Abstract
Mechanical forces are known to induce increases of [Ca2+]i in the aldosterone-sensitive distal nephron (ASDN) cells to regulate epithelial transport. At the same time, mechanical stress stimulates ATP release from ASDN cells. In this study, we combined ratiometric Fura-2 based monitoring of [Ca2+]i in freshly isolated split-opened ASDN with targeted deletion of P2Y2 and TRPV4 in mice to probe a role for purinergic signaling in mediating mechano-sensitive responses in ASDN cells. ATP application causes a reproducible transient Ca2+ peak followed by a sustained plateau. Individual cells of the cortical collecting duct (CCD) and the connecting tubule (CNT) respond to purinergic stimulation with comparative elevations of [Ca2+]i. Furthermore, ATP-induced Ca2+-responses are nearly identical in both principal (AQP2-positive) and intercalated (AQP2-negative) cells as was confirmed using immunohistochemistry in split-opened ASDN. UTP application produces elevations of [Ca2+]i similar to that observed with ATP suggesting a dominant role of P2Y2-like receptors in generation of [Ca2+]i response. Indeed, genetic deletion of P2Y2 receptors decreases the magnitude of ATP-induced and UTP-induced Ca2+ responses by more than 70% and 90%, respectively. Both intracellular and extracellular sources of Ca2+ appeared to contribute to the generation of ATP-induced Ca2+ response in ASDN cells. Importantly, flow- and hypotonic-induced Ca2+ elevations are markedly blunted in P2Y2 −/− mice. We further demonstrated that activation of mechano-sensitive TRPV4 channel plays a major role in the sustained [Ca2+]i elevation during purinergic stimulation. Consistent with this, ATP-induced Ca2+ plateau are dramatically attenuated in TRV4 −/− mice. Inhibition of TRPC channels with 10 µM BTP2 also decreased ATP-induced Ca2+ plateau whilst to a lower degree than that observed with TRPV4 inhibition/genetic deletion. We conclude that stimulation of purinergic signaling by mechanical stimuli leads to activation of TRPV4 and, to a lesser extent, TRPCs channels, and this is an important component of mechano-sensitive response of the ASDN.
Highlights
It is recognized that the distal part of renal nephron, which includes the connecting tubule (CNT) and the cortical collecting duct (CCD), is responsible for the final regulation of electrolyte (Na+, K+, Ca2+), water and acid-base balance
Purinergic stimulation caused a rapid (,20 sec rise time) Ca2+-spike followed by a sustained plateau with no obvious decline after 10 min of ATP application. [Ca2+]i returned to basal levels upon ATP removal
This study establishes a reciprocal link between mechanosensitivity and paracrine purinergic signaling in native aldosterone-sensitive distal nephron
Summary
It is recognized that the distal part of renal nephron ( termed the aldosterone-sensitive distal nephron, or ASDN), which includes the connecting tubule (CNT) and the cortical collecting duct (CCD), is responsible for the final regulation of electrolyte (Na+, K+, Ca2+), water and acid-base balance. Dynamic changes in renal tubular flow and fluid composition can be sensed by the cells of the ASDN [4,5]. It is proposed that the ASDN cells respond to these environmental changes by increasing [Ca2+]i [5]. An important physiological role of the primary cilium in flow-mediated cellular responses has been recently proposed [4]. Mutations in both PKD1 and PKD2 genes result in functional defects of the primary cilium accounting for all cases of autosomal dominant polycystic kidney disease [4]. Intercalated cells (IC), which are devoid of primary cilium, respond to flow changes with comparable increases in [Ca2+]i as principal cells (PC) [8]
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