High- and low-affinity binding sites for vasoactive intestinal peptide (VIP) in the rat kidney revealed by light microscopic autoradiography

The distribution of VIP binding sites in rat kidney and adrenal gland has been examined by light microscopic autoradiography. A fully characterized mono-iodinated molecular form of VIP (M-125-I-VIP) which maintains the biological activity of the native peptide, was used for this study. Two types of VIP binding sites, with high and low affinity, have been identified. High affinity sites are associated with (i) glomerular structures in the cortex, (ii) the inner stripe of the outer medulla, possibly corresponding to Henle's loops and distal tubules, (iii) radiated structures in the inner zone of the medulla, likely to represent labeling of collecting ducts and/or vascular bundles and (iv) the adrenal cortex. Autoradiographic grains associated with low affinity sites are present diffusely throughout the renal cortex, possibly corresponding to labeling of tubular and/or vascular structures, and throughout the adrenal gland. These observations further delineate a role of VIP in renal and neuroendocrine function.     

Prostaglandin receptors in rat kidney

The physiological effects of prostaglandins (PGs) are mediated through their interactions with specific binding sites (receptors) on effector cells. Since such receptors potentially regulate the action of PGs on the kidney, the distribution and properties of renal PG receptors in the rat were examined. The distribution of PGE2, PGE1, and PGF2 alpha receptors along the nephron was not uniform; the outer medulla had by far the greatest density of sites, followed by the inner medulla and cortex. Receptors were found exclusively in the particulate fractions, of which the 40,000g pellet had the highest specific activity. In the outer medulla, receptor density calculated from Scatchard plots was 2.12 pmol/mg for PGE2, 1.12 for PGE1, and 0.44 for PGF2 alpha; the KD's were similar for all prostaglandins. The conditions for optimal in vitro binding of PGE2 and PGF2 alpha by outer medullary membranes were investigated. In vivo administration of 16,16'-dimethyl-PGE2 resulted in a dose-dependent "down" regulation of PGE2 binding to outer medullary membranes due to changes in both the number and affinities of receptors. Changes in the numbers and/or properties of PG receptors may be an important mechanism for regulating the effects of PGs and renal function under normal and pathologic conditions.     

An autoradiographic study of prostaglandin E1 and/or its metabolites in mouse kidney tissue

The use of grain density autoradiography was evaluated as a means of quantifying the distribution of injected prostaglandin E₁ and/or its metabolites in the mouse kidney. Fifty microcuries of ³H-PGE₁ were injected into each of three mice which were sacrificed at 20 min, 40 min and 45 h after injection. Sections of frozen kidneys were mounted on prepared liquid emulsion slides. Following appropriate exposure and processing, the radioactivity in various regions was quantified by grain counting using an eyepiece grid superimposed over the field at 1000X. The highest grain density was found over the papilla and the second highest grain density occurred at the region of the inner zone of the cortex. In addition, the percentage decrease of grain density from tissue taken 40 min after injection when compared to that from tissue taken 20 min after injection implied that these two regions retained the radioactivity more so than the outer zone of the cortex, the outer zone of the medulla and the glomeruli.     

Ontogeny of atrial natriuretic peptide receptors in fetal rat kidney and adrenal gland

Summary With in vitro autoradiography, specific receptors for atrial natriuretic peptide (ANP) were localized in fetal rat kidney and adrenal glands. Receptors were present over renal vesicles, in the primitive renal medulla and throughout the adrenal gland as early as 16 days gestation. By 20 days gestation, several layers of developing renal corpuscles were present and ANP receptors were localized over developing glomeruli in each layer. Larger accumulations occurred over the juxtamedullary glomeruli. In the medulla, the receptors were localized in a reticular pattern near the pelivis. With emulsion coated sections, ANP receptors in developing renal corpuscles were seen primarily over the lower curve of S-shaped vesicles and around the periphery of the more mature corpuscles. In the renal medulla, receptors were localized over the interstitial cells. In the 16-day-old adrenal gland, ANP receptors were present throughout the cortical area but at 20 days gestation and 1 day postpartum receptors appeared more numerous in the peripheral region. these data suggest that ANP has important developmental effects in the kidney and adrenal gland and may be involved in regulation of body fluid homeostasis in the late gestation rat fetus.     

