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.     

Indicators of functional differentiation of the chick embryonic kidney

Relevant indicators of the functional capability of the embryonic kidney were tested in the chick mesonephros chosen as an ideal model accessible to direct observation in vivo. Evidence of glomerular filtration (GF) was checked up by the arterial injection of 2% lissamine green (LG) followed by measurement of the LG passage time on days 5, 6 and 7. Presence of the electrogenic transport was investigated by determining the transepithelial potential difference (TPD) which distinguished proximal and distal tubules of the 6-day nephrons. GF and tubular reabsorption could be demonstrated from day 5 by the storage of trypan blue (TB) in proximal tubules after intra-amniotic administration of the dye. The distribution of tubule staining corresponded to the proximal-distal gradient of the nephron differentiation. Activities of embrane enzymes, alkaline phosphatase and 5'-nucleotidase, were detected from day 4. They preceded the ultrastructural maturation in the differentiating proximal tubule epithelia. A semiquantitative evaluation of enzyme activities by the method of measuring of the minimum incubation time (MIT) together with the TB storage, appeared reliable and relevant indicators of the functional properties of mesonephric nephrons, suitable for distinguishing between more and less advanced stages of the nephron development.     

Nephron structure and immunohistochemical localization of ion pumps and aquaporins in the kidney of frogs inhabiting different environments

Amphibians inhabit areas ranging from completely aqueous to terrestrial environments and move between water and land. The kidneys of all anurans are similar at the gross morphological level: the structure of their nephrons is related to habitat. According to the observation by light and electron microscopy, the cells that make up the nephron differ among species. Immunohistochemical studies using antibodies to various ATPases showed a significant species difference depending on habitat. The immunoreactivity for Na+,K(+)-ATPase was low in the proximal tubules but high in the basolateral membranes of early distal tubules to collecting ducts in all species. In the proximal tubule, apical membranes of the cells were slightly immunoreactive to H(+)-ATPase antibody in aquatic species. In the connecting tubule and the collecting duct, the apical membrane of intercalated cells was immunoreactive in all species. In aquatic species, H+,K(+)-ATPase immunoreactivity was observed in cell along the proximal, distal tubule to the collecting duct. However, H+,K(+)-ATPase was present along the intercalated cells of the distal segments from early distal to collecting tubules in terrestrial and semi-aquatic species. In the renal corpuscle, the neck segment and the intermediate segment, immunoreactivities to ion pumps were not observed in any of the species examined. Taking together our observations, we conclude that in the aquatic species, a large volume of plasma must be filtered in a large glomerulus and the ultrafiltrate components are reabsorbed along a large and long proximal segment of the nephron. Control of tubular transport may be poorly developed when a small short distal segment of the nephron is observed. On the contrary, terrestrial species have a long and well-developed distal segment and regulation mechanisms of tubular transport may have evolved in these segments. Thus, the development of the late distal segments of the nephron is one of the important factors for the terrestrial adaptation.     

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

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

Modification of genital kidney nephrons for sperm transport in a plethodontid salamander,Eurycea longicauda

Salamanders possess kidneys with two distinct regions: a caudal pelvic portion and cranial genital portion. Nephrons of the pelvic region are responsible for urine formation and transport. Nephrons of the genital region transport sperm from testes to Wolffian ducts; however, nephrons of the genital region possess all the same functional regions found in pelvic kidney nephrons that are involved with urine formation and transport (renal corpuscles, proximal tubules, distal tubules, and collecting ducts). Morphological similarities between pelvic and genital regions stimulated past researchers to hypothesize that nephrons of genital kidneys possess dual function; that is, sperm transport and urine formation/transport. Considering size of glomeruli is directly related to the total amount of blood plasma filtered into the Bowman's space, we tested the hypothesis that nephrons of genital kidneys have reduced urine formation function by comparing glomerular size between nephrons of pelvic and genital kidney regions in Eurycea longicauda with general histological techniques. Light microscopy analysis revealed that glomeruli of pelvic kidneys were significantly larger than those measured from genital kidneys. Transmission electron microscopy analysis also revealed modifications in genital kidney nephrons when compared to pelvic kidney nephrons that suggested a decrease in urine formation function in genital kidneys. Such modifications included a decrease in basal and lateral plasma membrane folding in genital kidney proximal and distal tubules compared to that of pelvic kidney proximal and distal tubules. Genital kidney proximal tubules were also ciliated, which was not observed in pelvic kidney proximal tubules. In conclusion, although structurally similar at the histological level, it appears that nephrons of genital kidneys have decreased urine formation function based on glomerular size comparison and nephron ultrastructure.     

