Terrestrial adaptation and diversity of the kidney functions in the evolution of vertebrates, Amphibia]

The Amphibia bridge the phyletic gap between the aquatic fishes and the terrestrial vertebrates. This transition has involved many interesting changes of metabolisms. In this short review, we have attempted to summarize the kidney structure and functions on the osmoregulations in the Amphibia. Amphibians excrete the water absorbed through their skin as a dilute urine. Pronephros of tadpoles may start to work in the hatching stages and metanephros is well developed and functions. Glomerular filtration rate is relatively large and glomerular intermittency is important to regulate urine production. The proximal tubule reabsorbs approximately 20-45% of filtered water and sodium. Absorption is driven by the basolateral Na+, K(+)-ATPase common to all tubular cells. The diluting segment, early parts of distal nephron, highly develops basolateral interdigitation and reabsorbs approximately 40% of filtered Na+, K+, and Cl-, but is impermeable to water, thus this part results in the formation of hypo-osmotic tubular fluid. In the late distal tubule, the primary mechanism of reabsorption may be via a luminal NaCl synporter, driven by the ubiquitous Na+, K(+)-ATPase on basolateral membrane. In collecting tubule, there are two types of cells, the principal cells and the intercalated cells. Many hormonal and nervous regulations are involved in the glomerular filtration rate and reabsorptions in the amphibian nephrons.     

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.     

中华鳖肾脏的组织化学特性研究

利用光镜组织化学反应对中华鳖肾单位的结构和组织化学特性进行了详细的观察和分析。结果表明,中华鳖肾脏为分叶形的实质器官,肾小叶由被膜和实质组成,实质无髓质和皮质之分,但可以区分为外侧区和内侧区。外侧区嗜酸性,主要分布有近端小管和集合管。内侧区呈弱嗜酸性,肾小体、颈段、中间段和远端小管主要分布在内侧区。肾小球PAS反应呈阳性,但其琥珀酸脱氢酶(SDH)弱阳性,碱性磷酸酶(ALPase)、Na+/K+-ATPase和阿利新兰(AB)反应为阴性。足细胞酸性磷酸酶(ACPase)反应呈阳性。近端小管刷状缘嗜伊红,PAS反应以及ALPase、ACPase和Na+/K+-ATPase酶反应呈阳性,而SDH弱阳性。中间段、远端小管、集合管弱嗜酸性,SDH阳性。中间段Na+/K+-ATPase弱阳性。远端小管细胞侧面呈PAS阳性,腔面显示AB阳性。集合管胞质含有许多ACPase阳性颗粒,腔面呈PAS强阳性,AB阳性。甲苯胺兰(TB)染色可见集合管腔面有阳性颗粒,肾小管上皮含有亮、暗两种细胞。上述组化反应和分布结果表明,鳖的肾小管细胞类型较多,近端小管在原尿的重吸收中起主要作用,远端小管和集合管具有分泌黏液作用。中华鳖肾单位的结构与组化特性不仅与哺乳类和鸟类有一定差异,也与其他爬行动物不完全相同。       

Structure of the kidney in the crab-eating frog, Rana cancrivora

The structure of the nephron in the ranid frog, Rana cancrivora, was studied by light and electron microscopy. This frog is the only amphibian species to live in mangrove swamps of very high salinity. The nephron consists of the following parts: renal corpuscle, ciliated neck segment, proximal tubule, ciliated intermediate segment, distal tubule, connecting tubule, and collecting duct. The distal tubule is located in the ventromedial region of the kidney, and the other tubules are situated in the dorsolateral region. Renal corpuscles are found between the two regions. Some renal corpuscles have a wide Bowman's space because of the small glomerulus within them. The proximal tubules are composed of columnar cells with a dense luminal brush border of long microvilli and numerous apical vesicles and vacuoles. The initial part of the distal tubule consists of heavily interdigitated cells, characterized by a very regular palisade arrangement of mitochondria. In the terminal part of the distal tubule, shorter mitochondria of the infolding cells are situated irregularly around the nucleus. The connecting tubule consists of principal cells and canaliculus cells. The collecting duct consists of columnar or cuboidal cells; cytoplasmic organelles are relatively sparse. The canaliculus cells are intercalated between principal cells from the terminal distal tubule to the proximal part of the collecting duct. Our findings indicate that the kidney of R. cancrivora is structurally similar to kidneys of other amphibians. These findings are discussed with regard to probable correlations between ultrastructure and function in R. cancrivora.     