Production of urea from arginine in pars recta and collecting duct of the rat kidney

Urea production from arginine was studied in vitro in the kidney of normal rats in tubule suspensions of the four different renal zones (cortex, outer and inner stripe of outer medulla, and inner medulla), and in individual microdissected nephron segments. Tissue was incubated with L-[guanido-14C]-arginine to measure cellular arginase activity. Addition of urease to the incubate freed 14CO2 from the 14C-urea formed by arginase and released from the cells. CO2 was trapped in KOH and counted. These experiments revealed that significant amounts of urea are produced in the outer stripe and in the inner medulla. This intrarenal urea generation takes place mainly in the proximal straight tubule and in the collecting duct, with increasing activity in these two structures from superficial to deep regions of the kidney. Urea is known to play a critical role in the urinary concentrating process. The fact that some urea can be produced in the mammalian kidney, and that the two structures showing this capacity are straight portions of the renal tubular system descending along the corticopapillary axis suggest that this urea production might play a role in the formation and/or maintenance of the medullary urea concentration gradient.     

Morphological study of the development of the disseminate prostate in intact and castrated male pigs aged two to six months.

The light-microscopical and ultrastructural differentiation of the disseminate prostate was studied in 12 boars and 8 barrows, ranging in age from 8 to 28 and 12 to 27 weeks, respectively. The barrows had been castrated at the age of 8 weeks. In young boars the prostate is organized in an inner zone and an outer zone. The glandular tubules of the latter are more differentiated than those in the inner zone. Sexual maturation in the prostate is characterized by almost complete reduction of the inner zone and concomitant enlargement of the outer zone. This differentiation of the inner zone occurs progressively and is manifested by a higher density of the glandular tubules, by a decrease in the number of basal cells and by an increase in the secretory tubular cells containing more microvilli, organelles and secretory vesicles. A few weeks after castration, the characteristic morphology of the undifferentiated inner zone is found throughout the entire glandular layer. The number of glandular tubules in barrows has decreased, and their cells show minimal secretory activity, nuclear indentations and a decrease in their organelles. A further castration effect is vacuolization of the perinuclear cytoplasm in numerous glandular cells.     

In Situ Characterization of Renal Insulin Receptrors in the Rat

Abstract

Insulin regulates carbohydrate metabolism, and water, sodium, potassium, and phosphate reabsorption in the kidney by binding to specific receptors. Insulin receptors have been identified in the kidney using membrane preparations obtained from both glomeruli and tubules. In this study, an autoradiographic technique was used to characterize insulin receptors in the rat kidney. Frozen tissue sections were preincubated to remove endogenously bound insulin, incubated in a buffer containing 200 pM 12sl-Tyr-insulin, washed, and dried before exposure on Ultrofilm. Binding density was assessed by computerized microdensitometry. In the cortex, binding density was comparable in glomeruli and tubules. In the medulla, bound radioligand was found primarily in longitudinal structures traversing the outer portion, presumably corresponding to vascular bundles, and in the inner portion. Scatchard analysis of competition binding data resulted in curvilinear profiles, indicating either two classes of receptors with different affinity or the presence of a single class of receptors with a negative cooperative hormone-receptor interaction. Data analyzed for a two-site model showed one receptor site with Kd of 0.39f0.14 nmol/l and Bmax of 3. M. O × 1010 receptordmma and another site with Kd of 0.30fl.1 pmoVl and a Bmax of 3.2 × 1013 receptordmms. Thus, in situ autoradiography can be used to determine distribution and binding characteristics of insulin receptors in rat kidney and might be employed in receptor studies on rat models of human disease.     

