Morphogenesis and histogenesis of the connecting tubule in the rat kidney

Summary At the start of its morphogenesis the tubule of an S-shaped body always attaches to the terminal ampulla of the collecting duct. It remains attached there for some time while the terminal ampulla sprouts toward the renal capsule. The most distal part of the tubule that rises to the ampulla forms the connecting tubule, morphogenically the oldest segment of the tubulus nephroni. When younger S-shaped bodies join the same terminal ampulla, 2 to 3 connecting tubules are temporarily attached side by side to a single ampulla. Soon, however, the connecting tubule of the older nephron shifts its point of attachment away from the ampulla to the connecting tubule of the younger nephron. This process is repeated through successive nephron generations. Thus an arcade develops which at the close of morphogenesis includes the connecting tubules of the 3 to 4 oldest juxtamedullary and midcortical nephrons of the collecting duct and is always situated close to interlobular veins. The connecting tubules of the 2 youngest subcapsular nephrons are not incorporated into arcades, but join directly and permanently the collecting duct. — At the start of histogenesis, the undifferentiated tubule epithelium of the S-shaped body has a uniform structure over its entire length. At the junction of the connecting tubule and the terminal ampulla there is a cytologically sharp boundary between cuboidal, dark-stained connecting-tubule epithelium of the nephron and cylindrical, light-stained ampullary epithelium of the collecting duct. The epithelial differentiation begins in the oldest, juxtamedullary connecting tubules of an arcade and progresses upward to the terminal ampulla, so that immature connecting-tubule epithelium and immature ampullary epithelium are temporarily interposed between mature connecting-tubule epithelium and mature collecting-duct epithelium. The mature epithelium of the connecting tubule stains light, in which respect it behaves like the epithelium of the distal tubule. However, it also contains individual cells that are more strongly basophilic, stain dark and, though of nephrogenic origin, cannot be distinguished from the dark cells of the collecting duct by light microscopy. — Morphogenically and histogenically, the arcade-forming connecting tubules and the subcapsular connecting tubules arise from the nephrogenic blastema.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Histogenesis of the acquired cystic kidney disease: an immunohistochemical study.

The aim of this study was to characterize by immunohistochemistry the histogenesis of cysts in acquired cystic kidney disease (ACKD). Thirty renal tissues fixed in 10% formalin and embedded in paraffin from 20 cases of ACKD were studied. Vimentin was used to stain the Bowman's capsule epithelium, Lotus tetragonolobus agglutinin (LTA) and Leu M1 (CD15) for proximal tubules; Tamm-Horsfall protein (THP) for distal tubules; epithelial membrane antigen (EMA), cytokeratin 19 (CK19), Arachis hypogea agglutinin (PNA), and Glycine maximum agglutinin (SBA) for distal tubules and collecting ducts; and Ulex europaeus agglutinin (UEA-I) and Dolichos biflorus agglutinin (DBA) for collecting ducts. A histologically normal kidney, free of cystic disease, was used as a control for all the markers. Most of the cysts showed strong reactivity to LTA and CD15, an immunophenotype more characteristic of proximal tubules.     

Polycystic disease of the kidney. Evaluation and classification based on nephron segment and cell-type specific markers.

The usefulness of various segment and cell-type specific antibody, lectin and functional markers in the study of cystic renal lesions was evaluated. For this purpose, kidneys from recessive polycystic kidney disease (RPKD), thought to involve mainly the collecting ducts, and cystic kidneys of Meckel's syndrome (MS), which show dilation randomly along the nephron, were studied. The segment (and differentiation-stage)-specific anti-brush-border (specific for proximal tubules) antibodies stained morphologically normal proximal tubules, failed to react with cyst wall epithelium in RPKD, but readily stained some cysts in MS. Immunostaining for Tamm-Horsfall glycoprotein (distal tubules) similarly revealed normal tubular profiles, and also stained moderately dilated tubules, but not the large cysts in either disease type. Lectin markers of the distal tubules and collecting ducts (peanut agglutinin, Helix pomatia agglutinin and Dolichos biflorus agglutinin) reacted with both dilated tubules and with the cyst walls in RPKD and Meckel kidneys, suggesting that in RPKD, the dilations also occur in the distal nephron in addition to the collecting duct, and in MS in any part of the renal tubule. The cell type-specific functional marker of the collecting duct, anti-NaK-ATPase reactivity (found in principal cells) could be seen in RPKD but not in Meckel kidney cysts, suggesting a minor involvement of principal cells in MS. Consistent with this, only occasional carbonic anhydrase (found in intercalated cells) or band 3 (bicarbonate-chloride exchanger molecule of intercalated cells) of collecting ducts positive cells in the cysts could be seen, suggesting that intercalated cells are only sparsely seen in these lesions. The results show the usefulness of a panel of independent markers in studying the segment, cell-type and function-specific features of renal cystic lesions as a basis for their classification.     