Morphology of the kidney in larvae of Bufo viridis (Amphibia, Anura, Bufonidae)

This study deals primarily with the morphology and ultrastructure of the pronephros in the green toad Bufo viridis during prometamorphosis when the pronephros and the developing mesonephros function simultaneously. Furthermore, the mesonephros was studied during pro- and postmetamorphosis with emphasis on the distal segments of the nephron. The paired kidneys consist of two cranial pronephroi immediately behind the gill region and two more caudal elongated mesonephroi. Each pronephros consists of a single convoluted tubule which opens into the coelom via three nephrostomes. This tubule is divided into three ciliated tubules, three proximal tubule branches, a common proximal tubule and a distal tubule, which in turn continues into the nephric duct. No intermediate segment is present. The length of the pronephric tubule is 12 mm, including the three branches of the ciliated tubules and proximal tubules. Primary urine is formed upon filtration from an external glomerulus, which is a convoluted capillary lined by podocytes, a specialization of the coelomic epithelium. From the coelom the filtrate is swept into the ciliated tubules. In the collecting duct system of the developing mesonephric nephron epithelial cells with conspicuous, apical osmiophilic granules appear in larvae of 9-10 mm. Heterocellularity of mixed intercalated (mitochondria rich) cells and principal cells is observed in the collecting duct system and nephric duct from a larval body length of 14 mm. As the proliferation of mitochondria-rich cells proceeds, the osmiophilic granules disappear and are completely absent from the adult amphibian mesonephros.     

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.     

The pelvic kidney of male Ambystoma maculatum (Amphibia,urodela, ambystomatidae) with special reference to the sexual collecting ducts

This study details the gross and microscopic anatomy of the pelvic kidney in male Ambystoma maculatum. The nephron of male Ambystoma maculatum is divided into six distinct regions leading sequentially away from a renal corpuscle: (1) neck segment, which communicates with the coelomic cavity via a ventrally positioned pleuroperitoneal funnel, (2) proximal tubule, (3) intermediate segment, (4) distal tubule, (5) collecting tubule, and (6) collecting duct. The proximal tubule is divided into a vacuolated proximal region and a distal lysosomic region. The basal plasma membrane is modified into intertwining microvillus lamellae. The epithelium of the distal tubule varies little along its length and is demarcated by columns of mitochondria with their long axes oriented perpendicular to the basal lamina. The distal tubule possesses highly interdigitating microvillus lamellae from the lateral membranes and pronounced foot processes of the basal membrane that are not intertwined, but perpendicular to the basal lamina. The collecting tubule is lined by an epithelium with dark and light cells. Light cells are similar to those observed in the distal tuble except with less mitochondria and microvillus lamellae of the lateral and basal plasma membrane. Dark cells possess dark euchromatic nuclei and are filled with numerous small mitochondria. The epithelium of the neck segment, pleuroperitoneal funnel, and intermediate segment is composed entirely of ciliated cells with cilia protruding from only the central portion of the apical plasma membrane. The collecting duct is lined by a highly secretory epithelium that produces numerous membrane bound granules that stain positively for neutral carbohydrates and proteins. Apically positioned ciliated cells are intercalated between secretory cells. The collecting ducts anastomose caudally and unite with the Wolffian duct via a common collecting duct. The Wolffian duct is secretory, but not to the extent of the collecting duct, synthesizes neutral carbohydrates and proteins, and is also lined by apical ciliated cells intercalated between secretory cells. Although functional aspects associated with the morphological variation along the length of the proximal portions of the nephron have been investigated, the role of a highly secretory collecting duct has not. Historical data that implicated secretory activity concordant with mating activity, and similarity of structure and chemistry to sexual segments of the kidneys in other vertebrates, lead us to believe that the collecting duct functions as a secondary sexual organ in Ambystoma maculatum. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Quantitative immunogold localization of Na, K-ATPase along rat nephron.

