scholarly journals Renal neuraminidase. Characterization in normal rat kidney and measurement in experimentally induced nephrotic syndrome

1986 ◽  
Vol 239 (3) ◽  
pp. 705-710 ◽  
Author(s):  
W H Baricos ◽  
S Cortez-Schwartz ◽  
S V Shah
Keyword(s):  
Nephrotic Syndrome ◽  
Specific Activity ◽  
Rat Kidney ◽  
Puromycin Aminonucleoside ◽  
Glomerular Diseases ◽  
Neuraminidase Activity ◽  
Acetylneuraminic Acid ◽  
Normal Rat ◽  
High Concentrations ◽  
Triton X

Several lines of evidence suggest that increased neuraminidase activity may be responsible for the loss of glomerular N-acetylneuraminic acid (AcNeu) observed in various glomerular diseases. However, virtually no information is available on the activity of neuraminidase in glomeruli or the potential role of this enzyme in glomerular pathophysiology. Utilizing 2′-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (4MU-AcNeu) as substrate, we defined optimal assay conditions and characterized neuraminidase activity in glomeruli and, for comparison, in other renal fractions and liver. Neuraminidase activity in glomeruli, cortex and tubules was maximal at pH 4.4. The Km for 4MU-AcNeu was estimated to be 195 microM for glomeruli and 226 microM for cortex. Glomerular neuraminidase was inhibited by AcNeu (90% at 25 mM) and high concentrations of Triton X-100 (26% at 0.5%), but unaffected by CaCl2, EDTA or N-ethylmaleimide (each 1 mM). Neuraminidase activity (nmol/h per mg of protein; mean +/- S.E.M.) in normal rat kidney was: cortex, 14.47 +/- 0.76; medulla, 7.85 +/- 0.64; papilla, 2.64 +/- 0.11; tubules, 13.79 +/- 0.70; glomeruli, 5.57 +/- 0.28. In comparison, neuraminidase activity in rat liver was 2.58 +/- 0.14. Puromycin aminonucleoside (PAN)-induced nephrotic syndrome is a model of glomerular disease in which the loss of glomerular AcNeu is well documented. In two separate studies, we observed no change in the specific activity of neuraminidase in either glomeruli or cortex isolated from rats treated with PAN (15 mg/100 g, intraperitoneally) and killed at either the onset or the peak of proteinuria. Results were similar whether neuraminidase activity was expressed per mg of protein or per microgram of DNA.

scholarly journals Increased cathepsin D-like activity in cortex, tubules, and glomeruli isolated from rats with experimental nephrotic syndrome

1984 ◽  
Vol 223 (2) ◽  
pp. 393-399 ◽  
Author(s):  
W H Baricos ◽  
S V Shah
Keyword(s):  
Nephrotic Syndrome ◽  
Cathepsin D ◽  
Rat Kidney ◽  
Urinary Protein Excretion ◽  
Urinary Protein ◽  
Rat Tissues ◽  
Puromycin Aminonucleoside ◽  
Protein Excretion ◽  
Normal Rat ◽  
Functional Areas

We have examined the activity and distribution of cathepsin D (EC 3.4.23.5), a major renal lysosomal endoproteinase, in the various anatomical and functional areas of normal rat kidney. Cathepsin D-like activities (delta A280/h per mg of protein) in normal rat tissues were: cortex, 0.78 +/- 0.05, n = 37; medulla, 0.62 +/- 0.03, n = 12; papilla, 0.63 +/- 0.04, n = 12; tubules, 0.74 +/- 0.04, n = 28; glomeruli, 0.59 +/- 0.03, n = 28; and liver, 0.41 +/- 0.02, n = 28. Enzyme activity was maximal at pH 3.0-3.5 and inhibited more than 90% by pepstatin (6.7 micrograms/ml), suggesting that the enzyme is cathepsin D. In subsequent experiments we measured cathepsin D-like activity in cortex, tubules and glomeruli isolated from rats with puromycin aminonucleoside (PAN)-induced nephrotic syndrome. Treated animals (15 mg of PAN/100g body wt., intraperitoneally) developed proteinuria beginning 4 days after injection and exceeding 900 mg/24h on day 9. In two separate experiments involving 52 animals we observed a significant increase in cathepsin D-like activity in cortex (+82.7%), tubules (+109.6%) and glomeruli (+54.7%) isolated from PAN-treated rats killed during marked proteinuria (day 9, mean total urinary protein excretion: 937 +/- 94 mg/24h). This increase was observed whether the activity was expressed per mg of DNA or per mg of protein. Increased cathepsin D-like activity was first observed in cortex and tubules coincident with the onset of proteinurea (day 4, mean total urinary protein excretion: 112 +/- 23 mg/24h). In contrast with the significant elevation of renal cathepsin D-like activity, the activity (nmol/h per mg of protein) of alpha-L-fucosidase (EC 3.2.1.51), a non-proteolytic enzyme, was markedly decreased in the identical samples used for the measurement of cathepsin D-like activity: cortex (-46.4%); tubules (-46.1%); and glomeruli (-38.5%). In addition to changes in renal enzyme activities, PAN-treated rats excreted large amounts of cathepsin D-like activity in their urine (beginning on day 3) compared with nearly undetectable cathepsin D-like activity in the urine from control rats. The significant increases in glomerular and tubular cathepsin D activity may reflect an important role for this enzyme in the pathophysiology associated with PAN-induced nephrotic syndrome.

scholarly journals Glycoproteins of the lysosomal membrane.

