Ultrastructural Characterization of Pannexin 1 Expression Along the Rat Nephron

TL;DR

This study identifies where pannexin 1 is located in rat kidneys, providing insights into its role in kidney function and disease.

Contribution

The first ultrastructural characterization of pannexin 1 expression in the rat nephron.

Findings

Pannexin 1 is strongly expressed in glomerular podocytes, proximal tubules, and collecting ducts.

Moderate pannexin 1 expression is found in capillary endothelium and loop of Henle segments.

Weaker expression is observed in distal tubular epithelium.

Abstract

Pannexins are transmembrane glycoproteins that share structural and functional similarities with the gap junction proteins innexins and connexins. They play a critical role in paracrine and intracellular signalling, including purinergic signalling via the release of extracellular ATP. The role of pannexins in renal function and the pathophysiology of renal diseases is being intensely studied. However, there are no data on the subcellular localization of pannexin 1 expression in the rat kidney. We studied the distribution of pannexin 1 in the rat kidney, combining light microscopy with immunofluorescent immunohistochemistry and transmission electron microscopy with immunogold pannexin labelling. We found strong expression of pannexin in glomerular podocytes, proximal tubules and collecting ducts; moderate expression in the endothelium of glomerular and peritubular capillaries; thin descending and thick ascending limbs of the loop of Henle; and weaker pannexin 1 expression in the distal tubular epithelium. We described the detailed ultrastructural localization of pannexin 1 expression. This is the first study describing the ultrastructural distribution of pannexin 1 in the rat kidney, one of the most used preclinical models in renal physiology and pathology research. These results provide previously missing data on the precise distribution of pannexin 1 in the rat kidney, which is a prerequisite for a proper understanding of its role in renal physiology and pathophysiology.

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1. Introduction

Pannexins (Panx) are a family of chordate transmembrane glycoproteins identified by Panchin et al. [1]. Panx share structural and functional similarities with the gap junction proteins innexins and connexins, despite lacking significant sequence homology [2]. Shared topological features include four transmembrane domains, intracellular amino and carboxy termini, and two extracellular loops, the first of which contains conserved cysteine residues. Panx are heavily glycosylated on their extracellular loops, which may hinder their ability to interact with pannexin channels on neighbouring cells [3,4,5]. Nevertheless, these hemichannels play a critical role in paracrine and intracellular signalling due to their permeability to ions and small metabolites up to 1.5 kDa in size [6,7], with purinergic signalling via the release of extracellular ATP being the most extensively studied [2,8,9,10]. The human and murine genomes contain three orthologous pannexin-encoding genes, namely Panx1, Panx2 and Panx3 [1,11]. Panx1 is widely expressed in various cell types and organs, including the endothelium, epithelial cells, immune cells, red blood cells, platelets, adipocytes, skeletal muscle, brain, spleen, thymus, skin, cartilage, liver, kidney, bladder, lung, gonads, colon, and heart [2,3,11,12,13,14,15,16,17,18,19,20,21]. Although the Panx1 channels were initially described as hexameric [6,22], recent studies using cryo-electron microscopy have revealed that Panx1 forms heptameric pannexons [22,23,24,25]. Furthermore, the conventional understanding of Panx as non-gap junction proteins [2,3,4,12] has recently been called into question by electrophysiological recordings demonstrating that Panx1 mediates gap-junction-like direct intercellular communication [26].

Panx1 channel stimulation leads to transmembrane ATP efflux and the activation of P2X7 purinergic receptors [6,8]. Subsequently, Panx1 channels are implicated in a range of physiological and pathological processes associated with purinergic signalling, including blood pressure regulation [16], glucose uptake [21], skeletal muscle contraction [27], chemotaxis [18] and several types of cell death [19,28,29]. Furthermore, Panx1 stimulates an inflammatory response by activating leukocyte migration, releasing cytokines, activating NLRP3, and initiating inflammasomes [9,10,19,30]. Additionally, recent findings suggest that overexpression of Panx1 contributes to cancer progression and metastasis [31,32,33]. In addition to purinergic signalling, Panx1 affects cell polarity and stability, being preferentially concentrated in the apical domains of polarized epithelial and neuronal cells and interacting with several cytoskeletal proteins, including actin and microtubules [34,35,36,37].

