Ingrid Lajdova

Effects of Long-Term Cholecalciferol Supplementation on Mineral Metabolism and Calciotropic Hormones in Chronic Kidney Disease

Background: Data on the efficacy and safety of long-term vitamin D supplementation in chronic kidney disease (CKD) are scarce. We assessed the effects of the 12-month vitamin D3 treatment on mineral metabolism and calciotropic hormones in patients with CKD stages 2–4. Methods: Eighty-seven patients (mean age 66 years, men/women 33/54) were randomized to cholecalciferol treatment with either 5,000 or 20,000 IU/week. Serum calcium, phosphate, 25(OH)D3, 1,25(OH)2D3, PTH and urinary mineral concentrations were obtained at baseline and after 4, 8 and 12 months. Results: The median serum mineral concentrations were normal and not changed throughout the study. The number of hypercalciuric patients slightly increased with higher dose, but no sustained rise in calciuria was present. Vitamin D insufficiency/deficiency was revealed in 72 (83%) patients at baseline and 37 (43%) at month 12. The 25(OH)D3 levels increased more with higher dose; a rise in 1,25(OH)2D3 was less impressive. The parathyroid hormone (PTH) concentrations were reduced, but the number of subjects with PTH below the lower limit for CKD stage 3 increased equally with both doses. Conclusions: Vitamin D insufficiency/deficiency in CKD significantly improved after the 12-month cholecalciferol treatment, with higher dose being more effective and equally safe. Further studies of vitamin D3 effects on bone metabolism are warranted.

Intracellular calcium homeostasis in patients with early stagesof chronic kidney disease: effects of vitamin D3 supplementation

BACKGROUND Chronic renal failure has been referred to as a state of cellular calcium toxicity. The aim of this study was to investigate the status of free cytosolic calcium ([Ca(2+)](i)), intracellular calcium reserves and the capacitative calcium entry in peripheral blood mononuclear cells (PBMCs) of early-stage chronic kidney disease (CKD) patients, and to determine the effect of vitamin D(3) supplementation on these parameters. METHODS The study involved 44 patients with CKD stages 2-3; 27 of them were treated with cholecalciferol (5000 IU/week) for 12 months. [Ca(2+)](i) was measured using Fluo-3 AM fluorimetry. Intracellular calcium reserves were emptied by the application of thapsigargin (Tg), a specific inhibitor of endoplasmic reticulum Ca(2+)-ATPase. 2-Aminoethyl-diphenyl borate (2APB) was used to examine the capacitative calcium entry. RESULTS [Ca(2+)](i) of CKD patients was substantially higher in comparison with healthy subjects: 123 (115-127) versus 102 (98-103) nmol/l, P < 0.001. The calcium concentration of Tg-sensitive stores and the capacitative calcium entry were also significantly increased in CKD patients. After the 12-month vitamin D(3) supplementation, there was a marked decrease in [Ca(2+)](i) [105 (103-112) nmol/l, P < 0.001 versus baseline], independently of the increase in 25(OH)D(3) or the decrease in PTH levels. No significant changes in intracellular calcium reserves and the capacitative calcium entry were found. CONCLUSIONS Our results demonstrate that (1) [Ca(2+)](i), intracellular calcium stores and the capacitative calcium entry were significantly increased already in early stages of CKD; (2) long-term vitamin D(3) supplementation normalized [Ca(2+)](i) without any effect on intracellular calcium reserves or the capacitative calcium entry.

Purinergic P2X7 receptors participate in disturbed intracellular calcium homeostasis in peripheral blood mononuclear cells of patients with chronic kidney disease.

Background: P2X₇ receptors intervene with lymphocyte activation and are responsible for multiple processes, including calcium influx. Here, we studied the participation of P2X₇ receptors in disturbed intracellular calcium homeostasis regulation in early-stage chronic kidney disease (CKD). Methods: The study involved 20 healthy volunteers and 20 CKD stage 2–3 patients. The free cytosolic calcium concentration ([Ca²⁺]_i) was measured using fluorimetry. The P2X₇ pore function was evaluated by the fluorescent dye ethidium bromide. Results: In peripheral blood mononuclear cells (PBMCs) of patients, [Ca²⁺]_i, intracellular calcium stores and the capacitative calcium entry were increased when compared with healthy subjects. The agonist of P2X₇ receptor BzATP caused a sustained increase in [Ca²⁺]_i in both groups, but the effect was smaller in patients. The antagonist at the P2X₇ receptor KN-62 reduced [Ca²⁺]_i in patients, but had no effect in healthy subjects. In patients, the permeability of ethidium bromide through P2X₇ pores, as well as through BzATP-activated and KN-62-inhibited pores, was distinct from permeability in healthy volunteers. Conclusions: These results demonstrate that the calcium signaling pathway in PBMCs of CKD patients is defective already in CKD stage 2–3, and the pore-forming P2X₇ receptors are involved in these pathophysiological processes.

