Oxidative enzymes in the urinary apparatus of several freshwater fishes

Synopsis The distribution and activities of several oxidative enzymes in the urinary apparatus of five freshwater fish species (river lamprey, lobe finned eel, Prussian carp, rainbow trout and three-spined stickleback) have been studied. Species were selected from three main taxonomic groups: Cyclostomata, Polypterini, Teleostei. Distinctly positive enzyme reactions were only found in the tubular elements of the kidney and the collecting duct-archinephric duct system, with the exception of the generally weak staining intensities of lactate dehydrogenase. The distal tubule normally showed strong to very strong reactions for most of the enzymes investigated. In the epithelial cells of the collecting tubule-collecting duct system, stronger reactions were observed for most of the mitochondrial-bound enzymes, especially succinate dehydrogenase and NADH-diaphorase. For these enzymes, the cells of the archinephric duct reacted strongly positive in Lampetra, Carassius and Gasterosteus.The enzyme patterns of various types of urinary tubules and ducts are compared with results of several morphological studies. In addition, the histochemical findings are discussed in relation to kidney function in different vertebrate groups.     

Oxidative enzymes in the urinary apparatus of several marine fishes

Summary The distribution and activities of several oxidative enzymes in the urinary apparatus of seven marine fish species (hagfish, lesser spotted dogfish, electric ray, herring, marine catfish, cod, sea-horse) have been studied. Species were selected from three main taxonomic groups: Cyclostomata, Elasmobranchii and Teleostei. Distinctly positive enzyme reactions were found in the tubular elements of the kidney and the collecting duct-archinephric duct system, with the exception of the generally weak staining intensities for NADP-liked malate dehydrogenase. In the proximal tubule segment the second, more distal part (PII) reacted, in general, very strongly when compared with the first proximal part (PI). If present, the distal tubule in teleosts showed only weak reactions, while this segment in elasmobranchs exhibited moderate to strong enzyme activities. In the epithelial cells of the collecting tubule-collecting duct system stronger reactions were confined to the glomerular teleost species, the corresponding part of the elasmobranch kidney showing weak staining intensities. In the urinary duct system distinctly positive enzyme reactions were only to be found in the archinephric duct of the teleost species, except forPlotosus. The ureters of the elasmobranchs exhibited weak enzyme activities throughout.The enzyme patterns of the various types of urinary tubules and ducts are compared with observations from several morphological and physiological studies. The histochemical findings are discussed in relation to corresponding investigations of fresh water fishes and problems arising from phylogenetic divergence of marine fish groups.     

The kidney,adrenocortical homolog,and corpuscles of Stannius in the cockscomb pricklebackAnoplarchus purpurescens

The morphology of the kidney, adrenocortical homolog, and the corpuscles of Stannius was examined in the cockscomb prickleback,Anoplarchus purpurescens, a marine teleost which inhabits the intertidal zone. The paired kidneys of this fish are fused throughout most of their length, there is essentially a single posterior cardinal vein on the right side, they possess renal corpuscles, and there is no distal segment of the tubule. The tubule is specialized, in descending order, into ciliated neck and two proximal segments before entering the system of collecting tubules and ducts. The cells of the latter system are specialized for mucous secretion, as are cells of the main excretory ducts, the paired archinephric ducts. Tubulogenesis occurs in the kidneys in close apposition to the archinephric ducts. The presumptive adrenocortical homolog is located around the posterior cardinal veins in the head kidney while paired corpuscles of Stannius are confined to the posterior end of the kidney. All of the above features are consistent with those found in the kidneys of many other marine teleosts.     

Stimulation of an N-ethylmaleimide-sensitive ATPase in the collecting duct segments of the rat nephron by metabolic acidosis

A plasma membrane ATPase sensitive to inhibition by N-ethylmaleimide (NEM) and insensitive to inhibition by oligomycin and ouabain has been shown to be involved in acidification of urine in the turtle bladder. The activity of this NEM-sensitive ATPase was determined in four types of distal nephron segments of normal rats and in rats treated with ammonium chloride. The enzyme activity was determined by a fluorometric micromethod in which ATP hydrolysis was coupled to NADH oxidation. Significant activities (10-35 pmol ADP X min-1 X mm-1) of NEM-sensitive ATPase were present in the distal convoluted tubule (DCT) and in the cortical and outer and inner medullary collecting duct segments of normal rats. In metabolic acidosis produced by ammonium chloride treatment (plasma CO2 content = 15.3 +/- 0.8 mequiv./L), the NEM-sensitive ATPase activity was increased significantly (60-100%) in the collecting duct segments without showing a significant change in the enzyme activity in the DCT. Our data are consistent with the hypothesis that a plasma membrane H+-ATPase (inhibited by NEM but not by oligomycin or ouabain) is involved in H+ secretion in the mammalian collecting duct.     