1985 ◽  
Vol 100 (6) ◽  
pp. 1839-1847 ◽  
Author(s):  
V Lewis ◽  
S A Green ◽  
M Marsh ◽  
P Vihko ◽  
A Helenius ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Polyclonal Antibodies ◽  
Rat Kidney ◽  
Lysosomal Membrane ◽  
Integral Membrane Proteins ◽  
Kidney Cells ◽  
Rabbit Antibody ◽  
Membrane Glycoproteins ◽  
Normal Rat ◽  
Triton X

Three glycoprotein antigens (120, 100, and 80 kD) were detected by mono- and/or polyclonal antibodies generated by immunization with highly purified rat liver lysosomal membranes. All of the antigens were judged to be integral membrane proteins based on the binding of Triton X-114. By immunofluorescence on normal rat kidney cells, a mouse monoclonal antibody to the 120-kD antigen co-stained with a polyclonal rabbit antibody that detected the 100- and 80-kD antigens as well as with antibodies to acid phosphatase, indicating that these antigens are preferentially localized in lysosomes. Few 120-kD-positive structures were found to be negative for acid phosphatase, suggesting that the antigen was not concentrated in organelles such as endosomes, which lack acid phosphatase. Immunoperoxidase cytochemistry also showed little reactivity in Golgi cisternae, coated vesicles, or on the plasma membrane. Digestion with endo-beta-N-acetylglucosaminidase H (Endo H) and endo-beta-N-acetylglucosaminidase F (Endo F) demonstrated that each of the antigens contained multiple N-linked oligosaccharide chains, most of which were of the complex (Endo H-resistant) type. The 120-kD protein was very heavily glycosylated, having at least 18 N-linked chains. It was also rich in sialic acid, since neuraminidase digestion increased the pI of the 120-kD protein from less than 4 to greater than 8. Taken together, these results strongly suggest that the glycoprotein components of the lysosomal membrane are synthesized in the rough endoplasmic reticulum and terminally glycosylated in the Golgi before delivery to lysosomes. We have provisionally designated these antigens lysosomal membrane glycoproteins lgp120, lgp100, lgp80.

scholarly journals Intracellular localization of human Ins(1,3,4,5,6)P5 2-kinase

2007 ◽  
Vol 408 (3) ◽  
pp. 335-345 ◽  
Author(s):  
Maria A. Brehm ◽  
Tobias M. H. Schenk ◽  
Xuefei Zhou ◽  
Werner Fanick ◽  
Hongying Lin ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Rat Kidney ◽  
Intracellular Localization ◽  
Stress Granules ◽  
Site Directed Mutagenesis ◽  
Normal Rat ◽  
High Concentrations ◽  
Green Fluorescent ◽  
Sites Of Action

InsP6 is an intracellular signal with several proposed functions that is synthesized by IP5K [Ins(1,3,4,5,6)P5 2-kinase]. In the present study, we overexpressed EGFP (enhanced green fluorescent protein)–IP5K fusion proteins in NRK (normal rat kidney), COS7 and H1299 cells. The results indicate that there is spatial microheterogeneity in the intracellular localization of IP5K that could also be confirmed for the endogenous enzyme. This may facilitate changes in InsP6 levels at its sites of action. For example, overexpressed IP5K showed a structured organization within the nucleus. The kinase was preferentially localized in euchromatin and nucleoli, and co-localized with mRNA. In the cytoplasm, the overexpressed IP5K showed locally high concentrations in discrete foci. The latter were attributed to stress granules by using mRNA, PABP [poly(A)-binding protein] and TIAR (TIA-1-related protein) as markers. The incidence of stress granules, in which IP5K remained highly concentrated, was further increased by puromycin treatment. Using FRAP (fluorescence recovery after photobleaching) we established that IP5K was actively transported into the nucleus. By site-directed mutagenesis we identified a nuclear import signal and a peptide segment mediating the nuclear export of IP5K.

scholarly journals Studies on the isolation and properties of renin granules from the rat kidney cortex

