| The vertebrate kidney is an important excretory organ, which removes excess water and other metabolic waste by forming urine; acting as the organ regulating the water and salt concentration and playing an important role in ion balance. precisely, it performs vital homeostatic functions. On the other hand, the kidney also produces a variety of hormones. Therefore, its normal function is directly related to health and survival for animal. In the tree of evolution, the kidney of reptile was found fifth position in the vertebrates according to its structure and development. The soft-shelled turtle, an important aquatic animal, likes to live near the water or in the wet sand, thus its body had a high demand for water and the kidneys played an important role in preserving or regulating the water content of the body. In order to elucidate the histological and cytological features of this, the turtle’s kidney and that of the drought tolerant bactrian camel posed an obviously opposite case and ideal for the comparative studies. Thus, habitation the soft-shelled turtle lives is unlike that of the bactrian camel, the arid desert regions, where it survives chronic water shortages well by means of its unique water-saving kidneys. Therefore, the kidneys are important organs to regulate the urine output by reducing urine excretion and thus conserving the body water; and this virtue is especially unique to be greater addressed in detail.In this study, the kidneys of soft-shelled turtle were studied to know the cellular, fine structural characteristics and distribution of the urinary tubules in the kidney, then to compare the morphology and function with the camel kidneys. In addition, a variety of enzymes and other bioactive substances of renal in the Chinese soft-shelled turtle were also investigated in the two genders to know any possible gender difference. On this basis, the first experiment was to elucidate the distribution of angiotensin-converting enzyme 2 (ACE2) in the turtle kidney, compared with the bactrian camel. Analysis of ACE2 was based on renin-angiotensin system (RAS) in rats. Furthermore, from the perspective of molecular biology the presence of ACE2 gene in soft-shelled turtle kidney was also recognized and cloned. In cellular and molecular level, by comparing micro-features and analyzing functional significance of the turtle kidney, basis with the soft-shelled turtle breeding, protection and utilization of resources were made.Experiment I Research on comparative histology of the kidney in the soft-shelled turtle, Pelodiscus sinensis We used light microscopy observe and determine the structural parameters as compared with the bactrian camel(Camelus bactrianus) kidney were studied. The results showed that each kidney comprises 5-6 lobes; A lobe includes two major inner zones. There was no clear distinction between cortex and medulla, but roughly divisible into peripheral and the middle zones; with a thickness ratio of about 3:1. The nephrons of Pelodiscus sinensis were composed of a renal corpuscle (RC) and of a renal tubule that appears divided into five morphologically distinct segments:neck segment (NS), proximal tubule (PT), intermediate segment (IS), distal tubule (DT), and collecting duct (CD). The proximal tubules mainly, collecting duct and most of intermediate segment distributed in the peripheral zone, and renal corpuscles, most of distal tubules located the intermediate zone. The renal corpuscles had a diameter of 120μm, in the middle area of the lobe were distributed on both sides within the vein or 2-4 aggregate distribution characteristics. The neck segment was very short or absent, cells were low cuboidal, with narrow lumen. Eosinophilic cytoplasm of proximal tubules, brush border cells were obvious. In lumen surface, epithelial cells were made of the bright cells, weak eosinophilic, and dark cells, strong eosinophilic. The proximal tubule was highly coiled throughout its length, cells has a height of 20μm. The epithelium of intermediate segment consists of 9μm high cuboidal cells with centrally located nucleiwith little cytoplasm, the nucleus occupies larger portion than the cytoplasm, at the transition zone with the proximal tubules show densely arranged nuclei and weakly eosinophilic cytoplasm. Distal tubule cells are cube-shaped, thick in diameter, the epithelium of this tubule has a height of 12μm. The nuclei in distal tubule arranged unevenly, weakly eosinophilic cytoplasm and with the length second to the proximal tubule. The largest collecting duct was lined by columnar cells, eosinophilic cytoplasm sparse at the periphery; and lies between the proximal tubule that is slightly bent around the earlier. Lobular vein and artery lies in the middle of the central. Interstitium was very scant and almost all were replaced by the capillaries between renal tubules. The distribution and composition of the turtle nephron, and other reptiles like lizards, alligators, are identical, but the differences with the mammalian Bactrian camel are even apparent. Bactrian mammalian kidney is concentrated into thin cortex and thicker medulla, the thickness ratio being 4:1. The renal corpuscles has an average diameter of 220μm; long loop-type intermediate nephron and medullary nephrons are more than the short side loop-type multiple superficial nephron; cortical labyrinth far longer than the proximal tubule. Distal convoluted tubule lies in outer medullary region, has vasa recta and forms wide vasa recta bundle, and uriniferous tubule arrangement of anfractuosity. Inner medullary thickness higher than the outer medulla; outer medulla shows sub-renal pyramids. Camel kidney structure makes it possible