Turkish Journal of Zoology Turk J Zool (2019) 43: 224-228 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Short Communication doi:10.3906/zoo-1807-24
Histological and histochemical study on the mesonephric kidney of Pelophylax bedriagae (Anura: Ranidae)
Esra AKAT* Division of Zoology, Department of Biology, Faculty of Science, Ege University, İzmir, Turkey
Received: 12.07.2018 Accepted/Published Online: 17.01.2019 Final Version: 01.03.2019
Abstract: The purpose of this study was to determine the histological structure of the kidney of Pelophylax bedriagae and the distribution of hyaluronic acid (HA) in the kidney tissue. The kidneys of P. bedriagae are long, brown, ribbon-like structures covered with connective tissue. Nephrons, the functional units of the kidney, consist of the renal corpuscle, proximal and distal tubules, collecting tubule, and collecting duct. The renal corpuscle is composed of the glomerulus and Bowman’s capsule. There is no structure similar to Henle’s loop, which is found in higher vertebrates. The visceral layer of Bowman’s capsule is composed of podocytes surrounding the glomerular capillaries. The parietal layer of Bowman’s capsule is lined with a simple squamous epithelium. The proximal tubule is formed by cubic epithelial cells with a brush border, and the distal tubule is covered with a simple cubic epithelium without a brush border. The collecting ducts consist of columnar or cubic cells, and they are larger than the proximal and distal tubules. Many melanomacrophage centers were observed in the kidney parenchyma. The localization of HA was determined to be in the interstitium surrounding the collecting ducts. HA probably plays a significant role in renal water handling and electrolyte balance due to its ability to retain water and bind cations.
Key words: Amphibian, mesonephros, kidney, hyaluronic acid, collecting duct
In vertebrates, the kidneys play a prominent role in consist of 1–3 nephrons (Saxén and Sariola, 1987; Ma and the regulation of body fluid (Junqueira and Carneiro, Jiang, 2007). The pronephric kidney is one of the typical 2006). Additionally, the kidneys are involved in many metanephridial systems in invertebrates. Although the physiological processes such as the production of red blood mesonephric kidney is more complex than the pronephric cells, calcium metabolism, and blood pressure regulation kidney, it forms a link between the metanephridial system (Epstein, 1997). The functional and structural unit of the of invertebrates and the metanephric kidney of adult kidney is the nephron, which is generally composed of the amniotes (Drummond and Majumdar, 2003; Ichimura renal corpuscle, proximal and distal tubules, Henle’s loop, and Sakai, 2017). Therefore, understanding the amphibian and collecting tubule and duct (Junqueira and Carneiro, kidney system is essential for understanding vertebrate 2006). kidney structure and nephron function. During the ontogenesis of vertebrates, 3 distinct Hyaluronic acid (HA) is a linear glycosaminoglycan forms of kidney appear: pronephros, mesonephros, and composed of repetitive disaccharide units of glucuronic metanephros. The pronephros and mesonephros are acid and N-acetylglucosamine (Toole, 2001). HA has present and functional in the life cycle of amphibians. many important functions in many groups, from bacteria The amphibian excretory system, pronephros in larvae to vertebrates, including humans (Almond, 2007). HA was and mesonephros in both larvae and adults, is important previously considered a passive molecule, but in recent for osmoregulation and excretion (Saxén and Sariola, years, researchers have reported important functions of 1987; Møbjerg, 2000). The pronephric kidney is present HA, including proliferation, regulation of cell–cell and cell– in all vertebrates. However, the pronephric kidney matrix adhesion, repair, and regeneration (Zimmerman et of mammals is a nonfunctional transitory structure, al., 2002; Jiang et al., 2007). Previous studies on HA have which is displaced by first the mesonephros and then shown that HA is distributed in different parts of the body the metanephros. The metanephric kidney in mammals such as the skin, muscle, brain, lung, vitreous fluid of the may be composed of as many as 1,000,000 nephrons. eye, and synovial fluid, which lubricates joints of the body However, the pronephric kidneys of amphibians and fish (Kogan et al., 2007; Necas et al., 2008; Akat et al., 2014). * Correspondence: [email protected] 224
