Modifications of the genital kidney proximal and distal tubules for sperm transport in Notophthalmus viridescens (Amphibia,Urodela, Salamandridae)

Male salamanders use nephrons from the genital kidney to transport sperm from the testicular lobules to the Wolffian duct. The microstructure of the epithelia of the genital kidney proximal tubule and distal tubule was studied over 1 year in a population of Notophthalmus viridescens from Crawford and Pike counties in central Missouri. Through ultrastructural analysis, we were able to support the hypothesis that the genital kidney nephrons are modified to aid in the transportation of sperm. A lack of folding of the basal plasma membrane, in both the genital kidney proximal and distal tubules when compared to the pelvic kidney proximal and distal tubules, reduces the surface area and thus likely decreases the efficiency of reabsorption in these nephron regions of the genital kidney. Ciliated epithelial cells are also present along the entire length of the genital kidney proximal tubule, but are lacking in the epithelium of the pelvic kidney proximal tubule. The exact function of these cilia remains unknown, but they may aid in mixing of seminal fluids or the transportation of immature sperm through the genital kidney nephrons. Ultrastructural analysis of proximal and distal tubules of the genital kidney revealed no seasonal variation in cellular activity and no mass production of seminal fluids throughout the reproductive cycle. Thus, we failed to support the hypothesis that the cellular activity of the epithelia lining the genital kidney nephrons is correlated to specific events in the reproductive cycle. The cytoplasmic contents and overall structure of the genital and pelvic kidney epithelial cells were similar to recent observations in Ambystoma maculatum, with the absence of abundant dense bodies apically in the epithelial cells lining the genital kidney distal tubule. J. Morphol. 275:914–922, 2014. © 2014 Wiley Periodicals, Inc.     

Platinum localization and kidney ultrastructure in the frog after cisplatin administration

X-ray microanalysis was used to study the localization of platinum, sodium and potassium in the frog nephrons 3 days after cisplatin administration (50 mg per kg of body weight). In the frog, like in mammals, platinum is accumulated in the proximal tubules. As evidenced by electron microscopy, the platinum content is correlated with the degree of deterioration of cell ultrastructure. The large scattering of the platinum content of individual tubules (from 0.03 to 0.15%) is probably due to the difference between the initial and late parts of the proximal tubule which is in agreement with the ultrastructural findings.     

Comparative histological, histochemical and ultrastructural studies of the nephron of selected snakes from the Egyptian area

The present study was aimed to compare and contrast the histochemical, histological and ultrastructural variations (microanatomical differences) in the nephrons of selected snake species, Eryx jaculus (Boidae), Psammophis sibilans (Colubridae), Naja haje (Elapidae) and Echis pyramidum (Viperidae) from Egypt. The structural studies were carried out by conventional light and electron microscopy. The nephron, the renal unit of snakes, consists of renal corpuscle, proximal tubule, intermediate segment, distal tubule and collecting tubule. The renal corpuscles have large capillaries with clear and dark fenestrated endothelial cells. The proximal tubule showed long microvilli, cytoplasmic vacuoles, developed endoplasmic reticulum and abundant mitochondria. The intermediate segment was lined by ciliated cells. The lining cells of the distal tubules showed few microvilli, abundant dense mitochondria and clear vesicles of mucous appeared in the terminal portion. The collecting tubules consisted of mucous cells. In summary, the ultra-structure studies of nephrons revealed several interspecies similarities and also some intra-species differences in species of snakes.     

Indicators of functional differentiation of the chick embryonic kidney

Relevant indicators of the functional capability of the embryonic kidney were tested in the chick mesonephros chosen as an ideal model accessible to direct observation in vivo. Evidence of glomerular filtration (GF) was checked up by the arterial injection of 2% lissamine green (LG) followed by measurement of the LG passage time on days 5, 6 and 7. Presence of the electrogenic transport was investigated by determining the transepithelial potential difference (TPD) which distinguished proximal and distal tubules of the 6-day nephrons. GF and tubular reabsorption could be demonstrated from day 5 by the storage of trypan blue (TB) in proximal tubules after intra-amniotic administration of the dye. The distribution of tubule staining corresponded to the proximal-distal gradient of the nephron differentiation. Activities of embrane enzymes, alkaline phosphatase and 5'-nucleotidase, were detected from day 4. They preceded the ultrastructural maturation in the differentiating proximal tubule epithelia. A semiquantitative evaluation of enzyme activities by the method of measuring of the minimum incubation time (MIT) together with the TB storage, appeared reliable and relevant indicators of the functional properties of mesonephric nephrons, suitable for distinguishing between more and less advanced stages of the nephron development.     

