GABA-immunoreactive structures in rat kidney.

We examined the distribution of gamma-aminobutyric acid-like immunoreactivity (GABA-LI) in the rat kidney by light and electron microscopy. In vibratome sections, GABA-LI was present in both the renal medulla and cortex. The inner stripe of the outer medulla was most heavily and almost homogeneously labeled, whereas GABA-LI in the cortex was mainly confined only to some tubules. GABA-positive structures involved the epithelial cells of the thin and the thick ascending limbs of the loop of Henle, the connecting tubules, and the collecting ducts. In GABA-positive connecting tubules and collecting ducts the immunoreactivity was present in the cytoplasm of about half of the epithelial cells. As revealed by electron microscopy, the labeled cells in the collecting tubules were the light (principal) cells. No GABA-LI occurred in neuronal structures. These findings are consistent with the presence of a non-neuronal GABA system in the rat kidney. Furthermore, the specific distribution of GABA in the tubular epithelium suggests a functional significance of this amino acid in tubular transport processes.     

S-100 alpha-like immunoreactivity in tubules of rat kidney. A clue to the function of a "brain-specific" protein

The localization of the alpha and beta subunits of S-100 protein was studied in normal tissue where the identification of three subclasses of S-100 containing cells was derived: i) cells that contain both alpha and beta subunits; ii) cells that contain only the alpha subunit; and iii) cells that contain only the beta subunit. In this study monospecific antibodies against the S-100 alpha and beta subunits were used to characterize the S-100-like immunoreactivity in the rat kidney: Certain cells in the distal nephron, i.e., the connecting piece, collecting ducts, and the thin limb of Henle's loop, contained S-100 alpha immunoreactivity. Proximal tubules, the thick ascending limb of Henle's loop, the distal tubules, and the juxtaglomerular apparatus were negative. No S-100 beta immunoreactivity was found in kidney tubules. However, Schwann cells of renal pelvic nerves contained S-100 beta immunoreactivity. The presence of S-100 alpha antigen in certain cells of the kidney gives further support to the assumption that the alpha subunit of S-100a is related to cells that are highly involved in pH, electrolyte, and water regulation.     

Distribution of IGF-I mRNA and IGF-I binding sites in the rat kidney

Summary In the present study we have investigated the distribution of IGF-I mRNA and IGF-I binding sites in the rat kidney. The distribution of IGF-I mRNA was investigated using a simple and sensitive non-radioactive in situ hybridisation technique based on probe labelling with digoxigenin labelled-UTP followed by detection with conventional immunocytochemical techniques. IGF-I mRNA was found predominantly in medullary collecting ducts and sparsely in cortical collecting duct cells. In addition IGF-I mRNA was expressed in scattered proximal tubular cells in the cortex and in cells confined to the glomerular tuft. IGF-I binding sites were studied using radiolabelled IGF-I and conventional autoradiographical techniques on tissue sections. It was found that IGF-I binding sites were widely distributed throughout the entire kidney and that the specific binding was highest in the inner medulla. These findings add further complexity to the understanding of IGF-I production and action on renal structures.     

Distribution of IGF-I mRNA and IGF-I binding sites in the rat kidney.

In the present study we have investigated the distribution of IGF-I mRNA and IGF-I binding sites in the rat kidney. The distribution of IGF-I mRNA was investigated using a simple and sensitive non-radioactive in situ hybridisation technique based on probe labelling with digoxigenin labelled-UTP followed by detection with conventional immunocytochemical techniques. IGF-I mRNA was found predominantly in medullary collecting ducts and sparsely in cortical collecting duct cells. In addition IGF-I mRNA was expressed in scattered proximal tubular cells in the cortex and in cells confined to the glomerular tuft. IGF-I binding sites were studied using radiolabelled IGF-I and conventional autoradiographical techniques on tissue sections. It was found that IGF-I binding sites were widely distributed throughout the entire kidney and that the specific binding was highest in the inner medulla. These findings add further complexity to the understanding of IGF-I production and action on renal structures.     

