Marked reduction in intramembranous particle clusters in the terminal portion of inner medullary collecting ducts of antidiuretic rats

Summary Antidiuretic hormone (ADH) induces an aggregation of intramembranous particles (IMP) into discrete clusters in the luminal plasma membrane of rat renal papillary collecting duct cells (Harmanci et al. 1978). The correlation between an elevated dose of ADH, increased urine osmolality, and greater IMP cluster frequency has led to speculation that the water permeability of the luminal plasma membrane is reflected by the IMP cluster density (Harmanci et al. 1980). The present study indirectly evaluated this water permeability by quantitating collecting duct IMP cluster frequency from freeze fracture replicas in two regions of the renal papilla, at its base and at its tip, in antidiuretic and in water diuretic rats. During antidiuresis there was a high frequency of IMP clusters (189/100 m² of luminal plasma membrane) in cells from the papilla base but not at the papilla tip (9.0/100 m²). During water diuresis there were few IMP clusters in either cells from the papilla base (5.9/100 m²) or at the papilla tip (1.4/100 m²). Most significantly these results suggest that the water permeability of the terminal portion of the inner medullary collecting duct of antidiuretic rats is significantly lower than that of the collecting duct epithelium higher in the papilla.Preliminary findings of this study were presented at the Second International Congress of Cell Biology, West Berlin 1980     

Microheterogeneity of the collecting duct system in rabbit kidney as revealed by monoclonal antibodies

Summary This report describes the immunolocalization of three monoclonal antibodies along the collecting duct system in rabbit kidney. The antibodies were raised against antigens derived from a membrane fraction of homogenized papillary tissue. Western Blot analysis demonstrated that each of the antibodies recognized a single band of about 190000 (P_CD1), 210000 (P_CD2) and 50000 (P_CD3) daltons. In renal tissue, the antibodies bound specifically to the epithelia of the connecting tubule (CNT), the collecting duct (CD) and the papillary surface epithelium. Differences in the binding patterns of the antisera were limited to the cortex. p_CD1 labeled only a few scattered cells in the CNT, and exhibited a heterogeneous binding along the cortical collecting duct (CCD). P_CD2 and P_CD3 binding patterns were similar. In the CNT, these antibodies bound to the intercalated cells (IC-cells) but not to the CNT-cells proper. In the CCD, both IC-cells and principal cells were labeled. The binding to the medullary collecting duct by all three antisera was identical. The ureter was labeled only by P_CD2 and P_CD3, and none of the antisera bound to the bladder epithelium.The antibody binding patterns provide information concerning tubular axial heterogeneity and embryogenetic aspects of the CNT and the CCD. These antibodies may be used as differentiation markers in studies of the developing kidney and of renal tissue culture systems.These studies were supported by Deutsche Forschungsgemeinschaft, Forschergruppe Niere, Kr 546/5-1     

Effect of chronic vasopressin treatment on alcohol drinking of Brattleboro HZ and DI rats

Preference for alcohol was determined for three groups of male and female rats, 100–150 days old, comprised of: (1) Long Evans (LE); (2) LE-derived Brattleboro heterozygous (HZ); and (3) Brattleboro homozygous (DI) animals afflicted with diabetes insipidus due to vasopressin deficiency. Each alcohol drinking test was run over 11 days during which food, water and an ethyl alcohol solution, increased in concentration from 3% to 25%, were freely available. Following an initial preference screen, 100 milli-units of vasopressin tannate in oil was administered subcutaneously, during a second preference test, once per day to each animal. This treatment ameliorated the polydipsia-polyuria syndrome characteristic of the DI sub-strain of Brattleboro rat. Administration of the peptide to both the LE or HZ animals exerted no effect on g/kg intake nor on the proportional measure of alcohol to water. However, in the DI rat of either gender, vasopressin reduced the mean absolute gram intake of alcohol over concentrations to resemble that of the other LE and/or HZ groups. These results demonstrate that vasopressin serves to normalize the intake of alcohol in the DI rat by virtue of the elimination of the diabetic condition. However, since vasopressin fails to alter alcohol consumption of the HZ and LE rats, it would appear that this neuroactive peptide may play only a minor role in the CNS mechanisms governing the voluntary selection of alcohol.     

