Ultrastructural tracer studies on the permeability of the Malpighian tubule of the Pill Millipede,Glomeris marginata (Villers)

Summary The electron-dense tracers ferritin, and iron-dextran, and the protein horseradish peroxidase, have been used to investigate the ultrastructural basis of permeability in the upper and lower segments of the Malpighian tubules of Glomeris marginata. All these materials were able to cross the basal lamina and enter the tubule lumen of the upper segment, and it was established that horseradish peroxidase was able to enter the channels which interrupt the apical junctions.In the upper segment, ferritin, iron-dextran, and horseradish peroxidase are all taken up and accumulated within intracellular vesicles. In the lower segment ferritin and iron-dextran enter the cells but become generally distributed over the cyptoplasm, as well as entering membrane-bounded vacuoles. The behaviour of horseradish peroxidase could not be assessed owing to the presence of endogenous peroxidase activity in the cells.After fixation by direct application of glutaraldehyde to the undissected tubules, the extracellular spaces contained large numbers of membrane-bounded vesicles. The significance of these observations is discussed in relation to the physiological activities of the tubules.The authors are indebted to the Science Research Council for financial supportThe authors wish to thank Mrs. Margarita Petri for her technical assistance and advice     

Localization of carbonic anhydrase activity in the vertebrate nephron

Synopsis The localization of carbonic anhydrase activity in the vertebrate nephron has been examined with particular reference to the proximal tubule and collecting duct. In all species studied, activity was present in the proximal tubular epithelium. In the pigeon and turtle, distinctive and similar patterns of staining were observed in the glomerulus and first portion of the proximal tubule. In the rat and rhesus monkey, the entire proximal tubule exhibited activity; in these species it has been shown previously with micropuncture techniques that there is a high absorptive capacity of this nephron segment for bicarbonate. In contrast, large portions of the dog proximal tubule were inactive; similar studies in this animal have shown tubular concentrations of bicarbonate only slightly lower than plasma levels. In the rat and dog, the entire length of the collecting duct was diffusely and intensely active; in contrast, pigeon collecting duct showed no activity. An alternating pattern of inactive and intensely active cells was observed in the collecting ducts of the toad, turtle, rabbit and monkey. A similar pattern has been described in the turtle and toad bladder, tissues utilized forin vitro studies of ion transport and H⁺ secretion.     

Glycosidases and acid-invertase activities are not correlated with Phaseolus hypocotyl growth

Changes in α-galactosidase, β-galactosidase, β-glucosidase and acid invertase activities were examined in Phaseolus vulgaris hypocotyls treated with gibberellic acid (GA), naphthyl acetic acid (NAA) and distilled water (DW) (control) in light condition. The activities were estimated both in cytoplasmic and ionically wall-bound fraction. The upper segment showed considerable elongation growth while there was hardly any growth in lower segment. GA and NAA showed distinct promotion and inhibition respectively in hypocotyl growth in upper segment. The glycosidase activities were detected in both the fractions but the activity was more pronounced in cytoplasmic than in wall fraction. Acid invertase activity was present only in cytoplasmic fraction. In lower segment, in both cytoplasmic and wall fraction, the glycosidase activity, in general, showed a decreasing trend and no effect of treatment could be envisaged. In upper segment, though the trend was similar to the lower one, in α- and β-galactosidase NAA treated segment had more activity. Invertase activity also did not show a clear trend to implicate its function in hypocotyl elongation growth. The results are discussed in relation to establishing a correlation between an activity (glycosidase and invertase) and a physiological process (hypocotyl elongation). It is concluded that these wall-loosening enzymes have no role in elongation growth of Phaseolus vulgaris hypocotyls.     

Histological and enzyme histochemical changes in the kidney of male bullhead (Cottus gobio) during the spawning period

Histological and enzyme histochemical studies were carried out on the excretory kidney of the male bullhead ( Cottus gobio ). During the spawning season striking morphofunctional changes were observed in the second proximal segment of the kidney tubule. The tubular epithelium was greatly hypertrophied, strongly basophilic and produced a PAS-positive secretion. The enzyme histochemical pattern also changed conspicuously during this time: the alkaline phosphatase activity in the brush border was greatly reduced; the acid phosphatase and non-specific esterase activity in the cytoplasm was distinctly elevated.     