Differential expression of alpha and pi isoenzymes of glutathione S-transferase in developing human kidney

Polyclonal antisera to the alpha and pi isoenzymes of glutathione S-transferase have been used in immunohistochemical studies to determine the developmental expression of these isoforms in human kidney. Before 35 weeks of gestation, both isoenzymes were expressed by the collecting tubules and developing nephrons. After this time, expression of the alpha set was restricted to the proximal tubule and that of the pi set to the distal and collecting tubules and the loop of Henle.     

Protein absorption by tubular mesonephric and metanephric structures in the human embryo

Summary The presence of different serum proteins in the cells of the proximal tubule of both meso- and metanephric nephrons in human embryos (7th–12th week of intrauterine life) was investigated using immunohistochemistry. Endogenous lysozyme, alpha-1-antitrpysin and ferritin were detected in mesonephric proximal tubules and, starting from the 8th week, also in metanephric proximal tubules. Our observations provide information concerning the appearance and distribution of tubular protein reabsorption during the early stages of development.     

Transepithelial potential in mesonephric nephrons of 7-day-old chick embryos in relation to the histochemically detected sodium pump

In order to obtain basic information on the transport properties of differentiating embryonic nephrons, we examined the 7-day-old chick mesonephros by measuring the transtubular epithelial potential difference (TPD) and by histochemical detection of Na,K-ATPase activity. TPD as an indicator of the electrogenic transport was measured in individual segments of superficial nephrons in vivo. Their electric polarity was always lumen-negative. TPD was reduced by addition of 10 mM KCN applied to the mesonephric nephrons from the outside. In the proximal tubules, TPD was significantly lower (mean+/-SD: -1.0+/-0.5 mV) than in the distal and collecting tubules (-2.2+/-1.0 mV, p< or =0.05). Activity of the sodium pump was evaluated histochemically by detection of ouabain-sensitive potassium-dependent p-nitrophenyl phosphatase in cryostat sections of the mesonephros. The enzyme activity was demonstrated only in distal tubules and in the collecting ducts, but not in the proximal tubules. These findings have revealed significant differences between embryonic nephron segments: the distal tubule, in contrast to the proximal one, is supplied by the sodium pump and is able to generate higher TPD. Therefore, we consider that it is only the distal nephron, which possesses the ability of active transport.     

Histochemical evaluation of mouse and rat kidneys with lectin-horseradish peroxidase conjugates

Paraffin sections of mouse and rat kidney were stained with a battery of ten lectin-horseradish peroxidase conjugates and lectin binding was correlated with the ultrastructural distribution of periodate-reactive sugar residues as determined by the periodic acid-thiocarbohydrazide-silver proteinate technique. Various segments of the uriniferous tubule in both species showed differential affinity for labelled lectins. Significant differences were also evident between comparable tubular segments in mouse and rat kidneys. Neutral glycoconjugates containing terminal beta-galactose and terminal alpha-N-acetylgalactosamine were prevalent on the luminal surface of the proximal convoluted tubule in the rat, but alpha-N-acetylgalactosamine was absent in this site in the mouse. In both species, terminal N-acetylglucosamine was abundant in the brush border of proximal straight tubules but absent in proximal convolutions. Fucose was demonstrated in both proximal and distal segments of mouse kidney tubules but only in the distal nephron and collecting ducts in the rat. Lectin staining revealed striking heterogeneity in the structure and distribution of cellular glycoconjugates. Such cellular heterogeneity was previously unrecognizable with earlier histochemical methods. The marked cellular heterogeneity observed with several lectin-conjugates in distal convoluted tubules and collecting ducts of both species raises a prospect that lectins can provide specific markers for intercalated and principal cells in the mammalian kidney. Glycoconjugates containing terminal sialic acid and penultimate beta-galactose were present on vascular endothelium in both rodent kidneys, as were terminal alpha-galactose residues; but both species lacked reactivity for Ulex europeus I lectin in contrast to human vascular endothelial cells. The constant binding pattern of lectin conjugates allows convenient and precise differentiation of renal tubular segments and should prove valuable in the study of changes in kidney morphology promoted by experimental manipulation or pathologic changes.     

