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.     

Stereological studies on the ultrastructural markers of renal tubular transport during accelerations acting along the +Gz axis

The study was carried out on rats. Electron micrographs of the kidney obtained under +Gz accelerations were analysed. Using stereological methods the estimation of ultrastructural markers of active transport (mitochondria energy state), and passive transport (intercellular spaces and basal infolded channels) was performed in the proximal and distal tubules. The results obtained indicated that during the actions of accelerations active tubular transport was impaired as reflected by a lowering of mitochondrial energy states (similar to condensed) in the proximal and distal tubules. Widening of intercellular spaces and basal infolded channels was also observed, which suggests fluid stasis and thus impairment of passive transport.     

The renal response of sheep to a low dietary nitrogen intake

Renal functions were tested in sheep fed on a low nitrogen diet (LN sheep), with a daily N intake of 4.7 g (gross energy 17.76 . 10(6) J). Sheep given a high nitrogen diet (HN sheep) with 21.2 g N (24.12 . 10(6) J) acted as the control. The functions of the left kidney were measured in anaesthetized animals by the standard clearance technique. A comparison of HN and LN sheep showed that a low nitrogen intake led to a drop in the plasma urea level (from 5.91 +/- 0.35 to 2.87 +/- 0.36 mmol.1-1, (P less than 0.001), the glomerular filtration rate (GFR, from 36.6 +/- 3.6 to 20.7 +/- 2.4 ml.min-1, P less than 0.005), amount of urea excreted (from 106.7 +/- 18.1 to 15.7 +/- 3.3 mumol.min-1, P less than 0.001), fractional urea excretion (from 51.0 +/- 3.0 to 24.6 +/- 3.1 %, P less than 0.001) and the absolute tubular reabsorption of urea (Reaburea/GFR (from 3.06 +/- 0.27 to 2.12 +/- 0.28 mumol.ml-1, P less than 0.05), without a significant change in the effective renal plasma flow (182.6 +/- 20.0 and 138.5 +/- 21.0 ml.min-1, non-significant - N.S.) and in sodium and potassium excretory function. Free water clearance rose in LN sheep (from -0.53 +/- 0.11 to -0.19 +/- 0.06 ml.min-1, P less than 0.05) owing to inhibited urea excretion. A regression analysis of the relationship of the tubular reabsorption of urea to the amount of filtered urea (both normalized to the GFR) showed that the urea transport capacity of the tubules of LN sheep was significantly higher.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Immunohistochemical Localization of Arginase II and Other Enzymes of Arginine Metabolism in Rat Kidney and Liver

Arginine is a precursor for the synthesis of urea, polyamines, creatine phosphate, nitric oxide and proteins. It is synthesized from ornithine by argininosuccinate synthetase and argininosuccinate lyase and is degraded by arginase, which consists of a liver-type (arginase I) and a non-hepatic type (arginase II). Recently, cDNAs for human and rat arginase II have been isolated. In this study, immunocytochemical analysis showed that human arginase II expressed in COS-7 cells was localized in the mitochondria. Arginase II mRNA was abundant in the rat small intestine and kidney. In the kidney, argininosuccinate synthetase and lyase were immunostained in the cortex, intensely in proximal tubules and much less intensely in distal tubules. In contrast, arginase II was stained intensely in the outer stripes of the outer medulla, presumably in the proximal straight tubules, and in a subpopulation of the proximal tubules in the cortex. Immunostaining of serial sections of the kidney showed that argininosuccinate synthetase and arginase II were collocalized in a subpopulation of proximal tubules in the cortex, whereas only the synthetase, but not arginase II, was present in another subpopulation of proximal tubules. In the liver, all the enzymes of the urea cycle, i.e. carbamylphosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase and lyase and arginase I, showed similar zonation patterns with staining more intense in periportal hepatocytes than in pericentral hepatocytes, although zonation of ornithine transcarbamylase was much less prominent. The implications of these results are discussed.     

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.     

Xenopus Bicaudal-C is required for the differentiation of the amphibian pronephros

The RNA-binding molecule Bicaudal-C regulates embryonic development in Drosophila and Xenopus. Interestingly, mouse mutants of Bicaudal-C do not show early patterning defects, but instead develop polycystic kidney disease (PKD). To further investigate the molecular mechanism of Bicaudal-C in kidney development, we analyzed its function in the developing amphibian pronephros. Bicaudal-C mRNA was present in the epithelial structures of the Xenopus pronephros, the tubules and the duct, but not the glomus. Inhibition of the translation of endogenous Bicaudal-C with antisense morpholino oligomers (xBic-C-MO) led to a PKD-like phenotype in Xenopus. Embryos lacking Bicaudal-C developed generalized edemas and dilated pronephric tubules and ducts. This phenotype was caused by impaired differentiation of the pronephros. Molecular markers specifically expressed in the late distal tubule were absent in xBic-C-MO-injected embryos. Furthermore, Bicaudal-C was not required for primary cilia formation, an important organelle affected in PKD. These data support the idea that Bicaudal-C functions downstream or parallel of a cilia-regulated signaling pathway. This pathway is required for terminal differentiation of the late distal tubule of the Xenopus pronephros and regulates renal epithelial cell differentiation, which--when disrupted--results in PKD.     