High-Protein Carboxymethylase Activity and Low Endogenous Methyl Acceptor Proteins in Posterior Pituitary Lobe of Rats Lacking Neurophysin-Vasopressin (Brattleboro Rats)

Abstract: The activity of protein carboxymethylase and the endogenous protein methyl acceptor capacity were examined in the posterior, intermediate, and anterior lobes of the pituitaries of homozygous Brattleboro rats with diabetes insipidus and in heterozygous Brattleboro and Long-Evans control rats. Protein carboxyl methylation is selectively altered in the posterior pituitary lobes of homozygous Brattleboro rats. Protein carboxymethylase activity is higher (+40%) and endogenous methyl acceptor protein capacity is lower (-80%) with respect to heterozygous Brattleboro and Long-Evans control rats. This latter change is correlated with decreased methylation of proteins of a molecular weight of approximately 11K daltons, is selective for the posterior pituitary lobe, since it does not occur in the intermediate and anterior lobes, and probably reflects the absence of vasopressin-associated neurophysin in homozygous Brattleboro rats. Our results support a physiological role of protein carboxyl methylation in the neurosecretory process in the posterior pituitary gland.     

Tunicamycin,puromycin and brefeldin A influence the subcellular distribution of neuropeptides in hypothalamic magnocellular neurones of rat

Summary Magnocellular neurones in the supraoptic nuclei of normal Long Evans and homozygous Brattleboro rats were examined electron-microscopically after intracisternal injections of tunicamycin, puromycin, or brefeldin A. Moderate (50 g) or high (200 g) doses of tunicamycin caused the formation of electron-dense filamentous accretions in the endoplasmic reticulum (ER) cisterns of vasopressin neurones, but only the high dose of tunicamycin also caused accretions to form in the ER of some oxytocin neurones. Immunogold labelling of ultrathin sections from tunicamycin-treated rats revealed that, in about 5% of vasopressin neurones, the accretions could be immunogold-labelled for vasopressin and its associated neurophysin. However, in the majority of vasopressin neurones, the sections required trypsinisation before immunolabelling of the accretions could be detected. Small accretions in the ER of oxytocin neurones did not label for oxytocin or its neurophysin without prior trypsinisation, whereas larger accretions in other oxytocin cells could be labelled without prior trypsin treatment. Administration of puromycin resulted in the formation of small ER accretions in both vasopressin and oxytocin neurones. These accretions were immunolabelled with antisera, respectively, to vasopressin and oxytocin, but neurophysin-immunoreactivity was in most cases absent and was not revealed by treatment with trypsin, suggesting that neurophysin-immunoreactive epitopes were absent from truncated peptides forming the accretions. Brefeldin A caused dilatation of ER cisterns and disruption of the Golgi apparatus in both oxytocin and vasopressin neurones, but did not cause accretions to form in the ER.     

Expression of pigment-epithelium-derived factor during kidney development and aging

Inhibitors and stimulators of endothelial cell growth are essential for the coordination of blood vessel formation during organ growth and development. In the adult kidney, one of the major inhibitors of angiogenesis is pigment-epithelium-derived factor (PEDF). We have analyzed the expression and distribution of PEDF during various stages of renal development and aging with particular emphasis on the formation of functional glomeruli. We show that PEDF gene expression and protein levels in the kidney significantly increase with age. We have detected PEDF in the mesenchyme and endothelial cells at all developmental stages studied, in all regions of the nephrogenic zone in which the formation of new blood vessels is associated with the development of nephrons and collecting ducts, and in mature podocytes in the adult kidney. Our results are the first to suggest that PEDF is important in early renal postnatal development, that it could be relevant to the maturation of glomerular function and the filtration barrier formed by these cells, and that it may serve as an anti-angiogenic modulator during kidney development. Ana Luisa Pina and Marion Kubitza contributed equally to this work.     

The three-dimensional cytoarchitecture of the interstitial tissue in the rat kidney

Summary The cytoarchitecture of the interstitial tissue of the rat kidney was studied by combined scanning and transmission electron microscopy. The renal interstitium is composed of an elaborate network of stellate sustentacular cells. In the cortex, sustentacular cells radiate thin branching processes to form a fine reticulum, which supports intertubular spaces. In the medulla, these cells extend thick processes horizontally along the basal surfaces of the thin limbs or vasa recta, reinforcing their attenuate walls. The horizontal processes connect with each other at their terminals, compartmentalizing the interstitial space into thin layers. The medullary sustentacular cells contain abundant small lipid droplets. The network of sustentacular cells houses vasa recta, keeping them in parallel position to each other and to the tubules. The arterial vasa recta are accompanied by pericytes, which frequently contain lipid droplets larger in size than those in the sustentacular cells. Venous vasa recta extend numerous basal microvilli, which anchor the venous wall to adjacent tubules or vessels. Numerous free cells, round in shape, are found in the sustentacular cell network, especially in the cortex. They consist of macrophages and occasional lymphocytes. Some macrophages extend long pseudopodia, while others make intimate contact with lymphocytes, suggesting their high level of activity.     