Sodium transport-related proteins in the mammalian distal nephron – distribution, ontogeny and functional aspects

The mammalian distal nephron plays a pivotal role in adjusting urinary sodium excretion. Successive portions of the renal tubule are formed to adapt to this function, and an axial heterogeneity of the distal segments has been defined. The specific transport properties of these epithelia are accomplished by the expression of proteins (cotransporters, exchangers, channels) governing the movement of ions on either cell side. Molecular cloning of these proteins has had a marked impact on the study of their localization and function in the healthy and diseased kidney. Electroneutral cation-chloride cotransporters [Na(K)CC] have been localized to the thick ascending limb and the distal convoluted tubule using specific probes. Proteins implicated in the function of aldosterone target cells, such as the epithelial Na⁺ channel (ENaC), the mineralocorticoid receptor (MR) and 11β-hydroxysteroid dehydrogenase type 2 (11HSD2), an enzyme that confers mineralocorticoid specificity, have been found in the terminal portion of the nephron and the collecting duct. A mineralocorticoid-sensitive component of thiazide-sensitive NaCl transport has been identified in the distal convoluted tubule. Analysis of the ontogeny of these proteins in the maturing kidney has provided a detailed picture of epithelial differentiation and morphological specialization of the renal tubule. The study of mutations of the proteins related with NaCl transport has led to the identification of the molecular causes of inherited human diseases associated with hypo- or hypertension, and the respective sites of an impaired ion transport could be mapped to the renal tubule. Accepted: 13 April 1999     

A micropuncture study of urate excretion byCebus monkeys employing high performance liquid chromatography with amperometric detection of urate

Net renal reabsorption of endogenous urate was studied by the micropuncture technique inCebus monkeys in the absence of osmotic diuresis. Most of filtered urate (more than 70%) was reabsorbed in the proximal convoluted tubules. Samples from early distal tubules contained 9% of filtered urate; approximately 18% being reabsorbed between the late proximal and early distal segments. There was no detectable reabsorption along the distal tubule. Fractional delivery of urate to late distal tubules was greater than fractional excretion, implying reabsorption of some 4% of filtered urate in the collecting system. However, we cannot exclude nephron heterogeneity as the cause of the difference.The foregoing results were obtained using the method of Pachla and Kissinger for the determination of urate. Urate is separated by high performance liquid chromatography and detected by an amperometric technique. We found the method to be sufficiently sensitive, precise and specific for renal micropuncture samples.     

The elasmobranch kidney

Summary The nephron and collecting ducts of the little skate (Raja erinacea) and spiny dogfish shark (Squalus acanthias) have been investigated by light microscopy of semithin sections. Parts of the tubules (collecting ducts and distal segments) were identified after tubular injections with Microfil or carbon. The bundle zone was studied in serial sections. In the sinus zone transitions between the different segments were recorded. Thus, a complete reconstruction of the nephron, its subdivision into segments, and their localization in the kidney was accomplished.The nephron makes 4 loops. Beginning at Bowman's capsule, which sits between the bundle zone and sinus zone, the first loop is in the bundle zone. The nephron then extends into the sinus zone and turns back forming the second loop. This is followed by a third loop in the bundle zone which descends again into the sinus zone to form the last loop. The tail of the last loop (distal tubule) goes into the bundle zone and joins the collecting ducts. These collecting ducts are in the subcapsular connective tissue and progressively fuse to form a collecting tube. In the skate this tube traverses the thickness of the kidney between adjacent renal lobes to exit on the ventral kidney surface. In the shark, the large collecting ducts run on the surface of each lobe toward the medial margin of the kidney.Loops one and three and the early distal segment —all belonging to the same nephron - and a network of anastomosing capillaries form a bundle enclosed by a sheath of overlapping squamous cells termed peritubular sheath. This anatomical unit forms the renal countercurrent system of the marine elasmobranch. The tubular bundle has a straight portion in which the nephron segments are arranged in a highly parallel fashion. The remainder or the bundle and of the encasing peritubular sheath are convoluted. The sequence of the tubule morphology beginning at Bowman's capsule is: neck segment (early and late); proximal tubule (four portions), intermediate (six portions), distal tubule (early and late), collecting duct (early and late).Supported in part by National Institutes of Health AM 06345 and by Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 146)Alexander von Humboldt Fellow     