Functional morphology of the avian medullary cone

The organization of the renal medulla of the Gambel's quail, Callipepla gambelii, kidney was examined to determine the number of loops of Henle and collecting ducts and the surface area occupied by the different nephron segments as a function of distance down the medullary cones. Eleven medullary cones were dissected from the kidneys of four birds, and the tissue was processed and sectioned for light microscopy. In addition, individual nephrons were isolated on which total loop thin descending segment and thick prebend segment lengths were measured. The results show no correlation between the absolute number of loops of Henle and the length of the medullary cones. The number of thick and thin limbs of Henle and collecting ducts decrease exponentially with distance toward the apex of the cones and the rate of decrease is similar for cones of different lengths. Initially there is a rapid decrease in the number of thin limbs of Henle, indicating that most nephrons do not penetrate the cones a great distance. Thick descending limbs of Henle (prebend segment) ranged in length from 50 to 770 microm, and there was little correlation with the total length of the loop of Henle. However, the length of the thin limb of Henle correlated well with total loop length. The cell surface areas of the limbs of the loop of Henle and the collecting ducts decreased toward the apex of the cones.     

High-level expression of the 32.5-kilodalton calelectrin in ductal epithelia as revealed by immunocytochemistry

Calelectrins are a family of antigenically related Ca2+-binding proteins that have only recently been described. They have the important property of binding to membranes only in the presence of Ca2+. We systematically studied the tissue localization of one calelectrin, the 32.5-kilodalton species, in rats using immunocytochemistry. We found that high levels were exclusively present in the epithelial cells of bile and pancreatic ducts, renal collecting ducts, bronchial epithelia, and brain ependyma. In all of these organs, the other cells were not immunoreactive. In addition, strong immunoreactivity was found in the intercalated disks of myocardial cells, and mild immunoreactivity was observed in several endocrine tissues. In contrast, the cellular distribution of the 67-kilodalton calelectrin was more diffuse, involving most parenchymal cells in addition to the already-mentioned cells. Due to the presence of high levels of 32.5-kilodalton calelectrin in some cell types, this protein may be used as a histochemical marker for differentiated ductal epithelial cells, some specialized epithelia, myocardial cells, and Paneth cells.     

Organ-specific expression of IGF-I during early development of bony fish as revealed in the tilapia, Oreochromis niloticus, by in situ hybridization and immunohistochemistry: indication for the particular importance of local IGF-I

The cellular sites of insulin-like growth factor I (IGF-I) synthesis in the early developing tilapia (0-140 days post fertilization, DPF) were investigated. IGF-I mRNA and peptide appeared in liver as early as 4 DPF and in gastro-intestinal epithelial cells between 5-9 DPF. In exocrine pancreas, the expression of IGF-I started at 4 DPF and continued until 90 DPF. IGF-I production was detected in islets at 6 DPF in non-insulin cells and occurred throughout life. In renal tubules and ducts, IGF-I production started at 8 DPF. IGF-I production in chondrocytes had its onset at 4 DPF, was more pronounced in growing regions and was also found in adults. IGF-I mRNA and peptide appeared in the cytoplasm of skeletal muscle cells at 4 DPF. In gill chloride cells, IGF-I production started at 6 DPF. At 13 DPF, IGF-I was detected in cardiac myocytes. IGF-I-producing epidermal cells appeared at 5 DPF. In brain and ganglia, IGF-I was expressed in virtually all neurones from 6 to 29 DPF, their number decreasing with age. Neurosecretory IGF-I-immunoreactive axons were first seen in the neurohypophysis around 17 DPF. Endocrine cells of the adenohypophysis exhibited IGF-I mRNA at 28 DPF and IGF-I immunoreactivity at 40 DPF. Thus, IGF-I appeared early (4-5 DPF), first in liver, the main source of endocrine IGF-I, and then in organs involved in growth or metabolism. The expression of IGF-I was more pronounced during development than in juvenile and adult life. Local IGF-I therefore seems to have a high functional impact in early growth, metabolism and organogenesis.This study was supported by the SNF (NRP 50, project 4050-66580).     

Demonstration of prostaglandin synthesis in collecting duct cells and other cell types of the rabbit renal medulla.