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.     

Characterization of N-ethylmaleimide-sensitive proton pump in the rat kidney. Localization along the nephron

This study is aimed both at characterizing an ATPase activity in rat kidney equivalent to the proton pump described in bovine kidney medulla and at localizing this enzyme along the nephron. Membrane fractions isolated from kidney homogenates by differential and density gradient centrifugations were enriched 7-fold in ATPase activity sensitive to N-ethylmaleimide (NEM). These fractions also displayed ATP-dependent proton transport. ATPase activity and proton transport in vesicles had similar pharmacological properties as both were insensitive to vanadate and ouabain and had similar sensitivities toward NEM (apparent Ki = 20 microM) and N,N'-dicyclohexylcarbodiimide (apparent Ki = 50 microM). Proton transport was dependent on chloride availability as chloride addition to the extravesicular medium stimulated proton transport in a dose-dependent fashion (apparent K 1/2 = 7 mM). NEM-sensitive ATPase activity displaying similar pharmacological properties as proton transport in vesicles was also found in single segments of nephron. It was insensitive to vanadate and ouabain, was inhibited by similar concentrations of NEM (apparent Ki = 15-20 microM) and N,N'-dicyclohexylcarbodiimide (apparent Ki = 30 microM), and is therefore likely to be a proton pump. NEM-sensitive ATPase was localized in all the segments of the rat nephron; its activity was highest in proximal convoluted tubules; intermediate in proximal straight tubules, thick ascending limbs, and cortical collecting tubules; and lowest in outer medullary collecting tubules.     

Transport ATPase cytochemistry: ultrastructural localization of potassium-dependent and potassium-independent phosphatase activities in rat kidney cortex

A cytochemical method for the light and electron microscope localization of the K- and Mg-dependent phosphatase component of the Na-K-ATPase complex was applied to rat kidney cortex, utilizing p-nitrophenylphosphate (NPP) as substrate. Localization of K-N-ATPase activity in kidneys fixed by perfusion with 1% paraformaldehyde -0.25% glutaraldehyde demonstrated that distal tubules are the major cortical site for this sodium transport enzyme. Cortical collecting tubules were moderately reactive, whereas activity in proximal tubules was resolved only after short fixation times and long incubations. In all cases, K-NPPase activity was restricted to the cytoplasmic side of the basolateral plasma membranes, which are characterized in these neplron segments by elaborate folding of the cell surface. Although the rat K-NPPase appeared almost completely insensitive to ouabain with this cytochemical medium, parallel studies with the more glycoside-sensitive rabbit kidney indicated that K-NPPase activity in these nephron segments is sensitive to this inhibitor. In addition to K-NPPase, nonspecific alkaline phosphatase also hydrolyzed NPP. The latter could be differentiated cytochemically from the specific phosphatase, since alkaline phosphatase was K-independent, insensitive to ouabain, and specifically inhibited by cysteine. Unlike K-NPPPase, alkaline phosphatase was localized primarily to the extracellular side of the microvillar border of proximal tubules. A small amount of cysteine-sensitive activity was resolved along peritubular surfaces of proximal tubules. Distal tubules were unreactive. In comparative studies, Mg-ATPase activity was localized along the extracellular side of the luminal and basolateral surfaces of proximal and distal tubules and the basolateral membranes of collecting tubules.     

Lysosomal sulfoglycolipid storage in the kidneys of mice deficient for arylsulfatase A (ASA) and of double-knockout mice deficient for ASA and galactosylceramide synthase

