Immunohistochemical localization of sodium-dependent l-ascorbic acid transporter 1 protein in rat kidney

Recently, two l-ascorbic acid transporters were identified; sodium-dependent vitamin C transporter (SVCT) 1 and SVCT2. The previous study suggested that SVCT protein might be present on the apical membrane in the straight segment (S3) of proximal tubule. In the present study, SVCT1 immunoreactivity (IR) was observed in the brush border of proximal straight tubules in the medullary ray of renal cortex and the outer stripe of outer medulla, while SVCT2 IR was not localized in any region of the kidney. Since the mechanism of VC reabsorption in the kidney has not been fully elucidated up to the present time, it is meaningful to demonstrate the exact cellular distribution of SVCT protein in the kidney.     

Secretion of endogenous lithium in teleost kidneys as a probable reflection of Na+ and water secretion in their renal proximal tubules

Unlike mammals with renal reabsorption of lithium (Li+), in freshwater and, particularly, marine teleosts net secretion of this trace element by kidneys was discovered. The ratio of Li+ natural concentration (measured by mass spectrometric isotope dilution technique) in urine to that in blood plasma--(U/P)Li--lies in the range 2-6 in the freshwater species and between 5 and 14 in marine species, i.e. as a rule it is essentially higher than the inulin concentration index (U/P)In. It is supposed that the in vivo observed lithium net secretion in whole kidney reflects and quantitatively estimates Na+ and water secretion in renal proximal tubules of teleosts.     

Increased renal tubular synthesis of prostaglandins in the rabbit kidney in response to ureteral obstruction

The hydronephrotic rabbit kidney exhibits elevated basal prostaglandin synthesis and supersensitivity to peptide stimulation of vascular prostaglandin and thromboxane formation. In this study the distribution of the prostaglandin-forming cyclooxygenase in hydronephrotic and contralateral rabbit kidneys following one and four day ureteral obstructions was compared using immunohistofluorescence. No alterations were detected in the distribution or intensity of cyclooxygenase-positive fluorescence in the renal vasculature in response to ureteral obstructions. However, two significant differences were noted between hydronephrotic and contralateral kidneys in the staining of renal tubules: (a) the intensity of fluorescent staining in cortical and medullary collecting tubules of the hydronephrotic kidney was increased and (b) cyclooxygenase antigenicity appeared in the thin limbs of Henle's loop in the hydronephrotic organ. Although alterations in prostaglandin formation by the renal vasculature have been documented previously, our results indicate that ureteral obstruction also causes increased prostaglandin synthesis by renal tubules.     

Increased renal tubular synthesis of prostaglandins in the rabbit kidney in response to ureteral obstruction.

The hydronephrotic rabbit kidney exhibits elevated basal prostaglandin synthesis and supersensitivity to peptide stimulation of vascular prostaglandin and thromboxane formation. In this study the distribution of the prostaglandin-forming cyclooxygenase in hydronephrotic and contralateral rabbit kidneys following one and four day ureteral obstructions was compared using immunohistofluorescence. No alterasions were detected in the distribution or intensity of cyclooxygenase-positive fluorescence in the renal vasculature in response to ureteral obstructions. However, two significant differences were noted between hydronephrotic and contralateral kidneys in the staining of renal tubules: (a) the intensity of fluorescent staining in cortical and medullary collecting tubules of the hydronephrotic kidney was increased and (b) cyclooxygenase antigenicity appeared in the thin limbs of Henle's loop in the hydronephrotic organ. Although alterations in prostaglandin formation by the renal vasculature have been documented previously, our results indicate that ureteral obstruction also causes increased prostaglandin synthesis by renal tubules.     

Elevated Na+/K+-ATPase responses and its potential role in triggering ion reabsorption in kidneys for homeostasis of marine euryhaline milkfish (Chanos chanos) when acclimated to hypotonic fresh water