Histological and histochemical studies of the stickleback Gasterosteus aculeatus L. kidney. III. Activity of various phosphatases depending on secretion]

1. The activity of alkaline phosphatase is intensely positive in proximal tubule I and II during the breeding season. In the kidney of secretion producing the enzyme is detectable as against to kidney of winter on the whole proximal tubule II. 2. In the kidney what is able to build a nest, concentration and size of acid phosphatase granules are very increasing in proximal tubule II. 3. The detection of unspecific esterase was negatively already. 4, The reaction of glucose-6-phosphatase is slightly demonstrable in cells of proximal segment of secretion producing what are enlarged fourfold. 5; From the varied reaction of acid and alkaline phosphatase we conclude that both are to set in relation to excretory activity, but not to process of synthesis in kidney of late-spring fish.     

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.     

Localisation of immunoreactive kininogen and tissue kallikrein in the human nephron

The cellular localisation of kininogen and its relationships with tissue kallikrein containing cells was studied in the human kidney by the peroxidase-antiperoxidase method using antisera to human LMW kininogen and to human tissue kallikrein. Immunoreactive kininogen was localised in the principal cells of collecting ducts. Immunoreactive tissue kallikrein was detected in the connecting tubule cells, segment of the nephron preceding the cortical collecting ducts. The co-existence of tissue kallikrein and kininogen in the same transitional tubule, but in different cells, was established by the use of serial sections and double immunostaining. This anatomical relationship is in accordance with known studies that describe intermingling of principal cells and connecting tubule cells where connecting tubules merge into cortical collecting ducts in the human nephron. The close relationship between cells that contain tissue kallikrein and its substrate, kininogen, suggests that kinins could be generated in the lumen of distal cortical segments of the human nephron.     

Localisation of immunoreactive kininogen and tissue kallikrein in the human nephron

Summary The cellular localisation of kininogen and its relationships with tissue kallikrein containing cells was studied in the human kidney by the peroxidase-antiperoxidase method using antisera to human LMW kininogen and to human tissue kallikrein. Immunoreactive kininogen was localised in the principal cells of collecting ducts. Immunoreactive tissue kallikrein was detected in the connecting tubule cells segment of the nephron preceeding the cortical collecting ducts. The co-existence of tissue kallikrein and kininogen in the same transitional tubule, but in different cells, was established by the use of serial sections and double immunostaining. This anatomical relationship is in accordance with known studies that describe intermingling of principal cells and connecting tubule cells where connecting tubules merge into cortical collecting ducts in the human nephron. the close relationship between cells that contain tissue kallikrein and its substrate, kininogen, suggests that kinins could be generated in the lumen of distal cortical segments of the human nephron.     

Histochemistry of the juxtaglomerular apparatus in the toad Bufo bufo. Acid phosphatase activity of the epitheloid cells in the glomerular afferent arterioles

Histochemical techniques for acid phosphatase activity applied to kidney tissue of the toad Bufo bufo demonstrate that a high enzyme activity is present in the dense granules of the proximal tubule cells, but also in the media cells in the wall of the glomerular afferent arterioles. The acid phosphatase activity is confined to the characteristic granules in these juxtaglomerular cells, which therefore are lysosomal in nature.     

Seasonal changes in the ultrastructure of the kidney collecting tubule in the lizard Podarcis ( = Lacerta) taurica

In female Podarcis taurica the kidney collecting tubule always consists entirely of mucous-secreting cells. In males it has a seasonally variable sexual segment and a non variable mucous-secreting segment. In April the sexual segment is composed of columnar cells with cytoplasm rich in ribosomes and Golgi bodies and apical clusters of large vesicles with fibrous contents. The terminal region of the sexual segment also has pillar-shaped cells resembling those of the mucous-secreting segment. By May the accumulation of apical vesicles reaches a maximum, and many cells have apparently extruded their secretion into the lumen. In July all the cells are pillar shaped with dilated endoplasmic reticulum but with few apical vesicles. In September the sexual segment has some cells resembling those of the mucous-secreting segment and others the sexual segment pillar cells in April. It is suggested that during sexual activity in the spring the sexual segment secretes a spermatozoon-nutrient protein but subsequently reverts to mucous secretion. The non variable mucous-secreting regions in both males and females consist of mucous, intermediate, and dark cells. Mucous cells have apical masses of closely packed droplets, whereas dark cells have dense cytoplasm and small, loosely associated apical vesicles. Intermediate cells have some dark cell features but mucous cell apical vesicles. The dark, intermediate, and mucous cells probably represent activity states of a single type. The mucous secretion is interpreted as a protective material which lines the urinary passage and coats the secreted solid urates. Elaborated intercellular spaces in the mucous-secreting regions may indicate a water absorption capacity in urine concentration.     