The inherited deficiency of arylsulfatase A (ASA) causes lysosomal accumulation of sulfoglycolipids (mainly sulfo-galactosylceramide, S-GalCer ) and leads to metachromatic leukodystrophy in humans. Among visceral organs, kidneys are particularly affected. In the present study, the regional distribution and temporal development of sulfoglycolipid storage in kidneys of ASA-/- mice was investigated histochemically (alcian blue) and ultrastructurally. Furthermore, the sulfoglycolipid storage was examined in kidneys of double-knockout mice, which are incapable of: (a) degrading any sulfolipids (ASA-/-) and (b) synthesizing the major sulfolipid S-GalCer because of deficiency for galactosylceramide synthase (CGT), with the aim to search for additional ASA substrates. In ASA-/- mice, the nephron segments could be ranged in the order of decreasing sulfolipid storage: thin limbs of long-looped nephrons approximately thick ascending limbs > distal convoluted tubules > collecting ducts approximately short thin limbs. Macula densa and proximal tubules were unaffected. In ASA-/-/CGT-/- mice, the long thin limbs and distal convoluted tubules resembled those of ASA-/-/CGT+/+ mice, while the other segments showed less storage. The results suggest that the turnover of sulfolipids in general is highest in the distal nephron except macula densa, and that long thin limbs and distal convoluted tubules are the main sites for turnover of a minor sulfolipid species, which is known to be synthesized in the kidney of CGT-/- mice.     

Collectrin/tmem27 is expressed at high levels in all segments of the developing Xenopus pronephric nephron and in the Wolffian duct

Collectrin/tmem27 encodes a transmembrane protein that plays a critical role in amino-acid transport. Originally described as being expressed only in collecting ducts, it has subsequently also been shown to also be expressed in the S1 segment of the proximal tubule of mammalian metanephric nephrons. In this report we describe the expression of collectrin in the simple embryonic kidney of amphibians, the pronephros. Each pronephros contains a single large nephron with a proximo-distal segmentation very similar to that of mammalian metanephric nephrons. Analysis of collectrin expression in pronephroi at a variety of embryonic stages indicates that this gene is expressed at very high levels throughout the pronephric system, including proximal and distal segments and the Wolffian duct. Expression in the pronephros commences at Xenopus embryonic stage 28 which corresponds to when epithelialization begins within the pronephric mesenchyme. Like the Na+K+ATPase/atp1a1, another highly expressed pronephric marker, collectrin is also expressed in the cloaca but not in the cloacal derived posterior segment of the Wolffian duct, the rectal diverticulum. Unlike the Na+K+ATPase, which is expressed at lower levels in proximal portions of the pronephric nephron, expression of collectrin is even throughout all of the pronephric epithelia. This expression domain extends far beyond that shown to express amino-acid transporters and indicates collectrin may function in facilitating additional transport processes. Its high level of expression and broad distribution make it an excellent marker with which to examine pronephric kidney development.     

A microdissection study of Lampetra fluviatilis lamprey nephrons]

Each of the nephrons in the lamprey L. fluviatilis consists of three distinct segments-proximal, thin, and distal ones. Proximal segments are differentiated into a convoluted and a descending parts, whereas distal ones-into a convoluted and an ascending parts. Therefore microdissection studies indicate that the anatomical composition of a single nephron in the river lamprey is identical to that of a superficial nephron in mammals. Parallel arrangement of the proximal descending, thin, and distal ascending segments, as well as of the collecting tubules, also makes the kidney of the lamprey similar to the countercurrent system in the medulla of mammalian kidney. The data obtained imply that Henle's loop is present in the kidneys not only of higher Vertebrates, but of Cyclostomes as well.     

Kallikrein (kininogenase) in the mouse nephron: effect of dietary potassium

Kininogenase activity of kallikrein was measured in microdissected mouse nephron segments using kininogen from dog plasma and a radioimmunoassay for bradykinin. When single nephron segments were examined, results showed a large scatter. This was found to be due to heterogeneity of distal convoluted tubules (DCT) from different nephrons, since replicate measurements in pools of DCT structures did not show this degree of variation. Nearly 20% of activity was accessible to extracellular substrate when freshly dissected segments were incubated in isoosmotic media. Freezing and thawing which markedly releases activity of intracellular enzymes, did not significantly elevate kininogenase activity. On the other hand deoxycholate and trypsin treatment increased tubular kininogenase activity in an additive fashion. A detailed analysis of microdissected tubule fragments revealed that kallikrein is concentrated in late distal convoluted tubule before entering a branching point (connecting tubule). In contrast initial portions of distal convoluted tubules and cortical collecting tubules contained only little kallikrein activity. Potassium rich diet increased basal and total activity 5-fold, when compared to a potassium poor diet.     

