Abstract
Connexin43 (Cx43), pannexin1 (Panx1) and P2X7 receptor (P2X7R) are expressed in kidneys and are known to constitute a feedforward mechanism leading to inflammation in other tissues. However, the possible functional relationship between these membrane channels and their role in damaged renal cells remain unknown. In the present work, we found that MES-13 cells, from a cell line derived from mesangial cells, stimulated with angiotensin II (AngII) developed oxidative stress (OS, thiobarbituric acid reactive species (TBARS) and generated pro-inflammatory cytokines (ELISA; IL-1β and TNF-α). The membrane permeability increased progressively several hours before the latter outcome, which was a response prevented by Losartan, indicating the involvement of AT1 receptors. Western blot analysis showed that the amount of phosphorylated MYPT (a substrate of RhoA/ROCK) and Cx43 increased progressively and in parallel in cells treated with AngII, a response followed by an increase in the amount in Panx1 and P2X7R. Greater membrane permeability was partially explained by opening of Cx43 hemichannels (Cx43 HCs) and Panx1 channels (Panx1 Chs), as well as P2X7Rs activation by extracellular ATP, which was presumably released via Cx HCs and Panx1 Chs. Additionally, inhibition of RhoA/ROCK blocked the progressive increase in membrane permeability, and the remaining response was explained by the other non-selective channels. The rise of activity in the RhoA/ROCK-dependent pathway, as well as in Cx HCs, P2X7R, and to a minor extent in Panx1 Chs led to higher amounts of TBARS and pro-inflammatory cytokines. We propose that AngII-induced mesangial cell damage could be effectively inhibited by concomitantly inhibiting the RhoA/ROCK-dependent pathway and one or more non-selective channel(s) activated through this pathway.
Highlights
In chronic kidney disease, hypertension is one of the most common complications that generate predispositions to other health problems, affecting several organs
Angiotensin II (AngII) binding to AT1 receptor activates RhoA and Rho kinase (ROCK) [3], and Cx43 HCs can mediate changes in membrane permeability in different cells types [12,30], we decided to evaluate the activity of RhoA/ROCK and Cx43 HCs
To elucidate whether Cx HCs, Panx1 Chs and P2X7 receptors (P2X7Rs) participate in AngII-induced RhoA/ROCK activation, we evaluated the effects of selective blockers for each channel type—Gap27, PBC and A740003, respectively—applied during the last 24 h on the amount of phosphorylated MYPT in MES-13 cells treated with AngII (10−7 M) for 72 h
Summary
Hypertension is one of the most common complications that generate predispositions to other health problems, affecting several organs. Hypertensive nephropathy begins in the glomerulus by increasing intraglomerular pressure. These early events activate and damage mesangial cells, epithelial cells and podocytes in the glomerulus. These cells produce vasoactive and pro-inflammatory agents, which increase cell damage and promote fibrosis, reducing renal blood flow, permeability and, eventually, glomerular filtration [1]. Angiotensin II (AngII), a vasoactive peptide found in the renal tissue, stimulates aldosterone secretion, cellular infiltration, proliferation and migration, thrombosis, reactive oxygen species (ROS) production, and contributes to the induction of inflammatory responses common in nephropathy [1]
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