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.     

Modifications of the genital kidney proximal and distal tubules for sperm transport in Notophthalmus viridescens (Amphibia,Urodela, Salamandridae)

Male salamanders use nephrons from the genital kidney to transport sperm from the testicular lobules to the Wolffian duct. The microstructure of the epithelia of the genital kidney proximal tubule and distal tubule was studied over 1 year in a population of Notophthalmus viridescens from Crawford and Pike counties in central Missouri. Through ultrastructural analysis, we were able to support the hypothesis that the genital kidney nephrons are modified to aid in the transportation of sperm. A lack of folding of the basal plasma membrane, in both the genital kidney proximal and distal tubules when compared to the pelvic kidney proximal and distal tubules, reduces the surface area and thus likely decreases the efficiency of reabsorption in these nephron regions of the genital kidney. Ciliated epithelial cells are also present along the entire length of the genital kidney proximal tubule, but are lacking in the epithelium of the pelvic kidney proximal tubule. The exact function of these cilia remains unknown, but they may aid in mixing of seminal fluids or the transportation of immature sperm through the genital kidney nephrons. Ultrastructural analysis of proximal and distal tubules of the genital kidney revealed no seasonal variation in cellular activity and no mass production of seminal fluids throughout the reproductive cycle. Thus, we failed to support the hypothesis that the cellular activity of the epithelia lining the genital kidney nephrons is correlated to specific events in the reproductive cycle. The cytoplasmic contents and overall structure of the genital and pelvic kidney epithelial cells were similar to recent observations in Ambystoma maculatum, with the absence of abundant dense bodies apically in the epithelial cells lining the genital kidney distal tubule. J. Morphol. 275:914–922, 2014. © 2014 Wiley Periodicals, Inc.     

The distal nephron in the chick embryo as a target tissue for 1-alpha-25-dihydroxycholecalciferol

A histological and ultrastructural study as well as an autoradiographic analysis after injection of tritiated 1,25-dihydroxycholecalciferol (1,25-DHCC) were conducted on the distal convoluted tubules of chick embryos. Distal tubules in the embryo were shown to have the same spatial distribution as described for the adult kidney; they presented a convoluted portion located in the vicinity of the central intralobular veins and straight portions irradiating from this region towards the periphery. The epithelium in these tubules was well differentiated; its cells had numerous interdigitating folds in their lateral boundaries which were especially numerous at the basal ends. This device greatly increased the membrane surface available for interchange and was interpreted as an expression of active water and/or mineral transport. Nuclear concentration of radioactivity was found 2 h after injection of tritiated 1,25-DHCC in both the pars convoluta and the pars recta of the distal tubules. This concentration could be blocked by the previous administration of large amounts of nonradioactive 1,25-DHCC. These facts were interpreted as indicating that distal convoluted tubules in the chick embryo are functionally differentiated and contain target cells for 1,25-DHCC.     

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.     

Differential activation of CD95-mediated apoptosis related proteins in proximal and distal tubules during rat renal development

The CD95-mediated apoptotic pathway is the best characterized of the death receptor-mediated apoptotic pathways. The present study characterized localization and expression of proteins involved in CD95-mediated apoptosis during rat renal development. Kidneys were obtained from embryonic (E) 18 and 20-day-old fetuses and postnatal (P) 1-, 3-, 5-, 7-, 14-, and 21-day-old pups. Immunohistochemical characterization revealed that CD95, FasL and cleaved caspase-3 were strongly expressed in proximal tubules and weakly expressed in distal tubules, but that expression of caspase-8 in distal tubules was stronger than that in proximal tubules. Results from terminal deoxynucleotidyl transferase dUTP nick end labeling assays showed that levels of apoptosis in proximal tubules slowly increased after E18, while those of distal tubules slowly decreased after P5. Western blotting demonstrated that expression of CD95, FasL and FADD was very weak during embryonic development, but rapidly increased at P14. Expression of cleaved caspase-3 was maintained at high levels after P1, while caspase-8 expression gradually reached a peak at P7. Results from this study reveal that the CD95-mediated apoptotic pathway is a key driver of apoptosis in proximal tubules during late postnatal kidney development in rats and suggest that apoptosis in distal tubules is mediated by a different apoptotic pathway.     

