Association between Serum α-Klotho Levels and Behçet Disease

Abstract

Background

Endothelial dysfunction (ED) has a well-known role in promoting vascular inflammation in Behçet disease (BD). α-klotho is involved in regulation of endothelial function, and its reduction has been reported to be associated with ED. 

Objective

To assess serum α-klotho in patients with BD, compared with healthy control individuals.

Methods

In a cross-sectional study, 55 patients with BD and 30 age- and sex-matched healthy controls were enrolled, and their serum levels of α-klotho were measured.

Results

Common clinical symptoms in patients with BD were oral aphthous ulcers, uveitis, and genital ulcers. Median (IQR) serum α-klotho levels in the BD and control groups were 0.30 (0.20–0.70) and 1.00 (0.70–2.52) ng/mL, respectively. The difference was statistically significant (P = .005). No significant correlation was observed between serum α-klotho and age (r = 0.194; P = .14). Serum α-klotho levels in patients with uveitis were significantly lower.

Conclusion

α-klotho may have a role in the pathogenesis of ED and is a potential biomarker for uveitis in BD.

Behçet disease (BD) is a type of systemic vasculitis that mainly affects young adults. Recurrent oral aphthous and genital ulcers, skin lesions, and uveitis—as well as involvement of the central nervous system, musculoskeletal and vascular systems, and gastrointestinal tract—are common manifestations.¹ The etiology and pathogenesis of BD are unknown. However, the main pathological manifestation is infiltration of inflammatory cells in the perivascular area and the walls of the capillaries, veins, and arteries of various sizes.² BD is an inflammatory disease with autoimmune and autoinflammatory features that is caused by the activation of the immune system after exposure to environmental factors in genetically predisposed individuals.^3–5 Endothelial dysfunction (ED) is a hallmark of BD.^6,7 Endothelial activation has a well-known role in promotion of vascular inflammation and thrombosis in BD.^6,7

The α-klotho protein is an essential component of endocrine fibroblast growth factor (FGF) receptor complexes and exists in transmembrane and soluble forms.⁸ α-klotho primarily is known as an antiaging agent.⁸ Also, α-klotho is involved in the regulation of endothelial function; its reduction has been reported to be associated with ED⁹ and may have a role in the regulation of immune response.⁸ Several study reports^10–14 have presented the association between α-klotho and various rheumatic diseases, including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and systemic sclerosis (SSc).

The growing evidence regarding α-klotho dysregulation in patients with autoimmune diseases and ED has led to the hypothesis that these peptides can be used as a biomarker in BD. To our knowledge, there has been no investigation regarding the role of α-klotho in BD and its correlation with clinical manifestations and activity of disease. So, this work of research aimed to assess the serum α-klotho levels in patients with BD, compared with those levels in healthy subjects.

Methods

Subjects

In a cross-sectional study, 55 patients diagnosed with BD and 30 age- and sex-matched healthy controls were enrolled. Inclusion criteria were diagnosis of BD according to the International Criteria for Behçet’s Disease (ICBD)¹⁵ and age ≥16 years. Patients with BD were selected consecutively from the outpatient BD clinic of the Connective Tissue Diseases Research Center of Tabriz University of Medical Sciences, Iran, between February 2021 and December 2021. The exclusion criteria were impaired renal function, liver disease, diabetes mellitus, thyroid and/or 
parathyroid disorders, any other autoimmune diseases or overlap syndromes, malignant neoplasms, pregnancy and breastfeeding, ever smoking, and/or currently taking antioxidant supplements. The research protocol was approved by the ethics committee of Tabriz University of Medical Sciences, Tabriz, Iran; informed written consent was gained from all subjects before inclusion in the study. During the study, all personal information was kept confidential, and accordingly, we performed other steps to act according to ethical and humanitarian considerations.

Clinical and Biochemical Measurements

All participants were examined in a multidisciplinary clinic by a rheumatologist and, if necessary, by an ophthalmologist and other specialists. Data related to the demographic characteristics of participants and clinical manifestations of patients with BD were recorded. We collected 5 mL of venous blood specimens after 12-hour overnight fasting. The serum specimens were isolated from whole blood and were kept at −70°C until biochemical measurements were performed. Serum levels of α-klotho were measured using a human α-klotho ELISA kit in an ELISA plate reader (Model Stat Fax 2100, Awareness Technologies). The specimens collected from the studied groups were tested by A. Ghorbanihaghjo simultaneously, using the same reagent lot number.

Statistical Analysis

Statistical analysis was performed using SPSS software, version 16.0 (IBM). Normality of variables distribution was evaluated using the Kolmogorov-Smirnov test. Categorical and normally distributed quantitative variables were displayed as No. (%) and mean (SD), respectively. Variables not normally distributed were presented as median (IQR). Between-groups comparisons were made via χ ² testing, independent sample t-testing, and Mann-Whitney U testing, as appropriate. Correlations between variables were analyzed using the Pearson correlation coefficient; P < .05 was considered statistically significant.

