Rat intestinal ceramidase: purification, properties, and physiological relevance

2004 ◽  
Vol 287 (4) ◽  
pp. G929-G937 ◽  
Author(s):  
Maria Olsson ◽  
Rui-Dong Duan ◽  
Lena Ohlsson ◽  
Åke Nilsson
Keyword(s):  
Molecular Mass ◽  
Bile Salts ◽  
Apical Membrane ◽  
Rat Kidney ◽  
Intestinal Lumen ◽  
Renal Tubular Cells ◽  
Physiological Relevance ◽  
Sodium Taurodeoxycholate ◽  
Pancreatic Proteases ◽  
Renal Tubular

Neutral ceramidase activity has previously been identified in the intestinal mucosa and gut lumen and postulated to be important in the digestion of sphingolipids. It is found throughout the intestine but has never been fully characterized. We have purified rat intestinal neutral ceramidase from an eluate obtained by perfusing the intestinal lumen with 0.9% NaCl and 3 mM sodium taurodeoxycholate. Using a combination of acetone precipitation and ion-exchange, hydrophobic-interaction, and gel chromatographies, we obtained a homogenous enzyme protein with a molecular mass of ∼116 kDa. The enzyme acts on both [14C]octanoyl- and [14C]palmitoyl-sphingosine in the presence of glycocholic and taurocholic acid and the bile salt analog 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate but is inhibited by 2 mM or more of other bile salts. It is a glycosylated protein stable to trypsin and chymotrypsin exposure, is not influenced by Ca2+, Mg2+, or Mn2+, and is inhibited by Zn2+ and Cu2+. Mass fragmentographic analysis identified 12 fragments covering 17.5% of the sequence for neutral/alkaline ceramidase 2 purified (Mitsutake S, Tani M, Okino N, Mori K, Ichinose S, Omori A, Iida H, Nakamura T, and Ito M. J Biol Chem 276: 26249–262459, 2001) from rat kidney and located in apical membrane of renal tubular cells. Intestinal and kidney ceramidases also have similar molecular mass and ion dependence. Intestinal ceramidase thus is a neutral ceramidase 2 released by bile salts and resistant to pancreatic proteases. It is well suited to metabolize ceramide formed from dietary and brush border sphingolipids to generate other bioactive sphingolipid messengers.

scholarly journals Cellular Uptake and Localization of Polymyxins in Renal Tubular Cells Using Rationally Designed Fluorescent Probes

2015 ◽  
Vol 59 (12) ◽  
pp. 7489-7496 ◽  
Author(s):  
Bo Yun ◽  
Mohammad A. K. Azad ◽  
Cameron J. Nowell ◽  
Roger L. Nation ◽  
Philip E. Thompson ◽  
...  
Keyword(s):  
Cellular Uptake ◽  
Rat Kidney ◽  
Antimicrobial Activities ◽  
Core Structure ◽  
Confocal Imaging ◽  
Limiting Factor ◽  
Tubular Cells ◽  
Renal Tubular Cells ◽  
Renal Tubular ◽  
Apoptotic Effects

ABSTRACTPolymyxins are cyclic lipopeptide antibiotics that serve as a last line of defense against Gram-negative bacterial superbugs. However, the extensive accumulation of polymyxins in renal tubular cells can lead to nephrotoxicity, which is the major dose-limiting factor in clinical use. In order to gain further insights into the mechanism of polymyxin-induced nephrotoxicity, we have rationally designed novel fluorescent polymyxin probes to examine the localization of polymyxins in rat renal tubular (NRK-52E) cells. Our design strategy focused on incorporating a dansyl fluorophore at the hydrophobic centers of the polymyxin core structure. To this end, four novel regioselectively labeled monodansylated polymyxin B probes (MIPS-9541, MIPS-9542, MIPS-9543, and MIPS-9544) were designed, synthesized, and screened for their antimicrobial activities and apoptotic effects against rat kidney proximal tubular cells. On the basis of the assessment of antimicrobial activities, cellular uptake, and apoptotic effects on renal tubular cells, incorporation of a dansyl fluorophore at either position 6 or 7 (MIPS-9543 and MIPS-9544, respectively) of the polymyxin core structure appears to be an appropriate strategy for generating representative fluorescent polymyxin probes to be utilized in intracellular imaging and mechanistic studies. Furthermore, confocal imaging experiments utilizing these probes showed evidence of partial colocalization of the polymyxins with both the endoplasmic reticulum and mitochondria in rat renal tubular cells. Our results highlight the value of these new fluorescent polymyxin probes and provide further insights into the mechanism of polymyxin-induced nephrotoxicity.

