The Effect of Different Salinity Levels on Chloride Cells of Periophthalmus Waltoni’s Epidermis in Connection with NA + / K + / ATPase ion Transporter and NA + / K + / 2CL Cotransporter using Immunohistochemistry Technique

Background: The NA + / K + / ATPase and NA + / K + / 2CL cotransporter are two types of ions transporting proteins that are active in the secretion of chloride in bony sh. So, the level and activity of these transporting proteins are expected to increase in saline water. The aim of this study was to investigate the effect of different salinity levels on chloride cells of Periophthalmus waltoni’s epidermis in connection with NA+ / K+ / ATPase ion transporter and NA+ / K+ / 2CL cotransporter using immunohistochemistry technique. Results: Simultaneous localization of NA + / K + / ATPase and NA + / K + / 2CL cotransporter showed that both were simultaneously present in epidermal ion cells and were able to react to different salinity levels. Conclusion: The results of this study conrmed the model that states, NA + / K + / ATPase and NA + / K + / 2CL cotransporters are responsible for the secretion of chloride from the chloride cells of bony sh.


Background
Fish's skin, along with other important organs such as kidneys and lungs, plays a key and vital role in regulating the entry and exit of ions such as ammonium in various environmental conditions [1]. Ion cells also play an important role in the regulation and entry / exit of ions in the sh skin. In bony sh, the ion cells are also commonly known as chloride cells or mitochondrial-rich cells. These cells are the main cells that regulate osmosis in sh, and are found more in the epidermis of marine sh, especially euryhaline species, than the epidermis of freshwater sh [2]. Under optical microscope, ion cells are larger than their adjacent epithelial cells and have an acidophilic cytoplasm with a nucleus located in the middle or near the base of the cell. These cells extend throughout the epidermis thickness, and the cell apical is in contact with the outside environment [3].
The cytoplasm of this cell has a high density of mitochondria. In saline-adapted sh, the apical plasma membrane is locally serrated and forms a small crypt lled with mucus, often broken into smaller units by blades or ne folds. Crypt size usually decreases in freshwater-adapted sh, but the ne folds may be more developed. The small tubes branching into the cytoplasm vaguely reach the blind terminals in the apical region, which contain numerous vesicles. Deep folds in the plasma membrane often appear in the basolateral region of the cell, communicating directly with the space inside the cell. The ion cells are connected to adjacent epithelial cells by blocked-taped connections. Ionic movements in these cells are performed by various enzymes, the most important of which are NA + / K + / 2CL and NA + / K + / ATPase cotransporter [4].
All bony sh maintain an almost constant internal osmotic pressure, regardless of whether they are in sea water or fresh water [5]. NKA is present in the gills, kidneys and skin cells of bony sh. NKA in chloride cells has the highest known cell concentration, having more than 100 million molecules per cell whose job is to absorb ions in freshwater and secrete salt in saline water [6]. NKA is most likely involved in the basolateral release of sodium from the chloride cell into the blood [7].
The transporting protein of NKCC cotransporter ion has been immunohistochemically located in the basolateral membrane of chloride cells of saltwater euryhaline sh [8]. The immunohistochemical localization of NKCC cotransporter in saltwater Salmon Smolts has also been investigated [9].
NKCC is essential for the secretion of NaCl in saltwater sh [10].

Results
There were no deaths during the study. The results showed that chloride cells were present in the different areas of Priophthalmus Waltoni's skin epidermis and participated in ion exchange. In different groups, the immune response of Na + / K + / ATPase increased with a slight increase in salinity, but no signi cant difference was observed in the second group (BW). Chloride cells were multidimensionally stained for Na + / K + / ATPase in different layers of the epidermis and responded positively to this staining. The size, shape, and position of the cells that responded positively to Na + / K + / ATPase localization indicated that they were mitochondrial-rich chloride cells. Lack of Na + / K + / ATPase reaction was detectable in the nucleus and apical of the chloride cell. The staining pattern of basolateral section was not changed by salinity for Na + / K + / ATPase. Chloride cells of sh in all different salinity levels containing positive Na + / K + / ATPase were reported on the epidermis. The number of positive Na + / K + / ATPase chloride cells in the epidermis increased with increasing salinity and was higher compared to freshwater sh. The chloride cells of saltwater were larger than freshwater chloride cells ( Figs. 1 and 3). Freshwater chloride cells were smoother and more elongated (Fig. 1). However, the overall shape of chloride cells in the epidermis was not signi cantly altered by different salinity levels (Figs. 1-3). The staining pattern for NA + / K + / 2CL cotransporter was almost similar to that of Na + / K + / ATPase. All cells that were stained for Na + / K + / ATPase were simultaneously stained for NA + / K + / 2CL cotransporter, and the distribution of NA + / K + / 2CL cotransporter staining in chloride cells (staining throughout the cell except for the nucleus and most of the apical regions was similar to Na + / K + / ATPase staining. The NA + / K + / 2CL cotransporter stained areas of chloride cells was also equal to that of Na + / K + / ATPase. In other words, the number of NA + / K + / 2CL -positive cotransporter chloride cells of the epidermis increased with increasing salinity, and also was more frequent compared to freshwater sh. In the epidermis, only chloride cells were stained for Na + / K + / ATPase and NKCC, while other cells in the epidermis only had background staining (Fig. 1E). The distribution of Na + / K + / ATPase and the NA + / K + / 2CL cotransporter was limited to basolateral membrane of chloride cells. In addition, increasing salinity led to an increase in the number and area of chloride cells. Areas of the cell that responded positively to the simultaneous immunity of Na + / K + / ATPase and NA + / K + / 2CL cotransporter were calculated, which were positive for both proteins containing immunohistochemical stained ion (Table 1). In the present study, the proposed model of chloride absorption and secretion in the chloride cells of the epidermis of mudskipper sh is presented in Fig. 4.