Morphology of the kidney of adult bowfin, Amia calva, with emphasis on "renal chloride cells" in the tubule

The nephron of adult bowfin, Amia calva, was described using light and electron microscopic techniques. The kidney of the bowfin possesses an abundant supply of renal corpuscles with each consisting of a glomerulus and a Bowman's capsule of visceral (podocyte) and parietal layers. No juxtaglomerular apparatus is present. The epithelium of the tubule is continuous with the parietal epithelium and is divisible in descending order into neck, first proximal, second proximal, first distal, second distal, and collecting segments. The tubules drain into a complex system of collecting ducts that ultimately unite with the main excretory duct, the archinephric duct. Mucous cells are the dominant cell throughout the entire ductular system. Nephrostomes are dispersed along the kidney capsule. The neck segment has a ciliated epithelium, and while both proximal segments possess a prominent brush border, the fine structure of the first implies involvement in protein absorption and the second in the transport and reabsorption of solutes. The cells of the first distal segment are characterized by deep infolding of the plasma membrane and a rich supply of mitochondria suggesting the presence of a mechanism for ion transport. The second distal segment is composed of cells resembling the chloride cells of fishes and these cells are present in progressively decreasing numbers in the collecting segment and duct system so that only a few are present in the epithelium of the archinephric duct. The "renal chloride cells" possess an abundant network of smooth tubules and numerous mitochondria with a rich supply of cristae. Glycogen is also a conspicuous component of these cells. The presence of "renal chloride cells" in this freshwater holostean, in other relatively primitive freshwater teleosts, and in larval and adult lampreys is discussed with reference to both phylogeny and the need for a special mechanism for renal ion conservation through absorption.     

Presence of an extramitochondrial anion-stimulated ATPase in the rabbit kidney: Localization along the nephron and effect of corticosteroids

Summary To determine whether kidney membrane fractions contain an extramitochondrial anion-stimulated ATPase, we compared the pharmacological and kinetic properties of HCO₃-ATPase activities in mitochondrial and microsomal fractions prepared from rabbit kidney cortex and outer medulla. The results indicated that this activity differed markedly in each type of fraction. Microsomal HCO₃-ATPase was less sensitive than mitochondrial ATPase to azide, oligomycin, DCCD and thiocyanate, but was more sensitive to filipin and displayed different dependency towards ATP, magnesium and pH. Microsomal ATPase activity was stimulated by sulfite much more strongly than by bicarbonate, whereas mitochondrial activity was stimulated by both these anions to a similar extent. These results demonstrate the presence of an extramitochondrial HCO₃-ATPase in kidney membrane fractions. HCO₃-ATPase was also measured in single microdissected segments of the rabbit nephron using a radiochemical microassay previously developed for tubular Na, K-ATPase activity. An enzyme with the pharmacological and kinetic properties of the microsomal enzyme was detected in both proximal tubule, distal convoluted tubule and collecting duct, but the thick ascending limb was devoid of any detectable activity. Long-term DOCA administration markedly increased HCO₃-ATPase activity in the distal convoluted and collecting tubule. The insensitivity of microsomal HCO₃-ATPase to vanadate indicates that it belongs to the F₀–F₁ class of ATPases, and might therefore be involved in proton transport. This hypothesis is also supported by the localization of tubular HCO₃-ATPase activity at the sites of urinary acidification.     

Differential expression of Na+-Cl- cotransporter and Na+-K+-Cl- cotransporter 2 in the distal nephrons of euryhaline and seawater pufferfishes