Stationary microperfusion study of phosphate reabsorption in proximal and distal nephron segments

Summary Micropuncture studies demonstrate phosphate reabsorption in proximal tubules and between the late proximal and early distal convoluted tubule accessible to micropuncture. To further define the sites of phosphate reabsorption, the stationary microperfusion technique was applied to proximal and distal nephron segments. Phosphate reabsorption was evaluated in superficial loops of proximal tubules, descending segments beyond late proximal tubules accessible to micropuncture, ascending segments up to the point of micropuncture in the distal tubule, and superficial loops of distal tubules of thyroparathyroidectomized rats. Microperfusates of 1.3 or 2.6 nl (100 mmol/l mannitol, 100 mmol/l NaCl,³²P-phosphate and³H-inulin) were injected and then withdrawn after contact times of 2–108 s. Phosphate recovery relative to that of inulin was determined. A steep exponential decline of phosphate recovery (R) with increasing contact time (t) was observed in the superficial proximal tubule and descending segments. The slopes of the logarithmic regressions (¹⁰logR)/t, ±SEM) were: –1.68±0.33 and –1.21±0.24 min^–1 in superficial proximal tubules and descending segments respectively. In contrast, no significant decline in phosphate recoveries (–0.02±0.04 and +0.11±0.10 min^–1) was apparent in the ascending segments and distal tubule. It is concluded that phosphate is reabsorbed in the proximal convoluted tubule and adjacent descending segments of the superficial nephron and that there is no significant phosphate reabsorption in distal convoluted tubules and adjacent ascending segments.Study carried out during a one year fellowship at the Mayo Clinic. Present address: Institut für Physiologie, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria     

The structure of the kidney from the freshwater teleost Carassius auratus

Summary The structure of the kidney of the crucian carp (Carassius auratus; a freshwater teleost, Cypriniformes) was studied by means of reconstruction from serial paraffin and semithin sections. In C. auratus, the Wolffian duct traverses the entire kidney. At various levels collecting ducts of different length and thickness join the Wolffian duct at right angles. Each collecting duct accepts a large number of connecting tubules, which are established by the joining of many nephrons. A regular pattern concerning the distribution of nephrons and the fusion of renal tubules is not apparent. Four segments have been distinguished in renal tubules; 1) proximal tubule, 2) distal tubule, 3) connecting tubule and 4) collecting duct. A neck and an intermediate segment are absent. The proximal tubule is established by proximal tubule cells which bear a brush border and have a conspicuous apical cytoplasmic rim containing few cell organelles, ciliated cells, mucous cells and dark cells. In the first part of the proximal tubule the brush border and the apical cytoplasmic rim of proximal tubule cells are well developed. Ciliated cells are interposed between proximal tubule cells, decreasing in number toward the end of this part. In the second part ciliated cells are absent and dark cells are numerous. In the third part the brush border and the apical cytoplasmic rim of proximal tubule cells are scarcely developed. Ciliated cells reapear and increase in number toward the distal tubule. The distal and connecting tubule are similar in epithelial structure. Connecting tubules are joined distal tubules and thus they belong to two or more nephrons. The main cells of distal and connecting tubules contain abundant mitochondria, but have no brush border. The connecting tubule becomes a collecting duct before joining the Wolffian duct. The main cells of collecting ducts are characterized by division of their cytoplasm into a dark apical half and a light basal half.     

