecalciferol)

0-10

0

0

11-20

6

8.8

21-30

18

26.5

>30

44

64.7

25 (OH) Vitamin D (mean and standard deviation)

mean=38.87

SD=13.07

The female patients presented levels of 25 (OH) vitamin D lower than those of the male sex did (p=0.004). Patients with SD also had lower levels of 25 (OH) vitamin D when compared to those without SD (p=0.016) (Table 4).

Table 4. Comparison of 25 (OH) vitamin D levels among the variable sociodemographic, clinical and laboratorial tests

Variable

Mean

Standard deviation

p-value

Gender

0.004*

Male

43.14

16.61

Female

31.54

12.50

Race

0.410*

White

35.17

12.18

Non white

39.59

16.80

Diabetes mellitus

Yes

36.76

18.44

0.016*

No 

43.13

12.67

Arterial hypertension

0.439*

Yes

37.89

17.37

No

41.25

12.83

Corticoids use

0.611*

Yes

33.10

18.10

No

39.05

16.21

ANA

0.875*

Positive

38.81

16.37

Negative

40.33

12.29

ANCA

0.231*

Negative

40.08

16.39

Positive

35.27

13.28

*p<0.05 – Chi square test

There were no associations between the levels of 25 (OH) vitamin D and the ANA (p=0.875) and ANCA (p=0.321) levels. The clinical or sociodemographic characteristics also did not present a statistically significant association with the positivity of ANA and ANCA antibodies.

Discussion

Socio-demographic characteristics found in this study (sex, age, race and schooling) are in line with the Brazilian reality of patients with chronic kidney disease and with the European colonization of Santa Catarina [13,14]. Low schooling also reduces the patient's chances of understanding and adhering to medical recommendations, as well as giving an opinion on managing their illness.

The high prevalence of patients undergoing blood transfusion is a reflection of the socio-demographic profile of these patients. Due to the risk for HBV and HCV infection, as well as for the development of irregular antibodies against erythrocyte antigens and other autoantigens promoted by blood transfusion and/or blood derivatives, especially prior to 1993 when screening for HCV was not yet performed in donors, the recommendations for this procedure are quite discerning [15,16]. However, no statistical correlation was found between blood transfusion and autoimmunity markers.

Patients' reports regarding the etiology of kidney disease were inconclusive because 39.7% did not know how to answer this question. However, these data are not discrepant with the data of comorbidities, suggesting that, in the majority of patients, renal disease secondary to a previous metabolic disease.

As for the comorbidities reported by the patients, systemic arterial hypertension, diabetes mellitus and dyslipidemia were the most frequent, which has also been reported in other Brazilian studies [17,18]. Interestingly, the pathophysiology of renal disease is intertwined with the pathophysiology of these three entities, the main points being the deterioration of the glomerular barrier with loss of proteins and lipoproteins, and the renin-angiotensin and hepatic system reflex with increased synthesis of acute phase proteins and lipoproteins [19].

According to the recent positioning of the Brazilian Society of Clinical Pathology and Laboratory Medicine, and the Brazilian Society of Endocrinology and Metabolism, the reference range for dosing 25 (OH) vitamin D in patients with chronic kidney disease is between 30 and 60 ng/mL [20]. In this study, 64.7% of the patients had serum 25 (OH) vitamin D levels higher than 30 ng / mL, reflecting an important prevalence of 25 (OH) vitamin D deficiency [20]. At this point, it is interesting to note that the receptor for 25 (OH) vitamin D is present not only in hard tissues and parathyroid, but also in pancreatic islets, and that there is a clinical correlation between 25 (OH) vitamin D deficiency, obesity and risk of developing diabetes mellitus [21,22].

The detection of VDR in immunological cells such as macrophages, dendritic cells and T and B lymphocytes has also encouraged research that attempts to elucidate the role of 25 (OH) vitamin D in immunomodulation. In clinical practice, 25 (OH) vitamin D deficiency expresses a positive correlation with several autoimmune diseases [23]. It is known, however, that the occurrence of autoantibodies such as ANA and ANCA is a result of a multifactorial process, influenced by intrinsic factors such as immunomodulation and polymorphisms of histocompatibility molecules, and extrinsic factors such as viral, bacterial and alloantigen exposure and to drugs [24,25]. However, in our study, there was no association among 25 (OH) vitamin D deficiency, blood transfusion, hepatitis C infection, use of corticosteroids or interferon, and autoimmunity markers.

The ANA positivity, according to the standard and titration, can contribute to the diagnosis of several autoimmune conditions, whereas the ANCA is related to the diagnosis of three systemic necrotizing small vessel vasculitis: Microscopic Poliangiitis and its renal variant (Glomerulonephritis and Churg-Strauss syndrome and Wegener's granulomatosis [26,27]. The presence of these antibodies may promote the development or progression of renal disease by immune complexation and activation of the complement system [19]. We consider that this topic requires further studies, which include dilutions other than those recommended in the package inserts, to optimize the clinical interpretation of these autoantibodies and their predictive capacity.

