Localization of cathepsin L in rat kidney revealed by immunoenzyme and immunogold techniques

Summary Localization of cathepsin L in rat kidney was investigated by immunocytochemical techniques. Kidneys were fixed by perfusion and embedded in Epon or Lowicryl K4M without postomication. For light microscopy (LM), semi-thin sections of the Epon-embedded material were stained by the immunoenzyme technique after removal of epoxy resin. For electron microscopy (EM), ultra-thin sections of Lowicryl K4M-embedded material were stained by the protein A-gold technique. By LM, reaction deposits for cathepsin L were present in the cytoplasmic granules of proximal tubule cells, but little or no reaction product was noted in distal tubule, collecting tubule, and most of urinary tubules in the medulla. By EM, heavy gold label for cathepsin L was confined exclusively to lysosomes of the proximal tubule cells, but little or no label to those of the other segments. In immunocytochemical control sections, no reaction was observed. These results indicate that a main container of cathepsin L is lysosomes of the proximal tubule and suggest that the enzyme plays a role in the degradation of endocytosed proteins.     

Localization of cathepsin D in rat liver. Immunocytochemical study using post-embedding immunoenzyme and protein A-gold techniques

Light and electron microscopic localization of cathepsin D in rat liver was investigated by post-embedding immunoenzyme and protein A-gold techniques. By light microscopy, cytoplasmic granules of parenchymal cells and Kupffer cells were stained for cathepsin D. Weak staining was also noted in sinusoidal endothelial cells. In the parenchymal cells many of positive granules located around bile canaliculi. In the Kupffer cells and the endothelial cells, diffuse staining was noted in the cytoplasm in addition to granular staining. By electron microscopy, gold particles representing the antigenic sites for cathepsin D were seen in typical secondary lysosomes and some multivesicular bodies of the parenchymal cells and Kupffer cells. The lysosomes of the endothelial cells and fat-storing cells were weakly labeled. Quantitative analysis of the labeling density in the lysosomes of these three types of cells demonstrated that the lysosomes of parenchymal cells and Kupffer cells are main containers of cathepsin D in rat liver. The results suggest that cathepsin D functions in the intracellular digestive system of parenchymal cells and Kupffer cells but not so much in that of the endothelial cells.     

Light and electron microscopic localization of l-alpha-hydroxyacid oxidase in rat kidney revealed by immunocytochemical techniques

Summary Light and electron microscopic localization of l-alpha-hydroxyacid oxidase (l-HOX) in rat kidney was studied by means of immunocytochemical · techniques. Isozymes A and B of l-HOX were purified from rat liver and kidney, respectively. The apparent molecular weights of the subunits of the isozymes A and B were 35,800 and 33,500 daltons, respectively, by a slab gel electrophoresis. Antibodies to the isozymes were raised in rabbits. Anti(isozyme A) is not cross-reactive with the isozyme B and vice versa anti(isozyme B) not with the isozyme A. Using anti-isozyme B, semithin sections of Epon-embedded material and ultrathin sections of Lowicryl K4M-embedded material were stained by immunoenzyme and protein A-gold techniques, respectively. By light microscopy, fine discrete granular staining was noted in proximal tubules, but not in distal tubules including thick and thin limbs of Henle and collecting tubules. By electron microscopy, gold particles representing the antigen sites for l-HOX B were confined exclusively to peroxisomes, in which most of the gold particles were localized in electron dense peripheral matrix, but little in central matrix with low electron density. The results indicate that l-HOX B does not homogeneously distribute in peroxisomes of rat kidney but might be associated with some substructure within peroxisome matrix.     

Light and electron microscopic localization of L-alpha-hydroxyacid oxidase in rat kidney revealed by immunocytochemical techniques

Light and electron microscopic localization of L-alpha-hydroxyacid oxidase (L-HOX) in rat kidney was studied by means of immunocytochemical techniques. Isozymes A and B of L-HOX were purified from rat liver and kidney, respectively. The apparent molecular weights of the subunits of the isozymes A and B were 35,800 and 33,500 daltons, respectively, by a slab gel electrophoresis. Antibodies to the isozymes were raised in rabbits. Anti(isozyme A) is not cross-reactive with the isozyme B and vice versa anti(isozyme B) not with the isozyme A. Using anti-isozyme B, semithin sections of Epon-embedded material and ultrathin sections of Lowicryl K4M-embedded material were stained by immunoenzyme and protein A-gold techniques, respectively. By light microscopy, fine discrete granular staining was noted in proximal tubules, but not in distal tubules including thick and thin limbs of Henle and collecting tubules. By electron microscopy, gold particles representing the antigen sites for L-HOX B were confined exclusively to peroxisomes, in which most of the gold particles were localized in electron dense peripheral matrix, but little in central matrix with low electron density. The results indicate that L-HOX B does not homogeneously distribute in peroxisomes of rat kidney but might be associated with some substructure within peroxisome matrix.     

