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 kidney of stickleback Gasterosteus aculeatus L. I. Morphological changes of the kidney epithelial cells during the spawning period]

1; The tubule of kidney of Gasterosteus aculeatus L. consists of four histologically different regions: Proximal tubule I and III, connection segment and collecting tubule. 2. All of tubule segments inclusively the urinary duct out of the proximal tubule I are showing synthesis of secretion. 3. There are producing two various secretion in two distinct species of cells. From the cells of proximal tubule II are secreted and extruded a granular secretion and from the cells of abducted urinary ducts (connection segment, collecting duct and urinary duct) a hyalo-mucous secretion. 4. During the breeding season the morphological variationes were divided into three stadiums, the stadium of differentiation, of producing of secretion and of reproduction. In second stadium were differenced three phases, in particular characterizing by rhythmical variationes of nucleus structure and synthesis of secretion as extrustion. 5. There are discussing the parallels to synthesis of secretion in glandular cells.     

Enzymatic histochemistry of mouse kidney in plastic

Two-micrometer sections of methacrylate-embedded kidney were used to investigate the enzymatic activities of mouse kidney where the proximal tubule and Bowman's capsule from the same corpuscle were viewed in the same section. Alkaline phosphatase, acid phosphatase, 5'-nucleotidase, gamma-glutamyl transpeptidase, N-acetyl-beta-glucosaminidase, leucine aminopeptidase, alpha-naphthyl butyrate esterase, and adenosine triphosphatase activities were observed in the proximal tubule, but only 5'-nucleotidase, alpha-naphthyl butyrate esterase, and alkaline phosphatase were observed in the squamous portion of the parietal epithelium of Bowman's capsule. The use of methacrylate-embedded tissue allowed more precise localization of enzymatic activity than is possible with most frozen sections. This may provide interesting applications not only for characterization of kidney diseases but also for characterization of other normal and abnormal tissues.     

Influence of L-thyroxine upon enzymatic activity in the renal tubular epithelium of the rat under normal conditions and in mercury-induced lesions. I. Histochemical studies of alkaline phosphatase, acid phosphatase, adenosine- tri-phosphatase and leucine-aminopeptidase.

HgC12-induced renal tubular lesions in the rat present histochemically with a transitory decrease of alkaline phosphatase, adenosinetriphosphatase (ATPase), and leucine-aminopeptidase activity. The toxic alterations of enzyme activity were more pronounced in the pars recta of the proximal tubule and in the loop of Henle, as compared with the tubulus contortus I. L-thyroxine treatment leads to an accelerated reversal of that enzymatic defect, followinga characteristic pattern, and to a differentiating increase of acid phosphatase and ATPase activity in certain parts of the normal renal tubule. The observations are discussed with reference to the specific mode of action of sublimate and l-thyroxine upon the tubular enzymes and to the well-known metabolic and functional influences of thyroid hormone on the kidney.     

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。       

5′-Nucleotidase

Summary 5′-Nucleotidase and alkaline phosphatase activity was investigated in the developing kidney of the mouse by histochemical and electrophoretic methods. The growth of the kidneys was studied by determining the incorporation of radioactive thymidine by autoradiography. During development the isoenzyme patterns of 5′-nucleotidase and alkaline phosphatase behaved in a different way. In correlating the histochemical and electrophoretic changes, it has been found that the 5′-nucleotidase isoenzymes as well as the alkaline phosphatase isoenzymes are located in different parts of the kidney. In the convoluted part of the proximal tubule 5′-nucleotidase isoenzyme 3 and alkaline phosphatase isoenzyme 5 are present, while in the straight part of this tubule 5′-nucleotidase isoenzyme 5 and — upto three weeks — alkaline phosphatase isoenzyme 3 are located. So in tissue structures having different functional capacities, different isoenzymes of 5′-nucleotidase and alkaline phosphatase are found.     

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.     