Ultrastructural localization of Na, K-ATPase alpha-subunit along rat nephron segments was investigated quantitatively by immunogold electron microscopy on LR-White ultrathin sections using affinity-purified antibody against alpha-subunit of the enzyme. Ultrathin sections were incubated with the antibody at a saturation level and the number of gold particles bound per micron of the plasma membrane (particle density) of the tubular epithelial cells from the proximal tubule to the collecting duct was determined. In all the tubular epithelial cells, gold particles were located exclusively on the basolateral surface, and no significant binding of gold particles to the apical surface was observed. Distribution of gold particles on the basolateral membranes was quite heterogeneous; lateral membranes and infolded basal membranes were highly labeled, whereas the basal membranes which are in direct contact with the basal lamina were scarcely labeled. The average particle density on the basal surface was highest in the distal straight tubule cells (11.4 units), very high in the distal convoluted tubule cells (9.8 units), intermediate in the proximal tubule cells (3.3 units), in the connecting tubule cells (4.3 units), and in the principal cells of the collecting duct (5.6-3.8 units), low in the thin limb of Henle's loop (1.0 unit), and at the control level in the intercalated cells in the connecting and collecting duct. The relative number of gold particles/mm nephron segment and the relative number of gold particles in the various nephron segments were calculated using quantitative morphological data. The estimated distribution profile of the former was in good agreement with the Na, K-ATPase activity profile in rat nephron, which was determined biochemically with a microenzymatic method.     

Structure of the kidney in the coelacanth Latimeria chalumnae with reference to osmoregulation

The morphology of the nephrons of the coelacanth Latimeria chalumnae was investigated by light microscopy. Each nephron is composed of a large renal corpuscle with well‐vascularized glomerulus, non‐ciliated neck segment, proximal convoluted tubule divided into distinct first and second segments, non‐ciliated intermediate segment, distal tubule, collecting tubule and collecting duct. The parietal layer of the Bowman's capsule of the renal corpuscle is composed of low cuboidal cells. The short non‐ciliated neck segment is lined by cuboidal epithelium. The first and second proximal segments display a prominent brush border and contain amorphous material in their lumen. The second proximal segment differs from the first segment in having taller columnar epithelium and a relatively narrow lumen. The intermediate segment is lined by non‐ciliated columnar epithelium and its lumen appears empty. The distal tubule is narrow in diameter and its cuboidal epithelium is devoid of intercalated cells. A unique feature of L. chalumnae is having binucleate cells in the tubule and collecting duct epithelium. The renal arteries have poorly developed tunica media and its cells contain granular material. The structure of L. chalumnae nephrons correlates well with their osmoregulatory function and resembles those of euryhaline teleosts.     

Characterization and metabolism of canine proximal tubules, thick ascending limbs, and collecting ducts in suspension

Preparations of distinct nephron segments were obtained from dog kidneys by collagenase treatment. Four morphologically different tissues were isolated: glomeruli, proximal tubules, thick ascending limbs, and papillary collecting ducts. Each segment possessed a characteristic assay of membrane-bound and cytoplasmic enzymes. Specific metabolic characteristics also were found: gluconeogenesis and ammoniagenesis in proximal tubules, glycolytic aerobic metabolism in thick ascending limbs, and glycolytic anaerobic metabolism in papillary collecting ducts. The assay of Na+ -K+ ATPase, H+ -ATPase, and Ca2+ -ATPase activities in these nephron segments demonstrated a specific enrichment of Na+ -K+ ATPase in thick ascending limbs, and of H+ -ATPase in proximal tubules and papillary collecting ducts. Tubular respiration in the absence or presence of ouabain, 1,3-dicyclohexylcarbodiimide, or furosemide demonstrated that the respiration of each segment could be correlated to the activity of specific ion motive ATPases. Furthermore, a tight coupling between ion transport, ATP turnover, and substrate oxidation was demonstrated. These isolated tubular structures are thus viable and capable of transepithelial transport. Our preparation provides large amounts of defined population of tubules and are thus useful for the study of biochemical and functional heterogeneity along the nephron.     

