Intrarenal localization of the plasma membrane ATP channel pannexin1

Abstract

In the renal tubules, ATP released from epithelial cells stimulates purinergic receptors, regulating salt and water reabsorption. However, the mechanisms by which ATP is released into the tubular lumen are multifaceted. Pannexin1 (Panx1) is a newly identified. ubiquitously expressed protein that forms connexin-like channels in the plasma membrane, which have been demonstrated to function as a mechanosensitive ATP conduit. Here, we report on the localization of Panx1 in the mouse kidney. Using immunofluorescence, strong Panx1 expression was observed in renal tubules, including proximal tubules, thin descending limbs, and collecting ducts, along their apical cell membranes. In the renal vasculature, Panx1 expression was localized to vascular smooth muscle cells in renal arteries, including the afferent and efferent arterioles. Additionally, we tested whether Panx1 channels expressed in renal epithelial cells facilitate luminal ATP release by measuring the ATP content of urine samples freshly collected from wild-type and Panx1 Ϫ/Ϫ mice. Urinary ATP levels were reduced by 30% in Panx1 Ϫ/Ϫ compared with wild-type mice. These results suggest that Panx1 channels in the kidney may regulate ATP release and via purinergic signaling may participate in the control of renal epithelial fluid and electrolyte transport and vascular functions. ATP release; channel; immunofluorescence; kidney PURINERGIC SIGNALING IS INVOLVED in several major regulatory mechanisms in the kidney, including tubuloglomerular feedback, control of renin release, and tubular fluid and electrolyte transport (17). ATP concentrations consistent with P2 receptor activation have been found in the tubular lumen (40). Additionally, the identity of several P2X and P2Y receptors along the renal epithelium, as well as their effect on ion transport, has already been determined (17). However, the mechanisms by which ATP, the ligand, is released into the tubular lumen remain contentious.In the renal tubules, ATP may be released by both vesicular and channel-based release mechanisms. Several different transporters have been proposed as ATP conduits, including the cystic fibrosis transmembrane conductance regulator and the P2X7 receptor, but subsequently their ATP permeability has been challenged (24). It has also been suggested that connexin (Cx) proteins can form hemichannels, or connexons, which allow ATP release to the extracellular fluid (8,10,12,24). Recently, a study from our laboratory provided evidence that Cx30 is required for luminal ATP release in the cortical collecting duct and that this mechanism is involved in pressurenatriuresis (35). However, the issue of functional hemichannels formed solely by connexins under physiological conditions is controversial (37).A recently identified plasma membrane ATP channel is the pannexin channel (3-7). Pannexins are a class of proteins that also form hexameric transmembrane channels, similar to those formed by connexins (4, 5, 7). Despite having similar structures, connexins and pannexins have no sequence homology. ...

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“…For confocal immunofluorescence microscopy, kidney sections (5-μm thickness) were shipped to the laboratory of JP-P at the University of Southern California. These sections were processed by methods described previously [10,16,28]. Briefly, deparaffinized and rehydrated sections were heated for 2 × 10 min in a microwave with medium heat in PBS and allowed to cool for 40 min to retrieve antigen.…”

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“…More recently, pannexin 1 channels (Panx1), expressed at the apical membrane of ICs, has also been proposed to regulate ATP release as Panx1-deficient mice excrete less ATP than their wild-type littermates (15,46). Thus, ATP released via Cx30 and/or Panx1 is theoretically available for autocrine and paracrine signaling to ICs and PCs, respectively.…”

Functional roles of connexins and pannexins in the kidney

“…Although most studies have focused on the contribution of vascular Cx proteins to renal hemodynamics, accumulating evidence suggests an essential physiological role for these proteins in tubular epithelial function. Regarding the Panx family, our knowledge is still limited, as today there are only two studies reporting their expression throughout different renal compartments [17,18]. It should be noted though that Panx localization in some experiments was performed in paraffin-embedded tissues [17].…”

“…Indeed, urinary ATP levels were reduced by 30 % in Panx1 KO mice compared to wild-type mice. Since Panx1 was located at the apical membrane of various tubular segments, the authors suggested that Panx1-based channels may regulate ATP release and further participate in the control of renal epithelial fluid, electrolyte transport and vascular functions via purinergic signaling [18].…”

“…In addition, even though recent studies have associated high or decreased pannexin (Panx) expression with a wide range of human diseases, their role in kidney is poorly known [16]. Recently, two members of the Panx family have been identified in renal vasculature and the tubular compartment & Christos E. Chadjichristos christos.chadjichristos@upmc.fr [17,18]. However, the field of the Panx biology is quite young and the role of Panxs in renal function is not elucidated.…”