The kidney of the prussian carp, Carassius auratus gibelio (Cyprinidae). III. Glycogen-rich cells in the distal segment of nephronic tubule

Glycogen may be present in considerable amount in the distal segment of the nephronic tubule of a teleost freshwater fish, as revealed by light- and electron-microscopical observation. Adaptation of Crassiss auratus gibelio to increased salinity causes alterations of the cell fine structure (e.g. mitochondria and baso-lateral infoldings) but apparently does not influence deposition of glycogen. However glycogen storage is directly controlled by environmental temperature and may be part of an annual rhythm in carbohydrate metabolism.     

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 sexual segment of hemidactylus turcicus and the evolution of sexual segment location in squamata

The kidneys of the Mediterranean Gecko, Hemidactylus turcicus (Gekkonidae), were investigated using light and electron microscopy with the primary focus placed on morphology of the sexual segment of the kidney. The nephrons of male H. turcicus are composed of five distinct regions: 1) a renal corpuscle and glomerulus, 2) a proximal convoluted tubule, 3) an intermediate segment, 4) a distal convoluted tubule, and 5) the sexual segment of the kidney/collecting duct. Female H. turcicus is similar but lack a sexual segment of the kidney. The sexual segment of the kidney is hypertrophied during the months of March through August, which corroborates previous reports of reproductive activity. During inactive months, the sexual segment of the kidney is nondiscernable from the collecting ducts. The sexual segment consists of tall columnar epithelial cells with basally positioned nuclei. Perinuclear Golgi complexes and rough endoplasmic reticulum are present. Secretory granules, which fill the apices of the epithelial cells, are electron dense and released into the lumen by a merocrine secretory process. Narrow intercellular canaliculi separate each epithelial cell and are sealed by tight junctions at the luminal aspect. Basally, leukoctyes are observed within the intercellular canaliculi and outside the basal lamina. Mast cells can be found just outside the basal lamina in close association with renal capillaries. The sexual segment of the kidney of H. turcicus is similar to that of three unrelated lizards for which ultrastructure was investigated with secretion mode being the major difference Also, H. turcicus is similar to most other lizards in that complete regression occurs during reproductive inactivity, but differs in this trait from the skink, Scincella lateralis, and most snakes which display a hypertrophied sexual segment of the kidney throughout the entire year. Although some unique similarities appear during the optimization, no direct patterns or directions are observed, and only the molecular based phylogeny resolves the ancestral condition of the Squamata as the sexual segment of the kidney being observed in the distal convoluted tubule, collecting duct, and ureter. J. Morphol., 2011. © 2011 Wiley‐Liss, Inc.     

Comparative impact of cephaloridine on glutathione and related enzymes in LLC-PK1, LLC-RK1, and primary cultures of rat and rabbit proximal tubule cells

Among kidney tubular epithelial cell types, proximal tubule cells are one of the major renal targets for xenobiotics. Several in vitro culture models have been proposed for use of proximal tubule cells for in vitro pharmacotoxicology studies. This paper reports a comparative study of the response to cephaloridine exposure of two established cell lines from pig (LLC-PK1) and rabbit (LLC-RK1) kidneys and primary cultures of rat and rabbit proximal tubule cells. These cultured cells were first compared for their levels of activity of -methylglucopyranoside transport, alkaline phosphatase, succinate dehydrogenase, and NADPH cytochrome c reductase, their glutathione-dependent activity levels, and their adenylate cyclase response pattern to stimulation by PTH and AVP. The results presented show major phenotypic differences between these four cellular models. The differences observed in glutathione-dependent mechanism activities and regulation may in part be responsible for the variability of the responses of these four cellular models when exposed to cephaloridine.Abbreviations AVP arginine vasopressin - GGT -glutamyl transpeptidase - GRED glutathione reductase - GSH glutathione - GST glutathione S-transferase - PTC proximal tubule cells - PTH parathyroid hormone - SDH succinate dehydrogenase     

Use of phoA fusions to study the topology of the Escherichia coli inner membrane protein leader peptidase.

A topology of the Escherichia coli leader peptidase has been previously proposed on the basis of proteolytic studies. Here, a collection of alkaline phosphatase fusions to leader peptidase is described. Fusions to the periplasmic domain of this protein exhibit high alkaline phosphatase activity, while fusions to the cytoplasmic domain exhibit low activity. Elements within the cytoplasmic domain are necessary to stably anchor alkaline phosphatase in the cytoplasm. The amino-terminal hydrophobic segment of leader peptidase acts as a weak export signal for alkaline phosphatase. However, when this segment is preceded by four lysines, it acts as a highly efficient export signal. The coherence of in vitro studies with alkaline phosphatase fusion analysis of the topology of leader peptidase further indicates the utility of this genetic approach to membrane protein structure and insertion.     

