A Histological Study of the Kidney Structure of Van Fish (Alburnus Tarichi) Acclimated to Highly Alkaline Water and Freshwater

Marine and Freshwater Behaviour and Physiology

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A histological study of the kidney structure of Van fish (Alburnus tarichi) acclimated to highly alkaline water and freshwater

A.R. Oğuz

To cite this article: A.R. Oğuz (2015) A histological study of the kidney structure of Van fish (Alburnustarichi) acclimated to highly alkaline water and freshwater, Marine and Freshwater Behaviour and Physiology, 48:2, 135-144, DOI: 10.1080/10236244.2015.1004838 To link to this article: https://doi.org/10.1080/10236244.2015.1004838

Published online: 30 Jan 2015.

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A histological study of the kidney structure of Van ﬁsh (Alburnus tarichi) acclimated to highly alkaline water and freshwater A.R. Oğuz*

Faculty of Science, Department of Biology, Yüzüncü Yıl University, 65080 Van, Turkey (Received 7 May 2014; accepted 26 December 2014)

Kidneys are important organs that play a role in ion and water regulation in different aquatic environments. The Van fish (Alburnus tarichi, Güldenstädt, 1814) has acclimated to the alkaline waters of the largest saline soda lake in the world. In this study, changes in kidney tissue were examined histologically in alkaline and freshwater as the fish entered freshwater for reproduction. In addition, immunological changes were examined for Na+,K+, ATPase (NKA), an important transmembrane protein in kidneys. A histological comparison of the kidneys of fish taken from both environments was made. The glomerular volume was larger in fish acclimated to freshwater, and the collecting tubules were larger in diameter and had thicker walls. The fish acclimated to alkaline lake water had reduced numbers of glomeruli of smaller size. NKA enzyme was present in tubules within kidneys in both environments. It was, however, observed more frequently in fish acclimated to freshwater. Although plasma osmolality and Cl− values decreased in the fish acclimated to freshwater, hematocrit values increased (p < 0.05). No changes were observed in the muscle water content between alkaline water- and freshwater-acclimated fish. We clearly demonstrate that histologic and immunochemical changes take place in the kidney of the Van fish acclimated to the different physicochemical characteristics of alkaline and freshwater environments. Keywords: kidney; Na+;K+; ATPase; Van fish; alkaline acclimation; Alburnus tarichi

Introduction Gills, kidneys, and intestines are important organs that play a role in osmoregulation in teleost fish. These organs may show different physiology and histology in different habi- tats (freshwater and salt water). In hypo-osmotic freshwater, water input and ion loss occur passively in fish. The fish regains some of the ions it loses from gills and kidneys by reabsorption of filtered ions and products from diluted urine. In saltwater, just the opposite of these phenomena occurs (Evans et al., 2005; Marshal & Grosell 2006). Osmoregulation studies in fish mostly focus on gills, and studies on kidneys are limited. The kidneys of fish are not divided into separate zones such as the cortex and medulla found in mammals. However, they are composed of separate parts such as the head kidney where hemopoiesis takes place, as well as the endocrine gland, and trunk kidney where different functions are carried out. In teleosts, the kidney is in the dorsoventral and retroperitoneal regions of the body. In teleost fish, a nephron consists

*Email: [email protected]

© 2015 Taylor & Francis 136 A.R. Oğuz of four distinct regions: glomerulus, proximal tube, distal tube, and collecting tubule (Takashima & Hibiya 1995; Genten et al. 2009). Na+,K+, ATPase (NKA) is a transmembrane protein that plays a role in osmoregulation. NKA actively transports Na+ outside and K+ inside the cell in animal cells and maintains negative membrane potential. This important transmembrane protein is highly expressed in the gills and kidneys of different fish species (Teranishi & Kaneko 2010; Engelund & Madsen 2011). The presence and density of NKA in gills and kidneys is an indicator of ion transport activity. Lake Van is located in Eastern Turkey and is the largest lake in Turkey with a surface area of 3713 km2. It is the largest soda lake in the world with a volume of 576 km3 and is found at an altitude of 1646 m. The salinity of the lake is 2.2%, and the concentrations of chemicals that create the saline environment are 42% NaCl, 34% NaCO3, 16% Na2SO4, 3% KSO4, and 2.5% MgCO3 (Çiftçi et al. 2008; Reimer et al. 2009). The Van fish (Alburnus tarichi) is the only fish that lives in Lake Van, which has alkaline and brackish water (pH 9.8, alkalinity of 151.2 meq/kg, and salinity of 2.2%). The fish has great economic and biological importance for the area. The fish is endemic to Lake Van, and 10,000 tons of fish is harvested annually. The anadromous fish migrates to freshwater during the spawning period (April–July) and returns to the alkaline and brackish water after spawning. Within this short time, it acclimates to different aquatic environments. Studies on the acclimation of the fish are very limited (Danulat & Kempe 1992; Danulat 1995;Oğuz 2013). To our knowledge, no histologic and immunohistochemical studies have been performed on the impact of kidney function on the acclimation process of the Van fish. In this study, differences in the kidneys of Van fish from each area were examined histologically. NKA enzyme, which is very important in ion secretion and reabsorption, was measured in the kidney tissue, and plasma osmolality and Cl− were determined because they relate to osmoregulation, and hematocrit and muscle water content were determined.

