Immunohistochemical localization of atrial natriuretic peptide receptor in bovine kidney and lung

Receptors for atrial natriuretic peptide (ANP) were localized in the alveoli and bronchiolar smooth muscle cells of bovine lung and in podocytes of the kidney by immunofluorescence and immunoperoxidase methods. Two specific antisera were raised against the ANP receptor purified from bovine lung plasma membranes: anti-Rc 140 and anti-Rc 70. Anti-Rc 140 was raised against the 140 KD native receptor having a homodimeric structure, and anti-Rc 70 was elicited by immunizing a rabbit with the 70 KD reduced subunits. Essentially identical staining patterns were obtained with both antisera. Identification of ANP receptor sites would provide useful information in understanding the pulmonary and renal actions of ANP.     

Immunohistochemical localization of renin, NO synthase-1, and cyclooxygenase-2 in rodent kidney

The renin-angiotensin system (RAS) and tubuloglomerular feedback (TGF) are central to the maintenance of blood pressure and body fluid composition. Renin, NO synthase-1 (NOS-1), and cyclooxygenase-2 (COX-2) are key regulators of the RAS and TGF. In the present study, to investigate species-specific differences in the RAS and TGF, we immunohistochemically and morphometrically investigated the localization of renin, NOS-1, and COX-2 in the kidneys of various laboratory rodents and comparing males with females (DBA/2Cr mice, F344/N rats, Syrian hamsters, MON/JmsGbs gerbils and Hartley guinea pigs). In all animals, renin-positive immunoreactions were observed in the vascular walls of afferent arterioles. Renin immunoreactions appeared to be more widely distributed in mice. Mice had a greater number of renin-positive arterioles than other species. NOS-1-positive reactions were detected in the macula densa (MD) of all animals. Mice had the greatest number of NOS-1-positive MD cells. In addition to NOS-1-positive reactions, COX-2-positive reactions were observed in the MD of mice, rats, hamsters and gerbils. Interestingly, guinea pigs had no COX-2-positive MD cells. Rats had the greatest number of COX-2-positive MD cells. In nephron segments excluding the MD, the immunohistochemical localization of NOS-1 and COX-2 differed markedly among not only species but also sexes within the same species. In conclusion, we determined that localization of renin, NOS-1, and COX-2 showed large species- and sex-related differences. These data suggest that the regulation mechanisms of the RAS and TGF via renin, NOS-1, and COX-2 differ among rodents.     

Immunohistochemical localization, purification, and characterization of human urinary bladder glutathione S-transferases

This study describes immunohistochemical localization, purification and characterization of glutathione S-transferase (GST) of human urinary bladder. Even though all the three major classes of isoenzymes (alpha, mu, and pi) were expressed in human bladder, more than 90% of total GST activity was accounted for by a pi class anionic form. Human bladder alpha, mu, and pi class GSTs were immunologically related to respective isoenzymes of other human tissues. GST pi was present in all 13 samples analyzed, whereas GST alpha and mu were detected in nine and eleven samples, respectively. GST alpha of human bladder appeared to be unique, because unlike this class of GSTs of other human tissues, bladder enzyme had lower affinity for GSH linked to epoxy-activated Sepharose 6B affinity resin. Immunohistochemical staining indicated localization of GST alpha in epithelial surface cells, underlying submucosa and smooth muscle, whereas mu and pi class isoenzymes were predominantly distributed in epithelial surface cells. These results suggest that human bladder GSTs may play an important role in providing protection against xenobiotics because epithelium is considered a target for several carcinogens and all the three classes of isoenzymes are expressed in these cells.     