Although extensive research has been conducted on the role of purinergic signalling in various renal physiological mechanisms and pathologies, the involvement of Panx1, a major channel responsible for extracellular ATP release, has not yet been explored in these processes [38,39]. Evidence indicates that Panx1 is a significant factor in blood pressure control, renin secretion, and regulation of Ca^2+^ concentration [16,40], and is also linked to insulin resistance and diabetes mellitus [21,41,42]. The involvement of Panx1 in the pathological mechanisms of renal diseases has become the subject of studies in recent years.

The distribution of Panx1 has been thoroughly investigated in mouse kidneys. It has been observed in the endothelium of renal vessels, smooth muscle cells of renal arteries, including afferent and efferent arterioles, the juxtaglomerular apparatus, apical domains of renal tubule cells, including collecting ducts, thin and thick descending limbs of the loop of Henle, and proximal convoluted tubules [30,38,40,43], as well as in podocytes, both native and cultured [44]. Recently, our group examined the distribution of Panx1 expression among various cell populations in healthy postnatal human kidneys and during the stages of human embryonic and early fetal development.

In general, most data regarding the localization of Panx1 channels in the kidney have been derived from studies using mouse models, while research on Panx1 in rats has been limited. In our previous study, we observed Panx1 expression in distal tubular cells of diabetic rats, while none was found in non-diabetic controls. Given that distal tubule cells exhibited the most severe damage in this model, we concluded that Panx1 may contribute to distal tubule damage during diabetes [42]. In recent years, highly efficient rat models for contrast-induced, sepsis-induced, and “triple whammy” acute kidney injury have been established [45,46,47]. Furthermore, rat models have been developed for stem cell therapy for chronic kidney disease and ischaemia–reperfusion acute kidney injury caused by nephrotoxicity and diabetic nephropathy [48,49,50]. Although recent research has utilized rat models for these renal diseases, the site-specific cellular expression of Panx1 in rat kidneys has not been described in detail. Accordingly, the aim of this study was to localize Panx1 expression in the rat kidney. To achieve this, we used immunogold labelling to determine the ultrastructural localization of Panx1 in both the cortex and medulla using transmission electron microscopy.

2. Results

2.1. Immunofluorescent Immunohistochemistry

We studied the distribution of pannexin 1 expression in paraffin-embedded sections of the rat kidney using immunofluorescent immunohistochemistry. Green granular deposits in the tissue were considered pannexin 1 immunoreactivity. Histological sections of the renal cortex showed normal morphology (Figure 1A). The expression of pannexin 1 was widespread in different cells but was more concentrated in particular cell types (Figure 1, Figure 2 and Figure 3).

We found pannexin 1-positive puncta in various cells in the glomeruli, potentially mesangial cells or podocytes (Figure 1A–D). Pannexin 1 immunoreactive puncta were also present in the parietal epithelium of Bowman’s capsule (Figure 1B,C). We could clearly distinguish distal tubules in close proximity to the glomerulus (Figure 1A,B,D and Figure 2C). In the distal tubular epithelium, we also found pannexin 1 immunoreactive puncta (Figure 1D and Figure 2C).

The strongest pannexin 1 immunoreactivity in the renal cortex was observed in the epithelium of the proximal tubules (Figure 2A,B). As already mentioned, we also found pannexin 1 immunoreactivity in the epithelium of the distal tubules (Figure 2C). Further along the nephron, immunofluorescence for pannexin 1 was also visible in the epithelium of the thin descending limb of the loop of Henle (Figure 2D and Figure 3A). It is important to note that pannexin 1 immunoreactivity was evident in the endothelium of the peritubular capillaries in both the cortex (Figure 2B) and the medulla (Figure 2D). The strongest pannexin 1 immunoreactivity in the medulla was observed in the epithelium of the collecting ducts (Figure 3A), as well as in the epithelium of the thick ascending limb of the loop of Henle (Figure 3B).