Morphology of the Nephron in the Mesonephros of Bufo bufo (Amphibia,Anura, Bufonidae)

The present study deals with the morphology and ultrastruclure of the nephron in the mesonephros of the toad, Bufo bufo (Linnaeus, 1758). Based on serial sections in paraffin, Araldite and Epon, the position of the different segments of the nephron within the kidney tissue was determined, and a nephron subsequently reconstructed. The nephron consists of the following parts: Malpighian corpuscle, neck segment, proximal tubule, intermediate segment, early distal tubule, late distal tubule and collecting tubule. The late distal tubule was subdivided into three morphologically different sections. The total number of nephrons in the toad mesonephros was estimated at 6000 units. The length of the segments in the reconstructed nephron was calculated. The cytology of the epithelial cells constituting the segments was described using transmission and scanning electron microscopy. Heterocellularity was found in the late distal tubule section I and III and in the collecting tubule. The proportional distribution and number of intercalated (mitochondria-rich) cells in the late distal tubule and collecting tubule was calculated. Only one morphological type of intercalated cell could be distinguished. Late distal tubules were removed from fresh Bufo kidneys for preliminary studies of the intercalated cells with Nomarski optics.     

The testicular sperm ducts and genital kidney of male Ambystoma maculatum (Amphibia,Urodela, Ambystomatidae)

The ducts associated with sperm transport from the testicular lobules to the Wolffian ducts in Ambystoma maculatum were examined with transmission electron microscopy. Based on the ultrastructure and historical precedence, new terminology for this network of ducts is proposed that better represents primary hypotheses of homology. Furthermore, the terminology proposed better characterizes the distinct regions of the sperm transport ducts in salamanders based on anatomy and should, therefore, lead to more accurate comparisons in the future. While developing the above ontology, we also tested the hypothesis that nephrons from the genital kidney are modified from those of the pelvic kidney due to the fact that the former nephrons function in sperm transport. Our ultrastructural analysis of the genital kidney supports this hypothesis, as the basal plasma membrane of distinct functional regions of the nephron (proximal convoluted tubule, distal convoluted tubule, and collecting tubule) appear less folded (indicating decreased surface area and reduced reabsorption efficiency) and the proximal convoluted tubule possesses ciliated epithelial cells along its entire length. Furthermore, visible luminal filtrate is absent from the nephrons of the genital kidney throughout their entire length. Thus, it appears that the nephrons of the genital kidney have reduced reabsorptive capacity and ciliated cells of the proximal convoluted tubule may increase the movement of immature sperm through the sperm transport ducts or aid in the mixing of seminal fluids within the ducts. © J. Morphol., 2013. © 2012 Wiley Periodicals, Inc.     

Kidney Proximal Tubule Cells Originate from Approximately Four Progenitor Cells and Make Distinct Patches in Mouse Aggregation Chimeras

Giant lysosomal granules of bg^J/bg^J mutant mice were used as a marker to investigate the histological composition of kidney proximal tubule cells. Embryos of the bg^J/bg^J genotype and those of the +/+ genotype were aggregated, and lysosomes of their proximal tubule cells were histochemically stained with the β-glucuronidase activity. Tubules composed of bg^J/bg^J -type epithelial cells alone, tubules composed of +/+-type epithelial cells alone and tubules containing both types of epithelial cells were observed in cross sections. When the long straight portion of proximal tubules was reconstructed from serial sections, most of the tubules were of the mixed type, and distinct patches of bg^J/bg^J -type or +/+-type epithelial cells were detectable. These patches appeared to extend to the longitudinal rather than the circumferential direction. This suggests the clonal proliferation followed the mixing of embryonic progenitor cells for proximal tubule cells. Estimation from proportions of bg^J/bg^J -type proximal tubules in total examined proximal tubules gave a pool size of approximately four primordial precursor cells for proximal tubules in both right and left kindeys. The fact that the proportion of bg^J/bg^J -type components was comparable between the right and left kidneys of each chimera suggests that all proximal tubule cells in both right and left kidneys may originate from common primordial precursor cells.     