1979 ◽  
Vol 182 (2) ◽  
pp. 301-309 ◽  
Author(s):  
G A Sagnella ◽  
W S Peart
Keyword(s):  
Incubation Temperature ◽  
Specific Activity ◽  
Rat Kidney ◽  
Kidney Cortex ◽  
Low Molecular Weight ◽  
Enzyme Release ◽  
Rat Kidney Cortex ◽  
Dense Granules ◽  
Triton X ◽  
Primary Granule

The present study was undertaken to isolate and investigate some physicochemical properties of renin granules from the rat kidney cortex. Two preparations of subcellular organelles were used: a primary-granule fraction, which allowed the properties of lysosomes to be compared simultaneously with those of renin granules, and a semi-purified preparation of the latter. The specific activity of renin in the primary-granule preparations was about 4-fold higher than in the original homogenate; that of the semi-purified renin-granule preparation was about 18-fold higher than in the homogenate, and consisted mainly of electron-dense granules but some mitochondria were also observed. Renin and acid phosphatase release from the primary-granule preparation was increased by lowering osmolality, by a low-molecular-weight solute (glucose) and by Triton X-100 or digitonin. Enzyme release was decreased by lowering the incubation temperature (4 degrees C) or the presence of CaCl2. Renin release from the partially purified granule preparation was not affected by cyclic AMP, cyclic GMP and ATP.

scholarly journals Physicochemical modifications of lysosomal hydrolases during intracellular transport

1973 ◽  
Vol 132 (2) ◽  
pp. 267-282 ◽  
Author(s):  
Alfred Goldstone ◽  
Harold Koenig
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Lysosomal Enzymes ◽  
Intracellular Transport ◽  
Specific Activity ◽  
Rat Kidney ◽  
Basic Form ◽  
Acid Hydrolases ◽  
Lysosomal Hydrolases ◽  
Acetylneuraminic Acid

1. The following fractions were prepared from rat kidney and characterized ultrastructurally, biochemically and enzymically: (a) an ordinary rough microsomal (RM1) fraction; (b) a special rough microsomal (RM2) fraction enriched seven- to nine-fold in acid hydrolases over the homogenate; (c) a smooth microsomal (SM) fraction; (d) a Golgi (GM) fraction enriched 2.5-fold in acid hydrolases and 10-, 15- and 20-fold in sialyltransferase, N-acetyl-lactosamine synthetase and galactosyltransferase respectively; (e) a lysosomal (L) fraction enriched 15- to 23-fold in acid hydrolases. The frequency of Golgi sacs and tubules seen in the electron microscope and the specific activity of the three glycosyltransferases in these fractions increased in the order: RM2<RM1<SM<GM. 2. Five lysosomal hydrolases, acid phosphatase, β-N-acetyl-hexosaminidase, β-galactosidase, β-glucuronidase and arylsulphatase, were characterized in these fractions with respect to (a) solubility on freeze–thawing and (b) electrophoretic mobility in polyacrylamide gels. 3. In the RM2 fraction each of these hydrolases occurred largely or exclusively as a single bound basic form coincident with cationic glycoprotein bands in gels (Goldstone et al., 1973). 4. In the L fraction these hydrolases were present largely as soluble, acidic (anionic) forms. 5. The solubility, electrophoretic heterogeneity and anodic mobility of these hydrolases increased progressively in subcellular fractions in the order: RM2<RM1<SM<GM<L. 6. These findings, together with evidence cited in the text showing that N-acetylneuraminic acid residues are responsible for the solubility and electronegative charge of these acidic forms and incorporation of these residues into the Golgi apparatus, support the following scheme for the biosynthesis of lysosomal enzymes. Each hydrolase is synthesized as a bound basic glycoprotein enzyme in a restricted portion of the rough endoplasmic reticulum. The soluble, acidic forms are generated as the nascent glycoprotein enzymes migrate through the Golgi apparatus through the attachment of sugar sequences containing N-acetylneuraminic acid.

scholarly journals Antibodies to rat pancreas Golgi subfractions: identification of a 58-kD cis-Golgi protein.

1987 ◽  
Vol 105 (5) ◽  
pp. 2021-2029 ◽  
Author(s):  
J Saraste ◽  
G E Palade ◽  
M G Farquhar
Keyword(s):  
Polyclonal Antibodies ◽  
Rat Kidney ◽  
Myeloma Cells ◽  
Golgi Region ◽  
Golgi Staining ◽  
Normal Rat ◽  
Smooth Membrane ◽  
Golgi Protein ◽  
Triton X ◽  
In Cis

