Klotho Prevents NFκB Translocation and Protects Endothelial Cell From Senescence Induced by Uremia.

In patients with renal disease, uremia raises oxidative stress and senescence in endothelial cells, which can lead to endothelial dysfunction and cardiovascular disease. Klotho protein is a ß-glucuronidase capable of hydrolyzing steroid ß-glucuronides. This protein is recognized as an antiaging gene, that modulate both stress-induced senescence and functional response. The aim of the study was to investigate how senescence and oxidative stress induced by uremia in endothelial cells affects Klotho expression and whether intra or extracellular Klotho has effects on the response of these cells. Senescence and oxidative stress was obtained by exposure to uremic serum. Telomere length, the enzyme ß-galactosidase, and oxidative stress were studied by flow cytometry. Nuclear factor kappa B activity was determined by electrophoretic mobility shift assay. The expression of Klotho decreased with the uremia and preceded the manifestations of cell aging. Levels of intracellular Klotho decreases associated to endothelial senescence, and exogenous Klotho prevents cellular senescence by inhibiting the increase in oxidative stress induced by uremia and diminished the nuclear factor kappa B-DNA binding ability.

Endothelial microparticles mediate inflammation-induced vascular calcification.

Stimulation of endothelial cells (ECs) with TNF-α causes an increase in the expression of bone morphogenetic protein-2 (BMP-2) and the production of endothelial microparticles (EMPs). BMP-2 is known to produce osteogenic differentiation of vascular smooth muscle cells (VSMCs). It was found that EMPs from TNF-α-stimulated endothelial cells (HUVECs) contained a significant amount of BMP-2 and were able to enhance VSMC osteogenesis and calcification. Calcium content was greater in VSMCs exposed to EMPs from TNF-α-treated HUVECs than EMPs from nontreated HUVECs (3.56 ± 0.57 vs. 1.48 ± 0.56 µg/mg protein; P < 0.05). The increase in calcification was accompanied by up-regulation of Cbfa1 (osteogenic transcription factor) and down-regulation of SM22α (VSMC lineage marker). Inhibition of BMP-2 by small interfering RNA reduced the VSMC calcification induced by EMPs from TNF-α-treated HUVECs. Similar osteogenic capability was observed in EMPs from both patients with chronic kidney disease and senescent cells, which also presented a high level of BMP-2 expression. Labeling of EMPs with CellTracker shows that EMPs are phagocytized by VSMCs under all conditions (with or without high phosphate, control, and EMPs from TNF-α-treated HUVECs). Our data suggest that EC damage results in the release of EMPs with a high content of calcium and BMP-2 that are able to induce calcification and osteogenic differentiation of VSMCs.

Klotho modulates the stress response in human senescent endothelial cells.

Lack of Klotho expression in mice leads to premature aging and age-related diseases, including vascular diseases. The aim of this study was to determine how endothelial cell line senescence affects Klotho expression and whether intra- or extracellular Klotho has any effect on the response of senescent cells to oxidative stress. The study was performed using human endothelial cells (HUVEC); cell aging was obtained by prolongation of cell division to 42 population doublings (PD). Senescence was also obtained by exposure to TNFα, which causes cell changes resembling cellular senescence. The decline in Klotho preceded the manifestations of cell ageing: telomere shortening and ß-galactosidase expression. Klotho was also reduced in cells exposed to the proinflammatory cytokine TNFα. The addition of exogenous Klotho to aging cells did not modify the proportion of cells with short telomeres or any other feature of cell aging; however, exogenous Klotho prevented the changes resembling premature cellular senescence associated with TNFα, such as the decrease in telomere length and the increase in ß-galactosidase-positive cells. Likewise exogenous Klotho prevented the increases in reactive oxygen species (ROS) activity, mitochondrial potential and cell apoptosis induced by TNFα.

Bacterial DNA prolongs the survival of inflamed mononuclear cells in haemodialysis patients.

