Immunocytochemical localization of cathepsin B in rat kidney. I. Light microscopic study using the indirect immunoenzyme technique

Localization of cathepsin B in rat kidney was studied using immunocytochemical techniques. Cathepsin B was purified from rat liver and antibody to it was raised in rabbits. The antibody reacted with a lysosomal extract of rat kidney to form a single precipitin line in a double-diffusion test. Immunoblot analysis of lysosomal cathepsin B of rat kidney showed two species of 29K and 25K MW. After removal of Epon, semi-thin sections of glutaraldehyde-fixed tissue were stained by the indirect immunoenzyme technique. Dark-brown reaction product, indicating the antigenic sites for cathepsin B, was found in cytoplasmic granules throughout the nephron. Staining intensity and size of the positive granules varied widely in each segment of the nephron. In the glomeruli and distal tubules, a few small cytoplasmic granules were stained. In the proximal tubules, the S1 segment exhibited many large granules which were most heavily stained, whereas the S2 and S3 segments contained few positive granules. All segments of the distal tubules showed the smallest amount of positive granules. A few positive granules were also noted in the cortical and medullary collecting tubules. Control experiments confirmed the specificity of the staining. The results indicate that the major site for cathepsin B in rat kidney is the S1 segment of the proximal tubule which is known to actively take up proteins leaked through the glomerulus.     

Immunocytochemical localization of cathepsin B in rat kidney. II. Electron microscopic study using the protein A-gold technique

Thin sections of Lowicryl K4M-embedded materials were labeled with protein A-gold complex. Gold particles representing the antigen sites for cathepsin B were exclusively confined to lysosomes of each segment of the nephron. The heaviest labeling was noted in the lysosomes of the S1 segment of the proximal tubules. Labeling intensity varied considerably with the individual lysosomes. Lysosomes of the other tubular segments, such as the S2 and S3 segments of the proximal tubules, distal convoluted tubules, and collecting tubules were weakly labeled by gold particles. Quantitative analysis of labeling density also confirmed that lysosomes in the S1 segment have the highest labeling density and that approximately 65% of labeling in the whole renal segments, except for the glomerulus, was found in the S1 segment. These results indicate that in rat kidney the lysosomes of the S1 segment are a main location of cathepsin B. Further precise observations on lysosomes of the S1 segment revealed that apical vesicles, tubules, and vacuoles were devoid of gold particles, but when the vacuoles contained fine fibrillar materials, gold labeling was detectable in such vacuoles. As the lysosomal matrix becomes denser, the labeling density is increased. Some small vesicles around the Golgi complex were also labeled. These results indicate that the endocytotic apparatus including the apical vesicles, tubules, and vacuoles contains no cathepsin B. When the vacuoles develop into phagosomes, they acquire this enzyme to digest the absorbed proteins.     

Immunocytochemical localization of cathepsin H in rat kidney

Summary Light and electron microscopic localization of cathepsin H in rat kidney was studied using post-embedding immunocytochemical techniques. For ligh microscopy, Epon sections of the kidney were stained by immunoenzyme method after removal of Epon and for electron microscopy, ultrathin sections of the Lowicryl K4M-embedded material were labeled by protein A-gold (pAg) technique. By light microscopy, fine granular staining was found in throughout the nephron, but the staining intensity considerably varied. The strongest staining was noted in the S1 segment of the proximal tubules followed by the S2 and S3 segments and the medullary collecting tubules. The glomeruli, the distal tubules, and the cortical collecting tubules were weakly stained. By electron microscopy, a gold label was found exclusively in lysosomes, which showed various sizes and labeling intensity. The results were quite consistent with the light microscopic results. The labeling intensity tended to increase as the matrix of lysosomes was condensed. Quantitative analysis of the labeling density of lysosomes demonstrated that the highest labeling density is found in the S1 segment of the proximal tubules and the labeling density of other renal segments is significantly low levels. The results indicate that a main site for cathepsin H in rat kidney is the S1 segment of the proximal tubules.     

Crystallization of recombinant rat cathepsin B

A glycosylation-minus mutant of rat cathepsin B expressed in yeast has been purified and crystallized. X-ray diffraction data have been collected and molecular replacement for solving the structure is in progress. The space group for the recombinant rat cathepsin B was determined to be P2(1) with unit cell dimensions alpha = 62.2 A, b = 90.19 A, c = 47.07 A, and beta = 97.43 degrees. A unit cell contains 4 molecules and 2 molecules per asymmetric unit.     

Studies in fluorescence histochemistry

Summary Pretreatment of sections of fixed tissue with selective blocking reagents for indigenous thiol groups did not, it was found, impair the fluorescence subsequently obtainable with the acetic anhydridesalicylhydrazide-zinc technique (Stoward and Burns, 1967) for localizing C-terminal carboxyl groups of proteins. This suggests that thiol and S-acetyl groups do not participate in the complex reactions involved in the method. In further support of this suggestion it was found that artificially introduced thiol groups also do not take part.Sites containing either, glycogen or neutral periodate-reactive mucosubstances did not fluoresce in sections subjected to the technique. This indicates that O-acetyl groups, although probably formed, are not involved in the reactions giving rise to the fluorescence reaction in proteins.     