The milkfish (Chanos chanos) is an economic species in Southeast Asia. In Taiwan, the milkfish are commercially cultured in environments of various salinities. Na⁺/K⁺-ATPase (NKA) is a key enzyme for fish iono- and osmoregulation. When compared with gills, NKA and its potential role were less examined by different approaches in the other osmoregulatory organs (e.g., kidney) of euryhaline teleosts. The objective of this study was to investigate the correlation between osmoregulatory plasticity and renal NKA in this euryhaline species. Muscle water contents (MWC), plasma, and urine osmolality, kidney histology, as well as distribution, expression (mRNA and protein), and specific activity of renal NKA were examined in juvenile milkfish acclimated to fresh water (FW), seawater (SW 35‰), and hypersaline water (HSW 60‰) for at least two weeks before experiments. MWC showed no significant difference among all groups. Plasma osmolality was maintained within the range of physiological homeostasis in milkfish acclimated to different salinities, while, urine osmolality of FW-acclimated fish was evidently lower than SW- and HSW-acclimated individuals. The renal tubules were identified by staining with periodic acid Schiff’s reagent and hematoxylin. Moreover, immunohistochemical staining showed that NKA was distributed in the epithelial cells of proximal tubules, distal tubules, and collecting tubules, but not in glomeruli, of milkfish exposed to different ambient salinities. The highest abundance of relative NKA α subunit mRNA was found in FW-acclimated milkfish rather than SW- and HSW-acclimated individuals. Furthermore, relative protein amounts of renal NKA α and β subunits as well as NKA-specific activity were also found to be higher in the FW group than SW and the HSW groups. This study integrated diverse levels (i.e., histological distribution, gene, protein, and specific activity) of renal NKA expression and illustrated the potential role of NKA in triggering ion reabsorption in kidneys of the marine euryhaline milkfish when acclimated to a hypotonic FW environment.     

Effect of epsilon toxin-GFP on MDCK cells and renal tubules in vivo.

Epsilon toxin (epsilon-toxin), produced by Clostridium perfringens types B and D, causes fatal enterotoxemia, also known as pulpy kidney disease, in livestock. Recombinant epsilon-toxin-green fluorescence protein (epsilon-toxin-GFP) and epsilon-prototoxin-GFP were successfully expressed in Escherichia coli. MTT assays on MDCK cells confirmed that recombinant epsilon-toxin-GFP retained the cytotoxicity of the native toxin. Direct fluorescence analysis of MDCK cells revealed a homogeneous peripheral pattern that was temperature sensitive and susceptible to detergent. epsilon-Toxin-GFP and epsilon-prototoxin-GFP bound to endothelia in various organs of injected mice, especially the brain. However, fluorescence mainly accumulated in kidneys. Mice injected with epsilon-toxin-GFP showed severe kidney alterations, including hemorrhagic medullae and selective degeneration of distal tubules. Moreover, experiments on kidney cryoslices demonstrated specific binding to distal tubule cells of a range of species. We demonstrate with new recombinant fluorescence tools that epsilon-toxin binds in vivo to endothelial cells and renal tubules, where it has a strong cytotoxic effect. Our binding experiments indicate that an epsilon-toxin receptor is expressed on renal distal tubules of mammalian species, including human.     

Glomeruli and renal tubules are restricted to the cranial kidney of the adult estuarine Sphoeroides testudineus

The kidney of the pufferfish Sphoeroides testudineus , an abundant tropical euryhaline estuarine species of the western Atlantic Ocean found in southern Brazil (in salinities ranging from 0 to 34) has a large and laterally spread cranial red portion, and a very thin and pale caudal portion. When studied under light and transmission electron microscopy, the cranial kidney displayed glomeruli and renal tubules surrounded by haematopoietic tissue. These tubules appeared to drain into a single large convoluted collecting duct with a wide lumen and thick pseudostratified epithelium, the mesonephric duct, which constituted the sole structure of the caudal kidney. Apical microvillae were viewed in the renal tubules, as well as in the mesonephric duct. Basal mitochondria and membrane infoldings were observed in the renal tubules. Abundant more basally‐located mitochondria and electron‐dense vesicles, mainly in the apical cytoplasm, were observed along the entire length of the mesonephric duct. Aposomes (blebs) were frequently observed in the mesonephric duct, both by light‐ and electron‐microscopy. This euryhaline estuarine pufferfish has thus been revealed to possess a rare type of kidney.     

Morphometrical analysis with the light and electron microscope of the kidney of the anadromous three-spined stickleback Gasterosteus aculeatus,form trachurus,from fresh water and from sea water