Aprotinin uptake in the proximal tubules in the rat kidney. II. Uptake site relative to glomerulus

Glomerular filtration rates in whole kidney and in outer, middle and inner cortical zones have previously been estimated by measuring the amount of iodinated Aprotinin, filtered and taken up in the first two thirds of the proximal convoluted tubules, in part positioned more superficial than the parent glomerulus. Thus, an appreciable amount of the absorbed Aprotinin may be located superficial to its filtration site and lead to an underestimate of glomerular filtration in deep cortical layers. Therefore, in this study we have measured the distance from the glomerulus to the center of proximal convoluted tubular ball and the site of Aprotinin uptake. Measurements were made on photos of Microfil-injected tubules and on camera lucida drawings of tubular transections from autoradiographs of nephrons containing both Microfil and iodinated Aprotinin. Both techniques showed that the center of the tubular ball was localized more superficial in all cortical layers. The average distance, in percent of cortical thickness, from all proximal convoluted tubular transections to the parent glomerulus was 9% in deep and 13% in middle and superficial cortex. Corresponding distances for tubular transections containing Aprotinin were 7 and 12%. Grain density in five reconstructed proximal convoluted tubules showed a continuous and exponential fall of Aprotinin along the uptake segment. The results may be used to estimate single nephron filtration rate from Aprotinin uptake and glomerular density in outer, middle, and inner cortex.     

Ontogeny of atrial natriuretic peptide receptors in fetal rat kidney and adrenal gland

With in vitro autoradiography, specific receptors for atrial natriuretic peptide (ANP) were localized in fetal rat kidney and adrenal glands. Receptors were present over renal vesicles, in the primitive renal medulla and throughout the adrenal gland as early as 16 days gestation. By 20 days gestation, several layers of developing renal corpuscles were present and ANP receptors were localized over developing glomeruli in each layer. Larger accumulations occurred over the juxtamedullary glomeruli. In the medulla, the receptors were localized in a reticular pattern near the pelvis. With emulsion coated sections, ANP receptors in developing renal corpuscles were seen primarily over the lower curve of S-shaped vesicles and around the periphery of the more mature corpuscles. In the renal medulla, receptors were localized over the interstitial cells. In the 16-day-old adrenal gland, ANP receptors were present throughout the cortical area but at 20 days gestation and 1 day postpartum receptors appeared more numerous in the peripheral region. These data suggest that ANP has important developmental effects in the kidney and adrenal gland and may be involved in regulation of body fluid homeostasis in the late gestation rat fetus.     

Down-Regulation of Angiotensin II Receptor Subtypes and Desensitization of Cyclic GMP Production in Neuroblastoma N1E-115 Cells

Abstract: Murine neuroblastoma N1E-115 cells possess membranous receptors for the octapeptide angiotensin II (AngII) whose density is substantially increased by in vitro differentiation. Incubation of differentiated N1E-115 cells with AngII produced a rapid decrease in receptor density, but did not alter the affinity of these receptors for either ¹²⁵I-AngII or the high-affinity antagonist ¹²⁵I-[Sarc¹,Ile⁸]-AngII. This apparent down-regulation was dose related with an ED₅₀ of 1 nM, and maximal decreases of ～90% were obtained with 100 nM AngII. Receptor loss from differentiated cell membranes was unaffected by incubations of membranes obtained from agonist-exposed cells with non-hydrolyzable analogues of GTP for 60 min at 37°C to ensure dissociation of the ligand. Partial loss of AngII receptors was apparent within 5 min of agonist exposure, whereas maximal declines were not observed until 30 min. This temporal pattern resulted from a preferential decrease in the AT₁ receptor subtype during the first 5 min, followed by a decline in both AT₁ and AT₂ receptors with longer periods of agonist exposure. The loss of membranous receptors was reversible with partial recovery observed after 4 h, and with nearly full recovery observed 18 h after exposure of the cells to AngII. However, the long-term recovery of receptor density was blocked by the protein synthesis inhibitor, cycloheximide. The heptapeptide angiotensin III produced a similar down-regulation of receptors, and the high-affinity antagonist [Sarc¹, Thr⁸]-AngII blocked agonist-induced down-regulation. Finally, the apparent loss of cell surface Angll receptors decreased the ability of AngII to stimulate cyclic GMP production within intact N1E-115 cells. These results suggest that differentiated N1E-115 cells are an excellent cell line in which to examine the factors regulating the expression of AngII receptor subtypes in the nervous system.     