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HA is a highly charged hydrophilic molecule in nature, kidney parenchyma (Figure 1C). The localization of HA and it is capable of absorbing 1000 times its own weight was determined to be in the kidney tissue of P. bedriagae. in water. Therefore, HA is essential for maintaining tissue HA immunoreactivity indicated that it was located in structure and volume (Cowman and Matsuoka, 2005; the interstitium surrounding the collecting ducts (Figure Necas et al., 2008). 1D), but HA was absent around the renal corpuscle and The current study was carried out to examine the proximal and distal tubules (Figure 1A). histological and histochemical characterization and the Amphibians play a significant role as a study model for distribution of HA in the kidney of P. bedriagae in order to research on many biological processes (Feder and Burggren, understand its role. 1992). Studies related to the biology of amphibians The study was performed in accordance with the make a major contribution to many areas: morphology, guidelines of the Animal Research Ethics Committee at histology, embryology, ecology, endocrinology, genetics, Ege University (2018-077). Adult specimens of P. bedriagae and public health. However, there have been few studies (3 males and 3 females) were obtained in İzmir, Turkey on the microanatomy of amphibian kidneys. The present (38°50′N, 23°29′E). Animals were anesthetized and then study was conducted to examine the histological and euthanized by decapitation. The kidney tissues were fixed histochemical characterization and distribution of HA in 4% paraformaldehyde or Bouin’s fluid and embedded in the kidney of the anuran amphibian P. bedriagae. The in paraffin. Deparaffinized sections were stained with nephron is composed of a renal corpuscle, proximal hematoxylin and eosin (HE) and periodic acid–Schiff and distal tubules, and collecting tubule and duct. The (PAS). The distribution of HA was demonstrated by localization of HA was determined to be in the interstitium using biotinylated hyaluronic acid binding protein surrounding the collecting ducts. (B-HABP). Sections were deparaffinized and washed in The mesonephric kidneys of P. bedriagae were long, phosphate buffered saline (PBS) containing 2% bovine brown, ribbon-like structures. The renal capsule surrounded serum albumin (BSA) to suppress nonspecific binding of the kidney, and the distinction between medulla and cortex the antibody; sections were then incubated in B-HABP. was unclear. Morovvati et al. (2012) reported that the Sections were stained with diaminobenzidine (DAB) after kidney of Barbus pectoralis (Actinopterygii: Cyprinidae) being washed in PBS. To determine the specificity of HA’s is divided into 3 parts (the head, body, and caudal parts) immunoreactivity, we used Streptomyces hyaluronidase- as in other fish species, e.g., Ctenopharyngodon idella digested sections prior to treatment with the antibody. (Actinopterygii: Cyprinidae) (Morovvati et al., 2011), The sections were treated with B-HABP/HA for a positive Acipenser persicus (Actinopterygii: Acipenseridae), and control. Sections were examined with a Zeiss AxioScope Huso huso (Actinopterygii: Acipenseridae) (Charmi et A1 microscope attached to an Axiocam Erc 5s digital al., 2010). These parts were not observed in the kidney camera (Zeiss, Oberkochen, Germany). of P. bedriagae. Our results are in accordance with the The kidneys of P. bedriagae were long, brown, ribbon- observations of Capaldo et al. (2016) for Triturus carnifex like structures located retroperitoneally and adherent to (Amphibia: Urodela) and Møbjerg et al. (2004) for the dorsal wall of the animal. Connective tissue called Geotrypetes seraphini (Amphibia: Gymnophiona). During the renal capsule surrounded the kidney; the distinction the evolution of vertebrates, the development of renal between the medulla and cortex was unclear in P. bedriagae. anatomy appears to be quite conservative when compared The renal corpuscle consisted of Bowman’s capsule and the to the evolution of respiratory and cardiovascular systems. glomerulus. Bowman’s capsule was composed of 2 layers, Major anatomical changes in vertebrate kidneys include the visceral and parietal. The visceral or internal layer the arrangement of Henle’s loops, division of the kidney surrounded the glomerular capillaries through podocytes. into cortex and medullary portions, and an increase in The parietal or outer layer consisted of a simple squamous the total number of nephrons per kidney from fish to epithelium. The space between the visceral and parietal mammals (Long and Giebisch, 1979). layers was Bowman’s space, which receives glomerular The renal corpuscle is composed of Bowman’s filtrate (Figure 1A). The proximal tubule was formed by capsule and the glomerulus. Bowman’s capsule consists cubic epithelial cells with an apical brush border, which of the parietal and visceral layers, like that of any other was clearly seen with PAS staining. The distal tubule was amphibian. The visceral layer surrounds the glomerular composed of a simple cubic epithelium without a brush capillaries through podocytes. Podocytes, an epithelial border (Figure 1B). There is no structure similar to Henle’s cell type specialized for glomerular filtration, are found loop in P. bedriagae. The collecting duct was formed in all vertebrate species except for aglomerular teleost by a cubic or columnar epithelium, and the collecting fishes (Ichimura et al., 2009). Podocytes are involved in ducts were larger than the proximal and distal tubules. the regulation of glomerular filtration, immunological Melanomacrophage centers were also observed in the roles, and capillary support (Kerjaschki, 1990, 1994).