Immunohistochemical localization of sodium-dependent l-ascorbic acid transporter 1 protein in rat kidney

Recently, two l-ascorbic acid transporters were identified; sodium-dependent vitamin C transporter (SVCT) 1 and SVCT2. The previous study suggested that SVCT protein might be present on the apical membrane in the straight segment (S3) of proximal tubule. In the present study, SVCT1 immunoreactivity (IR) was observed in the brush border of proximal straight tubules in the medullary ray of renal cortex and the outer stripe of outer medulla, while SVCT2 IR was not localized in any region of the kidney. Since the mechanism of VC reabsorption in the kidney has not been fully elucidated up to the present time, it is meaningful to demonstrate the exact cellular distribution of SVCT protein in the kidney.     

Expression and localization of cysteine dioxygenase mRNA in the liver, lung, and kidney of the rat

Summary The expressions of cysteine dioxygenase (CDO) gene in the liver, lung, skeletal muscle, and kidney were studied byin situ hybridization with a cDNA probe from rat liver CDO under normal conditions. Significant expression of the CDO gene was detected in the liver, lung, and kidney, but not skeletal muscle. In the liver, the signal was confined to the cytoplasm of the hepatocytes. Furthermore, the signal was stronger in the periportal than that in the perivenous areas. In the lung, an intensive signal was found in the bronchiolar epithelium. As to the kidney, an intensive signal was observed in the distal convoluted tubules, while no signal was found in the proximal convultions.     

Immunohistochemical localization of tonin in rat salivary glands and kidney

Tonin has been localized in salivary glands and kidney by the indirect immunofluorescence technique of Coons and by the unlabeled antibody technique of Sternberger. Both techniques gave identical results. Immunoreactive tonin was localized in the cytoplasm of granular convoluted tubular cells and on the apical surface of striated duct cells and collecting duct cells of the submandibular gland. In the parotid and sublingual glands, which lack granular cells, tonin was only found on the apical surface of striated duct and collecting duct cells. In the kidney, immunoreactive tonin was found only associated with cells of the distal convoluted tubules. After fixation with Bouin fluid or with ethanol, tonin was found not only on the apical surface of the cells but also in the apical and perinuclear cytoplasm. This cytoplasmic staining has been attributed to artefactual diffusion since, after fixation with formol-picric acid, the enzyme could only be localized on the apical surface of the tubular cells.     

Immunohistochemical localization of tonin in rat salivary glands and kidney

Summary As a part of a microfluorometric investigation of the nucleoproteins of nuclei whose chromatin displays varying degrees of condensation, a comparison was made of mouse small thymocyte and hepatocyte nuclei stained with the acidic dye, brilliant sulfaflavine, at pH 2.8. These estimates of total protein content were compared with measurements obtained in similarly stained nuclei after extraction either with 0.4 N H₂SO₄ to remove all histones or with 0.35 M NaCl to remove nucleoplasmic proteins and some loosely bound non-histone chromosomal proteins. Treatment with 5% TCA at 60°C was used to remove nucleic acids and to reverse the effects of formaldehyde fixation. In all instances, the fluorescence of 2c hepatocyte nuclei greatly exceeded that of similarly treated thymocyte nuclei. While extraction with 0.4 N H₂SO₄ resulted in reductions of as much as 75% of the total fluorescence of small thymocyte nuclei, the losses of fluorescence in 2c hepatocyte nuclei amounted to only 20–30%. Nevertheless, the absolute values of fluorescence lost in both types of nuclei were very similar. After extraction in 0.35 M NaCl, thymocyte nuclei displayed slightly greater fluorescence than control thymocyte nuclei, while the total fluorescence of hepatocyte nuclei declined. In hepatocyte nuclei extracted with TCA, with and without treatment with 0.35 M NaCl, two populations of diploid nuclei were apparent: one corresponding to parenchymal cell nuclei and the other comprised of non-parenchymal cell nuclei. Only single diploid populations were visible in acid-extracted material. The ratios of 4c2c, 8c4c, and 8c2c hepatocyte nuclei in control, acid-extracted, and NaCl-extracted groups were generally lower than the expected 224 values. These results indicate that total nuclear histones may be estimated microfluorometrically by computing the difference between acid-extracted and unextracted preparations treated in otherwise equivalent ways. In addition, despite very similar absolute losses of fluorescence after removal of histones in thymocyte and 2c hepatocyte nuclei, the proportion of total protein ascribable to histones is much greater in thymocyte nuclei than in 2c hepatocyte nuclei — or, conversely, the percentage of total protein attributable to non-histone proteins is much greater in 2c hepatocyte nuclei than in thymocyte nuclei.     

