ZO‐1 expression in normal human macula densa: Immunohistochemical and immunofluorescence investigations

Abstract

The macula densa (MD) is an anatomical structure having a plaque shape, placed in the distal end of thick ascending limb of each nephron and belonging to juxtaglomerular apparatus (JGA). The aim of the present investigation is to investigate the presence of ZO‐1, a specific marker of tight juncions (TJs), in MD cells. Six samples of normal human renal tissue were embedded in paraffin for ZO‐1 expression analysis by immunohistochemical and immunofluorescence techniques. We detected ZO‐1 expression in the apical part of cell membrane in MD cells by immunohistochemistry. In addition, ZO‐1 and nNOS expressions (a specific marker of MD) were colocalized in MD cells providing clear evidence of TJs presence in normal human MD. Since ZO‐1 is responsible for diffusion barrier formation, its presence in the MD supports the existence of a tubulomesangial barrier that ensures a regulated exchange between MD and JGA effectors in renal and glomerular haemodynamic homeostasis.

ZO‐1 expression was detected in TJs located in the apical pole of the Macula Densa (MD) cells. In addition, ZO‐1 and nNOS expressions were colocalized in MD cells.

1. INTRODUCTION

The macula densa (MD) is an anatomical structure highly specialized contained at the distal end of the thick ascending limb in each nephron. It has a plaque shape consisting of 35–40 polarized cells (Lorenzi et al., 2020), strategically placed in the juxtaglomerular apparatus (JGA) (Cangiotti et al., 2018). This apparatus additionally includes renin‐producing granular cells, called juxtaglomerular cells placed in the thickness of afferent and efferent arterial walls as well as extraglomerular mesangial contractile cells. MD cells are sensors specialized in detecting the ionic composition of the tubular fluid and have a paracrine action on JGA effectors synthesizing and releasing chemical mediators. These characteristics are related to MD morphology. In fact, they have an apical primary cilium responsible for sensory function; small dense granules positioned in the basal cytoplasm and a well‐developed network of cellular processes, recently called maculapodia (Gyarmati et al., 2021), probably related to their secretory function still not fully defined (Barajas, 1979; Bell et al., 2003; Cangiotti et al., 2018; Fenton & Praetorius, 2015; Peti‐Peterdi & Harris, 2010; Schnermann & Homer, 2003). These MD morphological features are responsible of renal and glomerular haemodynamic regulation via tubulo‐glomerular feedback and of renin release from adjacent JG cells (Bell et al., 2003; Peti‐Peterdi & Harris, 2010; Schnermann & Homer, 2003; Shroff et al., 2021).

Zonula occludens‐1 is a member of the membrane‐associated guanosine monophosphate kinase family with ZO‐2 and ‐3 that are present in tight junctions (TJs) forming protein complexes with other proteins as occludin, claudins and tricellulin (Raleigh et al., 2010; Zihni et al., 2016). To date, there are no data on the presence of the ZO‐1 in the MD cells. Thus, the aim of the present investigation was to provide evidence of ZO‐1 expression in MD cells utilizing immunohistochemistry and immunofluorescence techniques.

2. MATERIALS AND METHODS

2.1. Tissue collection

The procedures followed for sample collection and handling were in accordance with the Helsinki Declaration of 1975, as revised in 2013. All patients (3 males and 3 females) provided their written informed consent, and all information regarding human material was managed using anonymous numerical codes. All normal‐looking tissue samples were selected from renal samples routinely processed for diagnostic purpose. All patients had a normal renal function at the time of sampling. Samples of normal‐looking kidney tissue were selected immediately after surgical resection of a tumour lesion (4 clear cell carcinomas, 1 chromophobe renal cell carcinoma and 1 oncocytoma). All surgical resection procedures were performed without renal ischemia. In particular, the normal‐looking kidney samples collected for this study were taken from healthy renal tissue surrounding the tumour lesion after confirmation of the absence of cancer cells by a pathologist (M.M.) using cryostat sections.

2.2. Light and immunofluorescent microscopy

For immunohistochemical and immunofluorescent analyses, normal‐looking kidney tissue samples were immediately fixed in 4% neutral buffered formalin at 4°C for 24 h and then washed in cold phosphate buffer pH 7.4 for 30 min. Thereafter, the specimens were dehydrated via a graded series of ethyl alcohol (50%, 75%, 96% and 100%), and two steps in xylene, at room temperature (RT). Then, samples were processed for paraffin embedding at 56°C as previously described (Tossetta et al., 2014).