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.     

Carbonic anhydrase activity in kidney and lower intestine of the European starling

A histochemical investigation of kidney and lower intestine of the European starling (Sturnus vulgaris) shows no carbonic anhydrase activity in proximal convoluted tubules, although activity is seen in similarly prepared sections of rat proximal tubules. Early distal tubule cells in the starling are stained throughout the cytoplasm and at the apical and highly infolded basolateral membranes. Late distal tubules lose apical activity and have reduced basolateral infolding, resulting in less intense staining. Darkly stained intercalated cells appear in the connecting tubules and cortical collecting ducts. Both of these segments also show intense basolateral staining. Medullary cones of the starling are highly organized, with central zones containing unstained thin descending limbs of loops of Henle, surrounded by both medullary collecting ducts with only scattered cells staining for enzyme, and by thick ascending limb segments. The latter contain many uniformly stained cells intermingled with occasional unstained cells. Scattered cells of the starling colonic villi demonstrate intense apical brush border membrane staining as well as cytoplasmic staining. Cells lining the cloaca stain less intensely. A biochemical assay for carbonic anhydrase was used to quantify enzyme activity in these tissues. Starling kidney contained 1.96 ± 0.33 (mean ± SEM) enzyme units/mg protein, less than half the activity seen in rat kidney. Stripped colonic epithelium contained 0.66 ± 0.15 enzyme units/mg protein. These quantitative results correlate well with the interpretations derived from the histochemical observations. The lack of proximal tubule carbonic anhydrase activity suggests that the avian kidney relies more on distal nephron segments to achieve net acidification of the urine.     

Colocalization of the (Pro)renin Receptor/Atp6ap2 with H+-ATPases in Mouse Kidney but Prorenin Does Not Acutely Regulate Intercalated Cell H+-ATPase Activity

The (Pro)renin receptor (P)RR/Atp6ap2 is a cell surface protein capable of binding and non-proteolytically activate prorenin. Additionally, (P)RR is associated with H⁺-ATPases and alternative functions in H⁺-ATPase regulation as well as in Wnt signalling have been reported. Kidneys express very high levels of H⁺-ATPases which are involved in multiple functions such as endocytosis, membrane protein recycling as well as urinary acidification, bicarbonate reabsorption, and salt absorption. Here, we wanted to localize the (P)RR/Atp6ap2 along the murine nephron, exmaine whether the (P)RR/Atp6ap2 is coregulated with other H⁺-ATPase subunits, and whether acute stimulation of the (P)RR/Atp6ap2 with prorenin regulates H⁺-ATPase activity in intercalated cells in freshly isolated collecting ducts. We localized (P)PR/Atp6ap2 along the murine nephron by qPCR and immunohistochemistry. (P)RR/Atp6ap2 mRNA was detected in all nephron segments with highest levels in the collecting system coinciding with H⁺-ATPases. Further experiments demonstrated expression at the brush border membrane of proximal tubules and in all types of intercalated cells colocalizing with H⁺-ATPases. In mice treated with NH₄Cl, NaHCO₃, KHCO₃, NaCl, or the mineralocorticoid DOCA for 7 days, (P)RR/Atp6ap2 and H⁺-ATPase subunits were regulated but not co-regulated at protein and mRNA levels. Immunolocalization in kidneys from control, NH₄Cl or NaHCO₃ treated mice demonstrated always colocalization of PRR/Atp6ap2 with H⁺-ATPase subunits at the brush border membrane of proximal tubules, the apical pole of type A intercalated cells, and at basolateral and/or apical membranes of non-type A intercalated cells. Microperfusion of isolated cortical collecting ducts and luminal application of prorenin did not acutely stimulate H⁺-ATPase activity. However, incubation of isolated collecting ducts with prorenin non-significantly increased ERK1/2 phosphorylation. Our results suggest that the PRR/Atp6ap2 may form a complex with H⁺-ATPases in proximal tubule and intercalated cells but that prorenin has no acute effect on H⁺-ATPase activity in intercalated cells.     