Conclusion

This study found no statistically significant correlation between ANCA, ANA, 25 (OH) vitamin D and laboratory markers of advanced chronic kidney disease, or clinical and socio-demographic aspects of these patients. However, this topic requires more studies, since the literature offers sufficient subsidence to propose that these autoantibodies participate actively in the pathophysiology of renal disease and may occur as a cause of kidney disease or in its natural course.

Conflict of interests

The authors declare no conflicts of interest.

Financial support

This work received financial support from the WAMA Diagnostic Company and the UNISUL Pharmacy Course for the acquisition of kits for the ANA and ANCA test, and Ortho Clinical Diagnostics for 25 (OH) vitamin D. reagents.

The project also used the infrastructure of the Clinical Analysis Laboratory and the UNISUL Didactic Laboratory.

References

  1. WHO (2012) Prevention & control of viral hepatitis infection: Framework for global action. World Health Organization.
  2. Batchoun RG, Al-Najdawi MA, Al-Taamary S (2011) Anti-ENA antibody profile in hepatitis C patients undergoing hemodialysis. Saudi J Kidney Dis Transpl 22: 682-688.
  3. Gacche RN, Kaid AM (2012) Epidemiology of viral hepatitis B and C infections in Ibb city, Yemen. Hepat Mon 12: 460-462.
  4. Paroli M, Iannucci G, Accapezzato D (2012) Hepatitis C virus infection and autoimmune diseases. Int J Gen Med 5: 903-907.
  5. Gentilini P, La Villa G (2008) Liver-kidney pathophysiological interrelationships in liver diseases. Dig Liver Dis 40: 909-919.
  6. Cabiedes J, Nunez-Alvarez CA (2010) Antinuclear antibodies. Reumatol Clin 6: 224-230.
  7. Langford C (2012) Clinical features and diagnosis of small-vessel vasculitis. Cleve Clin J Med 79 3: S3-S7.
  8. Weidner S, Geuss S, Hafezi-Rachti S, Wonka A, Rupprecht HD (2004) ANCA-associated vasculitis with renal involvement: an outcome analysis. Nephrol Dial Transplant 19: 1403-1411.
  9. Restrepo VCA, Aguirre AJV (2016) Vitamin D (25(OH)D) in patients with chronic kidney disease stages 2–5. Colom Med 47: 160–166.
  10. Bansal B, Bansal S, Mithal A, Kher V, Marwaha R (2012) Vitamin D deficiency in hemodialysis patients. Indian J Endocr Metab 16: 270-273.
  11. Esfandiari A, Gargari BP, Noshad H, Sarbakhsh P, Mobasseri M, et al. (2019) The effects of vitamin D3 supplementation on some metabolic and inflammatory markers in diabetic nephropathy patients with marginal status of vitamin D: A randomized doubleblind placebo controlled clinical trial. Diabetes Metab Syndr 13: 278-283.
  12. Caprio M, Infante M, Calanchini M, Mammi C, Fabbri A (2017) Vitamin D: not just the bone. Evidence for benecial pleiotropic extraskeletal effects. Eat Weight Disord 22: 27-41.
  13. Sesso RC, Lopes AA, Thome FS, Lugon JR, Martins CT (2017) Brazilian Chronic Dialysis Survey 2016. J Bras Nefrol 39: 261-266.
  14. Sesso R, Rodrigues-Neto JF, Ferraz MB (2003) Impacto of socioeconomic status on the quality of life of ESRD patients. Am J Kidney 41: 186-195.
  15. Pasqual B, Gonçalves DA (2014) Transfusão. J Bras Nefrol 36: 29-31.
  16. Ferreira BM, Paula Júnior MR (2015) Determinação da frequência de anticorpos irregulares pós-transfusionais. Univ Ci Saúde 13: 79-86.
  17. Mendonça AEO, Dantas JG, Andrade AD, Segato CT, Torres GV (2015) Perfil sociodemográfico e clinico de idosos submetidos à hemodiálise. Cogitare Enfermagem 2015: 20.
  18. Peres LAB, Bettin TE (2015) Dislipidemia em pacientes com doença renal crônica. Rev Soc Bras Clin Med 13: 10-13.
  19. Heloisa VR, Bouissou Morais SCM, Marcelo ST, Martins TM, Cristina Simoes SA (2011) Inflamação na doença renal crônica: papel de citocinas. J Bras Nefrol 33: 351-364.
  20. Posicionamento Oficial da Sociedade Brasileira de Patologia Clínica/Medicina Laboratorial e da Sociedade Brasileira de Endocrinologia e Metabologia 2018.
  21. Snijder MB, Vandam RM, Visser M, Deeg DJ, Dekker JM, et al. (2005) Adiposity in relation to vitamin D status and parathyroid hormone levels: a population-based study in older men and women. The Journal of Clinical Endocrinology and Metabolism, Springfield 90: 4119-4123.
  22. Arnson Y, Amital H, Shoenfeld Y (2007) Vitamin D and autoimmunity: new aetiological and therapeutic considerations. Ann Rheum Dis 66: 1137-1142.
  23. Marques CD, Dantas AT, Fragoso TS, Duarte AL (2010) The importance of vitamin D levels in autoimmune diseases. Rev Bras Reumatol 50: 67-80.
  24. Ercolini AM, Miller SD (2009) The role of infections in autoimmune disease. Clin Exp Immunol 155: 1-15.
  25. Paroli M, Iannucci G, Accapezzato D (2012) Hepatitis C virus infection and autoimmune diseases. Int J Gen Med 5: 903-907.
  26. Savige J, Dimech W, Fritzler M, Goeken J, Hagen EC, et al. (2003) Addendun to the International Consensus Statment on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies. Am J Clin Pathol 120: 312-318.
  27. Dellavance A, Andrade LEC (2007) Como interpretar e valorizar adequadamente o teste de anticorpo antinucleo. J bras patol med lab 43: 157-168.