to excrete concentrated urine thus conserving the body water. The results provide morphological data not only for the study of reptiles’ nephron structure but also for the relationship between the camels’ nephron structure and the concentrating capacity. As well, it provides morphological evidence to study of renal physiology.ExperimentⅡStudies on ultrastructure of nephrons comparative cytology in the soft-shelled turtle, Pelodiscus sinensis The comparative cytological study was performed using light microscopy and scanning electron microscopy. The results showed that the lumen wall was covered with epithelial cells with basal cell gap, and a finger-like protrusions distribution. Visceral podocytes contain small amounts of mitochondria, abundant rough endoplasmic reticulum and many glycogen granules. These organelles distributed to the end of the podocytes, and the later attached to the vascular basement membrane by means of large ball shaped structures. The podocyte cell body gave off large primary branches and many sub-branches of various widths, from which, in turn, emerged small but intensive tri-branches. These branches eventually attached to the capillary wall- a unique morphological characteristic that has never been reported in other vertebrates. Glomerular basement membrane was with the thick about 300-600nm, and expressed a few lenticels distributed over this region. Morphological characteristics of glomerular filtration barrier may be consistent with the lower reptile glomerular filtration rate. Proximal tubule microvilli were numerous and long and the cytoplasm contained lysosomes. The endocytosing structures were developed for the re-absorption of the main parts of urine. The intermediate segments comprised of a number of ciliated cells and non-ciliated cells.ⅠIts main role is to promote reabsorption of urine to the nephron and the transport segment; distal tubular cells have finger like projections within the fold and basal lamina embedded within the folds for material transport activity; the cells in upper layer of collecting duct show light cytoplasm containing many mucous granules. The nucleus near the Golgi bodies, and in collecting duct cells are electron dense, large dense mitochondria with lamellar cristae. Collecting duct cell gap between cells is covered by finger-like projections. Collecting duct cells secrete mucus of bright color in the lumen that combine with uric acid salts to form barriers to prevent water into the cell lumen.The soft-shelled turtle kidney is significantly different from that of camel; camel podocytes are rich in mitochondria, rough endoplasmic reticulum and Golgi bodies, and well developed microfilaments and microtubules. Podocyte primary branches are large, short sub-branches converge together to make the podocytes attached to the basement membrane. Proximal luminal surface microvilli are thick, distributed thoroughly and some form microvilli-like vacuoles. Proximal tubule has well-developed cytoplasmic microtubules and finger-like microvilli on inner face, and basal plasma membrane folds are more developed. Distal luminal surface microvilli are short with mitochondria scattered in cytoplasm, plasma membrane developed and with a sharp crease. The collecting tubules contained a large number of electron lucent cells with few mitochondria, while the electron dense cells rich in mitochondria. The cortex is in rich of capillary, with holes in its thin wall. The medulla straight small vessel developed with holes in the endothelium. Bactrian camel morphological characteristics of nephrons make it possible to produce concentrated urine. This study reveals the soft-shelled turtle fine structure of nephrons. Compared with the Bactrian camel, turtle has a low glomerular filtration rate and reabsorption of urine in proximal tubules. The structure of nephron has some differences with that in other reptiles, but significant differences with that in Bactrian camel.ExperimentⅢMicrostructure of kidney gender differences in soft-shelled turtle, Pelodiscus sinensis This was tested using toluidine blue semithin sections under transmission electron microscope and the male and female soft-shelled turtle kidney structure were compared. The results showed that in the proximal tubule, intermediate segment of male and female turtles there were some differences in epithelial cell structure. The baso-lateral intercellular space occurred in the soft-shelled turtle proximal tubule cells. while there was no space among the female turtle proximal tubule. In the male side of the middle section of epithelial cells are also gaps within the distribution of slender finger-like projections, but not in female. In addition, male proximal tubule epithelial cells composed with primary cells, bald cells and basal cells, as opposed, females only had two kinds of epithelial cells, the bright and dark. Male and female gender differences in kidney structure had not been reported in other vertebrates. In reptiles, the Squamata kidney had sexual segment as an enlarged male part of distal nephron. The secretions of it had something to do with reproductive activity. But no such segment was observed in the soft-shelled turtle kidney. Insteadly, the differences between male and female nephrons were found. Whether these differences go with the reproductive activity needs further research. Additionally, the size of the gaps may be important for the material transport, the male proximal tubular reabsorption capacity may be stronger than the female.ExperimentⅣCytochemistry characteristics of the kidney in soft-shelled turtle, Pelodiscus sinensis The light microscopy was used to find the chemical reactions of