The renal medulla has a high capacity for prostaglandin production and the interstitial cells, which contain abundant lipid inclusions have been suggested to be the site of synthesis. However, histochemical studies have indicated that the collecting ducts are the main site of production. The object of the present study was to study the distribution of prostaglandin synthetase in the rabbit renal medulla by direct, quantitative determination of the enzyme activity in different cellular fractions. Slices were cut from rabbit renal papilla and immersed in a hypertonic saline solution. 92% of the collecting duct cells were then removed from the slices by suction through a micropipette. The remaining dissected slices thus contained mainly three cell types, cells of Henle's loop, endothelial cells, and interstitial cells. The isolated collecting duct fraction, the corresponding dissected slices, from which the colelcting duct cells were removed, as well as intact slices were assayed for prostaglandin synthetase activity using a quantitative assay with [14C] arachidonate as substrate. Of the prostaglandin in synthetase activity 39% was found in the collecting ducts, 53% in the dissected slices, and 7% in the dissection medium. It is thus concluded that significant prostaglandin synthetase activity is present in collecting duct cells as well as in at least one other cell type of the medulla.     

Demonstration of prostaglandin synthesis in collecting duct cells and other cell types of the rabbit renal medulla

The renal medulla has a high capacity for prostaglandin production and the interstitial cells, which contain abundant lipid inclusions, have been suggested to be the site of synthesis. However, histochemical studies have indicated that the collecting ducts are the main site of production. The object of the present study was to study the distribution of prostaglandin synthetase in the rabbit renal medulla by direct, quantitative determination of the enzyme activity in different cellular fractions.Slices were cut from rabbit renal papilla and immersed in a hypertonic saline solution. 92% of the collecting duct cells were then removed from the slices by suction through a micropipette. The remaining dissected slices thus contained mainly three cell types, cells of Henle's loop, endothelial cells, and interstitial cells. The isolated collecting duct fraction, the corresponding dissected slices, from whcih the collecting duct cells were removed, as well as intact slices were assayed for prostaglandin synthetase activity using a quantitative assay with [¹⁴C] arachidonate as substrate.Of the prostaglandin synthetase activity 39% was found in teh collecting ducts, 53% in the dissected slices, and 7% in the dissection medium. It is thus concluded that significant prostaglandin synthetase activity is present in collecting duct cells as well as in at least one other cell type of the medulla.     

Immunohistochemical localization of atrial natriuretic peptide in the developing and adult mammalian kidney

The discovery, within the last decade, of atrial natriuretic peptide (ANP), a family of peptides with natriuretic/diuretic and vasorelaxant properties, has prompted much research into the mechanisms and sites of action of ANP within the kidney. In the present study, ANP was localized in the kidneys of several mammalian species by immunohistochemical techniques 1) to identify possible sites of synthesis; 2) to compare the localization of ANP to known physiological effects; 3) to determine species differences, if any, in ANP localization; and 4) to study the development of ANP immunoreactivity in the fetal and neonatal rat kidney. Using an antibody against rat ANP, IV, ANP was localized exclusively on the proximal convoluted tubule (PCT) brush border and within intercalated cells of the outer medullary and cortical collecting tubules and ducts of adult mouse, rat, pig, monkey, and human kidneys. The development of ANP immunoreactivity paralleled the differentiation and maturation of collecting duct epithelium in rat fetal kidney. Atrial natriuretic peptide found within intercalated cells of the cortical and outer medullary collecting ducts may be the result of endogenous synthesis and, following secretion, may be available to receptors in the inner medullary collecting ducts.     

Immunocytochemical demonstration of two vitamin D-dependent calcium-binding proteins in mammalian kidney

Distribution of vitamin D-dependent calcium-binding proteins (CaBPs) were studied in four mammalian species using monospecific antibodies raised against chick duodenal CaBP (D-CaBP), human cerebellar CaBP (L-CaBP), and rat duodenal CaBP (S-CaBP). The immunoperoxidase technique of unlabelled antibodies was employed. The distribution of D-CaBP/L-CaBP was identical in all the species studied except for the monkey. In the rat, pig, and human nephrons, D-CaBP/L-CaBP was seen in the cytoplasm of the cells of the distal convoluted tubules, initial segments of the collecting ducts and interspersed cells of the collecting ducts. Proximal convoluted tubules, glomeruli and maculae densae were negative. In the monkey, in addition to the cells of the distal convoluted tubules, the cells along the entire length of the collecting ducts were also strongly positive. S-CaBP was found to be species-specific, and hence positive results were obtained only in the rat nephron. The strongest positive reaction for S-CaBP was seen in the cells of the distal convoluted tubules. These same cells were also positive for D-CaBP/L-CaBP. S-CaBP was also detected in the cells of the thick ascending limb of the loop of Henle, along the entire length of the collecting ducts and in smaller amounts in cells of the macula densa. Intracellularly the S-CaBP was present only in the apical cytoplasm of positive cells. D-CaBP/L-CaBP stained the entire cytoplasm but the staining in the apical cytoplasm was denser.     