Studies in fluorescence histochemistry

Summary Tissue proteins believed to contain a relatively high concentration of C-terminal carboxyl groups emit an intense blue fluorescence after being treated with first a hot mixture of acetic anhydride and pyridine, second salicylhydrazide and last zinc acetate. Characteristically they do not fluoresce when the zinc treatment is omitted. Muscular tissues emit the strongest fluorescence, but normally neither mucosubstances nor loose connective tissues fluoresce at all.These and other results are consistent with Barrnett and Seligman's view that acetic anhydride in the presence of hot pyridine transforms the C-terminal carboxyl groups of proteins into methyl ketones. They do not support Karnovsky's more recent theory that hot acetic anhydride more or less exclusively converts side-chain carboxyl groups of proteins into mixed acid anhydrides instead.     

Fluorescent histochemical demonstration of cathepsin B in the rat yolk sac

Fluorescence demonstration of cathepsin B (E.C. 3.4.22.1) activity was performed in the yolk sac of rats near term (18th day of gestation). The enzyme demonstration was performed on freeze-dried and celloidin mounted yolk-sac sections using different substituted beta-naphthylamide derivatives as substrates and nitrosalicylaldehyde as coupling agent. The discrete reaction products are localized preferentially in the apical part of the visceral yolk-sac epithelium. There is little doubt that cathepsin B is contained here in the well developed lysosomal apparatus of the yolk-sac epithelium.     

Fluorescent histochemical demonstration of cathepsin B in the rat yolk sac

Summary Fluorescence demonstration of cathepsin B (E.C. 3.4.22.1) activity was performed in the yolk sac of rats near term (18th day of gestation). The enzyme demonstration was performed on freeze-dried and celloidin mounted yolk-sac sections using different substituted -naphthylamide derivatives as substrates and nitrosalicylaldehyde as coupling agent. The discrete reaction products are localized preferentially in the apical part of the visceral yolk-sac epithelium. There is little doubt that cathepsin B is contained here in the well developed lysosomal apparatus of the yolk-sac epithelium.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Demonstration of sensory cutaneous nerve fibers in guinea-pig lip using endogenous oxidase histochemistry

Summary We developed a new histochemical method based on endogenous oxidase activity for the detection of cutaneous nerves in guinea-pig lips. After the application of a reaction mixture containing 0.14 mM 3,3-diaminobenzidine(DAB) and 15 mM nickel ammonium sulfate in Tris-HCl buffer (pH 7.6), many blue-black sensory fibers were observed, i.e., dermal nerve fiber bundles, perifollicular plexuses, Merkel-cell endings, and Meissner-like nerve endings. The autonomic fibers of the skin were not stained. Electron microscopy revealed that the reaction product was mainly localized on mitochondrial cristae. The addition of 0.01 M sodium azide completely inhibited the reaction. This technique is simple and preferentially stains sensory nerves.     

Increased expression of mature cathepsin B in aging rat liver

Senescence has been proposed as an important safeguard against neoplasia. One of the hallmarks of cellular senescence in vitro as well as human aging in vivo is a reduced intracellular protein catabolism. The pathways affected and the mechanisms responsible for the decrease in overall protein turnover in aging cells are not well understood. Our aim was to determine whether or not expression of one of the major hepatic lysosomal cysteine peptidases, cathepsin B, changes during aging of Sprague-Dawley rats. Cathepsin B activity was assessed in whole rat liver homogenates, and was found to be increased fourfold (P< or =0.001) in aged livers compared with younger counterparts. This was paralleled by an at least a twofold increase in mature cathepsin B protein. Nonetheless, Northern blot analysis of total liver RNA revealed no change in steady-state levels of cathepsin B mRNAs. These findings seem to contradict the present dogma according to which aging tissues have a reduced intracellular capacity to catabolise proteins. We propose that our earlier observation of the accumulation of T-kininogen, a potent but reversible cysteine peptidase inhibitor, in aging rat liver may provide a plausible explanation for this discrepancy.     

The fluorescence and bright field microscopic demonstration of cathepsin B in human fibroblasts

Summary Cathepsin B was demonstrated cytochemically in human fibroblasts with Z-Ala-Arg-Arg-2-(4-methoxy)naphtylamide as substrate. The enzyme was visualized in the bright field microscope with the diazonium salt Fast Blue B as coupling reagent and in the fluorescence microscope with 5-nitrosalicylaldehyde. With both methods cathepsin B was found in small granules distributed throughout the cytoplasm.     

The fluorescence and bright field microscopic demonstration of cathepsin B in human fibroblasts

Cathepsin B was demonstrated cytochemically in human fibroblasts with Z-Ala-Arg-Arg-2-(4-methoxy)naphtylamide as substrate. The enzyme was visualized in the bright field microscope with the diazonium salt Fast Blue B as coupling reagent and in the fluorescence microscope with 5-nitrosalicylaldehyde. With both methods cathepsin B was found in small granules distributed throughout the cytoplasm.     