Localization and regulation of the renal kallikrein kinin system: Possible relations to renal transport functions

Summary The complete renal kallikrein kinin system has recently been localized in defined nephron segments. Kallikrein was found to be formed and secreted by connecting tubule cells in the late distal convoluted tubule, whereas kininogen and a novel kininase were located in collecting tubules. Kinins were shown to act on collecting tubule as well as medullary interstitial cells and the renal vasculature. The literature on interactions of this system with renal sodium transport is conflicting. Renal and urinary kallikrein was found to be increased under sodium restricted conditions, whereas kinin has a diuretic and natriuretic effect in the collecting tubule, when added from the basolateral surface. On the other hand renal kallikrein activity and connecting tubule cell morphology change in parallel with dietary potassium load indicating a coupling to potassium secretion. The possible role of the renal kallikrein kinin system in regulating collecting tubule function by tubular and vascular effects is outlined in spite of many open questions which remain to be answered.     

Lectin histochemistry of palatine glands in the developing rat

This study examined the binding pattern of lectins, soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), Vicia villosa agglutinin (VVA), Ulex europaeus agglutinin-I (UEA-I), peanut agglutinin (PNA), wheat germ agglutinin (WGA), and succinylated WGA (sucWGA) in the developing rat palatine glands. In adult rats, heterogeneous lectin binding patterns were revealed between the anterior and posterior portions of palatine glands, as DBA, VVA, and WGA were bound more intensely and broadly in the posterior portion. SBA, PNA, and sucWGA showed far less reactivity in the anterior than in the posterior portion. At embryonic day 18 (E18), weak labeling was observed with UEA-I and WGA at the basal membrane of terminal buds, UEA-I and PNA labeled the epithelial cord, and there was no apparent binding for SBA, DBA, VVA, and sucWGA. At E20, after acinar lumenization, all lectins were detected at the acinar cell basal membranes. After birth, all lectins detectably labeled at the mucous cell apical membranes and progressively, with maturation, extended from the apical to basal portions of the cytoplasm. Apparent serous cells were observed around postnatal day 10 (PN10) and bound UEA-I. Lectins reached peak reactivity at PN21 and the binding patterns became identical to those of adults around PN28.     

Specialized junction in the distal convoluted tubule of rat kidney.

The ultrastructure and permeability of the apical junctions between epithelial cells of the distal nephron have been studied in rat kidney using a collidal lanthanum tracer and uranyl acetate staining en bloc. The apical intercellular junctions of the macula densa and juxtaglomerular segment of the early distal convoluted tubule measure up to 0.5 mu in length and about 50 A in width. Lanthanum permeates the occluding portion of these junctions in a discontinuous manner, defining a series of closely spaced and parallel lines of fusion that run in a direction perpendicular to the apical-basal axis of the tubular cells. The apical junctions of the remainder of the distal convoluted and cortical collecting tubules are impermeable bolanthanum. This distinctive apical tight junction can account for the greater permeability to ions of the early distal convoluted vs. late distal convoluted and cortical collecting tubules.     

Developing nephrons in adolescent dogfish,Scyliorhinus caniculus (L.), with reference to ultrastructure of early stages,histogenesis of the renal countercurrent system,and nephron segmentation in marine elasmobranchs

Light and electron microscopy of the excretory kidney of adolescent dogfish, Scyliorhinus caniculus (L.), revealed immature and mature nephrons as well as four developmental stages of nephrons. At stage I the nephron was characterized by a condensed mass of mesenchymal cells in the center of several concentric layers of connective tissue. At stage II of the nephron, the S-shaped body was an elongate cyst with a high prismatic epithelium that was connected by a developing collecting tubule with the collecting duct system. At stage III, the developing nephrons already possess the essential features of the mature nephron but lack complete differentiation. Developing renal corpuscles had one afferent arteriole and two efferent vessels. Developing tubules ran four times between the lateral bundle zone and the mesial tissue zone before they joined the collecting duct system. A continuous sheath of flat cells, encompassing the collecting duct system, extended around the developing lateral bundle. A rudimentary central vessel ran from the developing lateral bundle to the venous sinusoid capillaries between the mesial convolutions. Developmental stage IV was similar to the mature nephron, however, renal corpuscles and tubular segments were smaller than those of mature nephrons. Conclusive evidence for morphological homology of elasmobranch nephron segments and collecting tubule-collecting duct system with those of other vertebrates is provided. The origin and nature of the central vessel and the bundle sheath is clarified. These specific structures of marine elasmobranch kidney supposedly are of great functional relevance for the renal countercurrent system that in turn is essential for ion- and osmoregulation.     