2.2. Transmission Electron Microscopy

We studied the ultrastructural distribution of pannexin 1 in the rat kidney, using immunogold staining and transmission electron microscopy. The distribution of pannexin 1 corresponded to that observed with immunofluorescent light microscopy.

In the glomerulus, gold deposits indicating panx 1 expression were observed in the podocytes (Figure 4A–D). Gold deposits were present in the primary and secondary foot processes, particularly in their lateral membranes (Figure 4B), but were especially dense in the foot process membrane facing the basal lamina (Figure 4C). In addition, we observed pannexin 1 gold deposits in the membranes of the endothelial cells in the glomerular basal membrane (visible in Figure 4B–D).

A strong and consistent expression of pannexin 1 was observed in the epithelium of the proximal tubules (Figure 5 and Figure 6). In the luminal part of the PTC, strong pannexin 1 expression was found in the membrane of the microvilli (Figure 5A–C), while less dense expression was also observed in the basolateral membrane of the PTC or nearby (Figure 5D), occasionally located in close proximity to the adherens junction (Figure 5C).

In the basal part of the PTC, pannexin 1 immunoreactivity was present in the mitochondria (Figure 6A–C). In addition, gold deposits were found in the membrane invaginations (Figure 6D–G). Pannexin 1 formations were also present in the basement membrane of the proximal tubules (Figure 6E,F). Moreover, pannexin 1 immunoreactivity was clearly visible in the membranes of the endothelial cells of the peritubular capillaries (Figure 6E,F).

In the loop of Henle, we observed very intense accumulations of gold in the apical part of the membrane and in its proximity to the thin-limb epithelium (Figure 7A,B). In addition, in the basal part of the membrane of the endothelial cells in the thin limb of the loop of Henle, we also found pannexin 1 immunoreactivity (Figure 7C,D).

In the thick limb of the loop of Henle, intense immunogold depositions were visible lining the apical membrane of the epithelial cells (Figure 8A–D), as well as the membranes of vacuoles found in the epithelial cells, which were especially dense in the apical parts of the cells (Figure 8A–D).

In the distal tubular epithelial cells (DTC), we found rare accumulations of gold compared to the proximal tubules and the thick limb of the loop of Henle (Figure 9 and Figure 10). However, when present, as in the PTC, pannexin 1 in DTC was located in the microvilli at the apical part of the cell (Figure 9A) and in the mitochondria (Figure 9A). We also observed pannexin 1 immunoreactivity in the basolateral membrane (Figure 9), including in close proximity to the adherens junctions (Figure 9A,B).

In the collecting duct epithelial cells, intense pannexin 1 immunogold accumulation was observed in the basal parts of the cells, especially in the mitochondria and the invaginations of the basal cell membrane (Figure 11A–D).

3. Discussion

We studied the distribution of pannexin 1 in the rat kidney, combining light microscopy with immunofluorescent immunohistochemistry and transmission electron microscopy with immunogold pannexin labelling. As expected from its designation, pannexin is widely present in various cells; however, its expression is more concentrated in specific cell types.

