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.     

Localization of S-adenosylhomocysteine hydrolase in the rat kidney.

S-adenosylhomocysteine (SAH) hydrolase is a cytosolic enzyme present in the kidney. Enzyme activities of SAH hydrolase were measured in the kidney in isolated glomeruli and tubules. SAH hydrolase activity was 0.62 +/- 0.02 mU/mg in the kidney, 0.32 +/- 0.03 mU/mg in the glomeruli, and 0.50 +/- 0.02 mU/mg in isolated tubules. Using immunohistochemical methods, we describe the localization of the enzyme SAH hydrolase in rat kidney with a highly specific antibody raised in rabbits against purified SAH hydrolase from bovine kidney. This antibody crossreacts to almost the same extent with the SAH hydrolase from different species such as rat, pig, and human. Using light microscopy, SAH hydrolase was visualized by the biotin-streptavidin-alkaline phosphatase immunohistochemical procedure. SAH hydrolase immunostaining was observed in glomeruli and in the epithelium of the proximal and distal tubules. The collecting ducts of the cortex and medulla were homogeneously stained. By using double immunofluorescence staining and two-channel immunofluorescence confocal laser scanning microscopy, we differentiated the glomerular cells (endothelium, mesangium, podocytes) and found intensive staining of podocytes. Our results show that the enzyme SAH hydrolase is found ubiquitously in the rat kidney. The prominent staining of SAH hydrolase in the podocytes may reflect high rates of transmethylation. (J Histochem Cytochem 48:211-218, 2000)     

Renal neuraminidase. Characterization in normal rat kidney and measurement in experimentally induced nephrotic syndrome.

Several lines of evidence suggest that increased neuraminidase activity may be responsible for the loss of glomerular N-acetylneuraminic acid (AcNeu) observed in various glomerular diseases. However, virtually no information is available on the activity of neuraminidase in glomeruli or the potential role of this enzyme in glomerular pathophysiology. Utilizing 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (4MU-AcNeu) as substrate, we defined optimal assay conditions and characterized neuraminidase activity in glomeruli and, for comparison, in other renal fractions and liver. Neuraminidase activity in glomeruli, cortex and tubules was maximal at pH 4.4. The Km for 4MU-AcNeu was estimated to be 195 microM for glomeruli and 226 microM for cortex. Glomerular neuraminidase was inhibited by AcNeu (90% at 25 mM) and high concentrations of Triton X-100 (26% at 0.5%), but unaffected by CaCl2, EDTA or N-ethylmaleimide (each 1 mM). Neuraminidase activity (nmol/h per mg of protein; mean +/- S.E.M.) in normal rat kidney was: cortex, 14.47 +/- 0.76; medulla, 7.85 +/- 0.64; papilla, 2.64 +/- 0.11; tubules, 13.79 +/- 0.70; glomeruli, 5.57 +/- 0.28. In comparison, neuraminidase activity in rat liver was 2.58 +/- 0.14. Puromycin aminonucleoside (PAN)-induced nephrotic syndrome is a model of glomerular disease in which the loss of glomerular AcNeu is well documented. In two separate studies, we observed no change in the specific activity of neuraminidase in either glomeruli or cortex isolated from rats treated with PAN (15 mg/100 g, intraperitoneally) and killed at either the onset or the peak of proteinuria. Results were similar whether neuraminidase activity was expressed per mg of protein or per microgram of DNA.     

Changes in the concentration of enolase isozymes and S-100 protein in degenerating and regenerating rat sciatic nerve