Effects of chronic kidney disease on blood cells membrane properties

Chronic kidney disease (CKD) is progressive loss of renal function associated among others with increased intracellular calcium concentration. The purpose of this study was to identify the effects of CKD on cell membrane properties such as human red blood cell Ca(2+) ATPase activity, lymphocyte plasma membrane P2X(7) receptor expression and function. This could help us in elucidating the origin of increased calcium concentration in blood cells. We found out Ca(2+) ATPase activity is decreased in early stage CKD patients resulting in altered calcium removal from cytoplasm. By means of flow cytometry we assessed that P2X(7) receptor expression on lymphocyte membrane is 1.5 fold increased for CKD patients. Moreover, we detected an increased uptake of ethidium bromide through this receptor in CKD at basal conditions. It means CKD lymphocyte membranes contain more receptors which are more permeable thus allowing increased calcium influx from extracellular milieu. Finally, we can state alterations in blood cell membranes are closely linked to CKD and may be responsible for intracellular calcium accumulation.

Calcium Transport across Plasma Membrane in Early Stages of Chronic Kidney Disease - Impact of Vitamin D3 Supplementation

Mini review summarizes the results of studies focused on elucidating pathophysiological mechanisms of altered intracellular calcium homeostasis in the peripheral blood mononuclear cells of patients with early stages of chronic kidney disease. The basic mechanisms of calcium entry as well as those of its removal are impaired by the disease. These disturbances cause an increase concentration of free cytosolic calcium which can result in a change of broad range of cellular processes and expression patterns of various signaling molecules. Vitamin D3 supplementation is the standard treatment of frequent vitamin D3 insufficiency or deficiency in these patients. It can be assumed that vitamin D3 through the pleiotropic effects may participate in modulation of intracellular calcium homeostasis. Vitamin D3 supplementation resulted in a reduction of cytosolic free calcium affecting some of the transport systems involved in cell calcium entry as well as calcium exit. Normalization of the cytosolic free calcium concentration can have a beneficial effect on intracellular signaling. The mechanisms for regulating and controlling intracellular calcium homeostasis in chronic kidney disease patients are currently still under investigation.

The Impact of Vitamin D3 Supplementation on Mechanisms of Cell Calcium Signaling in Chronic Kidney Disease.

Intracellular calcium concentration in peripheral blood mononuclear cells (PBMCs) of patients with chronic kidney disease (CKD) is significantly increased, and the regulatory mechanisms maintaining cellular calcium homeostasis are impaired. The purpose of this study was to examine the effect of vitamin D3 on predominant regulatory mechanisms of cell calcium homeostasis. The study involved 16 CKD stages 2-3 patients with vitamin D deficiency treated with cholecalciferol 7000–14000 IU/week for 6 months. The regulatory mechanisms of calcium signaling were studied in PBMCs and red blood cells. After vitamin D3 supplementation, serum concentration of 25(OH)D3 increased (P < 0.001) and [Ca2+]i decreased (P < 0.001). The differences in [Ca2+]i were inversely related to differences in 25(OH)D3 concentration (P < 0.01). Vitamin D3 supplementation decreased the calcium entry through calcium release activated calcium (CRAC) channels and purinergic P2X7 channels. The function of P2X7 receptors was changed in comparison with their baseline status, and the expression of these receptors was reduced. There was no effect of vitamin D3 on P2X7 pores and activity of plasma membrane Ca2+-ATPases. Vitamin D3 supplementation had a beneficial effect on [Ca2+]i decreasing calcium entry via CRAC and P2X7 channels and reducing P2X7 receptors expression.