Renal tubule of dogfish,Scyliorhinus caniculus: A comprehensive study of structure with emphasis on intramembrane particles and immunoreactivity for H+-K+-adenosine triphosphatase

The ultrastructure of renal tubule cells was studied in the European lesser spotted dogfish by the evaluation of thin sections and freeze fracture replicas. Computer-assisted three-dimensional reconstruction of entire nephrons was performed. The distinction of nephron segments and collecting tubule was made using results of previous histological work. The first proximal tubule segment (PI) consists of two subsequent portions, PIa and PIb. PIa is a component of the lateral countercurrent bundle, and PIb, which displays an apical tubulovesicular apparatus and an extended lysosomal compartment, is located in the vicinity of the glomeruli. Rod-shaped intramembrane particles were detected in PIa. The second proximal tubule segment (PII) is a special segment in elasmobranch and teleost fish. PII differs largely from PI in cell morphology and function. The apical cytoplasm was filled with small clear vesicles, and an apical endocytic apparatus was lacking. In the apical cell membrane, rod-shaped particles were revealed by freeze fracture. The apical tight junctions of PI and PII consisted of seven to ten meandering strands. The distal nephron was subdivided into two major segments: early distal tubule (EDT) in the lateral countercurrent bundles and late distal tubule (LDT) in the mesial tissue. The EDT showed marked amplification of basolateral cell membranes. The tight junctions displayed a low number of continuous parallel strands, which is also characteristically found in the diluting segments of other vertebrates. LDT cells showed cytoplasmic studs and rod-shaped intramembraneous particles at the apical cell membrane, thereby resembling type A intercalated cells of collecting duct. The collecting tubule (CT) emerged from the LDT and was part of the countercurrent arrangement inside the lateral bundles. Tight junctions of LDT and CT consisted of many meandering strands in a honeycomb pattern. With immunohistochemistry, binding sites of a polyclonal antibody against an extraplasmic portion of rat gastric H⁺-K⁺-adenosine triphosphatase (ATPase) were observed at the apical cell membrane of PIa, PII, and LDT. From the colocalization of binding sites for the antibody against the transport enzyme with rod-shaped intramembrane particles, we assume that these might be the morphological correlate of gastric H⁺-K⁺-ATPase-like enzyme in the renal tubule. © 1993 Wiley-Liss, Inc.     

The ultrastructural development of distal nephron segments in the human fetal kidney

Summary The ultrastructural development of the human distal nephron was studied in fetuses 14–18 weeks of gestational age. The three-dimensional course of the nephrons was traced in serial semi-thin sections. Single semi-thin sections containing defined distal nephron segments were then reembedded, thin-sectioned and analyzed by electron microscopy. In stage I (renal vesicle) and stage II (S-shaped body) epithelial cells were essentially similar in ultrastructure. In stage III there were only minor variations in cell ultrastructure between distal nephron segments, but distinct differences were observed between proximal and distal tubule cells, the former being the most differentiated. The segments which are present in nephrons of adult kidneys could be identified in stage IV and some ultrastructural differences recognized between the cells. However, the amplification of the baso-lateral membrane, which is prominent in iontransporting mature distal segments, was almost absent and the baso-lateral membrane area per unit tubule length was similar in all distal segments. Intercalated cells were present towards the end of the distal convoluted and in the connecting tubule in stage IV but the ampulla of the collecting tubule was composed of cells with a uniform ultrastructure. Cell ultrastructure varied again to some extent in the collecting tubule.The present observations demonstrate that distal nephron segments in the human kidney are structurally undifferentiated in the early fetal development and suggest that they only to a limited extent are capable of modifying the composition of the tubular fluid.     