The process of NaCl reabsorption in the distal nephron allows freshwater fishes to excrete hypotonic urine and seawater fishes to excrete urine containing high concentrations of divalent ions; the relevant transporters, however, have not yet been identified. In the mammalian distal nephron, NaCl absorption is mediated by Na(+)-K(+)-Cl(-) cotransporter 2 (NKCC2, Slc12a1) in the thick ascending limb, Na(+)-Cl(-) cotransporter (NCC, Slc12a3) in the distal convoluted tubule, and epithelial sodium channel (ENaC) in the collecting duct. In this study, we compared the expression profiles of these proteins in the kidneys of euryhaline and seawater pufferfishes. Mining the fugu genome identified one NKCC2 gene and one NCC gene, but no ENaC gene. RT-PCR and in situ hybridization analyses demonstrated that NKCC2 was highly expressed in the distal tubules and NCC was highly expressed in the collecting ducts of euryhaline pufferfish (mefugu, Takifugu obscurus). On the other hand, the kidney of seawater pufferfish (torafugu, Takifugu rubripes), which lacked distal tubules, expressed very low levels of NCC, and, in the collecting ducts, high levels of NKCC2. Acclimation of mefugu to seawater resulted in a 2.7× decrease in NCC expression, whereas NKCC2 expression was not markedly affected. Additionally, internalization of NCC from the apical surface of the collecting ducts was observed. These results suggest that NaCl reabsorption in the distal nephron of the fish kidney is mediated by NCC and NKCC2 in freshwater and by NKCC2 in seawater.     

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.     

Immunolocalization of C1-tetrahydrofolate synthase in the rat kidney

The distribution of the trifunctional enzyme C1-tetrahydrofolate synthase (C1-THF synthase) was examined in the rat kidney by immunolocalization with anti-C1-THF synthase serum using the peroxidase-antiperoxidase method. C1-THF synthase immunoreactivity was detected in both distal and proximal epithelial cells. Staining of the distal tubule epithelia was more intense and granular whereas staining of the proximal tubule epithelia was diffuse. All cells of the cortical collecting duct showed positive granular staining. In the outer medullary collecting duct, the intercalated cells showed intense granular cytoplasmic staining and the principal cells were either negative or weakly positive. The ascending thick limb of Henle's loop was also positive. Glomeruli and the inner medulla showed no staining for C1-THF synthase.     

Distribution of saccharide residues in glycoconjugates of the kidney in Gallus domesticus using peroxidase-conjugated lectins

A battery of seven different horseradish-peroxidase labelled lectins (DBA, PNA, SBA, UEA I, WGA, ConA, LTA) was used to study the distribution of sugar residues in the glycoconjugates along the nephron and the collecting duct of the kidney of Gallus domesticus. As far as the glomerular components are concerned, we have demonstrated that the podocytes and, with a lesser extent, the mesangial cells are characterised by the presence of D-mannose, D-galactose-(beta 1- greater than 3)-N-acetyl-D-galactosamine and sialic acid. The glomerular capillary wall shows the presence of the disaccharide D-galactose-(beta 1- greater than 3)-N-acetyl-D-galactosamine and sialic acid. With regards to the tubules, the proximal tubule, the descending limb of the loop of Henle, the connecting tubule and the collecting one, are characterised by N-acetyl-D-galactosamine, (1- greater than 6)-alpha-L-fucose, D-mannose, N-acetyl-D-galactosamine and D-galactose-(beta 1- greater than 3)-N-acetyl-D-glucosamine. The cells of the connecting and collecting ducts show the presence of intracellular sialic acid, found also as component of the mucous secretion. The ascending limb of the loop of Henle and the distal tubule contain only three saccharidic residues, i.e. (1- greater than 6)-alpha-L-fucose, D-mannose and N-acetyl-D-glucosamine. Lectin histochemistry was also useful to define the saccharidic components of the mucus, which is normally present within the connecting and collecting ducts of the kidney of the birds. The cellular variability of the connecting and the collecting ducts is similar to that found in the kidney of some mammals. Such a variability seems to suggest a possible cell specialization along a single kidney tubule.     

Structure Organization of Urinary System in the Yellow Spotted Mountain Newts （Salamandridae： Neurergus microspilotus）

This study deals with the histomorphology of the mesonephros in male and female Neurergus microspilotus. The slender and narrow kidneys are positioned in the retro peritoneal position up against the ventral aspect of vertebral column and may extend the length from the esophagus-stomach junction to cloaca. The kidney in both sexes is composed of sexual（anterior） and pelvic（posterior） parts. The duct of sexual kidney is a narrow duct which is lying alongside its lateral edge. In the female, it is connected to the ureters and then the duct of defi nitive kidney. Before entering the cloaca, two ureters are joined together and open to the apex of the cloaca. In the male, after entering the sexual kidney, the sperm leave the testis through efferent ducts, then these ducts join together and eventually form Bidder＇s duct. The Bidder＇s duct joins the Bowman＇s capsule of the nephrons in the sexual kidney and the nephrons make collecting ducts which are fi lled with both sperm and urine. After leaving the kidney, all the collecting ducts are connected to the Wolffi an duct. Wolffi an duct joins the ureters（merge from defi nitive kidney） just before entering the cloaca. Based on serial paraffi n sections, nephrons consist of a fi ltration unit, the Malpighian corpuscle, and a renal tubule, which can be divided into 4 morphologically distinct segments： proximal tubule（first and second segment）, distal tubule, and collecting tubule. Collecting tubules merge and form a branch system that opens into collecting ducts.     