Editorial Information

Editor-in-Chief

Yohei Miyamoto

Article Type

Research Article

Publication history

Received: March 05, 2020
Accepted: March 25, 2020
Published: March 28, 2020

Copyright

©2020 Nascimento D. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation

Nascimento D (2020) Prevalence of positive ANA, ANCA antibodies and 25 (OH) vitamin D levels in patients in hemodialysis. Nephrol Renal Dis 5: DOI: 10.15761/NRD.1000160.

Corresponding author

Deisy S. Fernandes Nascimento

Department of clinical immunology, professor of pharmacy course at Universidade do Sul de Santa Catarina, Brazil.

E-mail : bhuvaneswari.bibleraaj@uhsm.nhs.uk

Table 1. Sociodemographic Characteristics

Sociodemographic Characteristics

N

%

Gender

Female

25

36.8

Male

43

63.2

Race

White

57

83.8

Black

11

16.2

Age (years)

21-40

8

11.9

41-50

15

22.0

51-70

36

52.9

71-80

7

10.3

>80

2

2.9

Age (Mean ± standard deviation in years)

Mean=55.94

SD=14.79

Scholarity (completed)

0-4

23

33.8

5-8

27

39.7

9-11

14

20.6

12-16

Scholarity (mean ± standard deviation in years)

4

mean=6.81

5.9

SD=3.84

Table 2. Clinical characteristics of the patients

Clinical characteristics

N

%

Comorbidity

Arterial hypertension (HAS)

48

70.6

Diabetes Mellitus (DM)

31

45.6

Urinary infection of repetition

9

13.2

Kidney stone

6

8.8

Dyslipidemia

28

41.2

Hepatitis C

3

4.4

Hepatitis B

0

0

Transfusion

Never received

36

52.9

Received until 3 times

28

41.1

Received from 4 to 6 times

1

1.5

Received from 7 to 10 times

1

1.5

Received more than 10 times

2

3.0

Hemodyalisis’ time (in months)

1 – 24 months

43

63.2

25-50 months

10

14.7

51-80 months

7

10.3

81-100 months

2

3.0

101-180 months

6

8.8

Mean and standard deviation (SD) Primary causes of kidney injury 

mean=35.96

SD=38.92

Routine tests or doesn´t know the fact that related to the diagnostic of the kidney injury

27

39.7

Symptoms (pain, edema, weakness)

12

17.6

Hypertension

9

13.2

Infection of urinary tract

2

2.9

Diabetes

7

10.3

Vascular accident (angina, heart attack, stroke)

4

5.8

Hereditary / congenital

2

2.9

Kidney stone

2

2.9

Drug toxicity

3

4.4

Creatinine (pre-dialysis)

mean=9.93

SD=2.64

Urea

mean=156.44

SD=37.12

Platelets

mean=191313.24

SD=22.44

Table 3. Prevalence of positivity of ANA, ANCA, and levels of 25 (OH) vitamin D

Variable

N

%

ANA

positive

3

4.4

negative

65

95.6

ANCA

negative

51

75.0

mean cytoplasmic (c-ANCA)

6

8.8

Mean perinuclear (p-ANCA)

11

16.2

25 (OH) Vitamin D (25 OH cholecalciferol)

0-10

0

0

11-20

6

8.8

21-30

18

26.5

>30

44

64.7

25 (OH) Vitamin D (mean and standard deviation)

mean=38.87

SD=13.07

Table 4. Comparison of 25 (OH) vitamin D levels among the variable sociodemographic, clinical and laboratorial tests

Variable

Mean

Standard deviation

p-value

Gender

0.004*

Male

43.14

16.61

Female

31.54

12.50

Race

0.410*

White

35.17

12.18

Non white

39.59

16.80

Diabetes mellitus

Yes

36.76

18.44

0.016*

No 

43.13

12.67

Arterial hypertension

0.439*

Yes

37.89

17.37

No

41.25

12.83

Corticoids use

0.611*

Yes

33.10

18.10

No

39.05

16.21

ANA

0.875*

Positive

38.81

16.37

Negative

40.33

12.29

ANCA

0.231*

Negative

40.08

16.39

Positive

35.27

13.28

*p<0.05 – Chi square test