Localization of heparin and low-molecular-weight heparin in the rat kidney

2004 ◽  
Vol 91 (05) ◽  
pp. 927-934 ◽  
Author(s):  
Vivian Douros ◽  
Thomas Podor ◽  
Stephen Shaughnessy ◽  
Jeffrey Weitz ◽  
Edward Young
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Rat Kidney ◽  
Organic Anion ◽  
Molecular Size ◽  
Immunohistochemical Analysis ◽  
Immunoperoxidase Staining ◽  
Low Molecular Weight ◽  
Renal Tubular Cells ◽  
Renal Tubular

SummaryUnfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) are cleared, at least in part, by the kidneys through a poorly understood process. This study was undertaken to explore the mechanism of renal clearance of these drugs. Rats were given fluorescein-5-isothiocyanate (FITC)-labeled UFH or LMWH intravenously. At intervals after injection, rats were euthanized and the kidneys were harvested and subjected to immunohistochemical analysis and fluorescence microscopy. Both UFH and LMWH were localized to renal tubular cells and no immunoperoxidase staining or fluorescence was detected in glomeruli. Autoradiography demonstrated similar intracellular distribution of radio-labeled UFH suggesting that this phenomenon is independent of the method used to label heparin. Fluoresence in the tubules increased as a function of time after UFH injection, but reached a plateau after LMWH injection suggesting that the rate of renal tubular uptake depends on the molecular size of the heparin. When administered prior to FITC-labeled UFH or LMWH, probenecid, a renal organic anion inhibitor, decreased the renal tubular uptake of the heparins, whereas cimetidine, a renal organic cation inhibitor, had no effect. These findings suggest that renal excretion of UFH and LMWH primarily reflects tubular uptake via an organic anion transport mechanism.

scholarly journals Isolation, partial characterization, and localization of a rat renal tubular glycoprotein antigen. Antibody-induced birth defects.

1982 ◽  
Vol 156 (2) ◽  
pp. 372-384 ◽  
Author(s):  
C C Leung
Keyword(s):  
Birth Defects ◽  
Rat Kidney ◽  
Polyacrylamide Gel Electrophoresis ◽  
Yolk Sac ◽  
Affinity Column ◽  
Immunofluorescent Localization ◽  
Renal Tubular Cells ◽  
Visceral Yolk Sac ◽  
Renal Tubular ◽  
Endodermal Cells

A glycoprotein with an apparent 340,000 mol wt (gp 340K) was isolated from rat kidney saline-soluble extract by ammonium sulfate precipitation, DE 52 ion-exchange cellulose chromatography, concanavalin A affinity column, Sephacryl S-300 gel filtration, and discontinuous polyacrylamide gel electrophoresis (PAGE). The relative purity of gp 340K was examined by double immunodiffusion analysis, disc PAGE, and immunoelectrophoresis. Injection of rabbit gp 340K antiserum into pregnant rats during the organogenetic period induced abnormal embryonic development, fetal growth retardation, and embryonic death. Antiserum against the immunocomplexes isolated by immobilized protein A also produced the same embryotoxic effects. The biologic effects of the antisera appeared to be dose dependent. Defects such as anophthalmia, hydrocephaly, exencephaly, cleft palate, cleft lip, and some cardiovascular anomalies were observed. The most frequently observed anomaly was anophthalmia. Immunofluorescent localization studies indicated that gp 340K antibodies localized in vivo in the visceral yolk-sac endodermal cells and the embryonic endoderm. In vitro immunofluorescent localization studies revealed that gp 340K was a component of the renal tubular cells that cross-reacted with antigen in the visceral yolk-sac endodermal cells and embryonic endoderm. The underlying mechanism whereby gp 340K antibodies induce birth defects is not known. Three hypotheses were discussed.

scholarly journals Cleavage state of γENaC in mouse and rat kidney

2021 ◽  
Author(s):  
Gustavo Frindt ◽  
Shujie Shi ◽  
Thomas R Kleyman ◽  
Lawrence G Palmer
Keyword(s):  
Molecular Mass ◽  
Apical Membrane ◽  
Rat Kidney ◽  
Mouse Kidney ◽  
Apparent Molecular Mass ◽  
Na Channel ◽  
Extracellular Proteases ◽  
Salt Diet ◽  
Heterologous System ◽  
Membrane Bound