We found pannexin 1 in various cells within the glomeruli, including the parietal epithelium of Bowman’s capsule. These findings are consistent with our previous results in mice [51], rats [52] and human postnatal kidneys [41]. TEM revealed the ultrastructural distribution of pannexin 1 expression in podocytes, particularly in the membranes of the primary and secondary foot processes. It was dense in the lateral membrane, but even denser in the foot process membrane facing the basal lamina. Our finding of pannexin 1 in the membranes of podocytes is consistent with the results of Li and collaborators in murine podocytes, who discovered that Panx1 channels can mediate the conduction of anions across the podocyte plasma membrane and allow the transport of ATP [44]. In contrast, Hanner et al. failed to detect pannexin 1 in the glomerulus [38]. The difference between our findings and those of the latter study could be attributed to the higher sensitivity of the antibodies we used, as well as the fact that we employed transmission electron microscopy to support our light microscopy results. In agreement with our findings, Li and collaborators found expression of pannexin 1 in mouse podocytes using immunohistochemistry, confocal microscopy and Western blotting [44]. They found that in podocytes, pannexin 1 forms voltage-gated transmembrane channels permeable to anions, including the release of ATP into the extracellular space. This ATP can subsequently act as a pro-inflammatory signal. It is important to note that we found the strongest pannexin 1 expression in the foot processes of the podocytes, particularly in the membrane facing the basal lamina. This was consistent with the results of the aforementioned study, which found that pannexin 1 expression in podocytes colocalizes with nephrin, one of the main constitutive proteins in the slit membrane [44]. They demonstrated that pannexin 1 expression in podocytes is tonically inhibited by adiponectin and thus attenuated during obesity, which leads to obesity-induced podocytopathies.

The strongest pannexin 1 immunoreactivity in the renal cortex was observed in the epithelium of the proximal tubules. In the luminal part of the PTC, strong pannexin 1 expression was found in the membrane of the microvilli in the brush border membrane. Less dense expression was also found in the basolateral membrane of the PTC or in its proximity, occasionally close to the adherens junction. The presence and role of pannexin 1 in the PTC have been intensely studied, especially its role in the pathogenesis of AKI [43,53,54]. In the PTC, channels formed by pannexin 1 serve to release ATP into the lumen of the tubules, which subsequently participates in purinergic signalling in an autocrine/paracrine fashion and plays a role in the regulation of electrolyte transport and renal hemodynamics [38,53,54]. The prominent pannexin 1 expression that we found in the apical membrane of the microvilli in the brush border membrane of the PTC in the rat kidney is in accordance with the results of the study conducted by Hanner and collaborators (2012), who also demonstrated that pannexin 1 in the PTCs serves as a membrane channel that facilitates ATP release from the cell.

In the basal part of the PTC, pannexin 1 immunoreactivity was present in the mitochondria. We also found it in the basement membrane of the proximal tubule, where it was especially dense in invaginations of the cellular membrane. Our finding of pannexin 1 expression in the mitochondria of different renal cells, with the most intense expression in the proximal tubules, is in agreement with the results of recent studies [54,55]. In pathological conditions, pannexin 1 exacerbates kidney injury by activating death pathways [56]. It was previously found that increased activation of pannexin 1 channels in PTCs leads to increased ATP release from the cell accompanied by intracellular ATP depletion, which results in cellular damage [54]. PTC and endothelial pannexin 1 play a crucial role in the pathophysiology of AKI [43,53] via several mechanisms, including DAMP signalling [28], facilitation of leukocyte extravasation [57], and contributing to mitochondrial dysfunction in PTCs [54]. In agreement with the results of a recent study conducted on mice by Huang and collaborators [55], we found consistent expression of pannexin 1 in the contact sites of the mitochondria and endoplasmic reticulum in the rat kidney. The aforementioned study demonstrated that pannexin 1 within the endoplasmic reticulum of PTCs acts as a Ca^2+^ leak channel promoting cellular senescence [55], a function already known in other cell types [58,59].

In addition to the proximal tubules, we also found pannexin 1 immunoreactivity in the epithelium of distal tubules, but it was substantially less dense compared to the proximal tubules and other nephron structures. This finding is in accordance with the literature, including the results of our studies in mice [51] and human kidney [41]. However, as in the proximal tubule cells, pannexin 1 in distal tubule cells was present in the microvilli at the apical part of the cell and in the mitochondria, as well as in the basolateral membrane, including an area near the adherens junctions. The primary distinction between our study on rat kidneys and earlier studies is that we detected Panx1 immunoreactivity in distal tubule cells, which was not observed in some other studies in healthy mice and rats [38]. These discrepancies could be attributed to the higher sensitivity of the antibodies used and the very high resolution of transmission electron microscopy as a method. However, it is notable, in agreement with the literature, that the distal tubules are the part of the nephron with the lowest pannexin 1 expression. At the electron microscopy level, we observed substantially fewer pannexin 1 immunoreactive puncta compared to their density in other parts of the nephron, which might be more difficult to detect by light microscopy and may also be affected by the specific sensitivity of the antibody. However, in pathological conditions, in our rat DM1 model, we found very strong expression of pannexin 1 almost exclusively in the distal tubular cells, indicating their potential role in the pathogenesis of diabetic kidney disease [42].