Comparison of the distribution of tissue kallikrein and esterase A, a kallikrein-like enzyme, in rat kidney using specific monoclonal antibodies

Tissue kallikrein (E.C. 3.4.21.35) and arginine esterase A, another closely related, kinin-generating serine protease, have been localized by immunocytochemistry in rat kidney, using monoclonal antibodies that do not crossreact with other kallikrein-related enzymes or with tonin. Kallikrein was present primarily in the apical cytoplasm of the connecting tubule and the cortical collecting duct. Esterase A, on the other hand, was present primarily in the basolateral region of both proximal and distal straight tubules in the outer medulla and medullary rays. In addition, esterase A was demonstrable in distal convoluted tubules and, to a lesser extent, in proximal convoluted tubules. The presence of different kinin-generating enzymes at these sites would permit the formation of kinins from appropriate substrates on both the vascular and luminal poles of separate segments of the kidney tubule.     

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.     

Carbonic anhydrase in the monkey kidney

The distribution of carbonic anhydrase in the kidney of the cynomolgus monkey was studied by the histochemical method of Hansson. Glomeruli and Bowman's capsule were inactive. Convoluted proximal tubules showed high enzyme activity at the brush border and the basolateral membranes and the cytoplasm. Straight proximal tubules were less intensely stained. In nephrons with long loops of Henle, the descending thin limb contained weak enzyme activity, whereas the ascending thin limb was inactive. The thick limb of Henle's loop displayed most enzyme activity at the luminal cell border. In distal convoluted tubules enzyme activity was restricted to the basal part of the cells. In the late distal tubule, intercalated cells appeared among the "ordinary" distal cells and contained abundant cytoplasmic enzyme. Many intensely stained intercalated cells were also found in the cortical and outer medullary segments of the collecting duct, intermingled with more weakly stained chief cells. In the inner medullary segment of the collecting duct, enzyme activity gradually disappeared. Many capillaries were clearly stained for enzyme activity. The capillary staining apparently varied with that of the kidney tubules; virtually all capillaries in the cortex, but very few in the inner medulla, were stained. The distribution of carbonic anhydrase in the kidney tubules of the monkey is very similar to that in man and in the rat, but the primate kidney differs from the rat kidney by the presence of capillary enzyme activity. The functional importance of this difference is not clear at present.     

OXIDATIVE AND HYDROLYTIC ENZYMES IN THE NEPHRON OF NECTURUS MACULOSUS : Histochemical, Biochemical, and Electron Microscopical Studies

The distribution of oxidative and hydrolytic enzyme activities along the nephron of Necturus maculosus Rafinesque was studied histochemically. The proximal tubule possessed all the demonstrable enzyme activities associated with the hexose-monophosphate shunt and glycolysis, but lacked detectable succinic dehydrogenase and cytochrome oxidase activities. Krebs cycle enzymes other than succinic dehydrogenase were easily detectable. The distal tubule, on the other hand, possessed no detectable hexose-monophosphate shunt enzyme activities, but all demonstrable glycolytic and Krebs cycle enzymes and cytochrome oxidase were present in high activity. These data indicate that the proximal tubule of Necturus probably cannot depend, as can the distal tubule, on the Krebs cycle and cytochrome system to provide energy for its transport processes, an inference supported, in general, by available physiological evidence. The question of the importance of the hexose shunt to proximal tubular function arises. Evidence is presented that the proximal tubular blood supply is primarily venous in nature, a hypothesis which would correlate well with its anaerobic metabolic pattern. In addition, the absence of cytochrome oxidase and succinic dehydrogenase from the proximal tubular cells implies either that they possess very few mitochondria, or that their mitochondria have a very unusual enzymatic pattern. Electron microscopical observations and data obtained from the measurement of the enzyme activities of homogenates of Necturus kidney are presented which indicate that the second hypothesis is more probably correct.     

Effects of low-potassium diet on N-ethylmaleimide-sensitive ATPase in the distal nephron segments.

