Serum Levels of α-Klotho, Inflammation-Related Cytokines, and Mortality in Hemodialysis Patients.

It has been hypothesized that α-Klotho deficiency might contribute to chronic inflammation in patients with end-stage renal disease (ESRD), especially those on hemodialysis (HD). Serum Klotho levels by some authors are considered a potential predictor of cerebrovascular events. Therefore, we analyzed serum levels of α-Klotho with ELISA and inflammation-related cytokines in HD patients. Sixty-seven HD patients and 19 healthy people were recruited between November 2017 and June 2021. A Cytometric Bead Array (CBA) was used to determine the level of different cytokines: IL-12p70, TNF, IL-10, IL-6, IL-1ß, and IL-8. A human Klotho ELISA kit was used to determine the level of α-Klotho in the plasma samples of HD patients. There was no difference in serum levels of α-Klotho between HD patients and healthy people. Patients had increased serum IL-6 and IL-8. Significant positive correlations existed between the concentration of α-Klotho and the serum concentrations of IL-12p70, IL-10, and IL-1ß. However, in a multivariable linear regression analysis, only patients' age was associated independently with α-Klotho level. Serum α-Klotho was not associated with higher mortality risk in HD patients. While these results draw attention to potential relationships between α-Klotho proteins and inflammatory markers in HD patients, our cross-sectional study could not confirm the pathogenic link between α-Klotho, inflammation, and cardiovascular mortality.

The in vitro modulatory effect of TNFα on the mRNA expression and protein levels of zinc finger protein ZNF334 in CD4(+) lymphocytes of healthy people.

We have shown before that the expression of ZNF334 gene, coding for a newly described zinc finger protein of as yet unknown function, is extremely reduced in CD4(+) lymphocytes of rheumatoid arthritis (RA) patients regardless of their age, and thus can be considered a new molecular marker of the disease. Based on the promoter sequence of the gene we speculated that it might be regulated by TNFα. Here we have tested that hypothesis, studying the in vitro influence of TNFα on the ZNF334 gene expression and protein levels in resting and stimulated CD4(+) cells of healthy volunteers. We have confirmed that treatment with TNFα modifies the levels of ZNF334 expression in the CD4(+) cells ex vivo; however, the effect varied for different individuals and reduction of expression was seen only for those cell samples that initially exhibited high transcriptional activity of the gene, while for those exhibiting initially very low expression, some increase in the transcriptional activity was observed. Incubation with TNFα significantly reduced the amounts of two isoforms of ZNF334 protein (initially high in all subjects) in parallel to the reduced transcription. Finally, the expression of ZNF334 in CD4(+) lymphocytes isolated after various periods of anti-CD3 stimulation generally increased with longer culture times, and the effect of TNFα treatment was negligible. Concluding, our results obtained in vitro for helper lymphocytes of healthy individuals seem to mimic the regulatory effect of TNFα on the expression of ZNF334 in the cells of RA patients.

Disease activity in patients with long-lasting rheumatoid arthritis is associated with changes in peripheral blood lymphocyte subpopulations.

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic autoimmune disease and it is known that lymphocytes play a major role in its pathogenesis. However, there have been no comprehensive studies on the changes in peripheral blood lymphocyte (PBL) subpopulations expressing different clusters of differentiation (CD) in patients with long-lasting RA. OBJECTIVES: The aim of our study was to measure the main subpopulations of PBL, expression of costimulatory marker CD28, and activation status of CD4+ T cells depending on clinical disease activity in long-lasting RA. PATIENTS AND METHODS: The study comprised 60 patients with RA and 19 healthy volunteers. Disease activity, the proportion and number of the main PBL subpopulations (T, B, natural killer [NK], and NK T cells [NKT]), the expression of costimulatory marker CD28, and the activation status of CD4+ T cells were evaluated on the same day. A multicolor flow cytometry with marked monoclonal antibodies was used for the assessment of lymphocyte subpopulations. RESULTS: The percentage of CD3+CD4+, NKT, CD4+CD28-, CD8+CD28-, CD4+CD69+, CD4+CD25+, and CD4+HLA-DR+ was significantly higher in RA compared with the control group. A higher proportion of CD4+CD28- was associated with more active disease, while an inverse correlation was observed for B cells. The proportion of CD4+CD28- was not associated with disease activity. The number of CD4+CD69+ cells in RA patients increased with increasing DAS28, while the number of CD4+HLA-DR+ T cells showed no such association. CONCLUSIONS: Our results have shown for the first time an association between the phenotype patterns of PBL T, B, and NKT and RA activity in patients with long-lasting disease, which reinforces the hypothesis that PBL play an important role in modifying or maintaining the disease activity.

Varying expression of four genes sharing a common regulatory sequence may differentiate rheumatoid arthritis from ageing effects on the CD4(+) lymphocytes.

The CD28 gene is similarly down-regulated in CD4(+) lymphocytes from both healthy elderly people and patients with rheumatoid arthritis (RA) because of impaired protein-binding activity of the 'α' sequence in its promoter region. Other genes important for the CD4(+) cell function may share that sequence and may be similarly regulated and affected. We searched GenBank for possible 'α' homologues and then compared transcriptional activities of the respective genes in the CD4(+) cells of young and older healthy individuals and those with RA by real-time PCR. We show here that genes encoding one of the zinc finger proteins (ZNF334), the 'aging hormone' Klotho, the retinoid acid receptor ß2 (RARß2) and the T-cell adapter protein GRAP-2, contain sequences with various (exceeding 70%) degrees of homology to the 'α' sequence near their promoters. These genes are transcribed in human CD4(+) lymphocytes; the expressions of RARß2, KLOTHO and ZNF334 are significantly decreased in a correlated manner in the cells of patients with RA compared with those of healthy individuals. In RA patients, the extremely reduced expression of ZNF334 does not depend on the individual's age, apparently constituting a disease-related phenomenon; whereas that of RARß2 and KLOTHO occurs mostly in the cells of relatively younger patients, making them similar to the lymphocytes of healthy elderly in this aspect.

Klotho--a common link in physiological and rheumatoid arthritis-related aging of human CD4+ lymphocytes.

Human CD4(+) T lymphocytes undergo aging-related changes leading to decreased immunity to infections and neoplasms, and to increased frequency of autoimmune diseases including rheumatoid arthritis (RA). Certain changes, observed in the CD4(+) cells of RA patients, resemble those observed during physiological aging, but occur at earlier age. Underlying cellular mechanism(s) of these similarities are so far largely unknown. Here we show that KLOTHO, a beta-glucuronidase gene whose activity changes are associated with aging phenotype, is down-regulated at the mRNA, protein, and enzymatic (beta-glucuronidase) activity levels both in the healthy elderly and especially in RA CD4(+) lymphocytes. Although the exact role of Klotho activity for CD4(+) cell function is unknown, we propose here that it might be involved in anti-inflammatory processes occurring in the young and healthy individuals, but reduced in both healthy elderly and RA patients. To support this hypothesis, we show here that the reduction of Klotho expression and activity in both elderly and patients' lymphocytes occurs in concert with the down-regulation of T cell costimulatory molecule CD28, the latter known to be dependent on increased levels of TNF-alpha. Thus, a common mechanism of KLOTHO down-regulation, but executed at various times in life, may underlie both physiological and disease-related T cell aging. Klotho activity might become a target of anti-RA drug development as well as a tool to help increase the immune system efficiency in the elderly.