Localization of urate oxidase in the crystalline cores of rat liver peroxisomes by immunocytochemistry and immunoblotting

We investigated the immunocytochemical localization of urate oxidase by light and electron microscopy. Rabbits were immunized with urate oxidase prepared from rat liver and the resulting antibody was further purified by affinity chromatography. Immunoblotting of the antigen revealed a single band of Mr 32,500 daltons, consistent with a subunit of uricase. The same band was observed in immunoblots prepared from a total peroxisome fraction and in its subfraction containing the cores, but not in the matrix portion. Immunostaining of 1-micron sections with the antibody against uricase followed by protein A-gold-silver showed fine granules in hepatocytes, which exhibited distinct fluorescence when examined in a microscope equipped with epifluorescence illumination. Incubation of ultra-thin sections of rat liver, embedded in Lowicryl K4M, LR White, or Epon, with the anti-uricase antibody followed by protein A-gold showed prominent labeling of the crystalline cores, with no reaction in the surrounding peroxisomal matrix. In contrast, the core region was spared whereas the matrix was heavily labeled in sections incubated with an antibody against catalase. Direct incubation of cores, isolated by centrifugation, with the anti-uricase antibody followed by protein A-gold revealed gold particles on the surface of isolated cores, with rare particles within the lumen of the polytubular structures that make up the cores. Specificity of the immunolabeling was established in sections incubated with an IgG fraction from pre-immunized rabbits. These observations demonstrate that in normal rat liver urate oxidase is exclusively associated with the crystalline cores in peroxisomes.     

Localization of a phytohormone using immunocytochemistry

The localization of cytokinins in corn root tips was investigated using antibodies or antibody fragments directed against dihydrozeatin riboside and labeled with rhodamine or colloidal gold. Roots were sectioned at -30 degrees to -40 degrees for immunofluorescence or freeze-substituted in ethanol or acetone and embedded in plastic for electron microscopy. Meristematic cells surrounding the quiescent center as well as root cap cells were specifically labeled using direct immunofluorescence techniques, whereas cells of the quiescent center did not bind label. Tissue sections treated with colloidal gold-labeled antibody fragments had gold particles widely distributed in the cytoplasm. The results show that the quiescent center is not the major site of cytokinin localization in root tips.     

Localization of aminopeptidase A (angiotensinase A) in the rat and mouse kidney

Summary Aminopeptidase A (E.C.3.4.11.7; APA) can be demonstrated histochemically in the rat and mouse kidney by light microscopy (simultaneous azo coupling with -Glu-MNA as substrate and high-purity FBB as coupling agent) mainly in the brush borders, glomeruli and portions of the juxtaglomerular apparatus. Sex and species differences are found with regard to enzyme activity and localization. The relation of aminopeptidase A to angiotensinase A was established by inhibition experiments with angiotensin II and III. The following significant differences exist with respect to other aminopeptidases (aminopeptidase M and -glutamyl transferase), which were also demonstrated: APM shows no dependence on calcium ions; APM and -GT are not demonstrable in the glomerulus or juxtaglomerular apparatus.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Localization of TGF-beta signaling intermediates Smad2, 3, 4, and 7 in developing and mature human and mouse kidney.

Smad proteins are signaling intermediates of the TGF-beta superfamily and are involved in a range of biological activities including development and immune responses. We studied the expression of TGF-beta-receptor activated Smads (Smad2 and Smad3), the common partner Smad (Smad4), an inhibitory Smad (Smad7), and the activated (phosphorylated) Smad2 (pSmad2) in developing and adult kidneys of humans and mice. These studies demonstrate associated expression of these Smads in multiple renal cell types in all developmental stages and in mature non-diseased kidneys. Smad expression is in general most widespread at the earliest stages of nephron development and diminishes as components of the nephrons become more differentiated. Paucity of Smad expression in mesangial cells in contrast to widespread expression of these Smads in glomerular visceral epithelial cells in both developing and mature kidneys was remarkable. Divergent and less extensive expression of Smad4, compared with other Smad proteins, was also demonstrated in tubules of human kidneys. Based on the observed expression patterns, these findings demonstrate, for the first time, expression of the TGF-beta-receptor-activated Smad2 and Smad3, the common mediator Smad4, and the inhibitory Smad7 in the developing human fetal kidney, extending observations previously made in rodent systems to humans.     