Along the nephron, we also found pannexin 1 in the epithelium of the thin descending limb of the loop of Henle, consistent with findings in mice [38,51] and humans [41]. Very intense pannexin 1 expression was observed in the apical part of the membrane and in close proximity to the thin-limb epithelium. In addition, pannexin 1 immunoreactivity was detected in the basal part of the membrane of the endothelial cells in the thin limb of the loop of Henle. The role of pannexin 1 in the thin descending limb could be related to the release of ATP into the tubular lumen, which subsequently participates in purinergic signalling during the regulation of electrolyte and fluid transport [56].

Very strong pannexin expression was found in the thick ascending limb of the loop of Henle in rats. These findings are also in agreement with the expression pattern previously observed in human [41] and murine kidneys [51]. An ultrastructural study revealed intense pannexin 1 expression lining the apical membrane of the epithelial cells. It was also found lining the vacuole in the apical parts of the epithelial cells. We can assume that, as in other parts of the nephron, their role could be related to the release of ATP. However, their presence in the vesicular apparatus might indicate a potential connection with exo-or endocytotic processes.

The epithelium of the collecting ducts was the structure in which the densest pannexin 1 immunoreactivity was observed in the medulla. Intense pannexin 1 immunogold accumulation was seen in the basal parts of the cells, particularly in the mitochondria and invaginations of the basal cell membrane. We hypothesize that a strong expression of pannexin 1 in the collecting duct epithelium is probably related to its role in ATP release in the tubular lumen and its subsequent involvement in the regulation of water and electrolyte transport via purinergic signalling [56].

In addition to the nephron tubular structures, pannexin 1 immunoreactivity was also found in the endothelium of the peritubular capillaries in both the cortex and the medulla. Consistently, we previously found pannexin 1 expression in the endothelium of both mouse and human kidneys [41,51]. Ultrastructural studies revealed pannexin 1 expression in the membranes of the endothelial cells within the glomerular filtration membrane. Moreover, pannexin 1 immunoreactivity was clearly visible in the membranes of endothelial cells of the peritubular capillaries in proximal tubules, as well as in the basal part of the membrane of endothelial cells in the thin limb of the loop of Henle. The finding of pannexin 1 expression in the endothelium is in agreement with previous results by Lohman and colleagues, who observed consistent pannexin 1 expression in the endothelium along the arterial tree of mice, regardless of artery diameter, and concluded that pannexin 1 plays an important role in intercellular communication among vascular endothelial cells [20]. The role of pannexin 1 in endothelial cells has been intensively studied, and several mechanisms of action have been identified. The most important mechanisms in endothelial cells are ATP release and paracrine regulation of purinergic signalling in smooth muscle cells and leukocytes; facilitation of extracellular Ca^2+^ influx into the endothelial cell cytoplasm, which subsequently regulates inflammatory signalling; and regulation of the vascular tone by modulating vasoconstriction and vasodilation [56,60]. The latter function affects blood pressure and glomerular filtration rate in the kidneys. In addition, it has been shown that pannexin 1 in endothelial cells affects vascular permeability and facilitates the extravasation of phagocytic leukocytes [30,56,60]. As mentioned above, the role of endothelial pannexin 1 in the pathogenesis of acute kidney injury caused by ischaemia–reperfusion was well established [43].