Materials and methods Fish and water analyses Fish were caught from Lake Van and the Karasu River, which feed the lake during the spawning season, with trammel nets and casting nets, respectively. There was a wide range of body weights and lengths in the fish caught from the lake and river (42.5–111.9 g and 15.2–20.8 cm from the lake and 52.2–120.3 g and 10.9–18.6 cm from the river). Genders of the fish used in this study were not taken into consideration. The pH, temperature, conductivity, and dissolved oxygen levels were determined with a multiparameter device (Orion 5 Star; Thermo, Barrington, IL) from the localities where the fish were caught. The fish were brought to the laboratory in containers supplied with oxygen. The fish were not fed at any time. All study procedures were carried out according to national animal care regulations.

Plasma osmolality, Cl, hematocrit, and muscle water content analyses After the ﬁsh were anesthetized with MS-222 (40 mg/L, Sigma Chemical Co., St. Louis, MO), their blood was taken by caudal puncture with heparinized syringes. The blood was centrifuged at 1000 × g at 4 °C. From the obtained plasmas, osmolality was measured with an osmometer (Wescor 5520; Wescor, Logan, UT). Plasma Cl− Marine and Freshwater Behaviour and Physiology 137 concentration was determined using an autoanalyzer (Cobas Integra 800; Roche, Mannheim, Germany). The hematocrit value was determined as a percentage after 12,000-rpm centrifugation in 75-mm hematocrit tubes for 5 min. Muscle water content was measured gravimetrically. The muscles taken from the dorsal parts were weighed, dried for 48 h at 100 °C, and reweighed on a precision balance to determine total water lost. Values were expressed as percentage water content. For these analyses, 10 ﬁsh were used from each environment.

Histology and immunohistochemical staining After the fish were anesthetized, they were killed rapidly by decapitation. Kidneys were removed from the dorsal part of the body cavity, and tissues were maintained in Bouin’s fixative for 24 h and thereafter in 70% alcohol. For routine histology, tissues were passed through a graded series of ethanol and xylene and placed in paraffin. Sections (5 μm) were taken from paraffin blocks with a microtome (HM 325 Manual Microtome; MICROM International GmbH, Waldorf, Germany). Sections were stained with periodic acid-Schiff and hematoxylin (PAS&H) and then observed and photo- graphed under a light microscope. The diameter of glomeruli and diameter and thickness of the collecting tubules were measured according to the procedures of Wong and Woo (2006). All histomorphometric measures were carried out using a Leica DMI 6000B microscope (Leica, Germany) with a Leica DFC 490 camera. For immunohistochemical localization of NKA, tissues were fixed in 4% parafor- maldehyde for 12 h and then placed in a 30% sucrose solution containing sodium azide. Sections (8 μm) were taken from tissues using a cryostat microtome (Leica CM 1100, Germany). The sections were washed in phosphate-buffered saline (PBS) and incubated for 10 min with a blocking solution containing 1% bovine serum albumin and 0.1% gelatin in PBS. The slides were incubated overnight at room temperature with the spe- cific monoclonal mouse antibody (1:10) raised against the α subunit of the chicken NKA (Developmental Studies Hybridoma Bank, University of Iowa, USA). The slides were washed in PBS and were then incubated with the secondary antibody anti-mouse IgG (1:100) produced in goat (Alexa Fluor 488; Invitrogen, Carlsbad, CA) for 2 h under dark conditions in a humidified chamber. All slides were mounted with a coverslip and viewed with a digital camera attached to a microscope (Leica, Wetzlar, Germany). Null control sections were incubated under the same conditions without primary antibody and yielded no immunoreactivity.

Statistical analysis The data were expressed as mean ± standard error of mean (SEM). The data were ana- lyzed using Student’s t-test comparison to determine signiﬁcant differences between groups. Signiﬁcance was accepted at p < 0.05.