The mature mesonephric nephron of the rabbit embryo

Summary Transmission electron micrographs of the mesonephric nephron in 18 day rabbit embryos reveal major cytological structures reappearing in the nephron of the definitive rabbit kidney. The initial segment of the proximal tubule resembles (despite quite different cell proportions) the cell picture of the metanephric S₂-segment. The changes occurring at the end of the terminal proximal segment, the decrease in cell size, flattening of the nuclei, shortening of the brush border and reduction of Golgi profiles and endocytotic organelles largely parallel those between S₂ and S₃. The type of increased basolateral cell face of the proximal and distal tubule cells shows only quantitative differences to their metanephric counterparts. The distal tubule, which cannot be further subdivided (except the macula densa-region) exhibits varying degrees of cell interdigitations with vertically arranged and partially arching lateral ridges. This tubule matches closely the metanephric medullary straight part of the distal tubule, so that the sequence of the first mesonephric nephron segments is similar to the metanephric ones with the exception that the thin limb of Henle is absent. The large macula densa-region is characterized by its cell height and distended infranuclear spaces. The principal cells of the collecting tubule, with a few basal infoldings and intense short lateral interlockings resemble metanephric cells of the outer medullary collecting duct. The mitochondria-rich intercalated cells occur in dark and light contrasting forms and are more frequent than was evident from our SEM-study. The homogeneous cell population of the Wolffian duct is characterized by large glycogen deposits and comparatively smooth cell faces.     

Angiotensin II stimulates H?-ATPase activity in intercalated cells from isolated mouse connecting tubules and cortical collecting ducts

Intercalated cells in the collecting duct system express V-type H(+)-ATPases which participate in acid extrusion, bicarbonate secretion, and chloride absorption depending on the specific subtype. The activity of H(+)-ATPases is regulated by acid-base status and several hormones, including angiotensin II and aldosterone. Angiotensin II stimulates chloride absorption mediated by pendrin in type B intercalated cells and this process is energized by the activity of H(+)-ATPases. Moreover, angiotensin II stimulates bicarbonate secretion by the connecting tubule (CNT) and early cortical collecting duct (CCD). In the present study we examined the effect of angiotensin II (10 nM) on H(+)-ATPase activity and localization in isolated mouse connecting tubules and cortical collecting ducts. Angiotensin II stimulated Na(+)-independent intracellular pH recovery about 2-3 fold, and this was abolished by the specific H(+)-ATPase inhibitor concanamycin. The effect of angiotensin II was mediated through type 1 angiotensin II receptors (AT(1)-receptors) because it could be blocked by saralasin. Stimulation of H(+)-ATPase activity required an intact microtubular network--it was completely inhibited by colchicine. Immunocytochemistry of isolated CNT/CCDs incubated in vitro with angiotensin II suggests enhanced membrane associated staining of H(+)-ATPases in pendrin expressing intercalated cells. In summary, angiotensin II stimulates H(+)-ATPases in CNT/CCD intercalated cells, and may contribute to the regulation of chloride absorption and bicarbonate secretion in this nephron segment.     

Potassium channels along the nephron

The K+ channels that are present in three different nephron segments, the Necturus proximal, Amphiuma early distal (diluting segment), and rabbit collecting tubule have been examined. Ca2+-sensitive K+ channels were present in the apical membranes of the cells lining all these segments. The channels were all voltage-sensitive and their open probability increased with membrane depolarization. Because of the ubiquitous distribution, it is suggested that this channel is responsible for K+ secretion by the nephron and that the same intracellular regulators act throughout the various segments. Basolateral K+ channels have been examined only in Necturus proximal tubules. This channel is apparently insensitive to Ca2+; the voltage dependence is exactly opposite to that of the apical K+ channels; that is, hyperpolarizing potentials caused an increase in open probability. These differences in regulatory factors permit the independent regulation of apical and basolateral membrane K+ permeabilities that must occur in renal cells.     