Phosphatases and oxidative enzymes in the kidney of the Prussian carp (Carassius auratus gibelio Bloch) adapted to salt water

The distribution and activities of phosphatases and oxidative enzymes have been determined with the help of histochemical methods in the kidney of the Prussian Carp, a stenohaline freshwater-fish. In addition to fish maintained in freshwater aquaria, a group of the animals used has been adapted to seawater of moderate salinity. The following pattern of enzyme reaction intensities has been observed in the various kidney structures: Strong reactions of alkaline phosphatase in the nephron are confined to the glomerular capillary convolute and the brush border of proximal segments. Equally enzyme activities are observed in the connective tissue sheath of the collecting duct -- archinephric duct system. Acid phosphatase can be detected in all segments of the nephronic tubule, strong activities are found in the proximal segment (P I), in the epithelium of the archinephric duct, and, especially, in the interstitial tissue. ATPase reacts strongly positive in epithelial cells of the distal tubule and the collecting duct -- archinephric duct system. ATPase reactions are inhibited by Ouabain, and therefore can be regarded as reactions of Na--K-ATPase. Mitochondrially bound oxidative enzymes, connected with the citric acid cycle and the respiratory chain, show very strong reaction intensities in the distal tubule and the collecting duct- archinephric duct system, while the glomeruli generally exhibit negative reactions. Lactate -- and malate dehydrogenases are found to react weakly to negatively throughout the whole kidney. Maintenance in seawater does not deeply affect the enzyme pattern of the kidney of the Prussian carp, with exception of some oxidative enzymes, reacting weaker in the distal tubule and the collecting duct-archinephric duct system. In addition, the epithelial cells of the archinephric duct of seawater adapted fish show a marked apical localization of reaction products for these enzymes. Possible relations between enzyme histochemistry and fish kidney physiology are discussed, in connection with comparative aspects of the enzyme histochemistry of the vertebrate kidney. A short review of normal histology and function of the kidney of the Prussian carp is added.     

Localization of d-myoinositol 1:2-cyclic phosphate 2-phosphohydrolase in rat kidney

1. On subcellular fractionation of rat kidney homogenates by differential and density-gradient centrifugation, the bulk of the inositol 1:2-cyclic phosphate 2-phosphohydrolase activity remains with the alkaline phosphatase activity, suggesting localization in the brush borders of the proximal tubules. 2. Histochemical studies with a medium containing inositol 1:2-cyclic phosphate and Escherichia coli phosphomonoesterase show Gomori staining around the brush borders of the proximal tubules in the outer cortex only. 3. Serial sections across the kidney from cortex perimeter to papilla suggest that the inositol 1:2-cyclic phosphate 2-phosphohydrolase has a limited distribution along the proximal tubule of the nephron, probably being limited to the pars convoluta, whereas the alkaline phosphatase extends along the pars recta.     

Metabolic heterogeneity of the proximal and distal kidney tubules

Proximal and distal tubule suspensions were prepared from kidneys of Sprague-Dawley rats by an isolation procedure on a Percoll^R gradient. The marker enzymes alkaline phosphatase (brush border) and hexokinase (cytoplasmic) as well as p-aminohippurate transport capacity, gluconeogenic activity and electron microscopy were used to characterize the two kidney tubule suspensions. The results of this study indicate that cytochrome P-450 is localized to the proximal tubular cells and that the O-deethylation of 7- ethoxycoumarin was higher in the proximal than distal fraction. Both proximal and distal tubules showed glucuronidation and deacetylation capacities and a relatively equal distribution of non-protein sulfhydryls. These studies demonstrate metabolic heterogeneity of the nephron, the proximal tubule being the main site of renal xenobiotic metabolism. Understanding of metabolic heterogeneity of proximal and distal kidney tubules should provide important information regarding cell specific mechanisms of nephrotoxicity.     

Sodium-sensitive, probenecid-insensitive p-aminohippuric acid uptake in cultured renal proximal tubule cells of the rabbit.