Junctions between pericytes and the endothelium in rat myocardial capillaries: A morphometric and immunogold study

Summary Pericytes are cells of mesodermal origin which are closely associated with the microvasculature. Despite numerous studies little is known about their function. We have studied the relationship between pericytes and the endothelium in rat myocardial capillaries employing ultrastructural and immunogold techniques. 14% of the subendothelial cell membrane is covered by comparatively small pericytic cell processes. About half of these processes are completely embedded in baseement membrane material, whereas the remaining half forms closer contacts with the endothelium. These contacts are devoid of anti-laminin immunogold label, a marker for basement membranes. A small fraction of these contacts has been identified as tight junctions resembling those seen between endothelial cells in capillaries of the same tissue. The remaining majority of junctions reveals a cleft of approximately 18 nm between the apposed membranes in which a succession of cleft-spanning structures can often bedetected. It was also found that pericytic processes are preferentially located close to interendothelial junctions. We suggest that the high frequency of intimate junctions between pericytes and the endothelium and the preferential localisation near paracellular clefts may have functional significance.     

Vasopressin and ethanol preference. I. Effects of vasopressin and the fragment DGAVP on altered ethanol preference in Brattleboro diabetes insipidus rats

Preference for concentrations of ethanol between 2.2 and 10 percent versus tap water was studied in Brattleboro rats homozygous for diabetes insipidus (di/di), heterozygous (di/+) or normal (+/+). The di/di rats, totally lacking in vasopressin, had greatly reduced preference scores for all concentrations of ethanol. Their intake of ethanol (g/day) was higher than heterozygotes or normals, but only when 2.2 percent ethanol was offered as a choice. Administration of lysine vasopressin or the vasopressin fragment des-9-Glycinamide-[Arginine8] vasopressin (DGAVP) using osmotic minipumps enhanced ethanol preference scores, reduced ethanol (g/day) intake, and restored total daily fluid intake in di/di rats. When di/di and di/+ rats were first allowed to develop stable ethanol preference before treatment with DGAVP, the peptide had no effect on preference scores. Thus, no treatment was effective in dissociating polydipsia from reduced ethanol preference and increased ethanol intake. While these results cannot exclude a possible regulatory role for endogenous vasopressin in ethanol preference drinking, they more strongly suggest that reduced preference for ethanol and increased ethanol intake are epiphenomena secondary to a polydipsic state.     

乌梢蛇输精管道的组织学与免疫细胞化学观察

为探讨乌梢蛇(Zaocys dhumnades)输精管道结构与其功能之间的关系,该研究用一般光镜技术观察了乌梢蛇输精管道的显微结构及其年周期变化,并结合免疫细胞化学方法研究了雄激素受体(AR)、雌激素受体(ER)、孕激素受体(PR)和芳香化酶(Ar)在输精管和精巢中精子细胞表达的相关性.为验证该文在乌梢蛇输精管中观察到的大量精子和圆球状结构,用一般光镜技术还观察了黑眉锦蛇(Elaphe taeniura)、赤链蛇(Dinodon rufozonatum)与虎斑颈槽蛇(Rhabdophis tigrina lateralis)的输精管道.结果表明,乌梢蛇的输精管道主要由输出小管、附睾管与输精管构成;8-10月输出小管中有精子,8月—翌年1月附睾管中有精子,全年(除7月外)输精管中有大量精子;在输精管内首次观察到由多个精子细胞构成的圆球状结构,该结构与精巢中精子细胞的AR、ER、PR和Ar累计光密度值之间分别无显著差异.由于在乌梢蛇、黑眉锦蛇及赤链蛇的输精管内圆球状结构均可见精子细胞变态形成精子.因此,建议将蛇类输精管内圆球状结构命名为生精小球(seminiferous spherule).该文认为,蛇类精巢是精子形成的主要部位,而输精管内的生精小球是精子形成的另一个部位;附睾与输精管均可以储存精子,但输精管是精子储存的主要器官.     