Results Physicochemical features of Lake Van and the rivers feeding the lake are presented in Table 1. Among measured values, it was determined that salinity, osmolality, pH, dissolved oxygen, and conductivity differed between the two areas. Examination of the gross morphology of the kidneys of the Van ﬁsh revealed an elongated narrow organ united in the body cavity of the ﬁsh similar in disposition to 138 A.R. Oğuz

Table 1. General physicochemical characteristics of surface water of Lake Van and freshwaters which feed the lake. Lake Van Freshwater pH 9.6a, 9.8d 8.42a, 7.39d Conductivity (mS/cm) 29.07a, 25.5d 0.46a, 0.130d Salinity (ppt) 18.6a, 22.7d 0.2a Osmolality (mOsmol/kg) 543a, 551d 253a,9d Alkalinity (meq/kg) 151.2b – Dissolved oxygen 11.21a 6.8a Na+ (mmol/L) 296.032C, 337.9d 0.468c Cl−(mmol/L) 178.919C, 160. 60d 1.001c K+ (mmol/L) 8.78C, 10.90d 0.113c Mg2+ (mmol/L) 2.794C, 4.42d 0.389c Ca2+ (mmol/L) 0.365C, 0.11d 0.087c 2− d SO4 (mmol/L) 24.33 – 3− d PO4 (μmol/L) 3.51 – aPresent study. bDanulat and Selcuk (1992). cOğuz (2013). dDanulat (1995). that of carp. While it is a large structure in the anterior part of the fish, it starts to narrow posteriorly. Histologically, it was the mesonephric type and divided into two parts: the head kidney and the trunk kidney. The hemopoietic tissue covered a large area in addition to the renal tubules in the head kidney and continued to decrease in the trunk kidney. In the microscopic examinations with PAS&H staining, it was shown that the nephron consists of glomerulus, proximal, distal, and collecting tubules (Figure 1). The proximal tubules were covered with a glycocalyx cover (brush border) at the end of PAS staining and clearly differed from other tubules in this way (Figure 1, arrow). Distal tubules did not contain brush borders. Collecting tubules consisted of columnar epithelia with basal nuclei and did not contain brush borders. There were some histological differences between the alkaline lake and freshwater river samples (Figure 1(A) and (B)). The glomerular structure had collapsed in the fish that had acclimated to the lake, while in fish acclimated to the freshwater river, collecting canals had a thin and narrow lumen and the glomerular structures were larger. The diameter of the glomerulus and the diameter and thickness of the collecting tubules were significantly larger in freshwater river samples than in the alkaline lake samples (Table 2). Further, hyaline droplets in the kidney were observed in Van fish from both areas. Immunohistochemical localization of NKA in fish is shown in Figure 2. Although NKA was detected in all tubules, it was not detected in glomeruli (data not shown). Red blood cells in both glomeruli and other kidney capillaries showed, however, auto- fluorescent characteristics (Figure 2(A)). Comparison of the kidneys of fish acclimated to the different aquatic environments revealed that NKA was observed more intensely in freshwater samples. In addition, NKA was more intense in distal and collecting tubules than in the proximal tubule (Figure 2(B)). The mean hematocrit value in fish acclimated to the alkaline lake was 35.18 ± 2.66%. When the fish migrated to freshwater, the hematocrit values increased significantly to 48.31 ± 1.51% (Figure 3). There was no significant difference (p > 0.05) in the muscle water content of the fish acclimated to the alkaline lake water (75.27 ± 0.48%) compared to the fish acclimated to freshwater (77.11 ± 0.84%). (Figure 3). Plasma osmolality values Marine and Freshwater Behaviour and Physiology 139

Figure 1. (Colour online) Periodic acid-Schiff–hematoxylin staining of paraffin section of kidneys (A) Lake Van-acclimated fish and (B) freshwater-acclimated fish. (* hematopoietic tissue, G glomerulus, CD collecting duct, proximal tubule (arrows), distal tubule (arrow heads)). in the fish acclimated to lake water (560.0 ± 3.2 mOsm/kg) were higher than in the fish acclimated to freshwater (299.6 ± 1.1 mOsm/kg; p < 0.05). Plasma Cl− values also differed significantly between the two environments. Cl− values were 142.83 ± 2.24 (mmol/L) and 131.17 ± 0.95 (mmol/L) in lake water and freshwater, respectively. 140 A.R. Oğuz

Table 2. Morphometrics of glomeruli and collecting tubules in Van ﬁsh kidney acclimated to Van Lake and freshwater. Glomerular diameter Collecting tubules diameter Collecting tubules thickness (μm) (μm) (μm) Lake Van 56.22 ± 1.16 67.8 ± 1.79 23.48 ± 0.83 Freshwater 66.04 ± 1.40* 80.72 ± 1.86* 28.72 ± 0.80* Values are means ± SEM. (n:20). *Indicates signiﬁcant difference among lake and freshwater.