In the intact kidney, renal proximal tubule cells accumulate p-aminohippurate (PAH) via a basolateral, probenecid- and sodium-sensitive transport system. Primary cultures of rabbit proximal tubule cells retain sodium-glucose co-transport in culture, but little is known about PAH transport in this system. Purified proximal tubule cells from a rabbit were grown in culture and assessed for PAH and alpha-methyl-D-glucoside uptake capacities as well as proximal tubule marker enzyme activities. Control PAH uptake on collagen-coated filters (20 +/- 3 pmol/mg protein.min; n = 8) was not significantly different from uptake in the presence of 1 mM probenecid (19 +/- 4 pmol/mg protein.min; n = 8). Uptake from the basal side of the cell was 3.9 +/- 0.7 times greater than that from the apical side. In multi-well plate studies, the uptake was significantly reduced by removing sodium from the medium and stimulated by coating the wells with collagen. Glutarate (10 mM) had no effect on the uptake of PAH. Other differentiated proximal tubule characteristics were retained in culture, including the ability to form domes and to transport glucose by a phlorizin-sensitive system. Phlorizin-sensitive 1 mM alpha-methyl-D-glucoside uptake was 134 +/- 42 pmol/mg protein.min (n = 7; P less than 0.02). The proximal tubule marker enzymes alkaline phosphatase and gamma-glutamyltranspeptidase, increased in activity in the cultures after confluence. It was concluded that whereas some differentiated properties were retained during primary culture of rabbit proximal tubule cells, the PAH transport system was selectively lost or modified from that present in the intact kidney.     

Studies on the pathophysiology of acute renal failure. II. A histochemical study of the proximal tubule of the rat following administration of mercuric chloride.

Acute renal failure was induced in male rats by the subcutaneous injectioon of 4 mg HgC12 per kg body weight. Enzyme activities of the proximal tubule were studied histochemically at six time intervals from 15 min to 24 h. The enzyme studied were alkaline phosphatase, 5'-nucleotidase, acid phosphatase, alpha-glycerophosphate dehydrogenase (NAD-independent), malic dehydrogenase, succinic dehydrogenase, latic dehydrogenase, glucose-6-phosphate dehydrogenase and glucose-6-phosphatase. Decreases in activity were observed for alkaline phosphatase and 5'-nucleotidase after 15 min. Acid phosphatase was decreased after 30 min. These three enzymes returned to control levels after 3 h, but malic dehydrogenase and alpha-glycerophosphate dehydrogenase were decreased at this time interval. Succinic dehydrogenase was first decreased after 6 h. The earliest morphological changes detectable by light microscopy were observed in pars recta tubules in the medullary rays after 6 h, a time when all enzymes studied showed widespread decreased activity throughout the proximal tubule. After 24 h, the pars convoluta appeared morphologically normal but the pars recta was necrotic and exhibited calcification, whereas enzyme activity was decreased (absent in some cases) in both pars convoluta and pars recta. These results support the hypothesis that Hg++, when given in a sublethal dose, is associated with early histochemical changes in the brush border of the proximal tubule, which may be related to early changes in sodium reabsorption and to the subsequent development of acute renal failure. The observation that changes in plasma membrane-associated enzymes occur early and prior to alterations in enzymes of mitochondria and the endoplasmic reticulum suggests that Hg++ interacts initially with the plasma membrane.     

Peroxisome development in the metanephric kidney of mouse.