Distribution of anticancer antibiotic daunomycin in the rat heart and kidney revealed by immunocytochemistry using monoclonal antibodies

Two monoclonal antibodies (ADM-1-11 and 79-31 mAbs) were raised against daunomycin (DM) conjugated to bovine serum albumin via the cross-linker N-(gamma-maleimidobutyryloxy)succinimide. The monoclonal antibodies (mAbs) specifically detected DM as well as its analogs doxorubicin and epirubicin, but did not react with other anticancer antibiotics, including pepleomycin, mitomycin C, and actinomycin D. The mAbs reacted strongly with glutaraldehyde-conjugated DM in an enzyme linked immunosorbent assay (ELISA) used as a model system for immunocytochemistry as well as in appropriately pretreated sections of tissues from animals injected with DM. No staining occurred in tissues from uninjected animals. In order to perform DM ICC a number of tissue treatment conditions critical to the detection of low molecular weight substances were employed. Uptake of DM was studied in rats after a single i.v. or i.p. administration of the drug. In the heart, accumulation of DM occurred in nuclei and in the cytoplasm. In the kidney, DM immunoreactivity accumulated in all segments of the nephron except for the proximal tubules. Since the proximal tubules are known to be where a variety of transport systems including P-glycoprotein (Pgp) and organic anion-transporting polypeptides (OATPs) in drug interactions occur, the absence of DM accumulation in these segments may reflect a transport phenomenon depending upon such transporters. The availability of methods to study sites of accumulation of DM offers possibilities for understanding toxic side effects of this drug on the heart and kidney. Moreover, the immunocytochemical methodology developed may prove useful for the localization of other low molecular weight drugs that can be fixed in situ by glutaraldehyde.     

Localization of cytokinins in somatic and zygotic embryos of Tilia cordata using immunocytochemistry

Immunofluorescent and immunogold labeling was used to study the localization of cytokinins in developing somatic and zygotic embryos of Tilia cordata Miller. Broad-specificity polyclonal antibodies active against dihydrozeatin riboside (DHZR)/zeatin riboside (ZR)-type and isopentenyladenosine (iPR)-type cytokinins were used for immunolabeling. Immunofluorescent microscopy showed that these cytokinins were concentrated in highly cytoplasmic cells showing meristematic character. Cotyledon initials and primary meristems of heart-stage somatic embryos, as well as heart-stage zygotic embryos, were labeled. During elongation of embryos, cytokinin immunoreactive material was concentrated to areas having meristematic character. Root apex, shoot meristem, and cotyledon cells of somatic and zygotic cotyledonary embryos, as well as epidermal and subepidermal cell layers of the hypocotyl, showed the strongest immunoreaction. The nucleoli, especially, had a very strong signal. Results at the ultrastructural level with gold-conjugated protein A supported these conclusions. Gold particles were distributed in the nuclei, especially in the nucleoli and throughout the ground cytoplasm. They were occasionally associated with plastids and mitochondria, but seldom with other organelles.     

RT-PCR and immunocytochemistry studies support the presence of somatostatin, cortistatin and somatostatin receptor subtypes in rat Kupffer cells

The present study investigated the presence of somatostatin receptor subtypes (ssts) and the endogenous peptides somatostatin and cortistatin in rat Kupffer cells, since modulation of these cells by somatostatin may be important for the beneficial effect of somatostatin analogues in a selected group of hepatocellular carcinoma patients. Kupffer cells were isolated from rat liver in agreement with national and EU guidelines. RT-PCR was employed to assess the expression of somatostatin, cortistatin and ssts in Kupffer cells. Western blot analysis and immunocytochemistry were employed to assess the expression and the localization of the receptors, respectively. Quiescent Kupffer cells were found to express sst(1-4) mRNA, while immunocytochemical studies supported the presence of only the sst(3) and sst(4) receptors, which were found to be internalized. However, sst1 and sst(2A) receptors were detected by western blotting. RT-PCR and RIA measurements support the presence of both somatostatin and cortistatin. Stimulation of the cells with LPS activated the expression of the sst(2), sst(3) and sst(4) receptors. The present data provide evidence to support the presence of ssts and the endogenous neuropeptides somatostatin and CST in rat Kupffer cells. Both peptides may act in an autocrine manner to regulate sst receptor distribution. Studies are in progress in order to further characterize the role of ssts in Kupffer cells and in hepatic therapeutics.     

Localization of thymostimulin in mouse and rat thymuses

Anti-bovine thymostimulin serum reacts with reticulo-epithelial cells in the cortex and in the medulla of rat and mouse thymuses. The immuno-reaction is present in a smaller number of cells which have more pallid color than those found in calf thymus. Comparative examination as to the distribution of other thymic factors and as to species-specificity was conducted.     