Figure 2. (Colour online) Immunochemical localization of NKA (green) in (A) Lake water- acclimated fish and (B) freshwater-acclimated fish. Nuclei were detected by counterstaining with propidium iodide (red). Arrows indicate autofluorescence of red blood cells. (G glomerulus, PT proximal tubule, DT distal tubule, CD collecting duct, * hematopoietic tissue, scale bar 50 μm). Marine and Freshwater Behaviour and Physiology 141

Figure 3. (Colour online) Plasma osmolality (mOsm/kg), chloride concentration (mM), hematocrit (%), and muscle water content (%) of Van ﬁsh collected from Lake Van or freshwater (n =6, mean ± standard error. * indicates the statistical differences (p < 0.05)).

Discussion The diameter of glomeruli and collecting tubules in A. tarichi was larger in freshwater samples than lake water. Expanded glomerular structure and lumen width in the collecting tubules of the fish acclimated to freshwater indicate that this fish has high glomerular filtration rates. In contrast, fish acclimated to the alkaline lake had collapsed glomerular structure and shrunken lumen in collecting canals, indicating that filtration in the neph- rons is lower. Due to the drastic environmental changes in the two different aquatic environments, the fish excretes dilute urine in hypo-osmotic environments and reabsorbs needed ions. These changes led us to surmise that they are related to osmoregulation of the fish. Similar morphologic changes have been found in sturgeon, silver sea bream, and spotted scat (Krayushkina et al. 1996; Wong & Woo 2006; Ghazilou et al. 2011). Hyaline droplets were observed in Van fish kidneys sampled in both areas. Hyaline droplets in Lahontan cutthroat trout kidneys were significantly associated with increas- ing ionic concentrations in lakes differing in salinity and alkalinity (Galat et al. 1985). Further, different pollution factors have been shown to cause hyaline droplet formation (Silva & Martinez 2007). The appearance of hyaline droplets in Van fish kidneys may be due to high ion levels and contamination of the lake. Plasma osmolality and Cl− concentration were statistically lower in fish acclimated to freshwater than fish acclimated to lake water. Osmolality in the fish acclimated to the lake water is quite high and higher than in other teleost fish (370–480 mOsm/kg H2O) (Evans 1993). Plasma osmolality of the fish in the lake environment is almost equal to the osmolality of the lake water (Danulat & Kempe 1992). A. tarichi kidneys play a more important excretory role than in other teleost fishes because ammonia excretion via the gills is severely reduced in lake water (Danulat & Selcuk 1992). The reason that the osmolality of fish that migrate to freshwater decreases is due to the decrease in the 142 A.R. Oğuz plasma urea concentration and other inorganic ion content (Danulat & Selcuk 1992; Oğuz 2013). Since there is a strict relationship between the plasma osmolytes and the osmolytes of the water, the osmolality of the water will directly affect the osmolality of the fish (Marshall & Grosell 2006; Evans et al. 2005). Another parameter that is directly affected by the salinity change, similar to the change in plasma osmolality, is the plasma Cl− concentration. Similar responses were also observed when other euryhaline fish species were exposed to extreme salinities (Sampaio & Bianchini 2002; Lin et al. 2003; Prodocima et al. 2008). Hematocrit values of the fish acclimated to the freshwater were higher than in most fish species (32–43%). When hematocrit values were compared in fish acclimated to lake water and freshwater, higher hematocrit values were observed in the latter. In most fish acclimated to hypo-osmotic conditions, significant increases in hematocrit values were determined (Prodocimo et al. 2008; Serrano et al. 2010). Decreases in salinity cause an increase in extracellular water and consequently swelling of the red blood cells. The opposite of these impacts is observed with increases in salinity. It has been observed that muscle water content remained unchanged with acclimation to different halophilic areas in many euryhaline species (Jensen et al. 1998; Prodoc- imo et al. 2008; Tang et al. 2009). Muscle water control in crustaceans and fish may provide information about their osmoregulatory capacity, adaptation to different halophilic areas, and euryhalinity levels (Freire et al. 2008). In this study, it was determined that muscle water content did not change in either environment. Lin et al. (2003) showed that relative abundance and activity of NKA in fish acclimated to freshwater was higher than salt water and brackish water, and showed NKA in kidneys in distal tubules, proximal tubules, and collecting tubules. In this study, NKA was detected in all tubules of kidney samples from the two different environments. Staining was, however, more intense in freshwater samples compared to the lake samples. This observed NKA intensity indicates that there were low levels of ions in the hypotonic freshwater environment and that they were reabsorbed in the kidney. In naked carp (Gymnocypris przewalskii) that were acclimated to a saline–alkaline environment, as in Van fish, renal function was suppressed in the alkaline environment and kidney NKA activity decreased by 30% compared to that in freshwater (Wood et al. 2007).

Acknowledgement The author wishes to acknowledge the following persons for their contributions to this work: Burcu Ergöz and Fatma Aksuz (histological processing), and Elif KAVAL OĞUZ (microscopy).

Funding This work was supported by the Yüzüncü Yıl University Directorate of Scientiﬁc Research Projects [grant number 2006FED014].

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