The present study was undertaken to investigate whether or not potassium deficiency influences N-ethylmaleimide (NEM)-sensitive ATPase in the distal nephron segments of the rat. One group of animals was fed a low-K diet, whereas the normal K-group was given the same diet after supplementation with KCl. The nephron segments examined were: the medullary and cortical thick ascending limbs, the distal convoluted tubule, and the cortical, outer and inner medullary collecting ducts. NEM-sensitive ATPase activity in microdissected segments was measured by a fluorometric microassay. The plasma K+ concentration in the low-K group was 3.1 +/- 0.3 mEq/l compared with 4.2 +/- 0.1 mEq/l in the normal-K group. NEM-sensitive ATPase activity in the outer medullary collecting duct of low-K diet animals was significantly greater than in normal-K animals. There was no significant difference in NEM-sensitive ATPase activity between the two groups of animals in the other nephron segments examined. It is suggested that NEM-sensitive H-ATPase activity in the outer medullary collecting duct is modulated by the potassium status of the animal.     

Histological and enzyme histochemical studies on the nephrons of the freshwater fishes, Cyprinus carpio and Carassius auratus

The nephrons of carp (Cyprinus carpio) and goldfish (Carassius auratus) were examined histologically and also histochemically for enzymes. In both species the distal and collecting tubules have much wider lumens than do the other renal tubules; thus urine probably flows more slowly in these larger tubules. Enzyme histochemistry shows that epithelium of the neck and proximal and intermediate tubules respires anaerobically, whereas that of the distal and collecting tubules respires aerobically. The distribution of Na-K-ATPase in the distal and collecting tubules indicates that they also transport sodium actively. The slow flow of urine and the energy produced by aerobic metabolism probably increase the efficiency of active transport.     

Morphometric parameters of nutria kidney structures in postnatal ontogeny

Data on the morphometric parameters of the renal corpuscle, renal tubules, and collecting ducts of male and female nutrias in postnatal ontogenesis were obtained. It was found that the area of the renal corpuscle, glomerulus, the cavity and lumen of the capsule, and the proximal tubule diameter in the right and left kidney of female and male nutrias in the first year of life increase. The distal tubule diameter also increases; however, the dynamics of its changes becomes sinuous after 4.5 months. The collecting duct diameter varies depending on gender, age, and renal topography. The nuclear-cytoplasmic ratio in the cells of proximal and distal tubules and collecting ducts changes in a sinuous manner and depends on the gender and age of nutrias. The minimum mean value of the nuclear-cytoplasmic ratio was found in the proximal tubule cells in the left kidney of 12-month-old female nutrias (0.162 ± 0.002), and the maximum value was found in the distal tubule cells in the left kidney of newborn male nutrias (0.435 ± 0.007).     

Na-K]ATPase activity in proximal and distal tubules of the rat kidney: modification and application of a quantitative cytochemical technique

A modified cytochemical assay for [Na-K]ATPase in cryostat sections of kidney was further characterized and used to quantify activity in seven functionally distinct sites along the rat nephron. The activity of [Na-K]ATPase was defined as the difference in ATPase activity in specifically identified tubules contained in serial sections incubated with and without ouabain. Preincubation of sections with ouabain was required for maximal inhibition of [Na-K]ATPase activity in several distal sites. The concentration of ouabain necessary for maximal inhibition of activity was 3.0 mM and half-maximal inhibition was obtained in all regions with 30-100 microM ouabain. In distal sites, [Na-K]ATPase formed a higher proportion of total ATPase activity (60-80 per cent) than in proximal sites (20-40 per cent). Enzyme activity was quantified using two different methods. The first measured activity over the basal region of tubules and gave an index of the concentration of [Na-K]ATPase over the basal lateral infoldings of cells composing the tubule. The second read activity over the entire cross section of tubules and provided an estimate of [Na-K]ATPase per length of tubule. The highest activities over the basal basal region were obtained from tubules of the distal nephron including the inner (MALin) and outer (MALout) medullary ascending limb, distal convoluted tubule (DCT) and connecting segment (CS). Lower activities were obtained in proximal convoluted (PCT) tubules, proximal straight (PS) tubules and the papillary collecting duct (PD). Distal convoluted tubules contained the highest activity per length of tubule. Other sites contained lower levels of activity in the following order: MALin greater than MALout greater than PCT greater than PD greater than PS. The modifications introduced increase the sensitivity and precision of this assay and permit the application of this technique to studies of [Na-K]ATPase activity in the major functional regions of the rat nephron.