Expression of binding sites for Dolichos biflorus agglutinin at the apical aspect of collecting duct cells in rat kidney

Summary To identify precisely the structural and functional cell type in the collecting duct of the rat kidney expressing binding sites for Dolichos biflorus agglutinin (DBA), we stained serial paraffin sections of kidney with horseradish peroxidase-labeled DBA and with immunocytochemical methods for localizing (Na⁺+K⁺)-ATPase and carbonic anhydrase II (CA II), enzymes found preferentially in principal and intercalated cells, respectively. Most principal cells expressing a strong basolateral staining for (Na⁺ + K⁺)-ATPase showed binding sites for DBA at their luminal surfaces. However, a minority of cells rich in CA II and showing morphologic characteristics of intercalated cells also expressed DBA binding sites at their luminal surface and apical cytoplasm. These data suggest that DBA cytochemistrycan provide a useful tool for studying the functional polarity of the main cell types of the collecting duct of the rat kidney.     

Replication of segment-specific and intercalated cells in the mouse renal collecting system

The renal collecting system (CS) is composed of segment-specific (SS) and intercalated (IC) cells. The latter comprise at least two subtypes (type A and non-type A IC). The origin and maintenance of cellular heterogeneity in the CS is unclear. Among other hypotheses, it was proposed that one subtype of IC cells represents a stem cell population from which all cell types in the CS may arise. In the present study, we tested this stem cell hypothesis for the adult kidney by assessing DNA synthesis as a marker for cell replication. SS and IC cells were identified by their characteristic expressions of sodium- (epithelial sodium channel, Na-K-ATPase), water- (aquaporin-2) and acid/base- (H+ -ATPase, anion exchanger AE1) transporting proteins. Immunostaining for bromodeoxyuridine (BrdU) and for the proliferating cell nuclear antigen (PCNA) was used to reveal DNA synthesis in CS epithelium. BrdU- and PCNA-immunostaining as well as mitotic figures were seen in all subtypes of CS cells. Dividing cells retained the cell-type specific expression of marker molecules. Treatment of mice with bumetanide combined with a high oral salt intake, which increases the tubular salt load in the CS, profoundly increased the DNA-synthesis rate in SS and non-type A IC cells, but reduced it in type A IC cells. Thus, our data show that DNA synthesis and cell replication occur in each cell lineage of the CS and in differentiated cells. The replication rate in each cell type can be differently modulated by functional stimulation. Independent proliferation of each cell lineage might contribute to maintain the cellular heterogeneity of the CS of the adult kidney and may also add to the adaptation of the CS to altered functional requirements.     

Antioxidants attenuate diabetes-induced activation of peroxisomal functions in the rat kidney

Diabetes is a multifactorial disease that has now been recognized to involve overproduction of reactive oxygen species and pro-inflammatory cytokines. Peroxisomes are subcellular organelles with several important metabolic functions, and their role in the regulation of cellular oxidative stress is now well established. Despite having their own antioxidant system, peroxisomes undergo functional alterations during various conditions that are associated with free radical production such as inflammation, ischemia-reperfusion, carcinogenesis and diabetes. In this study we investigated the effect of diabetes on peroxisomal functions in rat kidneys and show for the first time that experimental diabetes induces redox-sensitive enhancement of peroxisomal activities. Streptozotocin-induced diabetes significantly increased (p<0.01) -oxidation of lignoceric acid and the enzymic activity of acyl coenzyme A oxidase. Catalase activity was significantly reduced (p<0.01) in the kidneys of diabetic rats, whereas the enzymic activity of DHAPATase (dihydroxyacetone phosphate acyltransferase) was not markedly affected by diabetes. Treatment of diabetic rats with antioxidants, thiocetic acid and vitamin C attenuated the diabetes-induced modulation of peroxisomal functions. The present study shows that the diabetes-induced effects on kidney peroxisomal functions are redox sensitive, and antioxidants might prove useful tools to alleviate nephropathy in diabetes.     

Transplantation experiments with the fat body of Calliphora: A morphometric study of induced ultrastructural changes

Summary Transplantation of fragments of the anterior fat body lobe of 4 day old feeding stage larvae into one day older hosts, which are at the end of feeding, leads to precocious induction of ultrastructural changes in the transplanted cells of the fat body. These changes include alterations in the mean relative and absolute areas of mitochondria, protein granules, lipid droplets and vacuoles. The extent of intracellular changes induced in the transplants depends on the physiological condition of the host. This confirms that alterations in the internal environment at termination of feeding induce premetamorphic changes in cells of the fat body.     