Histochemical evaluation of mouse and rat kidneys with lectin-horseradish peroxidase conjugates

Paraffin sections of mouse and rat kidney were stained with a battery of ten lectin-horseradish peroxidase conjugates and lectin binding was correlated with the ultrastructural distribution of periodate-reactive sugar residues as determined by the periodic acid-thiocarbohydrazide-silver proteinate technique. Various segments of the uriniferous tubule in both species showed differential affinity for labelled lectins. Significant differences were also evident between comparable tubular segments in mouse and rat kidneys. Neutral glycoconjugates containing terminal β-galactose and terminal α-N-acetylgalactosamine were prevalent on the luminal surface of the proximal convoluted tubule in the rat, but α-N-acetylgalactosamine was absent in this site in the mouse. In both species, terminal N-acetylglucosamine was abundant in the brush border of proximal straight tubules but absent in proximal convolutions. Fucose was demonstrated in both proximal and distal segments of mouse kidney tubules but only in the distal nephron and collecting ducts in the rat. Lectin staining revealed striking heterogeneity in the structure and distribution of cellular glycoconjugates. Such cellular heterogeneity was previously unrecognizable with earlier histochemical methods. The marked cellular heterogeneity observed with several lectin-conjugates in distal convoluted tubules and collecting ducts of both species raises a prospect that lectins can provide specific markers for intercalated and principal cells in the mammalian kidney. Glycoconjugates containing terminal sialic acid and penultimate β-galactose were present on vascular endothelium in both rodent kidneys, as were terminal α-galactose residues; but both species lacked reactivity for Ulex europeus I lectin in contrast to human vascular endothelial cells. The constant binding pattern of lectin conjugates allows convenient and precise differentiation of renal tubular segments and should prove valuable in the study of changes in kidney morphology promoted by experimental manipulation or pathologic changes.     

Lectin histochemistry of kidney tumours and its pathomorphological relevance

Thirty kidney tumours of various histological type were histochemically investigated under the light microscope by means of the ABC-method. We used four biotinylated lectins which are known to bind also to normal renal tubular epithelial cells of different nephron segments. The nuclear grade, the histological growth pattern, the cell type and the histogenesis of the tumours were studied. The lectin of Lotus tetragonolobus bound to nine out of ten renal cell carcinomas with low nuclear grade, but in contrast no binding was seen in eight poorly differentiated ones. Four carcinomas mimicking collecting duct epithelium were also negative after treatment with this lectin, but showed strong staining after treatment with peanut agglutinin, Dolichos biflorus agglutinin and soybean agglutinin. Five oncocytomas showed a high affinity only for Dolichos biflorus agglutinin and only one case was positive with Lotus tetragonolobus agglutinin; three tubulo-papillary adenomas of the renal cortex without any oncocytes were negative with all of the lectins used. The value of lectin histochemistry in tumour pathology and its significance in routine pathological examination of kidney tumours are discussed.     

Tubule-tubule and tubule-arteriole contacts in rat kidney distal nephrons. A morphologic study based on computer-assisted three-dimensional reconstructions.

BACKGROUND: Functional investigations of the tubulo-glomerular feedback mechanism have indicated the existence of a contact between the distal nephron and the macula densa region. The structural justification of such a contact is investigated. EXPERIMENTAL DESIGN: Tubule-tubule and tubule-arteriole contacts were investigated in distal nephrons from normal rat kidneys. Computer-assisted three-dimensional reconstructions of distal nephrons were made from serial sections of renal cortical tissue and selected sections were examined by electron microscopy. RESULTS: In 14 of 15 reconstructed nephrons, the distal convoluted tubule or the connecting tubule approached the macula densa region. A wall-to-wall contact between two tubules corresponding to a three-dimensional distance below 28 microns between the axes of the two tubules was found in only five of the reconstructed tubules. The distal nephron contacts to afferent and efferent arterioles of the same nephron were also examined. The efferent arteriole revealed no consistent contacts but the afferent arteriole contacted the distal convoluted tubule/connecting tubules consistently in all 10 of the superficial nephrons and in 3 of 5 midcortical nephrons. Electron microscopy confirmed a close contact between the distal tubule and the afferent arteriole in superficial nephrons and small nerves were often found at or near the site of contact, but the morphology at the site of contact was not unique. The arteriole contacts were made with late distal convoluted tubules, connecting tubules, or cortical collecting ducts. CONCLUSIONS: In conclusion, the present study shows that tubule-tubule contacts are inconsistent between the macula densa region and the distal nephron but that the tubule-afferent arteriole contact is consistent and close in superficial nephrons. This morphology is compatible with the existence of a feedback mechanism between the superficial distal nephron and the afferent arteriole, apart from the one located at the juxtaglomerular apparatus.     