Tandem scanning confocal microscopy (TSCM) of normal and ischemic living kidneys

Tandem Scanning Confocal Microscopy (TSCM) allows one to section optically into and record real-time images of living organs and tissues in a noninvasive fashion. In this paper, we will present some initial TSCM observations of subcapsular nephrons in the living, intact kidneys of Munich-Wistar rats and evaluate the nephron's responses to temporary ischemia and to intravenous infusion of mannitol. The rats were anesthetized with Inactin and a laparotomy performed to expose the kidneys. Using a TSCM equipped with a 20 x water-immersion objective, we optically sectioned through the intact kidney capsule and recorded real-time images of living subcapsular glomeruli and uriniferous tubules. The proximal tubule brush border was highly reflective and allowed us to distinguish between the first and second segments of the proximal tubules as well as the distal tubules. Cellular elements of the blood could be seen passing rapidly through peritubular capillaries and individual glomerular capillary loops. With fluorescent filters in place, intravenously injected carboxyfluorescein was seen to pass through the glomerular capillary loops and then progressively through the different segments of the uriniferous tubules. Ligation of the renal artery resulted in rapid swelling of proximal tubule cells into the tubular lumens, loss of reflectiveness of the microvillous brush borders, and closure of the peritubular capillary spaces. Upon release of the ligature, the proximal tubule lumens again became patent, often opening up abruptly and in a zipper-like fashion down the length of the tubules. Increasing the glomerular filtration rate by intravenous infusion of mannitol resulted in increases in tubular luminal and perimeter dimensions. Mannitol also acted as an effective impermeant osmotic agent and prevented most of the cellular swelling which was otherwise seen in response to renal ischemia.     

Membrane traffic after inhibition of endocytosis in renal proximal tubules

This study was performed to examine quantitatively the cellular organelles involved in membrane recycling after inhibition of luminal endocytosis in renal proximal tubules. Paraffin oil was microinfused into rat renal proximal convoluted tubules to prevent luminal endocytosis. After 1-2 hr the kidneys were fixed by perfusion and prepared for electron microscopy. Segment 1 proximal tubules infused with paraffin oil and control tubules from the same kidney were studied. In addition we examined proximal tubules from kidneys fixed by immersion 30 sec after removal of the kidney. In the oil-infused tubules the large endocytic vacuoles (greater than 0.5 micron) disappeared, the amount of small endocytic vacuoles (less than 0.5 micron) was reduced to about 10%, and the amount of dense apical tubules was significantly increased. The dense apical tubules were very seldom seen connected to the apical plasma membrane in controls but this was occasionally observed in tubules fixed by immersion and relatively often in oil-infused tubules. An ultrastructural morphometric analysis substantiated and extended the qualitative observations and provided quantitative estimates of volumes and surface areas for large endocytic vacuoles, lysosomes, mitochondria, small endocytic vacuoles, and dense apical tubules in control and experimental tubules. The results strongly support the suggestion that the dense apical tubules located in the apical cytoplasm represent the vehicle for the recycling of membrane from endocytic vacuoles back to the plasma membrane, and show that in renal proximal tubule cells small and large endocytic vacuoles are transformed into dense apical tubules when endocytosis is stopped.     

Ultrastructure and osmoregulatory function of the kidney in larvae of the Persian sturgeon Acipenser persicus

The localization of Na(+) , K(+) -ATPase (NKA) and the ultrastructural features of kidney were examined in larvae of the Persian sturgeon Acipenser persicus (L 31-41 mm total length and 182·3-417·3 mg). Investigations were conducted through light and electron microscopy and through immunofluorescence for NKA detection. The kidney nephrons consisted of a large glomerulus and tubules (neck, proximal, distal and collecting), which connected to the ureters. Posteriorly, ureters extended and joined together into a thin-walled ureter terminal sac. Ultrastructurally, the glomerular cells (podocytes) possessed distinctive pedicels that extended to the basal membrane. The proximal tubule (PT) showed two different cells. The cells lining the anterior part of PT possessed apical tall microvilli (c. 2·7 μm), a sub-apical tubular system, a basal nucleus and dense granules. Posteriorly in the cells, the sub-apical tubular system and granules were absent and round mitochondria associated with basolateral infoldings were found; the apical microvilli were reduced. Distal and collecting tubular cells showed the typical features of osmoregulatory cells, i.e. well-developed basolateral infoldings associated with numerous mitochondria. No immunofluorescence of NKA was detected in the glomeruli. A weak immunostaining was observed at the basolateral side of the cells lining the neck and PT. A strong immunostaining of NKA was observed in the entire cells of the distal tubules, collecting tubules and in some isolated cells of the ureters. In all immunostained cells, the basolateral region showed a much higher fluorescence and nuclei were immunonegative. In conclusion, the epithelial cells of kidney tubules had morphological and enzymatic features of ionocytes, particularly in the distal and collecting tubules. Thus, the kidney of A. persicus larvae possesses active ion exchange capabilities and, beside its implication in excretion, participates in osmoregulation.     