A 58-kD cis-Golgi protein has been identified by generating polyclonal antibodies against heavy (cis) Golgi subfractions. Total microsomes isolated from rat pancreatic homogenates were subfractionated to yield a rough microsomal fraction (B1) and three smooth membrane subfractions (B2-B4) enriched in cis-, middle, and trans-Golgi elements, respectively. The heavy (cis) subfraction, B2 (d = 1.17 g/ml), was fractionated by Triton X-114 phase separation, and the proteins recovered in the detergent phase were used to immunize rabbits. One of the anti-B2 antibodies obtained gave a "Golgi"-staining pattern when screened by immunofluorescence on normal rat kidney cells and mouse RPC 5.4 myeloma cells. In rat pancreatic exocrine cells the antibody reacted with the plasmalemma as well as elements in the Golgi region. By immunoelectron microscopy, the antigen recognized by anti-B2 IgG was found to be restricted to cis-Golgi elements in myeloma cells where it was concentrated in the fenestrated cis-most cisterna and in some of the tubules and vesicles located along the cis face of the Golgi complex. By immunoprecipitation and immunoblotting, the anti-B2 IgG exclusively recognized a 58-kD protein in myeloma cells. The anti-B2 IgG reacted with several proteins in solubilized pancreatic B2 membranes, including a 58-kD protein, but affinity-purified anti-58-kD IgG reacted exclusively with the 58-kD protein. These results suggest that the 58-kD protein is a specific component of cis-Golgi membranes.

Title Protective effects of rituximab on puromycin-induced apoptosis, loss of adhesion and cytoskeletal alterations in human podocytes

2021 ◽  
Author(s):  
Stefanie Jeruschke ◽  
Dana Künzl ◽  
Peter Friedrich Hoyer ◽  
Stefanie Weber
Keyword(s):  
Nephrotic Syndrome ◽  
Actin Cytoskeleton ◽  
Immunosuppressive Agents ◽  
Cellular Adhesion ◽  
Protective Effects ◽  
Puromycin Aminonucleoside ◽  
Glomerular Diseases ◽  
First Line Therapy ◽  
Steroid Dependent

Abstract Background Podocytes are highly specialized cells playing a key role in the filtration function of the kidney. A damaged podocyte ultrastructure is associated with a reorganization of the actin cytoskeleton and accompanied with a loss of adhesion to the glomerular basement membrane leading to proteinuria in many forms of glomerular diseases, e.g. nephrotic syndrome. If the first-line therapy with glucocorticoids fails, alternative immunosuppressive agents are used, which are known to have the potential to stabilize the actin cytoskeleton. A new option for preventing relapses in steroid dependent nephrotic syndrome is the monoclonal antibody rituximab, which, in addition to its B-cell depleting effect, is assumed to have direct effects on podocytes.Objectives We here provide data on the non-immunological off-target effects of the immunosuppressant rituximab on podocyte structure and dynamics in an in vitro puromycin aminonucleoside model of podocyte injury.Methods A conditionally immortalized human podocyte cell line was used. Differentiated podocytes were treated with puromycin aminonucleoside and rituximab. Our studies focussed on analyzing the structure of the actin cytoskeleton, cellular adhesion and apoptosis using immunofluorescence staining and protein biochemistry methods.Results Treatment with rituximab resulted in a stabilization of podocyte actin stress fibers in the puromycin aminonucleoside model, leading to an improvement in cell adhesion. A lower apoptosis rate was observed after parallel treatment with puromycin aminonucleoside and rituximab visualized by reduced nuclear fragmentation. Consistent with this data Western-blot analyses demonstrated that rituximab directly affects the caspase pathways by inhibiting the activation of Caspases-8 and − 3, suggesting that rituximab may inhibit apoptosis.Conclusions In conclusion, our results indicate an important role of the immunosuppressant rituximab in terms of stability and morphogenesis of podocytes, involving apoptosis pathways. This could help to improve therapeutical concepts for patients with proteinuria mediated by diseased podocytes.

The Morphology of the Rat Kidney Following Folic Acid Administration

1970 ◽  
Vol 28 ◽  
pp. 200-201
Author(s):  
Aline Byrnes ◽  
Elsa E. Ramos ◽  
Minoru Suzuki ◽  
E.D. Mayfield
Keyword(s):  
Folic Acid ◽  
De Novo ◽  
Specific Activity ◽  
Rat Kidney ◽  
Present Report ◽  
Intracellular Localization ◽  
Male Rats ◽  
Renal Hypertrophy ◽  
Electron Microscopic ◽  
Biochemical Alterations

Renal hypertrophy was induced in 100 g male rats by the injection of 250 mg folic acid (FA) dissolved in 0.3 M NaHCO3/kg body weight (i.v.). Preliminary studies of the biochemical alterations in ribonucleic acid (RNA) metabolism of the renal tissue have been reported recently (1). They are: RNA content and concentration, orotic acid-c14 incorporation into RNA and acid soluble nucleotide pool, intracellular localization of the newly synthesized RNA, and the specific activity of enzymes of the de novo pyrimidine biosynthesis pathway. The present report describes the light and electron microscopic observations in these animals. For light microscopy, kidney slices were fixed in formalin, embedded, sectioned, and stained with H & E and PAS.

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