four enzymes in the nephron and to analyse the distribution and characteristics of the two active substances. The results found different enzymes distributed throughout the kidney and showing varying reactions. Succinate dehydrogenase (SDH) as a sign of mitochondrial enzymes in cells, its main distribution in the intermediate segment, distal tubule, collecting duct epithelial cells and in the glomeruli, but in the proximal tubule the response was weekly positive. Alkaline phosphatase (ALPase), distributed only in brush border of the proximal tubule, its activity exists in the material exchange site. ATPase in the transmembrane transport of ions and the regulation of membrane potential plays a key role, mainly brush border of the proximal tubule, and in the intermediate segment though only weakly positive. Acid phosphatase (ACPase) as a sign of lysosomal enzyme is involved in intracellular transfer metabolism of phosphate groups. Its reaction in the proximal tubule brush border was strongly positive, podocytes were positive, and in the collecting tube cytoplasm contains a large number of ACPase positive particles. PAS reaction, used to identify the carbohydrates and the macromolecular glycoconjugates, is located throughout the kidney of soft-shelled turtle, mainly in the glomeruli, proximal tubules, collecting duct epithelial cells, and all tubular basement membrane. Luminal surface showed strong set of PAS-positive. endothelium of the glomerular capillaries, the distal tubule cells, and the brush border of proximal tubule cell were positive, while in other parts the response is weak. Alcian blue (AB) stained acidic mucin, which was only positive in lumen of the distal tubule and collecting duct. Toluidine blue (TB) stained the bright, dark cells of the proximal tubule, intermediate segment and collecting duct epithelium. The results showed that the glomerular endothelial cells and mesangial cells are rich in polysaccharides, providing energy for urinary glomerular filtration of materials transport. However, visceral podocytes have the ability of transporting phosphate groups. In the proximal tubule epithelium show a variety of material transport and the activity of enzymes related to active ingredients that play a major role in turtle proximal tubular reabsorption. Distal tubules and collecting duct cells are highly metabolic. The lumina in the collecting duct secretion acidic mucus may lubricate the lumen in order to keep water. High metabolic activity helps push urine of intermediate segment backward to the ciliated tubular segment. The chemistry of nephron is not only different than mammals and birds but also no same as other reptiles.ExperimentⅤImmunocytochemical study on angiotensin-converting enzyme 2 (ACE2) of the kidney in soft-shelled turtle, Pelodiscus sinensis Immunocytochemical reaction was used to know the difference of angiotensin-converting enzyme 2 (ACE2) in the Chinese soft-shelled turtle and two humped camel. The results for the first time show the distribution of ACE2 in the kidneys of reptiles. Further evidence of the specificity of ACE2 positive reaction was seen at the proximal tubule, podocytes, glomerular basement membrane, lobular vein of kidney in soft-shelled turtle, the intermediate segment and distal tubule, while collecting duct epithelium was weakly positive. ACE2 at different parts of nephrons in soft-shelled turtle showed the mainly in glomerulus and proximal tubule the original urine filtration and reabsorption are regulated. In contrast, ACE2 of kidney in bactrian camel distribution was more extensive in the cortex than the medulla. Cortical mesangial cells, proximal convoluted tubules and collecting tubules of Cortical were positive, while the reaction was weak in distal convoluted tubule. Both thin segment and vasa recta were negative. Medulla collecting tubule and the papillary duct were positive. These results indicate that ACE2 is widely distributed in bactrian camel kidney than Pelodiscus sinensis. By regulating the kidney renin-angiotensin system (RAS), ACE2 affects renal blood flow to regulate water and salt metabolism. The broad distribution of ACE2 of kidney in Bactrian camel might have something to do with the production of a highly concentrated urine.ExperimentⅥCloning and phylogenetic analysis of ACE2 gene of kidney in the soft-shelled turtle, pelodiscus sinensis ACE2 gene in Pelodiscus sinensis was cloned and identified, and further identified. ACE2 of the kidneys in soft-shelled turtle exists to provide for further study of the molecular basis for its function. The total RNA was extracted from the soft-shelled turtle’s kidney by using Oligo dT-Adaptor as the cDNA primer. According to the consensus sequence of ACE2 mRNA in the Homo sapiens, Danio rerio, Gallus, primers were designed. By polymerase chain reaction, some turtle ACE2 gene sequences were amplified and identified. ACE2 gene in soft-shelled turtle was measured 355 mRNA nucleotide fragments. The nucleotide sequence of ACE2 in Homo sapiens, Danio rerio, Gallus, gene get homologous to 70.3%,80% and 65.1% respectively. Amino acid homology was 63.8%,79.3% and 56.9% respectively. This obviously clarifies that renal ACE2 of the turtle, Homo sapiens, Gallus, ACE2 are highly homologous, whereas the Gallus shows the highest homology. The phylogenetic analysis showed that ACE2 of turtle has the closest relationship with the birds, and thus consistent with the theory of species evolution. Further more it indicated that ACE2 of kidney in the local RAS system may play an important role. The gene sequence had been submitted to GenBank, Log No:HM107424. |
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