Localization of carboxypeptidase A-like enzyme in rat kidney

In this study we demonstrate that carboxypeptidase A (CPA)-like enzyme is expressed in rat kidney. The major metabolites of angiotensin (Ang) I by the rat renal mesangial cell extract at 37 degrees C, pH 7.4, were Ang 1-9 and Ang II. Quinaprilat did not influence the formation of Ang 1-9, but it inhibited formation of Ang II. The formation of Ang 1-9 was inhibited by potato carboxypeptidase inhibitor, 1,10-phenanthroline or EDTA. Lowering the pH from 7.4 to 4.0 also inhibited the formation of this nonapeptide. These findings suggest that a metallocarboxypeptidase is responsible for Ang 1-9 production. Using monoclonal antibodies to CPA, Western blot showed the presence of CPA-like enzyme in the extracts prepared from the mesangial cells or kidney cortex of the rat. Immunohistochemistry showed that CPA-like enzyme is localized in the mesangial glomerular cells and adventitia of kidney blood vessels, whereas it was absent in the renal tubules. Our data suggest that a CPA-like enzyme could be added to a repertoire of enzymes present in the rat mesangial cells and adventitia of renal blood vessels.     

Localization of neurotensin binding sites in rat kidney

[3H]Neurotensin ([3H]NT) appears to bind specifically to a single class of sites in slide-mounted rat kidney sections (KD = 8.3 nM; Bmax = 31.6 fmol/mg tissue). Bound [3H]NT can be displaced by nonradioactive NT and a series of its fragments and analogues with relative potencies that correlate well (r = 0.91; p less than 0.005) to their potencies in the rat stomach strip bioassay. These results suggest that NT receptors are similar in both systems. However, they are probably slightly different from those present in the guinea pig atria (r = 0.78; p less than 0.1). We visualized these sites by using the tritium-sensitive LKB film technique analysed by computerized densitometry. [3H]NT binding sites are highly concentrated in the renal cortex while low levels are observed in the renal medulla. The possible physiological and/or pathophysiological significances of the presence of [3H]NT binding sites in the kidney are discussed.     

Localization of nucleotide pyrophosphatase in the rat kidney

Hydrolysis of NAD by a nucleotide pyrophosphatase of renal membrane fractions has been reported previously. The aim of the present study was to localize this enzyme in the rat kidney. Nucleotide pyrophosphatase was assayed in glomeruli, in three parts of the proximal tubule and in four parts of the distal tubule dissected form freeze-dried sections. Nucleotide pyrophosphatase activity, expressed in mumol X min-1 X mg protein-1, ranged between 9.8 and 32.3 in the proximal tubular segments and between 1.1 and 2.7 in the distal tubular segments. It was 3.4 in the glomeruli. The enrichment of the activity during the purification of brush border vesicles was measured. A ten-fold higher specific activity was found in the brush border vesicles as compared to the renal cortical homogenates. Thus, most of the renal nucleotide pyrophosphatase appears to be localized in the luminal membrane of the proximal tubule. A permeabilization of the membrane did not increase the activity of brush border vesicles. This indicates that all catalytic sites are accessible at the outer surface of the membrane.     

Localization of nucleotide pyrophosphatase in the rat kidney

Summary Hydrolysis of NAD by a nucleotide pyrophosphatase of renal membrane fractions has been reported previously. The aim of the present study was to localize this enzyme in the rat kidney. Nucleotide pyrophosphatase was assayed in glomeruli, in three parts of the proximal tubule and in four parts of the distal tubule dissected form freezedried sections. Nucleotide pyrophosphatase activity, expressed in mol·min^–1·mg protein^–1, ranged between 9.8 and 32.3 in the proximal tubular segments and between 1.1 and 2.7 in the distal tubular segments. It was 3.4 in the glomeruli. The enrichement of the activity during the purification of brush border vesicles was measured. A tenfold higher specific activity was found in the brush border vesicles as compared to the renal cortical homogenates. Thus, most of the renal nucleotide pyrophosphatase appears to be localized in the luminal membrane of the proximal tubule. A permeabilization of the membrane did not increase the activity of brush border vesicles. This indicates that all catalytic sites are accessible at the outer surface of the membrane.Supported by the Swiss National Foundation, grant nr. 3.813.084     

cGMP immunocytochemistry in aorta, kidney, retina and brain tissues of the rat after perfusion with nitroprusside