Immunocytochemical demonstration of carbonic anhydrase in human epithelial cells

Human tissues obtained early postmortem were immunostained to demonstrate carbonic anhydrase (CA) and, in some instances, to differentiate CA I and CA II, employing an immunoglobulin-peroxidase bridge method. Optimal immunostaining was obtained in tissues fixed a few hours in Carnoy's fluid or a buffered HgCl2 solution. Specimens fixed 1/2 to 2 hr with buffered formalin or Bouin's fluid stained less well but better than those fixed 24 hr with formalin. In tracheobronchial glands, serous acini and demilunes exhibited intense immunoreactivity demonstrative of the isozyme CA II. In kidney, all cells of the distal convoluted tubules were strongly positive for CA and cortical collecting tubule cells stained strongly but with some variability among individual cells. Cells in medullary collecting tubules ranged from intensely to negligibly reactive. Proximal convoluted tubules and thick ascending limbs showed moderate to light, uniform staining, but the thin limbs of the loop of Henle were negative. Renal cell immunoreactivity occurred only with antiserum to CA II. Seromucous acini in submandibular glands stained strongly and selectively for CA. Ducts in liver and pancreas showed strong selective immunostaining. The most superficial columnar cells lining the main lumen of the colon and appendix displayed strong reactivity, as did columnar cells lining the gall bladder.     

Stem cell marker TRA-1-60 is expressed in foetal and adult kidney and upregulated in tubulo-interstitial disease

The kidney has an intrinsic ability to repair itself when injured. Epithelial cells of distal tubules may participate in regeneration. Stem cell marker, TRA-1-60 is linked to pluripotency in human embryonic stem cells and is lost upon differentiation. TRA-1-60 expression was mapped and quantified in serial sections of human foetal, adult and diseased kidneys. In 8- to 10-week human foetal kidney, the epitope was abundantly expressed on ureteric bud and structures derived therefrom including collecting duct epithelium. In adult kidney inner medulla/papilla, comparisons with reactivity to epithelial membrane antigen, aquaporin-2 and Tamm–Horsfall protein, confirmed extensive expression of TRA-1-60 in cells lining collecting ducts and thin limb of the loop of Henle, which may be significant since the papillae were proposed to harbour slow cycling cells involved in kidney homeostasis and repair. In the outer medulla and cortex there was rare, sporadic expression in tubular cells of the collecting ducts and nephron, with positive cells confined to the thin limb and thick ascending limb and distal convoluted tubules. Remarkably, in cortex displaying tubulo-interstitial injury, there was a dramatic increase in number of TRA-1-60 expressing individual cells and in small groups of cells in distal tubules. Dual staining showed that TRA-1-60 positive cells co-expressed Pax-2 and Ki-67, markers of tubular regeneration. Given the localization in foetal kidney and the distribution patterns in adults, it is tempting to speculate that TRA-1-60 may identify a population of cells contributing to repair of distal tubules in adult kidney.     

Transforming growth factor-α immunoreactivity in a variety of epithelial tissues

Abstract. The immunohistochemical expression of transforming growth factor-alpha (TGFα) has been examined in a range of normal adult epithelial tissues from both man and rat using an anti-hTGFα monoclonal antibody (GF10). No differences in distribution were apparent between man and rat. In the continually renewing epithelium of the gastrointestinal tract, no staining was seen within the proliferative compartments, but strong immunoexpression was noted in various differentiated populations. In the testis, the spermatogonia were unstained, but the more luminally orientated germ cells were strongly positive. In the gastrointestinal tract, at least, any mitogenic action of TGFα must be mediated through a relatively long paracrine loop. In contrast, the differentiated parenchyma of kidney, salivary gland and liver remained unstained apart from collecting ducts in the kidney, striated ducts in salivary glands and bile ducts in the liver. The association of TGFα with tubule formation was reinforced by the very strong staining of newly forming bile ducts in a model of liver oval cell proliferation. Thus, in all the epithelia studied there was a distinct spatial pattern of TGFα immunoreactivity.     