Results

The study included 55 patients with BD and 30 healthy controls. Demographic and clinical characteristics of participants are presented in Table 1. There was no significant difference in age and sex between patients with BD and controls. The most common clinical symptoms in patients with BD were oral aphthous ulcers, uveitis, and genital ulcers.

We compared serum α-klotho levels in the BD and control groups (Figure 1). Median (IQR) serum α-klotho levels in BD and control groups were 0.30 (0.20–0.70) and 1.00 (0.70–2.52) ng/mL, respectively. The difference was significant (P = .005).

We assessed the correlation between serum α-klotho levels and age: no statistically significant correlation was observed (r = 0.194, P = .14) (Figure 2). Also, we assessed the association between serum α-klotho levels and categorical variables, including sex and the involvement of various organs, in patients with BD (Table 2). Serum α-klotho in patients with uveitis was significantly lower (Table 2). There was no significant correlation between serum α-klotho level and involvement of other organs (Table 2).

Discussion

ED is characterized by decreased vasodilation and predisposes patients to prothrombotic and proinflammatory states.¹⁶ Several study reports mentioned ED in various types of vasculitis, including BD.^14–16 ED is a well-known feature of BD.^17–21 Various factors, including oxidative stress and hyperhomocysteinemia, are reported to play a role in ED when it manifests in the context of BD.^17,22,23

The klotho proteins originally were known as antiaging proteins. However, it has been characterized that these proteins have an important role in protecting vasculature. Possible mechanisms include increased expression of superoxide dismutase 2 (SOD2) that facilitates the catalysis of superoxide generated by mitochondrial respiration into hydrogen peroxide²⁴; upregulating endothelial nitric oxide synthase, the enzyme that controls nitric oxide production in endothelial cells⁵; and activation of the PI3K/Akt pathway, which leads to increased expression of Nrf2 (a transcription factor that enhances the expression of genes encoding antioxidant defense proteins under oxidative conditions)²⁵; and inhibiting apoptosis of endothelial cells.²⁶ In addition to its 
protective role against oxidative stress, klotho reduces the expression of intracellular adhesion molecule–1 (ICAM-1) and vascular cell adhesion molecule–1 (VCAM-1) on endothelial cells.²⁷ Klotho also decreases the activation of NF-κB and suppresses the expression of TNF-α, IL-6, and IL-8.^27,28 Ushigusa et al¹² reported an association between reduction of α-klotho protein with ED and neuronal damage in patients with SLE.

In addition to the role of klotho proteins in the regulation of endothelial-cell function, these proteins may also play a role in modulating immune-system function.⁸ Data on the relationship between klotho proteins and the immune system are limited to animal models, in the literature.⁸ Odaka et al²⁹ reported underdevelopment of the immune system and impairment of B-cell differentiation in klotho-knockout mice. Liu et al²⁸ reported an anti-inflammatory property of α-klotho. It has been shown³⁰ that α-klotho suppresses TNF-α–induced proinflammatory cytokine production, such as monocyte chemoattractant protein–1, IL-6, and IL-8, in the kidneys in a mouse model of diabetes.³⁰

The present study results show downregulation of α-klotho in patients with BD. Also, we found lower serum α-klotho levels in patients with BD who have uveitis. Downregulation of α-klotho was reported in RA, SLE, and SSc.⁸ According to Witkowski et al,¹⁰ α-klotho was downregulated at the mRNA and protein levels in CD4⁺ lymphocytes from subjects with RA. In a cohort of patients with RA, higher plasma concentrations of α-klotho were associated with higher levels of autoantibodies: rheumatoid factor (RF), anticitrullinated peptide antibodies, and disease activity score of −28.¹¹ In another study report, Ushigusa et al¹² demonstrated that a lower level of soluble α-klotho in cerebrospinal fluid was an important factor for predicting neuropsychiatric SLE. In studies carried out by Talotta et al¹³ and Hajialilo et al,¹⁴ serum concentration of α-klotho was found to be lower in patients with SSc than in healthy controls. However, it was neither related to the severity of the disease nor to organ involvement.^13,14

To our knowledge, this is the first study in the literature on the serum level of α-klotho in BD. The limitations of the present study were the relatively small sample size and the cross-sectional design of the study. In conclusion, our findings showed that α-klotho could have a role in the pathogenesis of ED in BD—namely, that α-klotho may be a biomarker for uveitis in BD.

Abbreviations

Abbreviations
 
• BD

  Behçet disease

 
• ED

  endothelial dysfunction

 
• FGF

  fibroblast growth factor

 
• RA

  rheumatoid arthritis

 
• SLE

  systemic lupus erythematosus

 
• SSc

  systemic sclerosis

 
• ICBD

  International Criteria for Behçet’s Disease

 
• SOD2

  superoxide dismutase 2

 
• ICAM-1

  intracellular adhesion molecule–1

 
• VCAM-1

  vascular cell adhesion molecule–1

 
• RF

  rheumatoid factor

Acknowledgments

We thank L. Khabbazi who helped us in editing of the text.

Funding

This research was funded by the Connective Tissue Diseases Research Center of Tabriz University of Medical Sciences (grant No. 63671).

Conflict of Interest Disclosure

The authors have nothing to disclose.

References

Author notes