Immunohistochemical localization of alpha1-acid-glycoprotein in human liver parenchymal cells.

alpha1-Acid glycoprotein, a major human serum glycoprotein was detected and localized in human liver parenchymal cells of a biopsy specimen. A heavy metal salt containing fixative was required to retain sufficient antigen determinants of alpha1-acid glycoprotein in order to visualize this protein by the peroxidase-anti-peroxidase unlabeled antibody enzyme method.     

Immunohistochemical localization of epoxide hydratase in rat liver

An antibody produced against epoxide hydratase (EC 4.2.1.63) which had been purified to apparent homogeneity from hepatic microsomes of phenobarbital pretreated rats was employed in an unlabeled antibody peroxidase-antiperoxidase technique to localize the enzyme at the light microscopic level in the livers of untreated rats. Immunohistochemical staining for epoxide hydratase was detected in parenchymal cells throughout the liver lobule. Cells within the centrilobular regions, however, were observed to be stained more intensely than were those within the midzonal and periportal regions of the lobule. The results of this immunohistochemical study thus demonstrate that epoxide hydratase does not exhibit a uniform pattern of distribution within the liver lobule in untreated rats.     

Immunohistochemical localization of Klotho protein in brain, kidney, and reproductive organs of mice

Klotho mutant mouse (kl-/-), a mouse model for human aging, exhibits various phenotypes in a wide range of organs including arteriosclerosis, neural degeneration, skin and gonadal atrophy, pulmonary emphysema, calcification of soft tissues, and cognition impairment. Klotho mRNA, however, is expressed only in brain, kidney, reproductive organs, pituitary gland, and parathyroid gland. Therefore it remains to be elucidated how lack of Klotho protein in these limited organs leads to the variety of phenotypes. To shed light on mechanisms by which Klotho protein acts on distant targets, we examined localization of Klotho protein in brain, kidney, and reproductive organs, and analyzed brain and kidney in kl-/- mice searching for changes in target regions in these organs. In brain, Klotho proteins were localized at choroid plexus, where the proteins were dominantly localized at the apical plasma membrane of ependymal cells. In kl-/- brain, reduction of synapses was evident in hippocampus, suggesting a role of Klotho as a humoral factor in cerebrospinal fluid. Klotho proteins in kidney localized at distal renal tubules. Interestingly, in kl-/-mice, type IIa Na/phosphate (Pi) cotransporters, which function at the proximal renal tubules in reabsorption of phosphate ions, were translocated. This suggests that Klotho protein in kidney is implicated in calcium homeostasis which regulates localization of type IIa Na/Pi cotransporters via parathyroid hormone (PTH). Klotho proteins in reproductive organs were expressed only in mature germ cells, although in kl-/- mice germ cell maturation was arrested at earlier stages. Thus, Klotho proteins not only function as a humoral factor, but also are implicated in hormonal regulation, which may explain why mutation of klotho gene results in a variety of phenotypes.     

Immunohistochemical localization of antioxidant enzymes during hamster kidney development

