Essential Role Of Ca～(2+) Release Channels In Ca～(2+) Oscillations And Glomerular Mesangial Cell Contraction

Objective and background: The increased resistance of glomerulus as a result of contractive dysfunction of glomerular mesangial cells (GMCs) is, in part, associated with the reduction of gromerular filtration rate and the development of glomerulosclerosis. Evidences show GMCs contraction changes with intracellular Ca²⁺ concentration ([Ca²⁺]_i). Following with the new technology developing (such as confocal microscopy), Calium signaling research trends to intracellular micro-changing, such as calcium waves,calcium oscillations and calcium sparks. Calcium oscillations were found by the stimulation in GMCs. However, whether Ca²⁺ oscillations can trigger GMCs contraction and how it is regulated remain to be still unknown. So in this research, we will explore the mechanism of angiotensin II (Ang II )-induced Ca²⁺ oscillations and GMCs contraction. Methods: Primary GMCs of 3-month-old or 28-month-old rats were cultured to confluence in special glass-bottom microwell dishes, and then incubated with 2.5 uM fluo-3 plus 0.02% Pluronic F-127 in HBSS. GMCs were scanned for at least 30-40 mins after adding Ang II at different concentrations , then the following agents were added, including Ca²⁺ -free solution containing 5 mM EGTA, 2 uM TG, 20 uM 2-APB, 50 uM ET-18-OCH₃, or 100 μm ryanodine, for observing the changing of intracellular calcium oscillations. Mesangial cell planar area measurements with confocal microscopy and analysis of myosin light chainphosphorylation with western blot were for GMCs' contraction induced by Ang II. Confocal colocalization of InsP₃R and RyR subtypes in GMCs was detected using double immunofluorescence.Results: Ang II could induce representative Ca²⁺ oscillations and contraction of GMCs. Such a process could be completely inhibited by thapsigargin (TG), 2-APB, or ET-18-OCH₃, but be partly by ryanodine, and could not be inhibited in the absence of extracullular Ca²⁺. 1,4,5-trisphosphate (InsP₃) Receptors and ryanodine receptors displayed a strong colocalization contribution to the amplification of Ca²⁺ response. Myosin light chain phosphorylation and GMCs contraction were accompanied with Ca²⁺ oscillations except that global Ca²⁺ level changing. The frequency of Ca²⁺ oscillations was dependent on the Ang II concentration and correlated with the extent of GMCs' contraction, which could be attenuated by KN-93. The amplitude reduction of oscillations was correlated with the decrease of the contraction related to aging.Conclusions: [Ca²⁺]i response of GMCs to Ang II was characterized by repetitive spikes through the following cycles: Ca²⁺-releasing by PLC-InsP3 pathway, Ca²⁺ amplification by Ca²⁺-activated ryanodine receptors and re-uptake by the endoplasmic reticulum. Oscillations modulate GMCs contraction with a frequency-dependent mode for different Ang II concentration or an amplitude-dependent mode with aging.