Ouabain-insensitive, Na-ATPase activity in pure suspensions of rat kidney proximal tubules

The present work was undertaken to evaluate the distribution of the Na-ATPase activity in the different components of the rat kidney cortex. Suspensions of glomeruli, proximal and distal tubules were prepared following a collagenase digestion of outermost kidney cortex slices and a separation on a Percoll gradient. It was found that the Na-ATPase activity is higher in the fraction enriched in proximal tubules. The fraction enriched in glomeruli and in distal tubules show also a Na-ATPase activity, but it is lower.     

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.     

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.     

Transepithelial potential in mesonephric nephrons of 7-day-old chick embryos in relation to the histochemically detected sodium pump

In order to obtain basic information on the transport properties of differentiating embryonic nephrons, we examined the 7-day-old chick mesonephros by measuring the transtubular epithelial potential difference (TPD) and by histochemical detection of Na,K-ATPase activity. TPD as an indicator of the electrogenic transport was measured in individual segments of superficial nephrons in vivo. Their electric polarity was always lumen-negative. TPD was reduced by addition of 10 mM KCN applied to the mesonephric nephrons from the outside. In the proximal tubules, TPD was significantly lower (mean+/-SD: -1.0+/-0.5 mV) than in the distal and collecting tubules (-2.2+/-1.0 mV, p< or =0.05). Activity of the sodium pump was evaluated histochemically by detection of ouabain-sensitive potassium-dependent p-nitrophenyl phosphatase in cryostat sections of the mesonephros. The enzyme activity was demonstrated only in distal tubules and in the collecting ducts, but not in the proximal tubules. These findings have revealed significant differences between embryonic nephron segments: the distal tubule, in contrast to the proximal one, is supplied by the sodium pump and is able to generate higher TPD. Therefore, we consider that it is only the distal nephron, which possesses the ability of active transport.     

Immunocytochemical localization of Na+, K+-ATPase in the rat kidney

To determine if rat kidney Na+, K+-ATPase can be localized by immunoperoxidase staining after fixation and embedding, we prepared rabbit antiserum to purified lamb kidney medulla Na+, K+-ATPase. When sodium dodecylsulfate polyacrylamide electrophoretic gels of purified lamb kidney Na+, K+-ATPase and rat kidney microsomes were treated with antiserum (1:200), followed by [125I]-Protein A and autoradiography, the rat kidney microsomes showed a prominent radioactive band coincident with the alpha-subunit of the purified lamb kidney enzyme and a fainter radioactive band which corresponded to the beta-subunit. When the Na+, K+-ATPase antiserum was used for immunoperoxidase staining of paraffin and plastic sections of rat kidney fixed with Bouin's, glutaraldehyde, or paraformaldehyde, intense immunoreactive staining was present in the distal convoluted tubules, subcapsular collecting tubules, thick ascending limb of the loops of Henle, and papillary collecting ducts. Proximal convoluted tubules stained faintly, and the thin portions of the loops of Henle, straight descending portions of proximal tubules, and outer medullary collecting ducts did not stain. Staining was confined to basolateral surfaces of tubular epithelial cells. No staining was obtained with preimmune serum or primary antiserum absorbed with purified lamb kidney Na+, K+-ATPase, or with osmium tetroxide postfixation. We conclude that the basolateral membranes of the distal convoluted tubules and ascending thick limb of the loops of Henle are the major sites of immunoreactive Na+, K+-ATPase concentration in the rat kidney.     

Distribution of two aminotransferases and D-amino acid oxidase within the nephron of young and adult rats.

In the adult rat kidney, alanine aminotransferase (EC 2.6.1.2), aspartate aminotransferase (EC 2.6.1.1) and D-amino acid oxidase (EC 1.4.3.3) were measured in glomeruli, 4 parts of the proximal tubule, 2 parts of the distal tubule and in patches from the thin limb area and the papilla. These enzymes were measured in more limited parts of the nephron during postnatal development. Adult aspartate aminotransferase activities (percentage of the highest) ranged from 100 in the distal straight segment to 25 in the late part of the proximal straight segment to 10 in the thin limb and papillary area. Alanine aminotransferase (lower by a factor of 100 in absolute terms) was distributed as the mirror image of aspartate aminotransferase within proximal and distal tubules. D-Amino acid oxidase was 850-fold higher in proximal straight segments than in medullary structures. During development alanine aminotransferase increased 6-fold and D-amino acid oxidase, 4.5-fold in proximal straight tubules but aspartate aminotransferase increased in distal straight tubles 8-fold.     

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.