Distribution of saccharide residues in glycoconjugates of the kidney in Gallus domesticus using peroxidase-conjugated lectins

A battery of seven different horseradish-peroxidase labelled lectins (DBA, PNA, SBA, UEA I, WGA, ConA, LTA) was used to study the distribution of sugar residues in the glycoconjugates along the nephron and the collecting duct of the kidney of Gallus domesticus. As far as the glomerular components are concerned, we have demonstrated that the podocytes and, with a lesser extent, the mesangial cells are characterised by the presence of D-mannose, D-galactose-(beta 1- greater than 3)-N-acetyl-D-galactosamine and sialic acid. The glomerular capillary wall shows the presence of the disaccharide D-galactose-(beta 1- greater than 3)-N-acetyl-D-galactosamine and sialic acid. With regards to the tubules, the proximal tubule, the descending limb of the loop of Henle, the connecting tubule and the collecting one, are characterised by N-acetyl-D-galactosamine, (1- greater than 6)-alpha-L-fucose, D-mannose, N-acetyl-D-galactosamine and D-galactose-(beta 1- greater than 3)-N-acetyl-D-glucosamine. The cells of the connecting and collecting ducts show the presence of intracellular sialic acid, found also as component of the mucous secretion. The ascending limb of the loop of Henle and the distal tubule contain only three saccharidic residues, i.e. (1- greater than 6)-alpha-L-fucose, D-mannose and N-acetyl-D-glucosamine. Lectin histochemistry was also useful to define the saccharidic components of the mucus, which is normally present within the connecting and collecting ducts of the kidney of the birds. The cellular variability of the connecting and the collecting ducts is similar to that found in the kidney of some mammals. Such a variability seems to suggest a possible cell specialization along a single kidney tubule.     

Decreased Insulin-Like Growth Factor-I and Its Receptor Expression in the Hippocampus and Somatosensory Cortex of the Aged Mouse

Insulin-like growth factor-I (IGF-I) is a multifunctional polypeptide and has diverse effects on brain functions. In the present study, we compared IGF-I and IGF-I receptor (IGF-IR) immunoreactivity and their protein levels between the adult (postnatal month 6) and aged (postnatal month 24) mouse hippocampus and somatosensory cortex. In the adult hippocampus, IGF-I immunoreactivity was easily observed in the pyramidal cells of the stratum pyramidale in the hippocampus proper and in the granule cells of the granule cell layer of the dentate gyrus. In the adult somatosensory cortex, IGF-I immunoreactivity was easily found in the pyramidal cells of layer V. In the aged groups, IGF-I expression was dramatically decreased in the cells. Like the change of IGF-I immunoreactivity, IGF-IR immunoreactivity in the pyramidal and granule cells of the hippocampus and in the pyramidal cells of the somatosensory cortex was also markedly decreased in the aged group. In addition, both IGF-I and IGF-IR protein levels were significantly decreased in the aged hippocampus and somatosensory cortex. These results indicate that the apparent decrease of IGF-I and IGF-IR expression in the aged mouse hippocampus and somatosensory cortex may be related to age-related changes in the aged brain.     

Immunolocalization of cyclooxygenase-2 in the macula densa of human elderly.

To gain insight into the role of prostanoids in human kidney function, we examined the distribution of cyclooxygenase (COX) 1 and COX-2 by immunofluorescence and immunohistochemistry in human kidneys from adults of various age groups. COX-1 was detected in the collecting ducts, thin loops of Henle and portions of the renal vasculature. COX-2 was detected in the renal vasculature, medullary interstitial cells, and the macula densa. In addition, COX-2 immunoreactivity was noted in afferent arteries and the macula densa of the renal cortex and was more evident in the kidneys of older adults.     