Levels of enolase isozymes (alpha alpha, alpha gamma, and gamma gamma forms) and S-100 protein in rat sciatic nerves were determined during their degeneration and regeneration processes. The sciatic nerves were unilaterally crushed or severed. The rats were killed 1, 2, 6, and 8-9 weeks later, and both the proximal and distal portions of the damaged nerves were dissected. Control samples were obtained from the untreated contralateral hindlimbs. Enolase isozymes and S-100 protein in the nerve segments were determined with the enzyme immunoassay method. The control nerves contained about 40, 90, and 30 pmol/mg protein of alpha alpha, alpha gamma, and gamma gamma enolases, respectively, and 0.85 microgram/mg protein of S-100 protein. These levels were not affected by repetitive electrical stimulation of the nerve fibers in vivo. The levels of the nervous system-specific forms of enolase (alpha gamma and gamma gamma) and S-100 protein decreased markedly within a week in the distal portion of the crushed nerve (alpha gamma, 27 pmol/mg; gamma gamma, 5.5 pmol/mg; S-100 protein, 0.36 microgram/mg) with apparently no change in the concentration of alpha alpha enolase. These levels in the proximal portion of the crushed nerve remained unaltered. The sensory and motor functions impaired by the sciatic nerve crush showed a recovery more or less after 4-9 weeks. This recovery was accompanied by a gradual regaining of the specific proteins in the distal portion of injured nerves (alpha gamma, 64 pmol/mg; gamma gamma, 13 pmol/mg; S-100 protein, 0.63 microgram/mg at the 8-9th week).     

Superoxide Anion Production and Expression of gp91phox and p47phox Are Increased in Glomeruli and Proximal Tubules of Cisplatin‐Treated Rats

The chemotherapeutic drug cisplatin has some side effects including nephrotoxicity that has been associated with reactive oxygen species production, particularly superoxide anion. The major source of superoxide anion is nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase. However, the specific segment of the nephron in which superoxide anion is produced has not been identified. Rats were sacrificed 72 h after cisplatin injection (7.5 mg/kg), and kidneys were obtained to isolate glomeruli and proximal and distal tubules. Cisplatin induced superoxide anion production in glomeruli and proximal tubules but not in distal tubules. This enhanced superoxide anion production was prevented by diphenylene iodonium, an inhibitor of NADPH oxidase. Consistently, this effect was associated with the increased expression of gp91^phox and p47^phox, subunits of NADPH oxidase. The enhanced superoxide anion production in glomeruli and proximal tubules, associated with the increased expression of gp91^phox and p47^phox, is involved in the oxidative stress in cisplatin‐induced nephrotoxicity.     

Dopamine production by isolated glomeruli and tubules from rat kidneys

To locate the sites of dopamine (D) production in rat renal cortex, we separated glomeruli and proximal tubules by sieving or centrifugation in Percoll after collagenase digestion. After centrifugation layer I contained 60-80% glomeruli and 20-40% tubule fragments, half of which did not stain with alkaline phosphatase, layer II contained 0-5% glomeruli, 10-25% tubule fragments other than proximal tubules, and 70-85% proximal tubule fragments. Layer IV contained 85-95% proximal tubules. Gluconeogenic rates were (micromoles per hour per gram wet weight) as follows: I, 4 +/- 1; II, 7 +/- 2; and IV, 16 +/- 1. Norepinephrine (NE) content was (picomoles per gram wet weight) I, 310 +/- 30; II, 540 +/- 40; IV, 195 +/- 60. D content was (picomoles per gram wet weight) I, 26 +/- 6; II, 46 +/- 13; IV, 33 +/- 7. Surgical denervation 4-6 days previously reduced the norepinephrine content of layers I and II to 35 +/- 10 (p less than 0.001) and of IV to 60 +/- 20 (p less than 0.05) and the D content of layers I and II to 13 +/- 6 and 6 +/- 6 pmol/g, respectively (p less than 0.01); D content of layer IV was unchanged. D production from 10(-7) M 3,4-dihydroxyphenylalanine (DOPA) was (nanomoles per gram per minute) I, 0.2 +/- 0.03; II, 0.7 +/- 0.1; IV, 1.0 +/- 0.04. DOPA consumption was (nanomoles per gram per minute) I, 0.6 +/- 0.1; II, 1.4 +/- 0.3; and IV, 1.8 +/- 0.2. Denervation did not change D production or DOPA consumption.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased cathepsin D-like activity in cortex, tubules, and glomeruli isolated from rats with experimental nephrotic syndrome.