Summary The renal corpuscles, juxtaglomerular cells, nephronic tubules, and ureters of female sticklebacks were studied.In fresh water fishes, the diameter of the renal corpuscles is similar to that found in fishes obtained from the sea, whereas the diameter of the glomeruli and the nuclei of the podocytes are slightly larger. Furthermore, in fresh water the podocytes produce secretory globules, which show some of the histochemical characteristics of the substance constituting the glomerular basement membrane. In sea water animals, secretory phenomena are absent. Mesangial cells, which are scarce in fresh water fishes, are numerous in marine animals. Similarly, juxtaglomerular cells, hard to find in fresh water fishes, are prominent in specimens from the sea.The development of the epithelia of the nephronic tubules and of the ureters is better in fresh water. The cells and the nuclei are larger. In the first proximal tubule, which is involved in the reabsorption and the digestion—by lysosomes—of macromolecules, micropinocytosis vermiformis occurs.The results of stereological analysis of the fractional volume of the mitochondria and of the relative extent of the infoldings of the basal cell membranes—the location of the ion transport mechanisms—in the three different segments of the nephronic tubule and in the ureter, point to the existence of a structural gradient along the kidney tubules. In fresh water fishes the mitochondrial volume, per surface unit of basal cell membrane, is low in the first proximal segment and is increasingly higher in the other segments, while the highest value is found in the ureter. This structural gradient may be functionally linked with osmotic and ionic gradients, which exist in the renal tubules in fresh water. In the kidney tubules of marine sticklebacks, which do not show a major osmotic gradient, the structural gradient is small.The results are discussed on the basis of the known physiological differences in the function of the kidney of euryhaline teleosts in fresh water and in the sea.The author is indebted to Mr. J. Cappon and Mr. M. Veenhuis (Laboratory for Ultrastructural Biology, University of Groningen) for technical assistance.     

Mitochondria-rich (chloride) cells in the gill epithelia from four species of stenohaline fresh water teleosts

Summary The mitochondria-rich (chloride) cells have been found to be present in the gill epithelia of four species of stenohaline fresh water teleosts. The cytoplasm of these chloride cells contains an extensive network of cytoplasmic tubules which communicate with intercellular spaces bordering the lateral and basal cell surfaces. Numerous vesicles with fairly electron-dense interiors are also present in the apical cytoplasm of chloride cells. The apical surface of a chloride cell forms an apical pit, but the lumen of the pit does not appear to be in continuity with the interior of the apical vesicles and tubules inside the cell.When Carassius auratus were kept in 100, 200, 300, and 400 mOsm-diluted sea water for a month, no appreciable changes occurred in the number and fine structure of the chloride cells, except for a dilation of the apical vesicles and a slight decrease in diameter of the cytoplasmic tubules in these cells in the fishes kept in 300 and 400 mOsm.These results suggest that chloride cells may be a rather common occurrence in the gill epithelia of stenohaline fresh water teleosts, and may function in ion-transport in these fishes in fresh water environments.     

Heterogenous staining ofd-amino acid oxidase in peroxisomes of rat liver and kidney

Summary The localization ofd-amino acid oxidase (d-AAOX) in rat liver and kidney has been investigated using the cerium technique for electron microscopy and a recent modification of it for light microscopy. In the liver a mosaic pattern with strongly and weakly stained cells together with some completely negative hepatocytes is observed. The staining is stronger and more uniform in periportal than in perivenous regions of the liver lobule. In the kidney the reaction is confined to the proximal tubules of the renal cortex with the rest of the nephron being negative. At the ultrastructural level in both liver and kidney a marked heterogencity is obseved in the intensity of reaction in peroxisomes of some neighbouring cells. Moreover, in some cells heavily and weakly stained peroxisomes are seen side by side. When Pipes buffer is used in the incubation medium thed-AAOX reaction in kidney peroxiosomes is aggregated in the central region of the matrix with weaker staining of the periphery. A similar result is obtained when the enzyme is localized by immunocytochemistry confirming a recent report by Usuda et al. (1986). The heterogeneous staining of peroxisomes ford-AAOX suggests that subpopulation of this organelle with specialized functions may exist not only in different tissues and cells but even within the same cell.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

A method for preservation of soft tissues of marine teleosts for scanning electron microscopy

Soft, internal tissues of marine teleosts have been preserved for scanning electron microscopy in 2% glutaraldehyde in half-strength flounder saline; the osmolality of this fixative approximated the plasma osmolality of marine teleosts. Fixation was followed by washing, dehydration and Freon critical point drying. This procedure has revealed surface features of renal tubules of Anoplogaster and swimbladder of Melanonus     

Identification and apical membrane localization of an electrogenic Na⁺/Ca²⁺ exchanger NCX2a likely to be involved in renal Ca²⁺ excretion by seawater fish