4. Materials and Methods

All experimental procedures were conducted in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and were approved by the Ministry of Agriculture (approval no. UP/I-322-01/14-01/112; 525-10/0255-14-2). Tissues for this study were collected from an intact group of four female Sprague Dawley rats, weighing 270–300 g. General anesthesia was induced by intramuscular injection of ketamine/xylazine (90 mg/kg and 10 mg/kg, respectively). The animals were then perfused transcardially, first with 0.9% NaCl. The left kidney was then clamped and removed for further analyses, after which the animals were perfused with Zamboni’s fixative (0.2% picric acid in 4% buffered paraformaldehyde).

The right kidney was then post-fixed in Zamboni’s fixative. After fixation, the kidneys were washed in PBS, dehydrated in ethanol/water solutions, cleared with xylene and embedded in paraffin wax using a standard procedure [61]. Four-micrometre-thick sections were dewaxed in xylene and ethanol. Antigen retrieval was performed by soaking in citrate buffer (pH 6.5) and heating in a steam oven for 30 min. After cooling to room temperature, sections were washed in PBS. Non-specific secondary antibody binding was prevented by incubation with blocking buffer (ab64226, Abcam, Cambridge, UK) for 30 min. Rabbit anti-Pannexin 1/PANX1 (1:300, ABN242, Merck KGaA, Darmstadt, Germany) antibody was then applied and incubated overnight in a humid chamber at room temperature. After washing in PBS, the appropriate secondary antibody (Alexa Fluor^®^488 AffiniPure Anti-Rabbit lgG (H+L) 711-545-152; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA; 1:400 dilution) was applied and incubated for 1 h at room temperature. After subsequent washing in PBS, nuclei were stained with DAPI, sections were washed and coverslipped (ImmuMount, Shandon, Pittsburgh, PA, USA). Exclusion of the primary antibody resulted in no staining in the tissue. Stained sections were viewed and photographed using a BX51 microscope (Olympus, Tokyo, Japan) equipped with a cooled digital camera (DS-Ri2; Nikon, Tokyo, Japan) and NIS-Elements F software. The objectives used were: UPLFLN4X, UPLFLN10X2, UPLFLN40X, and UPLFLN100XO2 (all Olympus, Tokyo, Japan). Green granular deposits were interpreted as positive Panx1 immunoexpression. For presentation purposes, the background was subtracted from photomicrographs and contrast was slightly enhanced.

Pieces of renal tissue from the left kidney were immersed in McDowell fixative and, after overnight fixation, processed for immunogold staining and resin embedding, as previously described [61]. The samples were then washed in PBS. They were cut with a vibratome (Vibratome Series 1000, Pelco 101, Ted Pella, Inc., Redding, CA, USA) into 20 μm thick sections. After washing in PBS, the sections were incubated first in 50% ethanol for permeabilization and then in the primary antibody for 48 h at +4 °C: rabbit anti-Pannexin 1/PANX1 (1:300, ABN242, Merck KGaA, Darmstadt, Germany). Next, the sections were rinsed in PBS followed by overnight incubation with donkey anti-rabbit gold-conjugated antibodies (711-205-152; 12 nm; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA; 1:1000 dilution). The following day, the sections were rinsed in PBS, post-fixed in 1% osmium tetroxide (1 h), dehydrated in acetone, and embedded in Spurr resin (Sigma-Aldrich Inc., St. Louis, MO, USA). The sections were observed with a transmission electron microscope (JEOL JEM-1400 Flash, Jeol Ltd., Tokyo, Japan).

5. Conclusions

In summary, this is the first study to take advantage of transmission electron microscopy combined with the immunogold technique to reveal the ultrastructural distribution of pannexin 1 in the rat kidney, one of the most commonly used preclinical models in renal physiology and pathology research. Most of our findings were in accordance with previous studies that used light microscopy in murine or human specimens. However, the high resolution of transmission electron microscopy enabled us to observe the expression of pannexin 1 in parts of the nephron, including distal tubules and the thick limb of the loop of Henle, where light microscopy had previously failed to detect it. The latter could potentially be attributed to species differences. These results provide previously missing data about the precise distribution of pannexin 1 in the rat kidney, which is a prerequisite for a proper understanding of its role in renal physiology and pathophysiology.