We have examined the activity and distribution of cathepsin D (EC 3.4.23.5), a major renal lysosomal endoproteinase, in the various anatomical and functional areas of normal rat kidney. Cathepsin D-like activities (delta A280/h per mg of protein) in normal rat tissues were: cortex, 0.78 +/- 0.05, n = 37; medulla, 0.62 +/- 0.03, n = 12; papilla, 0.63 +/- 0.04, n = 12; tubules, 0.74 +/- 0.04, n = 28; glomeruli, 0.59 +/- 0.03, n = 28; and liver, 0.41 +/- 0.02, n = 28. Enzyme activity was maximal at pH 3.0-3.5 and inhibited more than 90% by pepstatin (6.7 micrograms/ml), suggesting that the enzyme is cathepsin D. In subsequent experiments we measured cathepsin D-like activity in cortex, tubules and glomeruli isolated from rats with puromycin aminonucleoside (PAN)-induced nephrotic syndrome. Treated animals (15 mg of PAN/100g body wt., intraperitoneally) developed proteinuria beginning 4 days after injection and exceeding 900 mg/24h on day 9. In two separate experiments involving 52 animals we observed a significant increase in cathepsin D-like activity in cortex (+82.7%), tubules (+109.6%) and glomeruli (+54.7%) isolated from PAN-treated rats killed during marked proteinuria (day 9, mean total urinary protein excretion: 937 +/- 94 mg/24h). This increase was observed whether the activity was expressed per mg of DNA or per mg of protein. Increased cathepsin D-like activity was first observed in cortex and tubules coincident with the onset of proteinurea (day 4, mean total urinary protein excretion: 112 +/- 23 mg/24h). In contrast with the significant elevation of renal cathepsin D-like activity, the activity (nmol/h per mg of protein) of alpha-L-fucosidase (EC 3.2.1.51), a non-proteolytic enzyme, was markedly decreased in the identical samples used for the measurement of cathepsin D-like activity: cortex (-46.4%); tubules (-46.1%); and glomeruli (-38.5%). In addition to changes in renal enzyme activities, PAN-treated rats excreted large amounts of cathepsin D-like activity in their urine (beginning on day 3) compared with nearly undetectable cathepsin D-like activity in the urine from control rats. The significant increases in glomerular and tubular cathepsin D activity may reflect an important role for this enzyme in the pathophysiology associated with PAN-induced nephrotic syndrome.     

Unique regional distribution of delta 4-3-ketosteroid-5 alpha-oxidoreductase and associated epididymal morphology in the marsupial, Didelphis virginiana

The epididymal epithelial ultrastructure has been described in the adult male North American opossum, Didelphis virginiana. Morphological results have suggested that absorptive activity is prominent in the proximal epididymal region by virtue of numerous microvilli, an endocytotic complex, dense granules, and multivesicular bodies in the apical cytoplasm. In contrast, the middle and distal epididymal regions exhibit ultrastructural features indicative of protein synthesis such as large invaginated euchromatic nuclei, large nucleoli, and increased amounts of granular endoplasmic reticulum. It is in the middle and distal epididymal regions where sperm head rotation and sperm pairing take place. Epididymal delta 4-3-ketosteroid-5 alpha-oxidoreductase (5 alpha-reductase) activity also has been measured. It has been found that the level of enzyme activity differs significantly (p less than 0.01) between the proximal, middle, and distal epididymal regions. Enzyme-specific activity has been found to be highest in the middle region (47.6 +/- 5.4 picomoles 5 alpha-reduced androgens formed/b/mg protein), lower in the distal region (18.3 +/- 0.7 picomoles 5 alpha-reduced androgens formed/b/mg protein), with little activity (2.4 +/- 1.2 picomoles 5 alpha-reduced androgens formed/h/mg protein) found in the proximal epididymal region. This regional distribution of enzyme activity differs markedly from that reported for eutherian mammals. Both the suggested epididymal protein synthetic and secretory activity and the level of epididymal 5 alpha-reductase activity appear to correlate regionally with the morphological changes that occur in the opossum spermatozoa as they transit the epididymis.     