Discussion
The functions of ion cells have been reviewed by various researchers. Usually, the function of ion cells in dehydration is to release ions into seawater, but in freshwater where sh are exposed to hydration from their surroundings, they are responsible for absorbing ions [11]. In Anadromous and Catadromous sh, the function of these cells temporarily changes from secretion to absorption and vice versa [2]. Ion cells have been shown to be responsible for the secretion of chlorine ions in seawater-adapted bony sh, whereas in freshwater bony sh, these cells are the place of calcium and chlorine ions ow into the epithelial tissue. Ion cells also play a separate role in the regulation of acid / alkali [11]. In Euryhaline species of bony sh, certain morphological changes occur in ionic cells during adaptation from freshwater to saline or seawater. In terms of properties, the number and size of these cells decrease during adaptation to seawater. Under special conditions where ion regulation in fresh water is imposed on sh, proliferation of these cells may also occur [12]. Ionic cells do not appear to be con ned to bony sh, as even primitive sh, such as hag sh that have a highly permeable skin to water and as a result their blood plasma osmolarity is equivalent to that of seawater sh, have mitochondrial-rich cells in their gills that are morphologically similar to the ionic cells of bony sh. These cells have also been found in the gills and skin of lampreys that migrate between freshwater and marine environments, and the cartilaginous sh [11].
Ionic movements in these cells are performed by various enzymes, the most important of which are NA + / K + / 2CL and NA + / K + / ATPase cotransporter [4]. In other words, NA + / K + / 2CL and NA + / K + / ATPase cotransporter proteins are known as ion transporters [5].
The distribution of NKCC protein in chloride cells of freshwater-adapted Atlantic salmon is completely overlapped with NA + / K + / ATPase protein in the basolateral membrane [9].
These ndings support the chloride secretion model by bone sh chloride cells, in which the presence of two ion transporting proteins of NA + / K + / 2CL and NA + / K + / ATPase cotransporter in the basolateral membrane of chloride cells as well as a CFTR chloride channel in the apical membrane of these cells, is shown.
The distribution of NA + / K + / ATPase and NKCC ion transporting proteins in the gills of Hawaiian goby was the same, so that except for the nucleus and apical of chloride cells, the rest of the chloride cells that include basolateral membranes responded positively to the simultaneous localization of these proteins [13].
NKCC is essential for the secretion of NaCl in saltwater sh, but its gene has not yet been cloned in bony sh.
Previous studies using electronic microscope have shown that NA + / K + / ATPase is widely present in the basolateral membrane but not in the most apical part of the cell [8].
Therefore, in the present study, high level of NA + / K + / ATPase and NKCC in chloride cells is likely to indicate their distribution in the basolateral membrane. Marshall et al. (2002) recently found that NKCC immunity occurs in seawater-adopted sh throughout the chloride cell, but in freshwater-adopted sh it has a more limited distribution, which is restricted to the basolateral membrane [14]. Pelis et al. (2001) found that in seawater-adopted sh and freshwater-adopted sh, NKCC immunity occurred throughout the chloride cell, but in freshwater-adapted sh, the number of stained chloride cells and the NKCC immunity was lower [9].
In most bony sh adapted to saltwater, chloride cells appear to contain NA + / K + / 2CL and NA + / K + / ATPase ion transporting proteins in the basolateral membrane.
Cotransporter occurs in two important isoforms; secretory isoform (NKCC1) and adsorbent isoform (NKCC2). There is a tendency for NKCC immunity in the chloride cells of Hawaiian goby gills to be secretory [13]. In most tissues, the secretory form is found only in the basolateral membrane of chloride cells, while the absorbent form is found only in the apical membrane [13]. The only exception to this general rule is the choroid plexus where both the NKCC1 and NA + / K + / ATPase are found in the apical membrane [15].
In the present study, the NKCC immunity throughout chloride cells indicates a basolateral distribution, which shows that this isoform is of secretory type. The large number of NKCCs in sh adapted to seawater indicates that the amount of this ion-carrying protein increases with the adaptation of sh to seawater, similar to the results found in other bony sh.