Ultrastructural organization of the transition from the distal nephron to the collecting duct in the desert rodent Psammomys obesus

Summary The transition from the nephron to the collecting duct is formed by three tubular segments (convoluted part of the distal tubule, connecting tubule, cortical collecting duct), which in the desert rodent, Psammomys obesus, transform gradually from one segment to the next, due to intermingling of their different cell types.The convoluted part of the distal tubule (DTC) starts abruptly, shortly beyond the macula densa and initially is homogeneously composed of characteristic DTC-cells. Subsequently, the DTC-cells intermingle with intercalated cells. The first appearance of the connecting-tubule cell, which gradually replaces the DTC-cell, is regarded as the beginning of the connecting tubule. The major portion of the connecting tubule is lined by connecting-tubule cells and intercalated cells. The first appearance of the principal cell between them defines the beginning of the cortical collecting duct, which in the medullary ray is lined by principal and intercalated cells only.Each cell type is described in detail and discussed in relation to the assumed function of the tubular segments.Interspecies differences in the cellular composition of the transitional zone from the nephron to the collecting duct are discussed in relation to the different organization of the collecting duct system.     

Histochemical localization of Mg2+-Na+-K+-adenosine triphosphatase in different stages of the sheep mesonephros.

Mesonephroi of sheep embryos ranging from 12 to 100 mm C.R. length were examined for the occurrence and localization of transport-ATPase. Native cryostat sections were incubated according to the technique of Guth and Albers for demonstrating the nitrophenylphosphatase activity of Mg2+-Na+-K+-adenosine triphosphatase. The basal cytoplasm of the collecting tubule of the narrow segment of the distal tubule exhibit strong activity, the wide segment of the distal tubule is moderately active. Glomeruli, proximal tubule, and Wolffian duct remain unstained. The basal labyrinths of the reactive nephron segments are believed to be the sites of a Na+-K+ exchange pump. In mature and regressing mesonephroi, the findings fully agree with biochemical data; in maturating mesonephroi, whose basal labyrinth is not yet fully established, the biochemical assay proves to be more sensitive. The specifity of the reaction was ascertained by diverse inhibitors and activating ions. The localization of Mg2+-ATPase is different to the above mentioned reaction pattern, as it shows moderate activity in the proximal tubule, too (mature mesonephros). Mesonephroi of very young embryos exhibit strongest Mg2+-ATPase activity in the proximal tubule; here the distal and collecting tubule stain only moderately.     

Immunocytochemical localization of cathepsin B in rat kidney. I. Light microscopic study using the indirect immunoenzyme technique

Localization of cathepsin B in rat kidney was studied using immunocytochemical techniques. Cathepsin B was purified from rat liver and antibody to it was raised in rabbits. The antibody reacted with a lysosomal extract of rat kidney to form a single precipitin line in a double-diffusion test. Immunoblot analysis of lysosomal cathepsin B of rat kidney showed two species of 29K and 25K MW. After removal of Epon, semi-thin sections of glutaraldehyde-fixed tissue were stained by the indirect immunoenzyme technique. Dark-brown reaction product, indicating the antigenic sites for cathepsin B, was found in cytoplasmic granules throughout the nephron. Staining intensity and size of the positive granules varied widely in each segment of the nephron. In the glomeruli and distal tubules, a few small cytoplasmic granules were stained. In the proximal tubules, the S1 segment exhibited many large granules which were most heavily stained, whereas the S2 and S3 segments contained few positive granules. All segments of the distal tubules showed the smallest amount of positive granules. A few positive granules were also noted in the cortical and medullary collecting tubules. Control experiments confirmed the specificity of the staining. The results indicate that the major site for cathepsin B in rat kidney is the S1 segment of the proximal tubule which is known to actively take up proteins leaked through the glomerulus.