A micropuncture study of several indices of renal function in rats during development]

The function of single superficial nephrons has been studied by means of several micropuncture methods in 22-, 30- and 42-day rats. It was shown that intratubular hydrostatic pressure, transit time of tubular fluid through a proximal tubule and Henle's loop, as well as local reabsorption in the proximal tubules measured by Gertz's split oil droplet method increase between the 22nd and the 30th days. The ration of tubular fluid and plasma (TF/P) inulin concentrations in late proximal and in early distal tubules increases with age. The values of TF/P for Na in early distal tubules are higher in 22- and 30-day rats than in older ones. TF/P for K does not change simultaneously with that for Na. These data are consistent with the assumption that the sodium load in the distal part of the nephron is higher in young rats than in adult ones.     

Localization of cathepsin L in rat kidney revealed by immunoenzyme and immunogold techniques

Localization of cathepsin L in rat kidney was investigated by immunocytochemical techniques. Kidneys were fixed by perfusion and embedded in Epon or Lowicryl K4M without postosmication. For light microscopy (LM), semi-thin sections of the Epon-embedded material were stained by the immunoenzyme technique after removal of epoxy resin. For electron microscopy (EM), ultra-thin sections of Lowicryl K4M-embedded material were stained by the protein A-gold technique. By LM, reaction deposits for cathepsin L were present in the cytoplasmic granules of proximal tubule cells, but little or no reaction product was noted in distal tubule, collecting tubule, and most of urinary tubules in the medulla. By EM, heavy gold label for cathepsin L was confined exclusively to lysosomes of the proximal tubule cells, but little or no label to those of the other segments. In immunocytochemical control sections, no reaction was observed. These results indicate that a main container of cathepsin L is lysosomes of the proximal tubule and suggest that the enzyme plays a role in the degradation of endocytosed proteins.     

Localization of cathepsin L in rat kidney revealed by immunoenzyme and immunogold techniques

Summary Localization of cathepsin L in rat kidney was investigated by immunocytochemical techniques. Kidneys were fixed by perfusion and embedded in Epon or Lowicryl K4M without postomication. For light microscopy (LM), semi-thin sections of the Epon-embedded material were stained by the immunoenzyme technique after removal of epoxy resin. For electron microscopy (EM), ultra-thin sections of Lowicryl K4M-embedded material were stained by the protein A-gold technique. By LM, reaction deposits for cathepsin L were present in the cytoplasmic granules of proximal tubule cells, but little or no reaction product was noted in distal tubule, collecting tubule, and most of urinary tubules in the medulla. By EM, heavy gold label for cathepsin L was confined exclusively to lysosomes of the proximal tubule cells, but little or no label to those of the other segments. In immunocytochemical control sections, no reaction was observed. These results indicate that a main container of cathepsin L is lysosomes of the proximal tubule and suggest that the enzyme plays a role in the degradation of endocytosed proteins.     

Expression of Bcl-2 and Bax in mouse renal tubules during kidney development

Bcl-2 and Bax play an important role in apoptosis regulation, as well as in cell adhesion and migration during kidney morphogenesis, which is structurally and functionally related to mitochondria. In order to elucidate the role of Bcl-2 and Bax during kidney development, it is essential to establish the exact location of their expression in the kidney. The present study localized their expression during kidney development. Kidneys from embryonic (E) 16-, 17-, 18-day-old mouse fetuses, and postnatal (P) 1-, 3-, 5-, 7-, 14-, 21-day-old pups were embedded in Epon. Semi-thin serial sections from two E17 kidneys underwent computer assisted 3D tubule tracing. The tracing was combined with a newly developed immunohistochemical technique, which enables immunohistochemistry on glutaraldehyde fixated plastic embedded sections. Thereby, the microstructure could be described in detail, and the immunochemistry can be performed using exactly the same sections. The study showed that Bcl-2 and Bax were strongly expressed in mature proximal convoluted tubules at all time points, less strongly expressed in proximal straight tubules, and only weakly in immature proximal tubules and distal tubules. No expression was detected in ureteric bud and other earlier developing structures, such as comma bodies, S shaped bodies, glomeruli, etc. Tubules expressing Bcl-2 only were occasionally observed. The present study showed that, during kidney development, Bcl-2 and Bax are expressed differently in the proximal and distal tubules, although these two tubule segments are almost equally equipped with mitochondria. The functional significance of the different expression of Bcl-2 and Bax in proximal and distal tubules is unknown. However, the findings of the present study suggest that the mitochondrial function differs between mature proximal tubules and in the rest of the tubules. The function of Bcl-2 and Bax during tubulogenesis still needs to be investigated.