2.3. Serial sections

The MD is about 40 μm long, and each cell, which composes MD, is 8–12 μm high and 7–8 μm wide (Lorenzi et al., 2020). Therefore, the same cell belonging to MD could be observed in two different serial sections, each with a thickness of 4 μm when the best conditions exist. So, we performed two serial sections for each sample to identify the same cell at least in two consecutive sections. The first section aimed to identify MD by studying neuronal nitric oxide synthase (nNOS), which is the immunohistochemical specific marker (Mundel et al., 1992), whereas the second section allowed to study the expression of ZO‐1, marker of TJs (Licini et al., 2016; Tossetta et al., 2014).

2.4. Immunohistochemistry

Immunohistochemical staining was performed as previously described (Fantone et al., 2020; Tossetta et al., 2019). Briefly, sections were deparaffinized and rehydrated through xylene and a graded series of ethyl alcohol. To inhibit endogenous peroxidase activity, sections were incubated with 3% hydrogen peroxide for 1 h at RT, then washed in phosphate‐buffered saline (PBS), immersed in 0.1 M citrate buffer pH 6 and subjected to high temperature treatment for 10 min at 98°C. After washed in PBS, all sections were incubated for 30 min at RT with Animal‐Free Blocker (Vector laboratories, Burlingame, CA) diluted 1:5 in PBS to block nonspecific background, and then overnight at 4°C with the primary antibodies mouse anti‐nNOS (1:100 sc‐17,825, Santa Cruz Biotechnology, Inc.) or rabbit anti‐ZO‐1 (1:200; HPA001636, Sigma) in PBS. After washing in PBS, sections were incubated for 1 h at RT with the appropriate secondary biotinylated antibody (Vector Laboratories) diluted 1:200 in PBS. The peroxidase avidin–biotin complex method (Vector Laboratories) was applied for 1 h at RT using 3′,3′ diaminobenzidine hydrochloride (DAB; Sigma) as chromogen. Sections were counterstained with Mayer's hematoxylin, dehydrated and finally mounted with Eukitt solution (Kindler GmbH and Co.).

Negative controls were performed by omitting the primary or the secondary antibody. Isotype mouse IgG (#I‐2000‐1) and rabbit IgG (#I‐1000‐5), both from Vector Laboratories, were used as isotypic control antibodies as a further negative control at the same dilutions and conditions of the primary antibodies.

2.5. Immunofluorescence

Immunofluorescence staining was performed as previously described (Licini et al., 2021). Briefly, sections were deparaffinized and hydrated with xylene and a graded alcohol series. Sections were then immersed in 0.1 M citrate buffer pH 6 and subjected to high‐temperature treatment for 10 min at 98°C. To reduce autofluorescence, samples were incubated with 0.1% Sudan Black B (Sigma) in 70% ethanol for 30 min and then washed with PBS incubated for 30 min at RT with Animal‐Free Blocker (Vector laboratories) diluted 1:5 in PBS to block non‐specific sites. Sections were then incubated with the primary antibodies mouse anti‐nNOS (1:100, sc‐17,825, Santa Cruz Biotechnology) and rabbit anti‐ZO‐1 (1:200, HPA001636, Sigma) in PBS overnight at 4°C. Sections were washed with PBS and incubated with donkey anti‐rabbit Alexa Fluor 594‐conjugated and donkey anti‐mouse Alexa Fluor 488‐conjugated secondary antibodies (both from Invitrogen) for 30 min at RT. Slides were then incubated with TOTO‐3 Iodide (1:3000; Invitrogen) for 10 min for nuclear staining, washed and mounted onto glass slides using Vectashield mounting medium (Vector Laboratories). Negative controls were performed by omitting the primary antibodies.

Sections were analysed with a motorized Leica DM6000 microscope (Leica Microsystems srl.), and fluorescence was detected with a Leica TCS‐SL spectral confocal microscope equipped with an Argon and a He/Ne mixed gas laser. Images (1024 × 1024 pixels) were obtained sequentially from two channels using a confocal pinhole of 1.1200 and stored as TIFF files. Negative controls were performed as described in the previous paragraph.

3. RESULTS

3.1. MD cells express ZO‐1 by immunohistochemistry

ZO‐1 was expressed on the apical cell membrane of MD cells forming a belt‐like structure in both distal tubule (DT) (Figure 1a) and collecting ducts (CD) (Figure 1b). In addition, ZO‐1 was detected in some cells of the proximal tubules (PT) (Figure 1c) and in the vascular endothelium as expected (not shown).

FIGURE 1.

ZO‐1 expression (arrows) in (a) distal tubules (DT), (b) collecting ducts (CD) and (c) some proximal tubules (PT). Scale bar = 50 μm.

Two serial sections were used to identify MD cells by nNOS staining expressing ZO‐1. In the first section, MD cells were identified by using a specific marker for this tissue, namely nNOS (Figure 2a). In the second section, the same MD identified by nNOS (in first section, Figure 2a) expressed ZO‐1 (Figure 2b, arrows), as shown in the positive internal control (Figure 1). ZO‐1 positivity was also present in some cells adjacent to MD, which morphologically belong to the DT, as well as in some cells of the capsular epithelium (Figure 2b, arrowheads).