Seawater (SW) contains ～10 mM Ca(2+), yet marine fish must drink seawater as their major water source. Thus marine teleosts fish need to excrete Ca(2+) to maintain whole body Ca(2+) homeostasis. In the intestine, seawater Ca(2+) interreacts with epithelial-secreted HCO(3)(-) by the intestinal epithelium, and the resulting CaCO(3) precipitates, which is rectally excreted. Recently the transporters involved in intestinal HCO(3)(-) secretion were identified. Ca(2+) is also excreted by the kidney, but the protein(s) involved in renal Ca(2+) excretion have not been identified. Here we identified a candidate transporter by using SW pufferfish torafugu (Takifugu rubripes) and its closely related euryhaline species mefugu (Takifugu obscurus), which are becoming useful animal models for studying molecular mechanisms of seawater adaptation. RT-PCR analyses of Na(+)/Ca(2+) exchanger (NCX) family members in various torafugu tissues demonstrated that only NCX2a is highly expressed in the kidney. Renal expression of NCX2a was markedly elevated when mefugu were transferred from freshwater to seawater. In situ hybridization and immunohistochemical analyses indicated that NCX2a is expressed in the proximal tubule at the apical membrane. NCX2a, expressed in Xenopus oocytes, conferred [Ca(2+)](out)- and Na(+)-dependent currents. These results suggest that NCX2a mediates renal Ca(2+) secretion at the apical membrane of renal proximal tubules and has an important role in whole body Ca(2+) homeostasis of marine teleosts.     

Advantages and limitations of the use of isolated kidney tubules in pharmacotoxicology

Among the cellular models used in in vitro renal pharmacotoxicology, isolated kidney tubules, used as suspensions mainly of proximal tubules, offer important advantages. They can be prepared in large amounts under nonsterile conditions within 1–2 h; thus, it is possible to employ a great number of experimental conditions simultaneously and to obtain rapidly many experimental results. Kidney tubules can be prepared from the kidney of many animal species and also from the human kidney; given the very limited availability of healthy human renal tissue, it is therefore possible to choose the most appropriate species for the study of a particular problem encountered in man. Kidney tubules can be used for screening and prevention of nephrotoxic effects and to identify their mechanisms as well as to study the renal metabolism of xenobiotics. When compared with cultured renal cell, a major advantage of kidney tubules is that they remain differentiated. The main limitations of the use of kidney tubules in pharmacotoxicology are (1) the necessity to prepare them as soon as the renal tissue sample is obtained; (2) their limited viability, which is restricted to 2–3 h; (3) the inability to expose them chronically to a potential nephrotoxic drug; (4) the inability to study transepithelial transport; and (5) the uncertainty in the extrapolation to man of the results obtained using animal kidney tubules. These advantages and limitations of the use of human and animal kidney tubules in pharmacotoxicology are illustrated mainly by the results of experiments performed with valproate, an antiepileptic and moderately hyperammonemic agent. The fact that kidney tubules, unlike cultured renal cells, retain key metabolic properties is also shown to be of the utmost importance in detecting certain nephrotoxic effects.     

Histochemical demonstration of l-amino acid-tetrazolium reductase

Summary A method for the histochemical demonstration of l-amino acid-tetrazolium reductase is described. The diformazan deposition was obtained with l-leucine as substrate and no precipitate was present when l-serine and l-lisine were used. In the kidney the reaction was positive in the podocytes, the cells of proximal and distal convoluted tubules, in the ascending limbs of Henle and in the cells of the collecting tubules. In the liver the reaction was positive in the cytoplasm of the hepatocytes. The reaction pattern suggests that it is predominantly extramitochondrial. The specificity of this method is discussed.     

A cytophotometric analysis of the activity of oxidative enzymes and Na, K-ATPASE in vertebrate nephrons at different levels of sodium transport in the kidney.]

Using quantitative cytochemistry, activities of Na, K-ATPase, succinate dehydrogenase (SDH) and alpha-keto-glutarate dehydrogenase (alpha-KDH) was investigated in cells of renal tubules at different levels of sodium reabsorption in the kidney. The activity of these enzymes in mammals and birds renal tubule cells was found to be higher than in the cells of corresponding renal tubules of cold-blooded vertebrates. This corresponds to the increased total amount of reabsorbed sodium in the kidney of warm-blooded animals. The summer frogs, as compared to the winter ones, exhibit higher activities of SDH and Na,K-ATPase in the proximal tubule cells where changes in sodium reabsorption are also noted. In the kidney of marine teleosts, a negative correlation between U/PNa and the activity of SDH and Na,K-ATPase in the cells of proximal and distal tubule was observed. Aldosterone was found to stimulate sodium reabsorption and to activate Na,K-ATPase.SDH and alpha-KDH mainly in the distal convoluted tubule. Furosemide was observed to inhibit sodium reabsorption and to reduce SDH and Na,K-ATPase activities in cells of the proximal tubule and Henle's loop. In the kidney of adrenalectomized rats, both sodium reabsorption and activities of Na,K-ATPase, SDH, alpha-KDH decreased in all the segments of the nephron. The data obtained suggest that changes in sodium reabsorption may be coupled with those in the activities of the investigated enzymes.     