The relationship of enzymatic activity to organelle development and organelle number during differentiation of the metanephric kidney in the mouse was approached from several experimental directions. Biochemical analyses of marker enzymes for peroxisomes (catalase and D-amino acid oxidase), mitochondria (cytochrome oxidase) and lysosomes (acid phosphatase) were performed on kidneys at ages from 17 days prenatal to adult. These data were correlated with a morphometric analysis of populations of peroxisomes and mitochondria in differentiating cells of the proximal tubule. Postnatal development of the metanephric kidney was found to be accompanied by a rapid increase in both the specific activity of catalase and the number of peroxisomes per 100 mu2 in the proximal tubule during the first 4 weeks of postnatal growth. Elaboration of the endoplasmic reticulum (ER) was seen to parallel the increase in number of peroxisomes to which segments of ER were often in close apposition. Extensive interactions between segments of ER and peroxisomes were readily visible in 0.5-mu sections viewed in the high voltage electron microscope. In contrast to peroxisomes, neither mitochondria nor lysosomes followed a similar pattern of net organelle increase, suggesting that a defined population density of mitochondria and lysosomes may exist in the proximal tubule at birth, prior to complete development of the kidney.     

Quantification of nephrotoxicity in rabbits by automated morphometry of alkaline phosphatase stained kidney sections

Summary Male and female rabbits were injected intravenously with a single dose of either cefroxadine or cefsulodin or cephaloridine. Quantitative determinations of the activity of two brush border membrane enzymes, aminopeptidase and alaline phosphatase, were made in homogenates of cortical kidney tissue, in the urine and morphometrically in proximal tubules of cryostat sections. Morphometry was done by classification and enumeration of proximal tubule sections with the same level of enzyme reaction product using a microscopic television analysis system. By comparison with the control values, no changes were detectable 24 h after the injection of up to 1.2 g cefroxadine or cefsulodin per kg body weight. By contrast, after 300 mg/kg cephaloridine, the concentrations of the two enzymes were decreased in a large number of proximal tubules, i.e. the bursh border membranes, and concomitantly cell degeneration and necrosis took place. Alkaline phosphatase activity in sections and tissue homogenates was reduced to a greater extent than aminopeptidase activity. A corresponding, significant increase in enzymic activity in the urine was only demonstrable in respect of aminopeptidase. The classification of proximal tubules in tissue sections by television analysis on the basis of alkaline phosphatase reaction product concentration appears to be a reliable measure for detecting and quantifying toxic effects on proximal tubules of kidney.Dedicated to Prof. Dr. Gerhard Pfleiderer on the occasion of his 60th birthday     

A microfluorometric method for alkaline phosphatase: Application to the various segments of the nephron

A microtechnique has been developed for the measurement of alkaline phosphatase in minute amounts of renal tissue. This microtechnique utilizes the known fluorescent property of 4-methylumbelliferyl phosphate following enzymatic hydrolysis. The reaction is sensitive and reproducible and is inhibited by l-bromotetramisole, a specific alkaline phosphatase inhibitor. The microdetermination of alkaline phosphatase activity in the various segments of the mouse nephron allowed the localization of the enzyme in the glomeruli, and in the proximal convoluted tubule where the activity progressively decreases from the capsule of Bowman to the more distal segments. The enzyme was absent from the pars recta or S3 and from the rest of the nephron. This technique is applicable to very small amounts (0.1 μg of protein) of any tissue containing alkaline phosphatase.     

Proton transport and Na+/H+ exchange in vesicles isolated from sockeye salmon (Oncorhynchus nerka) kidneys during migration from salt to fresh water.

Renal epithelial function, proton flux and sodium stimulated proton flux, was observed in vesicles isolated from the brush border of the proximal tubule of Sockeye Salmon (Oncorhynchus nerka) during migration. Brush border membrane vesicles (BBMV) were isolated from the body kidney of Sockeye Salmon using aggregation/differential centrifugation techniques. Vesicle purity was tested using a series of epithelial and basal lateral markers including alkaline phosphatase, maltase, gamma-glutamyl transferase (GGTP), Mg(2+)-activated ATP-ase, Na(+)+K(+)-activated ATPase, and 5'-nucleotidase and the lysosomal marker acid phosphatase. An enrichment/depletion factor for each marker was determined by comparison of purified BBMV with kidney homogenate. Vesicles exhibit an enrichment factor for alkaline phosphatase, GGTP, maltase, Mg(2+)-activated ATP-ase, Na(+)+K(+)-activated ATPase, and 5'-nucleotidase. A depletion factor was observed for acid phosphatase. Vesicle integrity was tested by measuring the time course of proton flux in the presence of a pH gradient. Amiloride sensitive sodium stimulated proton flux was observed in these vesicles. The presence of sodium caused a saturable increase in the rate of proton flux, indicating the activity of a sodium/proton antiport protein in BBMV.     