Localization of cathepsin D in rat liver

Summary Light and electron microscopic localization of cathepsin D in rat liver was investigated by post-embedding immunoenzyme and protein A-gold techniques. By light microscopy, cytoplasmic granules of parenchymal cells and Kupffer cells were stained for cathepsin D. Weak staining was also noted in sinusoidal endothelial cells. In the parenchymal cells many of positive granules located around bile canaliculi. In the Kupffer cells and the endothelial cells, diffuse staining was noted in the cytoplasm in addition to granular staining. By electron microscopy, gold particles representing the antigenic sites for cathepsin D were seen in typical secondary lysosomes and some multivesicular bodies of the parenchymal cells and Kupffer cells. The lysosomes of the endothelial cells and fat-storing cells were weakly labeled. Quantitative analysis of the labeling density in the lysosomes of these three types of cells demonstrated that the lysosomes of parenchymal cells and Kupffer cells are main containers of cathepsin D in rat liver. The results suggest that cathepsin D functions in the intracellular digestive system of parenchymal cells and Kupffer cells but not so much in that of the endothelial cells.     

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 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.     

Localization of cyclic AMP-dependent protein kinase subunits in rat hepatocyte nuclei by immunogold electron microscopy

We have applied the indirect colloidal immunogold technique to examine the ultrastructural localization of the catalytic subunit C and the regulatory subunits RI and RII of cyclic AMP-dependent protein kinase in rat hepatocyte nuclei before and after glucagon or dibutyryl cyclic AMP administration. The technique allowed the identification and localization of all three subunits in hepatocyte nuclei. Morphometric quantitation of the relative staining density of nuclear subunits indicated an increase of immunogold staining of nuclear catalytic subunit but not of the regulatory subunits after glucagon or dibutyryl cyclic AMP stimulation. The increase of catalytic subunit occurred in a biphasic manner with peak levels 2-30 min and 90-150 min after stimulation. Our experiments represent the first reported use of the immunogold procedure to identify and localize protein kinase subunits in the nucleus.     

Purification and tissue distribution of rat cathepsin L

Cathepsin L was purified to apparent homogeneity from rat kidney. The molecular weight of the enzyme was estimated to be 30,000, but part of the enzyme was found to consist of two polypeptide chains of Mr 25,000 and 5,000. Antibody against rat kidney cathepsin L did not cross-react with rat cathepsin B or H and detected only cathepsin L in crude rat tissue preparations on immunoblotted sheets. The concentrations of cathepsin L in various rat tissues and peripheral blood cells of rats were determined by a sensitive immunoassay, in which the minimum detectable amount of cathepsin L was 20 pg/assay. The concentration of cathepsin L was found to be highest in the kidneys, where it was more than 3 times higher than in the liver, spleen, lungs, and brain. Nervous tissues, especially the cerebellar cortex, also contained fairly high concentrations of cathepsin L, but the heart, skeletal muscle, and gastrointestinal tract contained low concentrations, as did peripheral blood cells. The cathepsin L content of macrophages was 20% of that of cathepsin B. The concentrations of cathepsin L in lymphocytes, neutrophils, and erythrocytes were 10%, 20%, and less than 0.2%, respectively, of those in resident macrophages.     

Localization of glucuronoxylans in Japanese beech visualized by immunogold labelling

Summary An antiserum against glucuronoxylans (GXs) has been raised from a mouse. The dot-blot immunoassay and competitive inhibition test indicated that the antibodies could bind specifically to GXs. Therefore, the antiserum was used for immunogold labelling to investigate the localization of GXs in Japanese beech. Labelling of GXs was seen only in the secondary walls of xylem cells, but not in the primary walls or the middle lamella. GXs were evenly distributed in the secondary walls except for the outer part of the outer secondary-wall layer in which they were less abundant. The labelling density in each secondary-wall layer (S₁, S₂, and S₃) increased during cell wall formation. This result strongly suggests that the deposition of GXs occurs in a penetrative way.     