Summary Immunolocalization studies of hamster kidney development were performed using polyclonal antibodies to antioxidant enzymes, including antibodies to copper, zinc and manganese superoxide dismutases, catalase, glutathione peroxidase and glutathioneS-transferases and their subunits. Antibodies to extracellular matrix proteins were also studied to determine the temporal sequence between expression of immunoreactive protein for basement membrane proteins, which serve as markers of embryonic induction of nephron development, and antioxidant enzyme expression in kidney development. Immunoreactive proteins for antioxidant enzymes were not detectable in the developing kidney until after extracellular matrix proteins had been deposited. However, immunoreactive proteins for the antioxidant enzymes copper, zinc and manganese superoxide dismutases, catalase, and α class glutathioneS-transferase Ya subunit were detected in renal tubules before birth. μ class glutathioneS-transferase subunits Yb₁ and Yb₂ stained transitional epithelium at high levels before birth. Our results indicate: (1) each type of kidney cell has a unique antioxidant enzyme profile, (2) antioxidant enzymes are expressed in different types of cell at different times during development, but antioxidant enzyme immunoreactive protein was not present until after immunoreactive proteins for extracellular matrix molecules were detected, and (3) certain antioxidant enzymes are present before birth, indicating that high oxygen tension present at birth is not crucial for induction of immunoreactive protein.     

Immunohistochemical detection of betaine-homocysteine S-methyltransferase in human, pig, and rat liver and kidney

Betaine-homocysteine S-methyltransferase (BHMT) has been shown to be expressed at high levels in the livers of all vertebrate species tested. It has also been shown to be abundant in primate and pig kidney but notably very low in rat kidney and essentially absent from the other major organs of monogastric animals. We recently showed by enzyme activity and Western analysis that pig kidney BHMT was only expressed in the cortex and was absent from the medulla. Using immunohistochemical detection, we report here that in human, pig, and rat kidney, BHMT is expressed in the proximal tubules of the cortex. Immunohistochemical staining for BHMT in human, pig, and rat liver indicate high expression in hepatocytes. The staining patterns are consistent with cytosolic expression in both organs.     

Immunohistochemical localization of glucagon-like peptide 1

Summary We report the use of poly-and monoclonal antibodies to study the immunohistochemical distribution of glucagon-like peptide-1 immunoreactivity (GLP-1-IR) in various tissues. The polyclonal antibodies against GLP-1 reacted with pancreatic A cells, enteroglucagon (L) cells in the gut, and some neurons in the central nervous system of all species tested. In pancreas and gut the monoclonal antibodies against GLP-1 exhibited a similar, but species specific distribution, relative to the polyclonal antibodies. The colocalization of GLP-1 and glucagon immunoreactivity in pancreatic, intestinal, and nervous tissues is in agreement with previously reported findings that both peptides are part of a single precursor molecule (preproglucagon).Supported by the DFG, SFB 90     

Immunohistochemical localization of carbonic anhydrase isoenzymes II and III in quail kidney

The immunohistochemical localization of carbonic anhydrase isoenzymes has never been investigated in avian renal tissue previously. Enzyme activity has largely been documented by histochemical and physiological reports. In this investigation, specific antisera were used to study the distribution of the cytosolic carbonic anhydrase II and III isoenzymes in the quail kidney. Comparison between the present findings and the corresponding histochemical patterns, previously obtained in the same species by a cobalt phosphate precipitation method, resulted in the bulk of renal carbonic anhydrase activity being attributed to the carbonic anhydrase II isoenzyme. Conversely, moderate carbonic anhydrase III immunostaining appeared to be confined to the smooth muscle cells of ureteral and arteriolar walls. Indirect evidence of the occurrence, in the quail kidney, of a membrane-associated carbonic anhydrase form, antigenically distinct from the II and III isoforms, was inferred.     

Purification and immunohistochemical localization of rat liver glutathione peroxidase

Glutathione peroxidase was purified from the rat liver to give a single protein band in polyacrylamide gel electrophoresis. Rabbits were immunized with this purified enzyme, and a highly specific anti-glutathione peroxidase antiserum was obtained. Using this antibody, an immunohistochemical technique (the indirect method of peroxidase-labeled antibody) was applied to study the localization of the enzyme in the liver cells.On immunohistochemical observation, glutathione peroxidase was localized exclusively in the cytoplasm of hepatocytes, and a stronger ‘immuno-staining’ was exhibited in the peripheries of the hepatic lobules than in the central zone.     