FIGURE 2.

Two serial sections of the same macula densa (MD) stained with nNOS (a) and ZO‐1 (b). Some ZO‐1 positive cells are localized in both MD and in adjacent DT cells (arrows). The ZO‐1 expression is also observed in some cells of the capsular epithelium (arrowheads). EM, extraglomerular mesangium. Scale bar = 50 μm.

3.2. ZO‐1 and nNOS colocalization confirmed ZO‐1 expression in MD cells

Confocal fluorescent microscopy analysis confirmed that ZO‐1 and nNOS were colocalized in the apical cell membrane of MD (Figure 3). Some DT cells adjacent to MD expressed ZO‐1 protein (Figure 3) as well as some cells of the capsular epithelium (Figure 3).

FIGURE 3.

Representative confocal microscopy images of normal human kidney showing (a–c) macula densa (MD) (expressing the nNOS marked in red) that also express ZO‐1 (green), a widely used marker of tight junction (arrows). ZO‐1 was also expressed in cells adjacent to MD (not shown). Some cells of the capsular epithelium are also positive for ZO‐1 (arrowheads). Nuclei were stained with TOTO3. The inset is the enlargement of some MD cells positive for ZO‐1. G, glomerulus. Scale bar = 50 μm.

4. DISCUSSION

TJs are protein complexes, located near the apical cell surface forming a semipermeable barrier in lining epithelia of different organ compartments (Zihni et al., 2016). These types of junctions play a vital role in the selective barriers of endothelial and epithelial tissues (Tokes et al., 2013). Endothelial TJs maintain intravascular volume and regulate both fluid and solutes flow between blood vessels and parenchyma (Rahimi, 2017). Epithelial TJs regulate gas exchange in lung; production of appropriately concentrated urine in kidney; absorption of nutrients and containment of bacteria through gastrointestinal tract (Anderson & Van Itallie, 2009). Dysfunction of endothelial and epithelial barriers may be responsible for nutrient malabsorption, intestinal bacteria translocation, capillary leak, interstitial oedema, oxygen loss and organ failure in many pathologies.

ZO‐1 distribution pattern in kidney varies markedly along the nephron. It is low in the PT, while its expression increases in the more distal segments, which exhibit a progressively more restrictive barrier phenotype (Gonzalez‐Mariscal et al., 2003; Raschperger et al., 2006). However, the gap junctions are absent (Cangiotti et al., 2018; Forssmann & Taugner, 1977; Lorenzi et al., 2020; Taugner et al., 1978; Yao et al., 2009) as well as desmosomes (Cangiotti et al., 2018; Lorenzi et al., 2020 and present study), unlike what Tisher et al. (1968) described in other kidney compartments. In particular, the presence of ZO‐1 in the apical rim of the cell membranes of MD supports the idea that this structure works as tubulomesangial barrier (Cangiotti et al., 2018; Lorenzi et al., 2020). This can be considered a diffusion barrier that assures the compartmentalization of two zones, that is the fluid present in the DT lumen and the JGA effector placed under the basal part of the MD cells. MD and JGA effectors crosstalk is assured by maculapodia that keep in touch these two structures via paracrine communication for systemic blood pressure homeostasis (Gyarmati et al., 2021). ZO‐1 presence in MD represents a structural basis for MD function since it is an important scaffold protein that connects TJ proteins and actin cytoskeletons (Gonzalez‐Mariscal et al., 2003; Gyarmati et al., 2021). In addition, our study confirmed that ZO‐1 is highly expressed in DT and CD, weakly expressed in PT and in whole capsule epithelium.

In conclusion, our study provided the evidence that ZO‐1 is present on or near the apical domains of the MD cells as in the other numerous barriers present in the organism playing a pivotal role in MD function. These data pave the way for new studies on how the alteration of ZO‐1 can affect MD function.

FUNDING INFORMATION

This work was supported by grant from Università Politecnica delle Marche (Grant number: RSA 2022 to M.M. and D.M.). The authors did not receive support from any organization for the submitted work. All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non‐financial interest in the subject matter or materials discussed in this manuscript.

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no conflict of interest. The authors have no competing interests to declare that are relevant to the content of this article.

Tossetta, G. , Fantone, S. , Senzacqua, M. , Galosi, A.B. , Marzioni, D. & Morroni, M. (2023) ZO‐1 expression in normal human macula densa: Immunohistochemical and immunofluorescence investigations. Journal of Anatomy, 242, 1184–1188. Available from: 10.1111/joa.13832

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.