Ghrelin receptors are expressed by distal tubules of the mouse kidney

Ghrelin, a peptide hormone from the stomach, has been recently discovered to reduce sodium excretion from the kidney. Although the effects on the kidney suggest actions in the distal nephron, the sites of expression of ghrelin receptors have not been localised. In the present work we have used a mouse that expresses green fluorescent protein under the control of the ghrelin receptor promoter to locate sites of receptor expression in the kidney. Receptor expression was confined to the straight parts of the distal tubules and the thin limbs of the loops of Henle. No expression was detected in other structures, including the glomeruli, proximal tubules and collecting ducts. Ghrelin receptors were not found in extra-renal or intra-renal arteries, despite observations that ghrelin is a vasodilator. The distribution revealed by in situ hybridisation histochemistry was the same as that revealed by the reporter. In conclusion, ghrelin receptors have a restricted distribution in the kidney. The location in the straight parts of the distal tubules accords with observations that ghrelin promotes sodium retention.     

Renal filtration and reabsorption of GFP in <Emphasis Type="Italic">Rana temporaria</Emphasis>: Effect of arginine-vasotocin

The renal tubular uptake of green fluorescent protein (GFP) in frog Rana temporaria was studied by laser confocal microscopy. The specific green fluorescence was revealed in cells of proximal tubules 30 min after intravenous GFP injection. The GFP fluorescence was located predominantly in the apical part of the cytoplasm in the form of intensively fluorescing vesicles. The GFP injections increased dose-dependently the GFP tubular uptake. This was confirmed by the quantitative assessment of intensity of the specific fluorescence, its relative vesicular density, and by correlation analysis. Preliminary administration of arginine-vasotocin into the dorsal lymphatic sack decreased significantly the GFP absorption. The effect of arginine-vasotocin was inhibited by pretreatment with an antagonist of vasopressin V_i-receptors. The results of this work together with literature data allow believing that a decrease in the GFP absorption in the frog kidney under effect of arginine-vasotocin is due to a fall of the AVT-dependent glomerular filtration rate and to a decrease in the input of protein into the lumen of tubules. The action of arginine-vasotocin seems to be mediated via the V_i-like receptors of preglomerular blood vessels.     

A microscopic study of kidney tissue in Familial Mediterranean Fever patients

Familial Mediterranean Fever (FMF) is an autosomal recessive hereditary disease leading mostly to renal failure and nephrotic syndrome. The ultrastructure of kidney has not been fully investigated in FMF associated renal disease. The aim of this study is to provide further evidence on the ultrastructure of kidney in patients with FMF who suffer from renal disease. Renal biopsies obtained from two patients who were diagnosed with FMF renal disease complications were examined. Examination of renal tissue by light and electron microscopy identified degenerations both in tubules and the filtration barrier. Foot processes were partly effaced. Amorphous material was found in thickened glomerular basement membranes. Fibrous material deposits in thick Bowman's capsule wall were also seen. Finally, degeneration in the form of folding of plasma membrane and vacuolization as well as fusion in mitochondria cristae, was observed. Accumulation of tissue remnants in the lumen was also found in tubules.     

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.     

Fish Infected with Sphaerospora spp. Thélohan (Myxosporea) from Waters Enzootic for Proliferative Kidney Disease of Salmonids1

ABSTRACT. Sphaerospores were found among three species of fish examined from waters known to be enzootic for proliferative kidney disease (PKD) of salmonids. They were detected in the renal tubules of both hatchery-reared rainbow trout (Salmo gairdneri) exposed to the infectious stage of PKD and in chubs (Gila bicolor) in the headwaters of a hatchery where PKD is enzootic. Sticklebacks (Gasterosteus aculeatus) collected near net pens where Pacific salmon had experienced a PKD epizootic were also found to harbor sphaerospores in the lumen of the kidney tubules. The latter two host species contained developmental stages of a myxosporidan in the blood and in the lumen of the kidney tubules which are similar to those of PKX, the causative agent of PKD in salmonid fish. The sphaerospores observed in the rainbow trout are the first to be observed in this species. The similarity to previously observed developmental stages, rarity, and presence of these sphaerospores in salmonid fish from a hatchery where PKD is enzootic suggest that they are the most mature stage of the PKX myxosporidan yet observed.