Immunohistochemical localization of vitamin D-dependent calcium-binding protein in duodenum,kidney, uterus and cerebellum of chickens

Summary Calcium-binding protein (CaBP) has been localized with the immunoperoxidase method using antiserum against purified chick duodenal CaBP. Different preparative procedures were employed to investigate the experimental conditions possibly responsible for the contradictory reports in the literature of the precise cellular localization of CaBP. Freeze substitution, frozen sections followed by fixation and coagulant and non-coagulant fixatives were used with appropriate control sections to demonstrate that the true localization of CaBP in the chick duodenum is in the absorptive cell cytoplasm. The goblet cell localization reported in the literature seems to be a diffusion artifact due to inadequate fixation. CaBP was also localized in several other tissues. In the hen uterus, the tubular glands beneath the surface epithelium showed intense reaction. In the kidney, CaBP was present in the cells of the straight and convoluted segments of distal tubules. The cortex of the chick cerebellum showed the CaBP in Purkinje cells. The entire dendritic trees contained the reaction product. No other neurons in the molecular or the granular layer were stained. In the deep cerebellar nuclei, all neurons were negative and these were outlined by deeply staining axons of the Purkinje cells and their synaptic endings.     

小鼠生长过程中肝、肺、肾胶原蛋白与MMP-1含量变化

利用高效液相色谱法(High performance liquid chromatography,HPLC)和酶联免疫吸附实验(Enzyme-linked immunoassay,ELISA)对小鼠生长过程中肝、肺、肾胶原蛋白与基质金属蛋白酶-1(Matrix metalloproteinase-1,MMP-1)含量变...     

Immunohistochemical localization of NADPH-cytochrome c reductase in rat liver.

NADPH-cytochrome $\text{c}$ reductase (NADPH-cytochrome reductase, EC 1.6.2.4), the flavoprotein which is responsible for the NADPH-dependent reduction of cytochromes P-450 in hepatic microsomes, has been localized immunohistochemically at the light microscopic level in rat liver. Localization was achieved through the use of sheep antiserum to rat hepatic microsomal NADPH-cytochrome $\text{c}$ reductase in an unlabeled antibody peroxidase-antiperoxidase technique. Parenchymal cells throughout the liver lobule were found to be stained positively for NADPH-cytochrome $\text{c}$ reductase, although the intensity of immunostaining was slightly greater in the centrilobular regions. Immunostaining for NADPH-cytochrome $\text{c}$ reductase was not detected in Kupffer cells, connective tissue cells, or in cells of the hepatic vasculature.     

Immunocytochemical localization of angiotensinogen in rat liver and kidney

Cell and Tissue Research - The renin substrate, angiotensinogen, was localized by immunocytochemistry in liver and kidney of normal rats by the use of an antiserum directed against pure rat...     

Immunohistochemical localization of glutathione S-transferase in preneoplastic and neoplastic lesions of the human uterine cervix

A mouse monoclonal antibody and a rabbit polyclonal antibody prepared against the placental form of the enzyme glutathione S-transferase (GST-pi) were used to immunohistochemically stain normal and neoplastic human uterine cervical tissues from 88 cases. Of 65 cases of preneoplastic squamous lesions and invasive carcinomas of the cervix, 94% stained with the monoclonal antibody and 100% with the polyclonal antibody. In the 23 benign tissues, staining of ectocervical squamous epithelium was generally not observed; however, areas of reserve-cell hyperplasia, immature squamous metaplasia and adjacent endocervical cells did show staining (68% with the monoclonal antibody and 95% with the polyclonal antibody). Many of the positive tissue types showed a variety of staining patterns and intensities. These findings do not support the concept that GST-pi staining can be used to distinguish preneoplastic lesions of the cervix from benign reactive or proliferative processes. These results are of interest in the investigation of cervical carcinogenesis since GST-pi may be involved in an early stage of neoplastic transformation of the cervical epithelium. The correlation of these findings with the results of human papillomavirus testing and DNA content analysis should be of interest in determining the relationship of this enzyme to cervical neoplasia.