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Figure 1. Light microscopic view of the kidney of P. bedriagae. A) Simple squamous epithelium of the parietal layer of Bowman’s capsule (black arrow), podocytes in the visceral layer of Bowman’s capsule (red arrow), Bowman’s space (*), glomerulus (G). B) Proximal tubule (PT), distal tubule (DT), glomerulus (G). C) Collecting duct (ellipse), melanomacrophages in the kidney parenchyma (black arrow). D) The localization of HA mainly in the interstitium surrounding the collecting ducts.
The glomerular capillaries lie between 2 arterioles, the loop, and the collecting tubule and duct (Junqueira afferent and efferent. Therefore, glomerular blood pressure and Carneiro, 2006). However, in P. bedriagae, there is is higher than that of other capillaries (Junqueira and no structure similar to Henle’s loop as found in higher Carneiro, 2006). vertebrates. Many adult amphibians absorb water via The proximal tubule is formed by cubic epithelial cells the skin instead of drinking water through their mouth with an apical brush border. Krayushkina et al. (1996) (Hoffman and Katz, 1999). The urinary bladder is also reported that proximal tubules are lined by columnar one of the most important osmoregulatory organs in epithelium with brush border in young individuals of H. many anurans (Suzuki et al., 2007). It is well known that huso and A. persicus. However, proximal tubules are lined P. bedriagae is an aquatic vertebrate. Therefore, there is with rounded squamous cells in A. naccani (Ojeda et al., probably no need to concentrate urine due to the large 2003). Sodium chloride, water, and glucose are absorbed volume of water entering the frog through its skin. by the proximal tubules. Sodium chloride also continues HA immunoreactivity indicated that it was located to be absorbed in the distal tubule, but water absorption in the interstitium surrounding the collecting ducts, but does not continue (Holden et al., 2013). The distal tubule HA was absent around the renal corpuscle and proximal is composed of a simple cubic epithelium without a brush and distal tubules. HA is known as a natural moisturizer, border in the kidney of P. bedriagae. and it is one of the most hydrophilic compounds in nature Generally, each nephron is composed of the renal (Necas et al., 2008). Corroborating our results, Hansell et corpuscle, the proximal and distal tubules, Henle’s al. (2000) reported that HA is present almost exclusively in
226 AKAT / Turk J Zool the renal medullary interstitium of rats and is not found in In conclusion, we provide a histological and the cortex. Additionally, HA is a highly negatively charged histochemical examination of the mesonephric kidney of P. molecule, and therefore it binds to cations (Plopper et al., bedriagae. The current study is the first report to reveal the 2013). The presence of HA in the interstitium surrounding distribution of HA in the amphibian kidney. Considering the collecting ducts provides the passive diffusion of water the presence of HA exclusively in the interstitium from the collecting ducts to the interstitium. Therefore, surrounding the collecting ducts and its ability to retain HA probably has an important role in renal water handling water and bind cations, HA probably plays a significant and electrolyte balance. role in renal water handling and electrolyte balance.
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