Immunohistochemical identification of kidney nephron segments in the dog, rat, mouse, and cynomolgus monkey

Kidney is a major target organ in preclinical studies. In recent years, intense research has been undertaken to characterize novel renal toxicity biomarkers. In this context, we studied nephron segment specific antibodies against aquaporin-1 (AQP-1), α-glutathione-S-transferase (alpha-GST), Tamm-Horsfall protein (TH), calbindin-D(28K) (CalD), and aquaporin-2 (AQP-2), using an immunoperoxidase method on formalin-fixed paraffin-embedded kidney tissues of dogs, rats, mice, and Cynomolgus monkeys. AQP-1 was specific for proximal tubules and thin descending limbs of Henle's loops and AQP-2 for connecting and collecting ducts in dogs, rats, mice, and Cynomolgus monkeys. Alpha-GST stained the straight part of proximal tubules in dogs and proximal convoluted tubule and straight part of proximal tubules in rats. TH was specific for thick ascending limbs of Henle's loops in mice, rats, dogs, and Cynomolgus monkeys and stained additionally scattered cells in cortical connecting/collecting ducts of dogs. CalD was found in distal convoluted tubules and cortical connecting and collecting ducts of dogs, rats, and mice and in distal convoluted tubules, connecting ducts, and cortical and medullary collecting ducts of Cynomolgus monkey. This panel of antibodies may be a helpful tool to identify renal tubules by light microscopy in preclinical studies and to validate new biomarkers of renal toxicity.     

Mineralcorticoid receptors along the nephron: [3H]aldosterone binding in rabbit tubules

To identify the site of mineralocorticoid action along the nephron, we measured the specific binding of [3H]aldosterone to nephron segments microdissected from aldosterone-deficient rabbits. Specific binding was defined as the difference between binding measured in the absence or in the presence of 2,000-fold excess of unlabeled hormone (in 10(-18) mol X cm tubule length-1 +/- SE). High specific binding capacity was found in the branched collecting tubule (108 +/- 4), the cortical collecting tubule (119 +/- 9), and the outer medullary collecting tubule (115 +/- 16), whereas specific binding was negligible in the proximal convoluted tubule (8 +/- 9), pars recta (2 +/- 6), medullary thick ascending limb (4 +/- 6), cortical thick ascending limb (6 +/- 2), and distal convoluted tubule (6 +/- 6). In cortical collecting tubules, Scatchard analysis of the specific [3H]aldosterone binding indicated a dissociation constant (KD) of 2.2 X 10(-9) M and a maximum number of binding sites of 157 X 10(-18) mol X cm tubule length-1. The steroid specificity was assessed from the competition of various steroids for [3H]aldosterone binding sites. Receptors from the cortical collecting tubule revealed the following sequence of affinities: aldosterone greater than DOCA greater than spironolactone greater than dexamethasone greater than 5 alpha-dihydrotestosterone = progesterone = 17 beta-estradiol, indicating that the binding sites in the collecting tubule are mineralocorticoid receptors. These results demonstrate significant [3H]aldosterone binding to receptors of high affinity and mineralocorticoid specificity only in the collecting tubule and suggest that this nephron segment is the target site of mineralocorticoid action in the rabbit kidney.     

Lectin histochemistry of Wilms' tumor. Comparison with normal adult and fetal kidney

The lectin histochemical staining patterns of nine surgically resected Wilms' tumors (WIT) (five classical, one rhabdomyomatous, one monomorphic tubular, and two blastematous) and four WIT heterotransplants in nude mice were compared with those of six normal adult and fetal kidneys using 11 biotinylated and fluorochrome-labeled lectins representing a spectrum of sugar specificities. Tubular epithelial cells of fetal and adult kidney demonstrated a complex pattern of glycosubstances, as detected by staining with most of the lectins. The dysplastic tubules of WIT and WIT heterotransplants resembled distal convoluted tubules and collecting ducts of adult kidney, as detected by staining with lectins that reacted exclusively with these portions of the nephron. A simpler lectin affinity profile was observed with blastema of normal fetal kidney and blastema of WIT and WIT heterotransplants. Thus, fetal, adult, and tumor tubules and fetal and tumor blastema resemble each other in terms of lectin binding patterns.