Magnesium ions in catecholamine fluorescence histochemistry

Summary The effects of high concentrations of magnesium ions in the cryostat and Vibratome procedures for visualization of catecholamine fluorescence in the central nervous system have been investigated. In cryostat sections, obtained from specimens perfused with a formaldehyde and glyoxylic acid containing buffer, the addition of high concentrations of MgSO₄ to the perfusion solution enhances the fluorescence intensity and reduces the unspecific background fluorescence and the diffusion of the catecholamine fluorophore. This improves the visualization of all portions of the central catecholamine-containing neurons. Similar effects are obtained in the formaldehyde-Vibratome technique by the introduction of an immersion bath containing MgSO₄ after the sectioning procedure. The use of the magnesium perfusion or immersion steps furthermore increases the reproducibility of the Vibratome and cryostat techniques. The paper describes the improved Vibratome and cryostat techniques used in our laboratory.     

Quantitative histochemistry of the lysosomal dipeptidyl aminopeptidase II in the proximal tubule of the rat kidney

Summary The activity of the lysosomal dipeptidyl aminopeptidase II (DAP II) was measured by quantitative histochemical methods in the S₁/S₂ segments of the proximal tubule using freeze dried and celloidin mounted cryostat sections (FDC sections) of rat kidney. The methodological studies show that there is a linear relationship between the amount of reaction product and reaction time for the first 5 min, as well as section thickness between 4 and 10 m. Maximal DAP II activities were demonstrated at pH 5.5. The K _m of DAP II was about 2.3 mM. — In addition to the methodological studies, DAP II activity was also measured in the proximal tubule (S₁/S₂ segments) of experimental animals (sham-operated and castrated male and female rats). Sham-operated females showed significantly higher DAP II activities than males. DAP II activity increased significantly in castrated males so that there were no significant differences between castrated males, sham-operated and castrated females. The quantitative histochemical results are largely in agreement with biochemical data published earlier.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)Dedicated to Prof. Dr. T.H. Schiebler, Chairman of the Institute of Anatomy of the University of Würzburg, on the occasion of his 60th birthday     

Quantitative histochemistry of the lysosomal dipeptidyl aminopeptidase II in the proximal tubule of the rat kidney

The activity of the lysosomal dipeptidyl aminopeptidase II (DAP II) was measured by quantitative histochemical methods in the S1/S2 segments of the proximal tubule using freeze dried and celloidin mounted cryostat sections (FDC sections) of rat kidney. The methodological studies show that there is a linear relationship between the amount of reaction product and reaction time for the first 5 min, as well as section thickness between 4 and 10 microns. Maximal DAP II activities were demonstrated at pH 5.5. The Km of DAP II was about 2.3 mM. In addition to the methodological studies, DAP II activity was also measured in the proximal tubule (S1/S2 segments) of experimental animals (sham-operated and castrated male and female rats). Sham-operated females showed significantly higher DAP II activities than males. DAP II activity increased significantly in castrated males so that there were no significant differences between castrated males, sham-operated and castrated females. The quantitative histochemical results are largely in agreement with biochemical data published earlier.     

Quantitative fluorescence histochemistry of combined formaldehyde-chloral-induced fluorescence of amino-terminal tryptophyl-peptide in model experiments and in the pars intermedia of the rat hypophysis

The relationship between the intensity of combined formaldehyde-chloral vapour-induced fluorescence and the concentration of amino-terminal tryptophyl-peptide in model experiments was found to be non-linear. At a certain concentration the intensity began to increase more slowly than the concentration, and when the concentration further increased the intensity even began to decrease. Based on the studies previously reported and on the above findings it seems that fluorescence induced by combined formaldehyde-chloral vapour, glyoxylic acid vapour and possibly also other combined formaldehyde and carbonyl compounds in the hypophyseal cells containing amino-terminal tryptophyl-peptides is quenched in normal conditions due to the high local concentration. Thus, small to moderate changes in the amounts of amino-terminal tryptophyl-peptides cannot be observed by measuring the fluorescence intensity. In tissue experiments the intensity of combined formaldehyde-chloral vapour-induced fluorescence in the rat pars intermedia was measured after reserpine treatment, which decreases the number of hormone storage granules as demonstrated electron microscopically. The fluorescence intensity measurements were combined with an estimation of the amounts of amino-terminal tryptophyl-peptides extracted from hypophyses and separated in thin-layer chromatography, and subsequently demonstrated by combined formaldehyde-chloral vapour and a protein stain (amido black). Reserpine treatment decreased the fluorescence intensity in the pars intermedia and in thin-layer chromatography, and the staining of the fluorescent band with amido black was also decreased. Amino-terminal tryptophyl-peptides appeared to be depleted from the pars intermedia cells together with endorphins and other hormones of the ACTH/MSH cells containing tryptophan.