Demonstration of lysosomal and extralysosomal sites for acid phosphatase in mouse kidney tubule cells with p-nitrophenylphosphate lead-salt technique.

Dual localization of acid phosphatase in lysosomal and extralysosomal sites of the tubule epithelial cells of normal mouse kidney was observed at the light and electron microscope level using a modified Gomori lead-salt method with p-nitrophenylphosphate (pNPP) as substrate. Based on previous biochemical and cytochemical findings, we developed optimal conditions for the enzyme activity in extralysosomal sites. The conditions used for the light microscopic level consisted of 1.5 mM PNPP, 2.0 MM Pb(NO3)2 and 0.05 M acetate buffer (pH 5.8). Those for the electron microscopic study required 3.0 mM PNPP, 3.6 MM Pb(NO3)2 and 0.1 M acetate buffer (pH 5.8). This modified lead-salt technique was highly specific and provided a suitable method for the demonstration of nonlysosomal as well as lysosomal sites of acid phosphatase activity in the tubule epithelial cells of normal mouse kidney. As expected, the enzyme activity appeared in the lysosomes, but the prominent reaction in the brush border, the rough endoplasmic reticulum and basal infolding plasma membranes was not anticipated. We were able to demonstrate in situ organelle precursors of microsomal acid phosphatase such as endoplasmic reticulum, plasma membrane and basal infolding membranes showing the same substrate preference, which had been observed previously in biochemical studies in our laboratory. Since the possible participation of alkaline phosphatases, K+-pNPPase or Na+-K+-adenosine triphosphatase was ruled out by use of appropriate inhibitors, the enzyme-reactive sites can be interpreted as reflecting nonspecific acid phosphatase.     

The kidney of Spinachia spinachia (L.) Flem. (Gasterosteidae, pisces) 1. Investigations of juvenile sticklebacks: anatomy, circulation and fine structure

The opisthonephric kidney of juvenile fifteen spined stickleback (Spinachia spinachia (L.)) has been studied by light and electron microscopical investigation. The renal portal system and microcirculation has been demonstrated by casts of coloured plastic Microfil. Glomeruli are well vascularized as revealed by perfusion fixation. They contribute to the formation of urine in collaboration with a proximal tubule segment I that shows resorptive capacity. Epithelial cells of the proximal tubule segment II, which make up the bulk of the nephronic tubular mass are supposed to constitute very active secretory elements. The possible role of mucus secretion in the collecting duct-urinary duct system is discussed. Juxtaglomerulcar cells have been seen in various stages of secretion.     

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.     

Fine structural localization of alkaline phosphatase in the granular pneumonocytes of hamster lung.

The fine structural localization of nonspecific alkaline phosphatase was studied in the granular pneumonocytes (type II alveolar epithelial cells) of hamster lung by incubating sections of glutaraldehyde-fixed tissues in a medium containing lead ions and sodium beta-glycerophosphate or alpha-naphthyl acid phosphate. The specificity of the reaction was tested by exposing the sections to inhibitors of alkaline phosphatase. The results showed that alkaline phosphatase activity was present in the inclusion bodies of granular pneumonocytes. The enzyme reaction was strong in the membrane lining the inclusion bodies and a weaker reaction was generally detectable in the inclusion contents. Although only a proportion of the inclusion bodies showed enzyme activity, there was no obvious correlation between the reactivity of the inclusions and their intracellular position or size. The other organelles were unreactive. The finding of alkaline phosphatase activity within the inclusion bodies of granular pneumonocytes is an enigma as these organelles are generally considered to be lyosomes.