Immunocytochemical localization of γ-glutamyl-transferase on isolated renal cortical tubular fragments

Summary The localization of -Glutamyltransferase (-GT, E.C.2.3.2.2.) was studied on isolated tubular fragments from rat kidney cortex immunocytochemically. Monospecific antibodies raised in the goat against rat kidney -GT were used. Antigoat immunoglobulin from the rabbit conjugated with ferritin was used for visualisation of the antibody binding sites. The enzyme was found to be localized at the brush border membrane of proximal tubules, the luminal membrane of distal tubules and collecting duct segments.The enzyme could further be localized on the antiluminal or basolateral cell membranes of proximal and distal tubular fragments, whereas no such localization was verified for collecting duct segments. The role of this basolateral -GT localization in context with the kidney's ability to extract over 83% of the renal arterial glutathione (GSH) input during a single passage is discussed.     

Three-dimensional architecture of the golgi apparatus in Sertoli cells of the rat.

Glutaraldehyde-fixed testes were stained "en bloc" with the Ur-Pb-Cu technique of Thiéry and Rambourg ('76) or post-fixed and stained with the osmium tetroxide-potassium ferrocyanide method of Karnovsky ('71). Thin or thick (up to 3 micron) sections were examined with the Philips (301 or 400) EM or the high voltage EM. Stereopairs were prepared with photographs of tilted specimens (+/- 7 degrees). At low magnification, in thick sections (0.5-3 micron) stained with Ur-Pb-Cu, the whole Golgi apparatus formed a single network of interconnected wavy ribbon or platelike structures extending from the juxtanuclear region toward the apex of the cell. At higher magnifications, with the two staining techniques, this Golgi network showed two distinct types of regions: the "saccular region" corresponding to the conventional stack of saccules and the "intersaccular connecting region" made up of anastomotic tubules which bridge adjacent stacks. In the saccurlar regions, there was, on the cis-face of the stack, a tight polygonal meshwork of anastomotic tubules (osmiophilic element). Underlying it there were three to seven closely apposed saccules perforated with pores of various diameters, and finally, on the trans-face, a network of tubules was usually connected to the last saccule of the stack, which seemed to peel off" from the pile. The intersaccular connecting regions showed proximal and distal zones with regard to the associated stacks. The proximal zone was made up of superimposed and parallel polygonal networks of membranous tubules which were continuous with corresponding saccules of the stack. In the distal zone they interdigitated, intertwined, anastomosed and bridged adjacent saccular regions; others turned at right angles and established connections with tubular extensions arising at various levels of the same stack. While cisternae of endoplasmic reticulum were contiguous with tubules or saccules located on the transface of the Golgi apparatus, a close association between the ER cisternae and the cis-face of the stacks was not usually observed.     

Effect of uroguanylin on potassium and bicarbonate transport in rat renal tubules

The effect of uroguanylin (UGN) on K+ and H+ secretion in the renal tubules of the rat kidney was studied using in vivo stationary microperfusion. For the study of K+ secretion, a tubule was punctured to inject a column of FDC-green-colored Ringer's solution with 0.5 mmol KCl/L+/-10(-6) mol UGN/L, and oil was used to block fluid flow. K+ activity and transepithelial potential differences (PD) were measured with double microelectrodes (K+ ion-selective resin vs. reference) in the distal tubules of the same nephron. During perfusion, K+ activity rose exponentially, from 0.5 mmol/L to stationary concentration, allowing for the calculation of K+ secretion (JK). JK increased from 0.63+/-0.06 nmol.cm-2.s-1 in the control group to 0.85+/-0.06 in the UGN group (p<0.01). PD was -51.0+/-5.3 mV in the control group and -50.3+/-4.98 mV in the UGN group. In the presence of 10(-7) mol iberiotoxin/L, the UGN effect was abolished: JK was 0.37+/-0.038 nmol.cm-2.s-1 in the absence of, and 0.38+/-0.025 in the presence of, UGN, indicating its action on maxi-K channels. In another series of experiments, renal tubule acidification was studied, using a similar method: proximal and distal tubules were perfused with solutions containing 25 mmol NaHCO3/L. Acidification half-time was increased both in proximal and distal segments and, as a consequence, bicarbonate reabsorption decreased in the presence of UGN (in proximal tubules, from 2.40+/-0.26 to 1.56+/-0.21 nmol.cm-2.s-1). When the Na+/H+ exchanger was inhibited by 10(-4) mol hexamethylene amiloride (HMA)/L, the control and UGN groups were not significantly different. In the late distal tubule, after HMA, UGN significantly reduced JHCO3-, indicating an effect of UGN on H+-ATPase. These data show that UGN stimulated JK+ by acting on maxi-K channels, and decreased JHCO3- by acting on NHE3 in proximal and H+-ATPase in distal tubules.     