Extracellular proteases can activate the epithelial Na channel (ENaC) by cleavage of the g subunit. Here we investigate the cleavage state of the channel in the kidneys of mice and rats on a low-salt diet. We identified the cleaved species of channels expressed in FRT cells by co-expressing the apical-membrane bound protease CAP1 (prostasin). To compare the peptides produced in the heterologous system with those in the mouse kidney we treated both lysates with PNGaseF to remove N-linked glycosylation. The apparent molecular mass of the smallest C-terminal fragment of gENaC (52 kDa) was indistinguishable from that of the CAP1-induced species in FRT cells. Similar cleaved peptides were observed in total and cell surface fractions of rat kidney. This suggests that most of the subunits at the surface have been processed by extracellular proteases. This was confirmed using non-reducing gels, in which the N- and C-terminal fragments of gENaC are linked by a disulfide bond. Under these conditions the major cleaved form in rat kidney had an apparent molecular mass of 56 kDa, ~4 kDa lower than that of the full-length form, consistent with excision of a short peptide by two proteolytic events. We conclude that the most abundant gENaC species in the apical membrane of rat and mouse kidney on a low-Na diet is the twice-cleaved, presumably activated form.

Renal hydrolysis of absorbed protein: influence of load and lysosomal pH

1984 ◽  
Vol 247 (4) ◽  
pp. F656-F664 ◽  
Author(s):  
M. J. Camargo ◽  
B. E. Sumpio ◽  
T. Maack
Keyword(s):  
Rat Kidney ◽  
High Capacity ◽  
Lysosomal Ph ◽  
Renal Tubular Cells ◽  
Weak Bases ◽  
Uptake Rates ◽  
Cyt C ◽  
Renal Tubular ◽  
Hydrolysis Of

The kinetics of intracellular hydrolysis of administered protein and the effect of alkalinization of lysosomal pH on this process were studied in the isolated perfused rat kidney (IPK). Cytochrome c (CYT c) was used as a probe protein, and its hydrolysis was determined by measuring the efflux of radioactivity from IPK preloaded in vivo with [14CH3]CYT c and various doses of unlabeled CYT c. The nature of radioactivity absorbed by the kidney and released to the perfusate was analyzed by Sephadex chromatography. Administered CYT c is absorbed and hydrolyzed by the kidney, and the resulting amino acids are returned to the perfusate. At low uptake rates, the half time of hydrolysis of absorbed CYT c is about 20 min. The disposal of absorbed CYT c is a saturable function of its concentration in kidney with a Vmax = 0.60 mg CYT c X h-1 X g kidney-1 and an apparent Km = 0.55 mg CYT c/g kidney. To alkalinize the lysosomal pH, IPK were perfused in the presence of NH4Cl (10 mM) or chloroquine (0.1 mM). These lysosomotropic weak bases almost completely inhibit in a reversible manner the hydrolysis of absorbed CYT c. The results demonstrate that renal catabolism of absorbed protein is a saturable process of high capacity compared with the normal filtered loads of protein. The data are consistent with the view that normal lysosomal function is required for an adequate disposal of absorbed proteins in the kidney. It is postulated that abnormal deposition of protein absorption droplets within renal tubular cells may result from high absorbed loads and/or a deficient acidification of lysosomes.

scholarly journals Oxalate-inducible AMBP gene and its regulatory mechanism in renal tubular epithelial cells

2005 ◽  
Vol 387 (3) ◽  
pp. 609-616 ◽  
Author(s):  
Jasjit S. GREWAL ◽  
Jeng Y. TSAI ◽  
Saeed R. KHAN
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Rat Kidney ◽  
Serine Proteinase ◽  
Precursor Protein ◽  
Mobility Shift ◽  
Renal Tubular Cells ◽  
Supershift Assay ◽  
Mobility Shift Assay ◽  
Renal Tubular

The AMBP [A1M (α1-microglobulin)/bikunin precursor] gene encodes two plasma glycoproteins: A1M, an immunosuppressive lipocalin, and bikunin, a member of plasma serine proteinase inhibitor family with prototypical Kunitz-type domain. Although previously believed to be constitutively expressed exclusively in liver, the present study demonstrates the induction of this gene by oxalate in porcine proximal tubular LLC-PK1 cells and rat kidney. In liver, the precursor protein is cleaved in the Golgi network by a furin-like enzyme to release constituent proteins, which undergo glycosylation before their export from the cell. In the renal tubular cells, A1M and bikunin co-precipitate, indicating lack of cleavage of the precursor protein. As the expression of the AMBP gene is regulated by A1M-specific cis elements and transcription factors, A1M protein was studied as a representative of AMBP gene expression in renal cells. Oxalate treatment (500 μM) resulted in a time- and dose-dependent induction of A1M protein in LLC-PK1 cells. Of the four transcription factors, HNF-4 (hepatocyte nuclear factor-4) has been reported previously to be a major regulator of AMBP gene expression in liver. Electrophoretic mobility-shift assay, supershift assay, immunoreactivity assay and transfection-based studies showed the presence of an HNF-4 or an HNF-4-like protein in the kidney, which can affect the expression of the AMBP gene. In situ hybridization and immunocytochemical studies showed that the expression of this gene in kidney was mainly restricted to cells lining the renal tubular system.