Time-resolved fluorescence monitoring of cholesterol in peripheral blood mononuclear cells

Precise evaluation of intracellular cholesterol distribution is crucial for improving diagnostics of diseased states associated with cholesterol alteration. Time-resolved fluorescence techniques are tested for non-invasive investigation of cholesterol in living cells. Fluorescent probe NBD attached to cholesterol was employed to evaluate cholesterol distribution in peripheral blood mononuclear cells (PBMC) isolated from the human blood. Fluorescence Lifetime Imaging Microscopy (FLIM) was successfully applied to simultaneously monitor the spatial distribution and the timeresolved characteristics of the NBD-cholesterol fluorescence in PBMC. Gathered data are the first step in the development of a new perspective non-invasive diagnostic method for evaluation of cholesterol modifications in diseases associated with disorders of lipid metabolism.

Enalapril Inhibits Growth and Proliferation of Various Tissues in Rat Normotensive Four–Sixths Kidney Ablation Nephropathy

Most experimental studies on kidney proliferation and its attenuation by angiotensin–converting enzyme inhibitors were performed in the rat hypertensive remnant–kidney model with a five–sixths kidney ablation. The developing hypertension rose the objections on the hypertension and its treatment in control rats. A normotensive four–sixths remnant–kidney model (Nx) was elaborated, compared with sham–operated (S) animals, and a subantihypertensive dosage of enalapril (E) was administered for 4 weeks of intensive kidney tissue proliferation (NxE). The pair–fed groups increased their body weight and blood pressure comparably. Moderately increased plasma creatinine and urea concentrations were found in the Nx group; markedly increased levels in the NxE group. Nx increased proteinuria, and E attenuated its increase. The remnant–kidney weight (Nx 912±31 vs. S 1,111±36 mg, p<0.001) was still lower, but collagen (Col; Nx 164±2 vs. S 148±5 mg/100 g, p<0.05) and tubular protein/DNA ratio (Nx 26.2±10.8 vs. S 9.8±1.0, p<0.05) increased markedly in the Nx group; E attenuated the kidney growth (NxE 719±31 vs. Nx 912±31 mg, p<0.01) and decreased the tubular protein/DNA ratio remarkably (NxE 15.3±10.5 vs. Nx 26.2 ±10.8), but E did not inhibit the Col accumulation. Nx decreased the heart (Nx 1,002±28 vs. S 1,130±41 mg, p<0.05), but not liver weights and did not influence Col concentrations or protein/DNA ratios either in heart or liver. E potentiated the weight decrease of heart (NxE 862±20 vs. Nx 1,002±28 mg, p<0.01) and liver (NxE 8.3±0.44 vs. Nx 10.3±0.51 g, p<0.001) and Col accumulation (heart: NxE 113±6 vs. Nx 92±5 mg/100 g, p<0.01; liver: NxE 134±8 vs. Nx 101±9 mg/100 g, p<0.01). Nx did not influence either the soleus muscle weight or its Col accumulation, but it increased its protein/DNA ratio (Nx 66.3±4.7 vs. S 35.5±2.8 mg/100 g, p<0.01). E increased the Col concentration in muscle (NxE 141±3 vs. Nx 110±5 mg/100 g, p<0.01), while it attenuated the increase in protein/DNA ratio (NxE 36.6±2.1 vs. Nx 66.3±4.7, p<0.01). In conclusion, kidney ablation nephropathy stimulating kidney proliferation evokes only minor changes in heart, liver and striated muscle. E inhibits markedly the kidney proliferation and functional recovery, but does not prevent the Col accumulation. E evokes antiproliferative changes also in the heart and surprisingly even in the liver. Alterations in soleus muscle are only borderline.

Flavin fluorescence lifetime imaging of living peripheral blood mononuclear cells on micro and nano-structured surfaces

Fabricated micro- and nano-structured surfaces were evaluated for use with living cells. Metabolic state was tested by means of endogenous flavin fluorescence of living peripheral blood mononuclear cells (PBMC) positioned on a coverslip, non-covered, or covered with micro- or nano-structured surfaces (OrmoComp polymer structures produced by 2-photon photopolymerisation, or Zinc Oxide (ZnO) layer fabricated by pulsed laser deposition). Confocal microscopy and Fluorescence Lifetime Imaging Microscopy (FLIM) were employed to gather flavin fluorescence lifetime images of living PBMC on structured surfaces. Gathered data are the first step towards monitoring of the live cell interaction with different micro/nano-structured surfaces and thus evaluate their potential applicability in the biomedical field.