Choline acetyltransferase immunocytochemical staining of the rat supraoptic nucleus and its surroundings

Summary Two different monoclonal antibodies raised against choline acetyltransferase were used, together with preembedding immunocytochemical techniques, to visualize the possible cholinergic innervation of the supraoptic and paraventricular nuclei of the rat hypothalamus. Light microscopy confirmed the presence of a group of bipolar and multipolar immunoreactive neurones in the hypothalamus dorsolateral to the supraoptic nucleus as well as numerous immunopositive fibers. Electron microscopy showed that the immunopositive cell bodies contained the usual perikaryal organelles while most immunoreactive fibers appeared dendritic; immunonegative terminals made synaptic contact onto these profiles. Immunopositive terminals making synaptic contact onto dendritic profiles were also noted in this area. In contrast, light microscopy showed no immunoreactivity to choline acetyltransferase in the magnocellular nuclei themselves. Electron microscopy revealed some immunopositive profiles along the boundaries of both nuclei, along the optic chiasm adjacent to the supraoptic nucleus and in the ventral glial lamina but not within the nuclei proper. Surprisingly, these immunopositive profiles appeared dendritic and were often contacted by one or more immunonegative synapses. Our observations thus indicate that cell bodies and dendrites in the supraoptic and paraventricular nuclei are not directly innervated by cholinergic synapses. The functional significance of the putative cholinergic dendrites in close proximity to magnocellular neurones remains to be determined.     

Localization of vasopressin,serotonin and angiotensin II in endothelial cells of the renal and mesenteric arteries of the rat

Summary The localization of vasopressin, serotonin and angiotensin II in the endothelial cells of renal and mesenteric arteries was investigated using the pre-embedding peroxidase-antiperoxidase technique for electron microscopy. Vasopressin-and serotonin-positive endothelial cells were present in both renal and mesenteric arteries while angiotensin II-positive cells were observed in the mesenteric artery exclusively. Both arteries showed less than 10% immunoreactive cells. The lack of angiotensin II in the endothelial cells of the renal artery suggests that there may be subtle physiological differences between the renal and mesenteric arteries with respect to the local control of blood flow.     

A scanning electron-microscopic study of the peripolar cell of the rat renal glomerulus

Summary The interior of Bowman's capsules of rat kidneys has been examined by scanning electron microscopy, and a distinctive population of cells around the exposed vascular poles of glomerular tufts were identified. The cells were situated in the annular groove at the root of the glomerulus, between the parietal epithelial cells and the podocytes. These peripolar cells were dendritic cells with long processes embracing the glomerular arterioles. Up to three peripolar cells were present at each vascular pole and they were mainly distributed in the glomeruli of the outer third of the renal cortex. This first detailed study of the surface morphology of the glomerular peripolar cell supports the suggestion that changes in the diameter of the polar region of the glomerular tuft may cause variations in stretching of the cuff of peripolar cells, and hence modulation of their secretory activity.     

Retinoic acid increases the length and volume density of ducts in the rat embryonic pancreas

In this study, the role of all-trans retinoic acid (RA) on the proliferation of rat embryonic pancreas ducts and on the proportion of insulin cells was investigated. All-trans RA (10-6 m) was added to Ham's F12.ITS serum-free medium in which 12.5 day rat dorsal pancreatic buds were cultured on Matrigel. Control explants were cultured on Matrigel in Ham's F12.ITS alone or in Ham's F12.ITS containing ethanol (the diluent for RA). After a 7 day culture period, explants were incubated with bromodeoxyuridine (BrdU) for assessment of cell proliferation. Explants were processed for both morphometry and immunocytochemistry. The length density and volume density of the pancreatic ducts were assessed using an image analysis system. Cells positive for insulin, BrdU and glucagon were localized on adjacent serial sections. RA treatment caused a statistically significant increase in the volume density (P < 0.007) and length density (P < 0.008) of the ducts, as well as a 1.2-fold increase (P < 0.0001) in the proportion of insulin to glucagon cells, compared to both control groups. Few insulin cells were BrdU positive, indicating that cells had a low proliferation rate. The increased proportion of insulin cells may relate to the increased volume density and length density of the ducts in RA-treated explants. It is suggested that RA stimulated the production of additional progenitor cells and not proliferation of existing insulin cells.