Lack of anion effect on volume expansion natriuresis in the developing canine kidney

The renal response to volume expansion with sodium chloride or sodium bicarbonate was studied in 15 newborn and 13 adult dogs. Proximal and distal nephron function were estimated using the technique of distal nephron blockade. Fractional sodium reabsorption was 99.0 +/- 0.3% in newborn and 96.6 +/- 0.06% in adult during the NaCl expansion (P less than 0.01) and 98.1 +/- 0.7% in the newborn and 93.2 +/- 0.7% in the adult during NaHCO3 expansion (P less than 0.001). With either anion the higher fractional sodium reabsorption in the newborn was due to reabsorption of a greater fraction of the load presented to the distal nephron segment. The percent of the distal sodium load that was reabsorbed was 98.0 +/- 0.6% in the newborn and 92.2 +/- 1.0% in the adult during NaCl expansion, and 96.1 +/- 1.3% in the newborn and 81.5 +/- 2.4% in the adult during NaHCO3 expansion. Differences in distal nephron chloride, potassium and bicarbonate reabsorption among the groups support the hypothesis that the enhanced distal sodium reabsorption in the newborn occurred largely in the ascending loop of Henle with NaCl expansion, while it occurred in the late distal and cortical collecting tubules with NaHCO3 expansion. There was no difference between the natriuretic responses to NaCl or NaHCO3 in the newborn (P greater than 0.20); however, the natriuretic response to NaCl was less than that to NaHCO3 in the adult (P less than 0.001). This suggests that the bulk of the sodium that escaped reabsorption in Henle's loop during NaHCO3 expansion was reabsorbed in the late distal tubule in the newborn, but not in the adult.     

A micropuncture study of several indices of renal function in rats during development]

The function of single superficial nephrons has been studied by means of several micropuncture methods in 22-, 30- and 42-day rats. It was shown that intratubular hydrostatic pressure, transit time of tubular fluid through a proximal tubule and Henle's loop, as well as local reabsorption in the proximal tubules measured by Gertz's split oil droplet method increase between the 22nd and the 30th days. The ration of tubular fluid and plasma (TF/P) inulin concentrations in late proximal and in early distal tubules increases with age. The values of TF/P for Na in early distal tubules are higher in 22- and 30-day rats than in older ones. TF/P for K does not change simultaneously with that for Na. These data are consistent with the assumption that the sodium load in the distal part of the nephron is higher in young rats than in adult ones.     

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

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

Entry of nephrons into the collecting duct network of the avian kidney: A comparison of chickens and desert quail

The avian kidney contains a population of nephrons with and without loops of Henle. How the collecting ducts of this heterogeneous population of nephrons merge to exit as single ducts from the medullary cones has been uncertain. The results of this study show that the collecting duct tree begins with the coalescence of the distal tubules of pairs of loopless nephrons. These primary collecting ducts receive output from only loopless nephrons. Primary collecting ducts fuse in pairs and become secondary collecting ducts. They receive the distal tubules of transition nephrons. Pairs of secondary collecting ducts fuse and become tertiary collecting ducts. Tertiary collecting ducts receive the distal tubules of looped nephrons. Thus, the fluid from all nephron types comingles as it passes through the medullary cone. The results of this study also show that the anatomical arrangement of medullary cones does not permit the output from one medullary cone to enter a second medullary cone. Thus, all the medullary cones function as parallel units. This anatomical organization of the avian kidney affects its ability to produce a urine hyperosmotic to the plasma. © 1993 Wiley-Liss, Inc.     

Localization of thyroxine 5'-monodeiodinase activity in the renal proximal tubules in rabbits and rats

To determine the localization of T4 5'-monodeiodinase activity in rabbit and rat nephron segments, the formation of tri-iodothyronine (T3) from thyroxine (T4) was measured in kidney homogenate and in isolated nephron segments obtained by the microdissection method. In order of decreasing activity, homogenates of rabbit renal cortex, outer medulla and inner medulla were capable of converting T4 to T3. In the isolated nephron segments of the rabbit cortex, the activities were noted in both proximal convoluted and proximal straight tubules. On the other hand, the activities were not detected in segments including the cortical thick ascending limb of Henle's loop, the distal convoluted tubule, the connecting tubule, and the cortical collecting tubule. It is concluded that both the convoluted and the straight tubules are the sites of T3 production in the kidney.