Ultrastructural changes in the rat kidney after single dose of cyclophosphamide—Possible roles for peroxisome proliferation and lysosomal dysfunction in cyclophosphamide-induced renal damage

2011 ◽  
Vol 30 (12) ◽  
pp. 1924-1930 ◽  
Author(s):  
Premila Abraham ◽  
Bina Isaac
Keyword(s):  
Electron Microscopy ◽  
Single Dose ◽  
Renal Damage ◽  
Rat Kidney ◽  
Ultrastructural Changes ◽  
Peroxisome Proliferation ◽  
Time Intervals ◽  
Subcellular Organelles ◽  
Renal Tubular Cells ◽  
Renal Tubular

Electron microscopy was used to examine changes in the subcellular organelles of the rat kidney at different time intervals after a single exposure to cyclophosphamide (CP). The morphological changes were studied at different time points (6 hrs, 16 hrs and 24 hrs) after a single-dose administration of CP. Six rats were killed at each time intervals after the administration of CP. Saline-treated rats served as controls. CP administration resulted in alterations in various subcellular organelles including peroxisomes, lysosomes, mitochondria, and the endoplasmic reticulum (ER) of the renal tubular epithelium as well as damage to the glomerulus. The basement membrane of the glomerulus was thickened. Many podocytes were destroyed. The nucleoplasm of the endothelial cell showed fewer granularities. The tubules were distorted and the brush border was destroyed. Two striking features in the renal tubular cells are increase in number and size of the peroxisomes (peroxisome proliferation) and decrease in the number of lysosomes. The mitochondria were elongated and the number was increased in the tubules of CP-treated rats. The ER was dilated. Cell necrosis was also seen. This study is an evidence of changes in morphology of rat kidney after induction of renal damage by a single dose of CP. Since transmission electron microscopy is the highest magnification tool at present, it can be useful in estimating the degree of injury and outcome of alternative treatment strategies in the management of CP-induced renal damage after establishing a scoring system.

Adenosine transport in perfused rat kidney and renal cortical membrane vesicles

1984 ◽  
Vol 246 (6) ◽  
pp. F794-F803 ◽  
Author(s):  
M. E. Trimble ◽  
R. Coulson
Keyword(s):  
Rat Kidney ◽  
Membrane Vesicles ◽  
Transport Systems ◽  
Bicarbonate Buffer ◽  
Renal Tubular Cells ◽  
Adenosine Receptor Agonist ◽  
Perfused Rat Kidney ◽  
Adenosine Transport ◽  
Renal Tubular ◽  
Phenylisopropyl Adenosine

Adenosine is a modulator of renal function but little is known about transport of this compound by renal tubular cells. Transport of exogenous adenosine was studied in isolated perfused rat kidney and in luminal (L) and antiluminal (AL) membrane vesicles isolated from rat renal cortex. Most experiments were performed in the presence of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine. Kidneys were perfused in a recirculating system with Krebs-Henseleit bicarbonate buffer containing 6 g albumin/dl and adenosine. Net secretion of adenosine occurred at perfusate adenosine concentrations greater than 40 microM, and net reabsorption was seen at concentrations less than 40 microM. N6-(L-2-phenylisopropyl)adenosine (PIA), a nondeaminated adenosine receptor agonist, also showed net reabsorption when unbound PIA concentrations were 10-20 microM. Influx or efflux of [3H]adenosine in vesicles was measured using a rapid filtration technique. Transport into both L and AL vesicles was saturable. L vesicles demonstrated both high Km (43 +/- 4 microM) and low Km (4.4 +/- 0.6 microM) transport systems. Only a low Km (5 +/- 1 microM) system could be demonstrated in AL vesicles. Results indicate that at concentrations in the physiological range (less than 1 microM) adenosine undergoes mediated transport across both L and AL membranes and that net transport across the L membrane is in the direction of reabsorption.

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