The distribution of cyclic guanosine 3',5' monophosphate (cGMP) producing cells in various organs of the rat were studied immunocytochemically using antibodies raised against formaldehyde-fixed cGMP. Sodium nitroprusside (SNP), a direct activator of guanylate cyclase and vasodilator, was used to enhance cGMP levels. In order to reach all organs optimally, whole body perfusion was performed using a modified Krebs-Ringer buffer at 37 degrees C, aerated with 5% CO2/95% O2, also containing isobutyl methyl xanthine (IBMX); a phosphodiesterase inhibitor. After 15-min pre-perfusion, SNP was added to the perfusate, followed by fast fixation with ice-cold 4% paraformaldehyde-phosphate buffer. After vehicle perfusion, only the retina showed cGMP immunoreactivity in the photoreceptor and ganglion layer, while other organs lacked cGMP immunoreactivity. After 15-min perfusion with SNP (10 microM), enhanced cGMP immunostaining was seen in smooth muscles of the aorta, amacrine-like cells in the retina, glomeruli of the kidney cortex, blood vessels in the dura mater, as well as cells in the pineal and in the median eminence. The results indicate that the distribution and the reactivity of cGMP producing cells, situated outside the blood brain barrier, can be studied by immunocytochemistry after pharmacological manipulations of the intact tissue with a nitrovasodilator using whole body perfusion.     

cGMP immunocytochemistry in aorta,kidney, retina and brain tissues of the rat after perfusion with nitroprusside

Summary The distribution of cyclic guanosine 3',5' monophosphate (cGMP) producing cells in various organs of the rat were studied immunocytochemically using antibodies raised against formaldehyde-fixed cGMP. Sodium nitroprusside (SNP), a direct activator of guanylate cyclase and vasodilator, was used to enhance cGMP levels. In order to reach all organs optimally, whole body perfusion was performed using a modified Krebs-Ringer buffer at 37° C, aerated with 5% CO₂/95% O₂, also containing isobutyl methyl xanthine (IBMX); a phosphodiesterase inhibitor. After 15-min pre-perfusion, SNP was added to the perfusate, followed by fast fixation with ice-cold 4% paraformaldehyde-phosphate buffer. After vehicle perfusion, only the retina showed cGMP immunoreactivity in the photoreceptor and ganglion layer, while other organs lacked cGMP immunoreactivity. After 15-min perfusion with SNP (10 M), enhanced cGMP immunostaining was seen in smooth muscles of the aorta, amacrine-like cells in the retina, glomeruli of the kidney cortex, blood vessels in the dura mater, as well as cells in the pineal and in the median eminence: The results indicate that the distribution and the reactivity of cGMP producing cells, situated outside the blood brain barrier, can be studied by immunocytochemistry after pharmacological manipulations of the intact tissue with a nitrovasodilator using whole body perfusion.     

Localization of dehydropeptidase-I, an enzyme processing glutathione, in the rat kidney

An antibody prepared against dehydropeptidase-I, one of the glutathione processing enzymes, from the renal membrane fraction of rats was employed to localize at the light microscopic level the enzyme in the kidney using the avidin/biotin-peroxidase complex method. Dehydropeptidase-I was found to be present on both the brush border and the basolateral membranes of proximal tubular cells. Furthermore, the enzyme activity in the isolated brush border and basolateral membrane fractions was also determined. Distribution profiles of the enzyme activity showed good agreement with the results of the immunohistochemical observations.     

Localization of S-adenosylhomocysteine hydrolase in the rat kidney.

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

Localization of the thioredoxin system in normal rat kidney

Components of the thioredoxin system were localized in normal rat kidney using immunoperoxidase techniques at the light microscopic level and immunogold techniques at the ultrastructural level. Results from both methods were similar. Thioredoxin, thioredoxin reductases, and peroxiredoxins showed cell-type-specific localization, with the same cell types (proximal and distal tubular epithelial, papillary collecting duct, and transitional epithelial cells) previously identified as having high amounts of antioxidant enzyme immunoreactive proteins and oxidative damage products also having high levels of proteins of the thioredoxin system. In addition, peroxiredoxins II and IV were found in high levels in the cytoplasm of red blood cells, identified in kidney blood vessels. While thioredoxin and thioredoxin reductase 1 were found in all subcellular locations in kidney cells, thioredoxin reductase 2 was found predominantly in mitochondria. Thioredoxin reductase 1 was identified in rat plasma, suggesting it is a secreted protein. Peroxiredoxins often had specific subcellular locations, with peroxiredoxins III and V found in mitochondria and peroxiredoxin IV found in lysosomes. Our results emphasize the complex nature of the thioredoxin system, demonstrating unique cell-type and organelle specificity.