Enhancement of maternal and fetal nephrotoxicity of salicylate by zinc deficiency. Morphological, enzyme histochemical and immunohistochemical studies

An oral dose of 700 mg/kg salicylic acid was given to normal and Zn-deficient rats at day 16 of gestation. Maternal and fetal kidneys were studied at day 19 of gestation. Zn-deficiency did not cause any lesions detectable by semi-thin section light microscopy, electron microscopy, enzyme histochemistry and immunohistochemistry. Salicylate may lead only to small morphological, enzymatic and cytoskeletal defects in the maternal and fetal kidney. However, enzyme activities decreased in plasma membranes, mitochondria, lysosomes, endoplasmic reticulum and peroxisomes in all segments of the tubular apparatus when salicylate was given to Zn-deficient rats. Cytoskeletal proteins such as keratin in the glomerular cells and epithelial lining of the collecting ducts and vimentin in vascular endothelial cells of the maternal kidney were also affected. In addition, the epithelial cells of the collecting ducts, which were comparatively less damaged, accumulated high amounts of fat. In severe cases, the enzymatic and cytoskeletal lesions were accompanied by hematuria and tubular necroses including and collecting ducts in the renal papilla. In less severe cases reduced activities of brush border hydrolases were the only sign of disturbed renal function in maternal rats indicating that membrane alteration and loss of membrane-bound enzymes are the primary defects. In the fetal kidneys, mitotic activity of the cells of the nephron anlagen and collecting ducts was reduced and enzymatic and morphological differentiation were disturbed. As a consequence less mature nephrons and collecting ducts occurred.     

Regenerating endothelial cells express insulin-like growth factor-I immunoreactivity after arterial injury

Summary In the present study the expression of insulin-like growth factor I (IGF-I; somatomedin C) immunoreactivity was examined in endothelial cells during repair after injury to the intima in the femoral artery of adult rats. Two types of injury were examined: (1) endothelial denudation induced by the use of a catheter, and (2) vessel compression by short-term ligation. In untreated rats, arterial endothelial cells showed no or, only infrequently, low IGF-I immunoreactivity in their cytoplasm. Endothelial cells at the border to the denuded area showed increased IGF-I immunoreactivity one day after injury to the intima of the femoral artery. Thrombocytes and fibrin deposits as well as vital endothelial cells, covered by clots, were immunonegative. The maximal intensity of IGF-I immunoreactivity was reached within 3 days after insult. The IGF-I immunoreactivity in the endothelial cells remained elevated for at least 4 weeks, compared to the controls. Intimai thickenings appeared within a week after injury and many cells in these thickenings showed intense IGF-I immunoreactivity as did the covering endothelial cells. Smooth muscle cells in the media were generally immunonegative during control conditions and after endothelial denudation. Spontaneously hypertensive rats (SHR) showed, similarly to their matched controls (WKY), approximately the same patterns of IGF-I immunoreactivity in their endothelial cells both under normal conditions and after injury. It is concluded that IGF-I is likely to be involved in the repair of the intima in injured arteries.     

Immunohistochemical localisation of glucose-6-phosphatase in developing human kidney

The objective of our study was to determine the cellular localisation of glucose-6-phosphatase in developing human kidney using monospecific antiserum and a standard immunohistochemical method (peroxidase-antiperoxidase, PAP) on formalin fixed and paraffin embedded tissue. In embryonic and early fetal development of the metanephric kidney, glucose-6-phosphatase is located primarily in derivatives of the ureteric bud such as the pelvis, calyxes and collecting ducts. In mid-fetal life as nephrons evolve and develop they become increasingly immunoreactive to glucose-6-phosphatase, such that in mature metanephric kidney the proximal tubules are highly reactive for glucose-6-phosphatase with other elements of the nephron also immunopositive albeit at lower reactivities. In addition the parietal layer of Bowman's capsule and some cells of the visceral layer are immunopositive. Only with the development of nephrons does the early predominance of glucose-6-phosphatase immunoreactivity to ureteric bud derivatives change: in mature kidney the reactivity in the collecting ducts is a small proportion of the total. In proximal tubular cells the distribution of glucose-6-phosphatase immunoreactivity is relatively uniform throughout development in contrast to collecting ducts where in fetal life this reactivity is displaced to the apices and basal areas by intracellular glycogen deposits. The mesonephric kidney has a similar pattern of glucose-6-phosphatase immunoreactivity to that of metanephric kidney. The availability of monospecific antiserum to glucose-6-phosphatase and immunohistochemical methods now allows an alternative approach to cellular localisation. Many of the difficulties in the fixation of tissue and assay of glucose-6-phosphatase activity inherent in conventional histochemical methods are avoided by such methods.