Histochemical analysis of molluscan stomach and intestinal alkaline phosphatase: A sialoglycoprotein

Summary Though sialoprotein nature of alkaline phosphatase of certain mammalian organs has been suggested by biochemical investigations, no histochemical techniques have yet been applied to elucidate this concept. With this view, the alkaline phosphatase of stomach and intestine of a mollusc—Semperula maculata—was analysed histochemically to elucidate its sialoglycoprotein nature. The localisation of alkaline phosphatase and sialic acid was investigated by employing well known and standard histochemical techniques.Alkaline phosphatase was localised selectively in the brush border of the mucosa of stomach and intestine, it was Mg⁺⁺ nonsensitive but showed a structure-linked sensitivity to phenylalanine. The sialomucins were selectively localised in the brush border, whereas the goblet cells contained both the sialomucins and sulfomucins, and the connective tissue of lamina propria contained sulfomucins. The localisation of alkaline phosphatase and sialomucins in the brush border uniquely coincided with each other. The alkaline phosphatase activity in the brush border was completely lost after neuraminidase treatment at 37.5° C for 16 h. Such effect of neuraminidase on alkaline phosphatase activity was pH dependent and controlled by velocity of reaction. Heat-inactivated neuraminidase showed no effect on alkaline phosphatase activity.These histochemical results have been interpreted as suggesting a sialoglycoprotein nature of alkaline phosphatase in the brush border, and sialic acid somehow seems to be essential for enzyme activity. These results, thus, indicate necessity of visualising some of the sialo-glycoproteins as macromolecules with catalytic activity.     

Ultrastructure of differentiated malpighian tubules from cockroach nymphs during the molting cycle

Differentiated Malpighian tubules of Periplaneta americana nymphs consist of four distinct regions. The distal, middle, and proximal regions are similar to the same regions in adult tubules. However, the transparent portion of the middle region was found to have ultrastructural characteristics different from those of the longer opaque segment of the middle region and the two other tubule regions. This newly distinguished region is called the lower middle region. Transitional zones, areas where cells show characteristics of two adjacent regions, are apparent between the distal and middle regions and between the middle and lower middle regions. The middle region of primary tubules undergoes an increase in autophagic activity and a modification of its basal infoldings and microvilli shortly before each molt. An increase in autophagic activity is also observed in the lower middle region near the time of molting.     

Histochemical,autoradiographic and electron microscopic investigations of the renal proximal tubule of male and female rats after castration

Summary After castration of 90-day-old male and female rats, changes appear in the renal proximal tubule. A distinction can be made between early changes (up to 10th postoperative day) and later changes (20th–30th postoperative day). Between the 3rd and 5th day after castration the kidney of the females shows an increase in free estrogen receptors (biochemical studies) which are localized in the pars contorta of the proximal tubule (autoradiographic studies), while the male kidney shows a marked increase in urinary protein excretion up to the 10th day after castration. Proximal tubule changes detectable histochemically and electron microscopically do not appear until day 20 or 30 after castration. The results of castration are similar in segments S₁ and S₂. By days 20 and 30 after castration there is a decrease in the activity of lysosomal enzymes (acid phosphatase, acid -galactosidase). Electron microscopy shows a conspicuous decrease in the number of giant lysosomes (mainly in females) and apical vacuoles (mainly in males). A marked increase in the number of lysosomes is found in the S₃ segment; females always have more lysosomes than males. The number of peroxisomes is also greatly increased; they appear circular in the females but can assume bizarre shapes in the males. Lipid droplets appear in the basal region of the tubule cell of segments S₂ and S₃ in the males. The sex differences are preserved in all segments even after castration and become even more pronounced in the S₃ segment.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)Dedicated to Prof. Dr. W. Graumann, Department of Anatomy, University of Tübingen, on the occasion of his 65th birthday     

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.     

Differentiation of epithelial cells in human jejunum: Localization and quantification of aminopeptidase,alkaline phosphatase and aldolase isozymes in tissue sections

Summary Sections from human jejunum were stained histochemically for aminopeptidase and alkaline phosphatase and the aldolase isozymes were detected with the mixed aggregation immuno-cytochemical technique. All enzyme concentrations increased from the bottom to the upper part of the crypt. The concentration of aldolase-A per cell was the same in the upper part of the crypt and the villus, whereas the concentration of the other three enzymes was still higher. Therefore, high amounts of aldolase-B, aminopeptidase and alkaline phosphatase are present in cells highly active in absorption in a fashion similar to that found in the proximal tubule cells of kidney. The relatively undifferentiated cells of the crypts contained both aldolase-A and aldolase-B. Alkaline phosphatase gains its full activity later than aminopeptidase. The synthesis of microvillar membrane enzymes comes to an end earlier than that of the cytosol enzymes.