Studies of tissue electrical admittance: relation to tissue sodium for different zones of rabbit kidney

Tissue electrical admittance (reciprocal impedance) and Na+ concentration were determined in slices of rabbit renal cortex, outer medulla, inner medulla and the papilla. In each zone admittance was highly and significantly correlated to tissue Na+ (r = 0.71 to 0.91, p less than 0.001). The cortex admittance proved a relatively insensitive index of tissue electrolyte concentration. The highest sensitivity was observed for the outer medulla: values for the inner medulla and papilla were slightly lower. The data confirm the usefulness of admittance measurement for dynamic assessment of the cortico-papillary electrolyte gradient but show that the values measured in the outer medulla cannot be directly compared with those for the inner medulla and the papilla.     

Purinergic receptors are part of a signalling system for proliferation and differentiation in distinct cell lineages in human anagen hair follicles

We investigated the expression of P2X₅, P2X₇, P2Y₁ and P2Y₂ receptor subtypes in adult human anagen hair follicles and in relation to markers of proliferation [proliferating cell nuclear antigen (PCNA) and Ki-67], keratinocyte differentiation (involucrin) and apoptosis (anticaspase-3). Using immunohistochemistry, we showed that P2X₅, P2Y₁ and P2Y₂ receptors were expressed in spatially distinct zones of the anagen hair follicle: P2Y₁ receptors in the outer root sheath and bulb, P2X₅ receptors in the inner and outer root sheaths and medulla and P2Y₂ receptors in living cells at the edge of the cortex/medulla. P2X₇ receptors were not expressed. Colocalisation experiments suggested different functional roles for these receptors: P2Y₁ receptors were associated with bulb and outer root sheath keratinocyte proliferation, P2X₅ receptors were associated with differentiation of cells of the medulla and inner root sheaths and P2Y₂ receptors were associated with early differentiated cells in the cortex/medulla that contribute to the formation of the hair shaft. The therapeutic potential of purinergic agonists and antagonists for controlling hair growth is discussed.     

Zur Entwicklung und funktionellen Anatomie des Phylloids vonLaminaria hyperborea

The persistent thallus of the phaeophyceanLaminaria hyperborea produces annually a new phylloid, formed by a meristematic zone between stipe and old phylloid. The growing frond is nourished with assimilates from the old one. Young and old phylloid are linked by a collar. Frond and stipe are of very similar anatomical structure. The frond is coated by a one-layer-meristoderm, which forms the external cortex. Using the position of slime ducts as border, the parenchymatous cortex can be subdivided into outer and inner cortex. Between inner cortex and medulla numerous transitions exist. The medulla itself is mainly composed of longitudinally arranged trumpet cells and hyphae. Frond enlargement is caused by the activity of both meristoderm and cortex. Not only cell divisions but also elongation in medulla and inner cortex contribute to growth of the frond. The frond diameter is decreased by this elongation process. Corresponding stretching occurs in the network of slime ducts. Swelling of longitudinal primary walls, accompanied by incorporation of alginate, facilitates the separation of cell strands, and across the medulla hyphae and crosslinks of both inner-cortex-layer cells form. Cells of the inner cortex continously differentiate to elements of the medulla. Trumpet cells within the outer part of the medulla are often branched and connected by hyphae to parenchymatous cells of the inner cortex. Toward the central part of the medulla, trumpet cells elongate and finally attain a length of 1000 µm; they form thick secondary longitudinal walls with ringshaped thickenings, reducing the lumen diameter. Crosswalls and whole cells are often plugged with callose and have, apparently, ceased to translocate.     