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.     

Dissolution of granules in the Malpighian tubules of Musca autumnalis DeGeer,during mineralization of the puparium

The dissolution of mineralized granules stored in the larval Malpighian tubules of the face fly, Musca autumnalis DeGeer, was studied both in situ and with isolated granules in vitro. The release of calcium, phosphorus, and magnesium from granules increased exponentially as the pH of the bathing medium was decreased. The pH measured in the distal region of the Malpighian tubules was 8.08 while that of the proximal region was 7.35. Thus, the decrease in pH of lumen contents from distal to proximal regions of the tubules appears to be a major effector of granule dissolution. Loss of structural integrity of the granules accompanied mineral release and also increased as pH of the bathing medium was lowered in vitro. This structural disintegration was similar to that observed in naturally dissolving granules isolated from the proximal region of the Malpighian tubules. The larval tubules, therefore, appear to have regional specialization in that granules are formed and stored in the distal lumen and dissolution takes place as granules move into the more acidic proximal region. No granules were found in the larval hindgut contents also indicating that dissolution and transport take place in the proximal region of the tubules. However, granules of similar composition were found in the meconium and in the most distal regions of adult Malpighian tubules.     

Parathyroid hormone stimulates ATP-dependent calcium pump activity by a different mode in proximal and distal tubules of the rat.

A new technique was developed to isolate basolateral membrane vesicles individually from proximal and distal tubules of the rat cortex. This new technique enabled us to study differences in their kinetics and mechanisms of hormonal regulation of Ca pump between proximal and distal tubules. The Ca pump in distal tubule has very high affinity (42.6 nM Ca2+) and the one in proximal tubule has relatively low affinity (75.6 nM Ca2+). Parathyroidectomy (PTX) decreased the Vmax of Ca pump activity in proximal tubule (4.68 +/- 0.99 vs. 9.08 +/- 2.21 nmol 45Ca2+/min per mg protein BLMV, P less than 0.05), while it increased Km in distal tubule (93.1 +/- 11.0 vs. 35.1 +/- 16.1 nM Ca2+, P less than 0.05). Restoration of serum Ca2+ concentration by 1,25(OH)2D3 supplement could not reverse these changes by PTX in Ca pump activity in either the proximal or the distal tubule. In conclusion, this study strongly suggested that parathyroid hormone stimulated Ca pump activity by increasing the Vmax in proximal tubule and by increasing the affinity in distal tubule. 1,25(OH)2D3 does not have a direct effect on the basolateral membrane Ca pump activity.     

Thromboxane receptors in human kidney tissues.

Thromboxane (TX) A2 effects in the kidneys include contraction of glomerular mesangial cells and intrarenal vascular tissue. A kidney cDNA encoding a TX receptor expressed in rat renal glomeruli and rat renal arterial smooth muscle cells has been reported. However, TXA2 receptors in human kidneys have not been documented. The purpose of this study was to identify and characterize TXA2 receptors in glomeruli and intrarenal arteries isolated from human kidneys. Normal kidneys, not used for transplant because of technical reasons, were kept at -70 degrees C and used for research purposes. The glomeruli and intrarenal arteries were isolated from renal cortical tissue by a mechanical sieving technique. The equilibrium dissociation constant and receptor number were determined by nonlinear analysis of binding inhibition data. The data were generated in radioreceptor assays using [125I]-BOP, a stable analog of TXA2. The dissociation constants (mean +/- SEM) for binding of I-BOP to human glomeruli and intrarenal arterial membranes were 6.6 +/- 1.1 nM (n = 7) and 20 +/- 6 nM (n = 7), respectively (p < 0.05). The receptor number was 311 +/- 91 fmol/mg protein (n = 7) in glomeruli and 74 +/- 16 fmol/mg protein (n = 7) in intrarenal arterial membranes (p < 0.04). The order of specificity of TXA2 analogs for [125I]-BOP binding sites was similar in glomeruli and in arterial membranes and was I-BOP > or = U46619 > or = pinane TXA2 > or = carbocyclic TXA2 > or = PGH2. These findings provide direct evidence for the presence of specific, high-affinity [125I]-BOP binding sites in human renal glomeruli and extraglomerular vascular tissue. These data also indicate that the human binding sites have higher affinity for the TXA2 agonist I-BOP than for PGH2.     