Localization of Na(+)-dependent active type and erythrocyte/HepG2-type glucose transporters in rat kidney: immunofluorescence and immunogold study

Glucose is actively taken up from the glomerular filtrate into the tubule cells by the Na(+)-dependent active glucose transporter (GT), and passively crosses the basolateral membrane via facilitated diffusion GT. With the use of antibodies directed against two types of GTs, we show the immunocytochemical localization of the Na(+)-dependent active GT (SGLT1) and the erythrocyte/HepG2-type facilitated diffusion GT (GLUT1). For light microscopic observation, frozen sections were stained by the rhodamine labeling method. Counterstaining with fluorescein-phalloidin and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) was employed to facilitate cell type identification. Immunogold staining was carried out on ultra-thin frozen sections for electron microscopy. The antibody to SGLT1 reacted with a 77 KD protein in immunoblotting of a kidney lysate. By immunocytochemistry, SGLT1 was localized in the microvillous plasma membrane in the apical brush borders of the cells of all three proximal tubule segments (S1, S2, and S3). The antibodies to GLUT1, a member of the facilitated diffusion GT family, were raised against human erythrocyte GT or synthetic oligopeptides derived from HepG2 GT, which reacted with a 48 KD protein in immunoblotting of the kidney lysate. GLUT1 was found at the basolateral plasma membranes of S3 proximal tubule cells, cells of the thick limb of Henle's loop, and collecting duct cells. Combined with known physiological data, our findings suggest that SGLT1 in the apical plasma membrane of the proximal tubule cells is responsible for the Na(+)-dependent active reabsorption of glucose from the glomerular filtrate. GLUT1 in the basolateral plasma membrane of S3 cells may transport reabsorbed glucose to the blood vessels. GLUT1 in the basolateral plasma membranes of cells of the thick limb of Henle's loop and of the collecting duct, on the other hand, may nourish these metabolically active cells by facilitating the diffusion of extracellular glucose provided from blood through the basolateral side of the cells.     

Localization of rat cathepsin K in osteoclasts and resorption pits: inhibition of bone resorption and cathepsin K-activity by peptidyl vinyl sulfones.

We have localized cathepsin K in rat osteoclasts and within exposed resorption pits by immuno-fluorescence microscopy. Intracellular staining using an antibody raised against recombinant mouse cathepsin K was vesicular and uniformly distributed throughout the cell. Confocal microscopy analysis did not reveal an accumulation of cathepsin K containing vesicles opposing the ruffled border and the resorption lacuna. Exposed resorption pits exhibited a uniform distribution of cathepsin K, and no differences were observed between the edges and the centers of the pits. The immunostaining of resorption pits with anti-cathepsin K antibodies demonstrates that the protease is secreted into the sub-osteoclastic compartment. Cathepsin K-specific inhibition using peptidyl vinyl sulfones as selective cysteine protease inactivators reduced bone resorption by 80% in a dose-dependent manner at sub-micromolar concentrations. No reduction of bone resorption was observed at those low concentrations using a potent cathepsin L, S, B-specific inhibitor. That the inhibition of bone resorption can be attributed to cathepsin K-like protease inhibition was corroborated by the selective inhibition of the osteoclastic Z-Gly-Pro-Arg-MbetaNA hydrolyzing activity by the cathepsin K, L, S, B-inhibitor, but not by the cathepsin L, B, and S inhibitor. Z-Gly-Pro-Arg-MbetaNA is efficiently hydrolyzed by cathepsin K but only poorly by cathepsins L, S, and B. On the contrary, the intracellular hydrolysis of the cathepsin B-specific substrate, Z-Arg-Arg-MbetaNA, was prevented by both types of inhibitors. The identification of cathepsin K in resorption pits and the inhibition of bone resorption and intracellular cathepsin K activity by selective vinyl sulfone inhibitors indicate the critical role of the protease in osteoclastic bone resorption.     

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     

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 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.     

Amino acid sequence of rat liver cathepsin L

The complete amino acid sequences of the heavy and light chains of rat liver cathepsin L (EC 3.4.22.15) were determined at the protein level. The heavy and light chains consisted of 175 and 44 amino acid residues, respectively, and their Mr values without glycosyl groups calculated from these sequences were 18941 and 5056, respectively. The amino acid sequence was also determined from the N-terminal sequences of the heavy and light chains, and the sequences of cleavage fragments of the heavy chain with lysylendopeptidase and cyanogen bromide. The fragments were aligned by comparison with the amino acid sequence deduced from the sequence of cDNA of rat preprocathepsin L. The sequence of rat liver cathepsin L determined at the protein level was identical with that deduced from the cDNA sequence except that in the heavy chain, residues 176-177 (Asp-Ser) were not present at the C-terminus and alanine was replaced by proline at residue 125. Asn-108 in the heavy chain is modified with carbohydrate.     

Demonstration of cathepsin B in the rat kidney using fluorescence histochemistry

The procedure of mounting freeze-dried sections with celloidin was adapted for the fluorescent-histochemical demonstration of cathepsin B in the rat kidney. A good localization of reaction products was shown in freeze-dried, 5-micron sections which had been mounted free floating with 1.5% celloidin solution on albuminized slides. Using this procedure, the reaction products were localized in the lysosomes, particularly those of the convoluted proximal tubule.