MP015
THE EFFECT OF VITAMIN D₃ SUPPLEMENTATION ON P2X₇ RECEPTOR FUNCTION IN EARLY STAGES OF CHRONIC KIDNEY DISEASE Free

Introduction and Aims: Chronic low-grade inflammation is common in chronic kidney disease (CKD) patients. The P2X₇ receptor (P2X₇R) is increasingly recognized as an important cell surface regulator of several key inflammatory molecules. P2X₇R activation by extracellular ATP results in opening the cation channel followed by forming a non-specific pore. Channel opening induces Na⁺ and Ca²⁺ influx and K⁺ efflux leading to plasma membrane depolarization, increase of intracellular Ca²⁺ level and activation of Ca²⁺ signalling cascades. This results in a variety of biological responses, mainly related to inflammation, cell proliferation and tissue damage. The aim of the study was examine the effect of vitamin D₃ supplementation on P2X₇R function and expression in early stages of CKD.

Methods: The study involved 20 healthy volunteers and 16 non-diabetic patients with stage 2-3 CKD. CKD patients were supplemented with cholecalciferol 7000 - 14000 IU/week orally for 6 months. Cytosolic Ca²⁺ measurements were performed by Fluo-3 fluorimetry in isolated peripheral blood mononuclear cells (PBMCs). To determine the P2X₇R function, a highly selective antagonist (AZ11645373) and the most potent agonist (BzATP) were used. The function of P2X₇ pores was measured by ethidium uptake at basal conditions, and with BzATP stimulation or AZ11645373 inhibition. The expression of surface P2X₇Rs was evaluated by flow cytometry using the antibody (anti-P2X₇ extracellular).

Results: Free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) was increased in PBMCs of CKD patients when compared with healthy subjects (120 ± 1.6 v.s. 102 ± 1 nmol/l, P < 0.001). Vitamin D₃ supplementation reduced [Ca²⁺]_i to 106 ± 1.1 nmol/l (P < 0.001). The application of P2X₇R antagonist led to reduction in [Ca²⁺]_i from 120 ± 1.6 to 112 ± 2.2 nmol/l (P < 0.001), but had no effect after vitamin D₃ supplementation. Moreover, the effect of an antagonist on stimulated PBMCs was significantly decreased after vitamin D₃ supplementation (36.4 ± 7.1 v.s. 17.5 ± 2.9 nmol/l, P < 0.01). The permeability of P2X₇ pores in PBMCs of CKD patients was significantly increased in comparison with healthy volunteers. Vitamin D₃ supplementation did not change the permeability of P2X₇ pores after the application of either the agonist or antagonist. The expression of surface P2X₇Rs was 1.5-fold grater on PBMCs from CKD patients when compared to healthy donors. Vitamin D₃ supplementation reduced the P2X₇R expression by about 55%.

Conclusions: These results demonstrate the inhibitory effect of vitamin D₃ supplementation on pro-inflammatory P2X₇R channels and P2X₇Rs expression already in early stages of CKD. This might be one of the mechanisms of an immunomodulatory effect of vitamin D.