ELECTRON MICROSCOPE STUDIES ON THE SURFACE COAT OF THE NEPHRON

Attempts to make visible the carbohydrate coat at the free cell surface of glomeruli as well as the tubules of rabbit kidney were undertaken. The ruthenium red procedure was performed, according to Luft, at various pH values. Moreover, the colloidal iron and the colloidal thorium methods were used. Neuraminidase digestion was also performed. In the ruthenium red procedure the luminal face of the epithelial cells of the nephron was coated distinctly with reaction product. The results obtained revealed that some of the differences at various levels of the nephron depended on the pH values. In glomeruli and proximal convoluted tubules the optimum pH value was 7.4; in the ascending limb of Henle loops and distal convoluted tubules the optimum pH value was 6.8. The ruthenium red-positive surface coat was either closely connected with, or appeared as a part of, the outer leaflet of the unit membrane. The slit pores of glomeruli were also covered by a coat continuous with the surface coat of the adjacent foot processes. The coat lining the microvilli of proximal convoluted tubules completely filled the intervillous spaces. Also, both the colloidal iron method and the colloidal thorium method evidenced the presence of surface coat. Pre-treatment with neuraminidase abolished the effect of the Hale reaction. These results may indicate that the surface coat of the epithelia of the nephron shows the presence of glycoproteins containing siliac acid residues.     

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.     

Catecholamine production along the nephron.

The present work proposes an extra neural site of catecholamine production along the nephron. LLC-PK(1), MDCK, and mIMCD-3 (proximal and distal tubules and inner medullary collecting duct, respectively) presented the following amine concentrations in the cell homogenates: Norepinephrine = 275+/-34, 56+/-16 and 255+/-21; Epinephrine = 161+/-20, 83+/-17 and 53+/-7; and Dopamine = 63+/-15, 39+/-6 and 36+/-7 pg/mg cell protein (Means +/- SEM), respectively. The culture medium showed Norepinephrine = 168+/-25, 22+/-3 and 135+/-8; Epinephrine = 32+/-6, 152+/-17 and 39+/-5; and Dopamine = 27+/-9, 241+/-34 and 26+/-5 pg/mg cell protein, respectively. The synthesis enzymes as tyrosine hydroxylase, dopa decarboxylase and dopamine beta-hydroxylase were detected by Western blotting. Biopterin, the enzymatic cofactor of tyrosine hydroxylase, was quantified in the intracellular and medium of mIMCD-3 cells (17+/-4 and 24+/-3 nmol/mg cell protein, respectively) and in the medium of MDCK cells (19+/-4 nmol/mg cell protein). The data confirmed that the proximal tubule is an important source of dopa decarboxilase and Dopamine and epithelial cell along the nephron express the biochemical pathway for catecholamine production.     

Renal immunolocalization of kallikrein in cisplatin nephrotoxicity in rats

Summary Treatment of rats with cisplatin (4 mg kg^-1body wt i.p. injection) induced variations of urinary kallikrein excretion (UKE). Three phases were observed: a transient increase of UKE one day after injection, followed by a decrease up to 10 days suggesting an altered biosynthesis and a recovery phase with return to normal control values, 21 days after injection. Early morphological lesions were observed in proximal tubule cells on day 1; severe changes and tubular necrosis were observed in the following days. Less marked changes were also present in distal tubules but the vacuolated and desquamated cells appeared in the lumen of the tubules. By immunocytochemical methods, kallikrein was observed in connecting tubule cells, but also in some proximal tubule cells and along the endothelial side of the glomerular basement membrane and urinary space of glomeruli. An intense labelling was present in desquamated epithelial cells in dilated lumen of tubules. This study provides evidence of the presence of immunoreactive kallikrein in the glomerulus, already reported during acute failure, and confirms the use of urinary kallikrein measurements as a useful non-invasive index to assess a possible nephrotoxic effect at the distal level.