BACKGROUND: Chronic kidney disease (CKD) patients show evidence of chronic inflammation with mononuclear cell activation which is mainly caused by uraemia itself and is exacerbated by haemodialysis. Small fragments of bacterial DNA (DNAb) are ubiquitous contaminants, which are capable of passing through dialyser membranes causing the stimulation of cells of the immune system. The aim of this study was to evaluate whether DNAb contamination may have an effect on apoptosis of activated monocytes from CKD-5 patients. METHODS: To test the ability of DNAb to stimulate the inflammatory response, mononuclear cells from 10 chronic kidney disease patients who had not begun haemodialysis (ND-CKD-5) and 10 patients undergoing regular dialysis (HD) were cultured in the presence and absence of DNAb. Ten healthy subjects were used as controls. RESULTS: The percentage of IL-1beta cells was higher in HD patients than in ND-CKD-5 (33.9 +/- 3.0% vs 20.0 +/- 2.3%, P < 0.001) and controls (9.4 +/- 2.1%, P < 0.001). The presence of DNAb induced an increase in the percent of cells expressing IL-1beta in controls, ND-CKD5 and HD patients. In addition, the DNAb also increased the release of cytokines in all groups, the effect was more marked in ND-CKD5 and HD than in controls. DNAb only inhibited apoptosis of activated mononuclear cells from, ND-CKD (17.5 +/- 2.8% vs 12.3 +/- 2.6%, P < 0.01) and HD patients (27 +/- 2.5% vs 14.6 +/- 2.9%, P < 0.01). CONCLUSIONS: DNAb enhances cytokine production and promotes the survival of inflammatory mononuclear cells from CKD patients. These results strongly suggest that DNAb fragments play an important role in maintaining chronic inflammation in patients on haemodialysis.

Antibody-mediated pure red cell aplasia (PRCA): ensuring future progress by collecting data from a registry.

The recent emergence of antibody (Ab)-mediated pure red cell aplasia (PRCA) resulting from administration of certain erythropoiesis-stimulating agents (ESAs) has heightened awareness of the potential for this disorder in patients receiving ESAs for anaemia associated with chronic kidney disease (CKD). The Spanish Society of Nephrology sponsored an independent registry for analysis of patients who developed epoetin-induced, Ab-mediated PRCA in Spain. Twelve patients from 11 regional hospitals were included in the Spanish PRCA registry from November 2000 to December 2002 that met the criteria for Ab-mediated PRCA. Patients were reported using a standardized form specifically developed for PRCA, and serum samples were analysed in a central laboratory at Reina Sofia University Hospital in Cordoba, Spain. The characteristics of these patients, their serological and haematological results, and the outcomes of immunosuppressive therapies are presented. The Spanish PRCA registry serves as a model for establishing an independent global PRCA registry sponsored by the various nephrology societies in European Union countries, and elsewhere and coordinated by key investigators from the respective countries.

The imbalance in the ratio of Th1 and Th2 helper lymphocytes in uraemia is mediated by an increased apoptosis of Th1 subset.

BACKGROUND: In uraemia there is a reduction in the total number of T lymphocytes and an imbalance in the ratio of Th1/Th2 T-helper (Th) lymphocytes. A higher rate of apoptosis in T lymphocytes has been reported in haemodialysis patients. The aims of the present study were to assess the Th1/Th2 pattern in uraemia and to evaluate whether a relative increase in Th1 apoptosis may explain the Th1/Th2 imbalance observed in uraemic patients. METHODS: Seventeen non-dialysed uraemic patients were evaluated; eight healthy volunteers served as controls. Intracellular interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) were measured by direct intracellular immunofluorescence and flow cytometry. Apoptosis was determined by flow cytometry using annexin V or TUNEL. Mechanisms of apoptosis were assessed by determination of Fas and Bcl-2 expression. RESULTS: Cell production of cytokines is significantly higher in uraemic patients than in controls. In addition, in uraemic patients only 5.1+/-2.1% of the T lymphocytes contained IFN-gamma (Th1 cells) while 61.9 +/- 14.8% contained IL-4 (Th2 cells) (P < 0.0001). The percentage of apoptosis was 29.6 +/- 6.3% and 4.7 +/- 1.6% in Th1 and Th2 lymphocytes, respectively (P < 0.001). Fas expression was higher in Th1 than in Th2 cells and the expression of Bcl-2 was lower in Th1 than in Th2 cells. The apoptosis induced by anti-Fas antibodies was similar in both types of lymphocytes. CONCLUSIONS: In uraemia there is a reduction in the proportion of Th1 lymphocytes due to a higher rate of apoptosis in this subset of lymphocytes. Th1 from uraemic patients show a higher expression of Fas and a lower expression of Bcl-2 than Th2. This makes uraemic Th1 cells more susceptible to apoptosis. The Th1/Th2 imbalance may contribute to alterations in cellular immunity observed in chronic kidney disease patients.