Conclusions
The mitochondrial-rich cells are present in the different region of epidermis of Periophthalmus waltoni.
Results of the present study show that NA + / K + / ATPase and NA + / K + / 2CL cotransporters are attended in the mitochondrial-rich cells of Periophthalmus waltoni's epidermis and participate in ion regulation.
Number of the mitochondrial-rich cells, NA + / K + / ATPase and NA + / K + / 2CL cotransporters are more in the sh adapted to sea water compared with lower salinities. Also, NA + / K + / ATPase and NA + / K + / 2CL cotransporters occupy a larger area of the mitochondrial-rich cells in the high salinities.

Methods
Fifteen adult sh with an average weight of 6.76 ± 0.42 g and an average lenght of 16.62 ± 1.10 cm were used in this study. Samples were obtained from the shores of Persian Gulf. Samples were purchased fresh and alive from local shermen. After the usual measurements, the sh in 3 groups including group 1 with salinity of 1 PPT (FW group), group 2 with salinity of 15 PPT (BW group), and group 3 with salinity of 35 PPT (SW group) were adapted for 2 weeks. Samples were xed in 4% paraformaldehyde for 24 hours. Slides obtained from different parts of the skin and epidermis were immunohistochemically studied. For immunohistochemical study, the slides were rst washed in an acid and alcohol solution with a concentration of 70% HCl in 1% EtOH at 60° C for 15 minutes using an Ultrasonic Cleaner machine. They were then washed in water for 15 minutes and exposed to distilled water for another 15 minutes.
They were dried gradually at 37° C for 24 hours and after 2 days, the slides were placed in a solution containing 245 ml of acetone and 5 ml of 3-aminoisoquinoline triethoxysilane. After placing the tissues on the coated slides, they were placed in xylol, and then in a decreasing concentration of alcohol, and nally in distilled water. The slides were then boiled in 0.05% Citraconic Anhydride solution for 30 minutes and were placed in distilled water for 10 minutes to cool down. Then, they were placed in an incubator at 37° C for 1 hour to dry. After that, the slides were immersed rst in SDS solution for 5 minutes and then, in TPBS solution for 5 to 10 minutes, after which 75 µl of buffer block was added to each section. Later on, the slides were placed in a damp room. Two different primary antibodies were added to each section. Rabbit αR1 antibody and mouse T4 antibody were used simultaneously on one section as the primary antibody. After doubling the primary antibody in each section, the slides were placed in a damp room and refrigerated overnight at 4° C. The next morning the slides were placed inside the TPBS.
Secondary antibody was added to all sections even in the control group. For this purpose, 50 µl of secondary antibody was added to each section. Blocking buffer was also used to dilute the secondary antibody. For every 500 µl of solution containing secondary antibody, 1 µl of Rabbit secondary antibody and 1 µl of mouse secondary antibody were used. After adding 50 µl of secondary antibody to all sections, they were incubated at 37° C (wet room) for 1 hour.
After that, the samples were placed in TPBS for 5 minutes, and then 60 ml of TPBS was mixed with 5 µl of DAPI and added to the sections. DAPI induced the molecular staining of nucleus, and was attached to genes within the nucleus. Availability of data and materials The datasets generated and analyzed during the current study are not publicly available due the con dentiality of their information but are available from the corresponding author on reasonable request. Immunohistochemical double labeling image of sh epidermis adapted to (FW) PPT = 1. A; the nucleus has blue staining, B; Na + / K + -ATPas has green staining, C; NKCC cotransporter has red staining, and D; epidermal background tissue that reacts negatively to immunohistochemical localization. Image E shows the merging of all 4 images in Figure 1. Immunohistochemical double labeling image of sh epidermis adapted to (BW) PPT = 15. Image A; the nucleus has blue staining, image B; Na + / K + -ATPas has green staining, image C; NKCC cotransporter has red staining, and image D; epidermal background tissue that reacts negatively to immunohistochemical localization. Image E shows the merging of all 4 images in Figure 2. Immunohistochemical double labeling image of sh epidermis adapted to (SW) PPT = 35. Image A; the nucleus has blue staining, image B; Na + / K + -ATPas has green staining, image C; NKCC cotransporter has red staining, and image D; epidermal background tissue that reacts negatively to immunohistochemical localization. Image E shows the merging of all 4 images in Figure 3.