Renal medullary electrolytes: effects of furosemide assessed by studies in vivo of electrical admittance

In anaesthetized rabbits electrical admittance (a reciprocal of impedance) of the kidney in situ was recorded using electrodes located in the cortex, outer medulla, inner medulla and papilla. Renal haemodynamics, clearances and Na+ concentration in tissue slices were also determined. Admittance changes in response to i.v. furosemide, 1.5 or 3 mg/kg body weight, and to 15% mannitol infusion, reflected changing interstitial electrolyte concentration and, indirectly, changes in tubular reabsorption of NaCl. The large dose of furosemide and mannitol infusion decreased admittance in all renal zones whereas the small dose affected only the inner medulla and papilla. The rapid onset of the fall in admittance of the inner medulla, even in absence of changes within the outer medulla, suggests that the drug's action is not confined to the thick ascending limb but includes the thin ascending segment.     

Gene expression in the normal adult human kidney assessed by complementary DNA microarray

The kidney is a highly specialized organ with a complex, stereotyped architecture and a great diversity of functions and cell types. Because the microscopic organization of the nephron, the functional unit of the kidney, has a consistent relationship to the macroscopic anatomy of the kidney, knowledge of the characteristic patterns of gene expression in different compartments of the kidney could provide insight into the functions and functional organization of the normal nephron. We studied gene expression in dissected renal lobes of five adult human kidneys using cDNA microarrays representing approximately 30,000 different human genes. Total RNA was isolated from sections of the inner and outer cortex, inner and outer medulla, papillary tips, and renal pelvis and from glomeruli isolated by sieving. The results revealed unique and highly distinctive patterns of gene expression for glomeruli, cortex, medulla, papillary tips, and pelvic samples. Immunohistochemical staining using selected antisera confirmed differential expression of several cognate proteins and provided histological localization of expression within the nephron. The distinctive patterns of gene expression in discrete portions of the kidney may serve as a resource for further understanding of renal physiology and the molecular and cellular organization of the nephron.     

Influence of diet on the development of nephrocalcinosis in the rat

A histological examination of the effect of a purified diet containing 20% alpha protein (an alkali-treated soyprotein) on the development of nephrocalcinosis induced by intraperitoneal injections of 0.5 neutral (pH 7.4) sodium phosphate was carried out in female weanling rats. Animals that were fed a standard commercial laboratory diet and given daily injections of phosphate for six or ten days developed a form of nephrocalcinosis that consisted mainly of intraluminal (intratubular) calcification at the junction of the outer and inner stripes of the outer medulla and in the inner stripe of the outer medulla. By contrast, rats that were fed the alpha protein diet and given injections of phosphate for six or ten days developed a form of nephrocalcinosis that was characterized primarily by a type of tubular basement membrane calcification at the junction of the inner stripe of the outer medulla and the inner medulla. The differences in nephrocalcinosis between the two dietary groups and the fact that an alpha protein diet by itself can cause renal calcification, leads to the suggestion that some component(s) or factor(s) in the alpha protein diet strongly influence(s) the development of nephrocalcinosis induced by injected neutral sodium phosphate.     

Renal actions of dendroaspis natriuretic peptide in rabbits

Dendroaspis natriuretic peptide (DNP) is one of four members of the natriuretic peptide family sharing functional and structural properties. The purpose of the present study was to elucidate the physiological role of DNP on renal functions and its cellular mechanism in the rabbit kidney. DNP (5 μg/kg/min) infused intravenously increased urine volume and urinary excretion of electrolytes. These renal actions induced by DNP were more pronounced than those caused by atrial natriuretic peptide (ANP). We compared profiles of (125)I-ANP and (125)I-DNP by reverse-phase HPLC during incubation in rabbit plasma at 37°C for 1, 2, and 4h. While (125)I-ANP was quickly degraded within 1h, (125)I-DNP was still stable in plasma for 4h. DNP induced the greatest cyclic guanosine monophosphate (cGMP) production in the glomeruli in a dose-dependent manner, when compared to other renal structures including cortical tubules, outer medullary tubules, and inner medullary tubules. Affinity cross-linking analysis revealed NPR-A is selective receptor for DNP in glomeruli. Forskolin, a stimulator of adenylyl cyclase, significantly decreased cGMP production in the renal glomeruli but not in the renal medulla. In summary, DNP is a more effective activator of renal functions than ANP, possibly because of the degradation resistance of DNP against the endogenous peptidases in plasma or tissues. These findings suggest that DNP plays a pivotal role as a renal regulating peptide via specific natriuretic peptide receptors with a guanylyl cyclase domain.     

An ultrastructural study of the renal medulla in experimental acute pyelonephritis.

Experimental acute pyelonephritis was produced in rats by a combination of intravenous administration of Escherichia coli, strain IMRU-54, and temporary unilateral mechanical ureteral obstruction. Structural alterations of the renal medulla were studied by light and electron microscopy. Major cellular alterations occurred in the vasa recta. Tubular and interstitial cells demonstrated minimal alterations after the brief period of acute inflammation. Polymorphonuclear leukocytes within tubular lumina contained structures resembling E. coli in nonprotoplasts-like form. Numerous protoplast-like organisms, to the exclusion of any other structural forms, were detected within the interstitium of the inner medulla. Nonprotoplast-like structures resembling E. coli were rarely observed in interstitium of the inner medulla. Following relief of ureteral obstruction, clearance of acute inflammation was rapid. In conclusion, hemoatogenous acute pyelonephritis induced by E. coli, IMRU-54, is able to inflict cytological and ultrastructural damage to structural elements of the inner and outer medulla of rats. Vasa recta incurred prominent alterations in endothelia and basement membranes, whereas tubular epithelia and interstitial cells had relatively good structural preservation. The data suggest that intravenously administered E. coli is capable to revert to a protoplast-like structure in the inner medulla.     

Localization of 125I-atrial natriuretic factor (ANF)-binding sites in rat renal medulla. A light and electron microscope autoradiographic study

Using light and electron microscope autoradiography in vivo, the localization of 125I-(Arg 101-Tyr 126) atrial natriuretic factor (ANF)-binding sites was studied in the renal medulla of rats. At the light microscopic level, the autoradiographic reaction was mainly distributed in patches in the outer medulla, and followed the tubular architecture in the innermost part of the inner medulla. At the electron microscopic level, binding sites were mainly found in the outer medullary descending vasa recta and inner medullary collecting ducts. These results suggest that, in rats, the renal medulla may participate in the natriuresis and diuresis produced by ANF through vascular and tubular effects; the former by changing medullary blood flow at the level of descending vasa recta and the latter by acting on electrolyte and water transport at the level of collecting ducts.     

Aldose reductase and sorbitol dehydrogenase distribution in rat kidney.

The sorbitol pathway catalyzes the conversion of glucose to fructose via the intermediate sorbitol. It consists of aldose reductase (AR) and sorbitol dehydrogenase (SDH). In adult (44 day) kidney zones, AR was highest in the outer medulla. In substructures AR was highest in distal convoluted tubule. The AR was greatest in newborn and 8-day zones of developing rat kidney. Acute alloxan diabetes was associated with decreased AR in small arteries, but not glomeruli. The SDH was lowest in outer medulla. It was most active in glomeruli and distal convoluted tubules. The diabetic state leads to no change of SDH in arteries but an increase in glomeruli. SDH increased with development. This study demonstrates AR and SDH in substructures of the kidney. The pathway is present in developing kidney. In diabetes the enzymatic changes would tend to decrease accumulation of sorbitol.