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Revised immunolocalization of the Na+-D-glucose cotransporter SGLT1 in rat organs with an improved antibody.

Previously, we characterized localization of Na(+)-glucose cotransporter SGLT1 (Slc5a1) in the rat kidney using a polyclonal antibody against the synthetic COOH-terminal peptide of the rat protein (Sabolić I, Skarica M, Gorboulev V, Ljubojević M, Balen D, Herak-Kramberger CM, Koepsell H. Am J Physiol Renal Physiol 290: 913-926, 2006). However, the antibody gave some false-positive reactions in immunochemical studies. Using a shortened peptide for immunization, we have presently generated an improved, more specific anti-rat SGLT1 antibody (rSGLT1-ab), which in immunochemical studies with isolat

Oral administration of potassium dichromate inhibits brush border membrane enzymes and alters anti-oxidant status of rat intestine.

Potassium dichromate (K2Cr2O7) is a soluble hexavalent chromium compound that is widely used in several industries. In the present work the effect of administration of K2Cr2O7 on rat intestinal brush border membrane(BBM) enzymes and anti-oxidant system was studied. Rats were given a single oral dose of K2Cr2O7 (100 mg/kg bodyweight) and sacrificed 6, 12, 24, 48 and 96 h after the treatment.Control animals were not given K2Cr2O7. The administration of K2Cr2O7 resulted in a reversible decline in the specific activities of several BBM enzymes. The decrease in the activities of these enzymes was d

Effect of acute increases in filtered HCO3- on renal hydrogen transporters: II. H(+)-ATPase.

Adaptive increases in renal bicarbonate reabsorption occur in response to acute increases in filtered bicarbonate (FLHCO3). In a previous study, we showed that an increase in FLHCO3 induced by plasma volume expansion increased the Vmax for Na+/H+ exchange activity in renal cortical brush border membrane vesicles (BBMV), providing a potential mechanism for the adaptive increase in HCO3- reabsorption. The present studies were undertaken to determine whether the increase in FLHCO3 induced by plasma expansion also stimulates the other major H+ transporter in cortical BBMV, the H(+)-ATPase. H(+)-AT

Effects of iodinated x-ray contrast media on renal epithelial cells in culture.

RATIONALE AND OBJECTIVES: To study cellular mechanisms that cause contrast media nephropathy, an in vitro system for proximal and distal tubular cells has been established to evaluate the influence of x-ray contrast media on tubular function. METHODS: Confluent cell cultures of the two renal cell lines, proximal tubule (LLC-PK1) and distal tubule (MDCK), were exposed for 20 hours to 0 to 100 mg iodine/mL of the ionic monomer metrizoate, the ionic dimer ioxaglate, and the non-ionic monomer iohexol. Toxicity was assessed by electron microscopy, cell viability, and biochemical assays of brush-bo

Molecular cloning and expression of a cDNA encoding the rabbit ileal villus cell basolateral membrane Na+/H+ exchanger.

A cDNA clone encoding a rabbit ileal villus cell Na+/H+ exchanger was isolated and its complete nucleotide sequence was determined. The cDNA is 4 kb long and contains 322 bp of 5'-untranslated region, 2451 bp of open reading frame and 1163 bp of 3'-untranslated area, with 70%, 91% and 40% identity to the human sequence, respectively. Amino acid sequence deduced from the longest open reading frame indicated a protein of 816 residues (predicted Mr 90,716) which exhibits 95% amino acid identity to the human Na+/H+ exchanger. The two putative glycosylation sites in the human Na+/H+ exchanger are c

Cystic fibrosis transmembrane regulator mRNA expression relative to ion-nutrient transport in spontaneously differentiating human intestinal CaCo-2 epithelial cells.

The relative abundance of cystic fibrosis transmembrane regulator (CFTR) message at various stages of postconfluence development was compared with evolving cellular ion-nutrient transport properties of CaCo-2 human intestinal cells. Initially these cells demonstrate electrogenic Cl secretion manifested by secretagogue-induced changes in short-circuit current. Over time, however, the secretory characteristics of CaCo-2 monolayers diminish, and brush border hydrolase activities and glucose-dependent and amiloride-sensitive Na transport increase. With a polymerase chain reaction-derived cDNA prob

Castor-oil induced diarrhoea in ponies: a model for acute colitis.

A reproducible, reversible model of colitis induced in ponies by administering castor oil (2.5 ml/kg bodyweight [bwt] per os) was characterised by abdominal pain, fever, watery diarrhoea, dehydration, hypovolaemia, toxaemia, leucopenia, decreased serum Cl, Na and K levels and metabolic acidosis. The signs were most severe between 24 and 48 h post induction, stabilisation was frequently observed after 72 h, although diarrhoea could persist beyond 96 h. Morphological and in vitro transport studies (right ventral colon) were conducted on tissues from animals destroyed at 24, 48 and 72 h. In the c

Clinical aspects of gut enzymology.

Enzymological alterations in functional disturbances and in diseases of the intestine are reviewed. Examples are given for diagnostic significance (e.g. in Hirschsprung's and Crohn's diseases), for pathogenetic considerations (e.g. in hypolactasia and in celiac disease), and for secondary involvement of the liver (e.g. in intestinal tumors and after bypass surgery) and are discussed in more detail.

The role of vitamin E in the prevention of zoledronic acid-induced nephrotoxicity in rats: a light and electron microscopy study.

IntroductionBisphosphonates are widely used in metastatic cancer such as prostate and breast cancer, and their nephrotoxic effects have been established previously. In this study we aimed to evaluate both the nephrotoxic effects of zoledronic acid (ZA) and the protective effects of vitamin E (Vit-E) on this process under light and electron microscopy.Material and methodsA total of 30 male Sprague-Dawley rats were divided into 3 groups. The first group constituted the control group. The second group was given i.v. ZA of 3 mg/kg once every 3 weeks for 12 weeks from the tail vein. The third group

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… Figure 3 A – In group 1 showing healthy nucleus (N), prominent nucleolus (n), preserved apical microvilli (↑), basal infoldings (▲) consisting of elongate mitochondria (M) (uranyl acetate-lead citrate, original mag. 4646×). … Degeneration of the PCT was characterized by total/partial loss of the long and straight microvilli, cell swelling and vacuolization, and occlusion of the tubular lumen. …

Characterization of Divalent Metal Transporter 1 (DMT1) in Brugia malayi suggests an intestinal-associated pathway for iron absorption.

Lymphatic filariasis and onchocerciasis are neglected parasitic diseases which pose a threat to public health in tropical and sub-tropical regions. Strategies for control and elimination of these diseases by mass drug administration (MDA) campaigns are designed to reduce symptoms of onchocerciasis and transmission of both parasites to eventually eliminate the burden on public health. Drugs used for MDA are predominantly microfilaricidal, and prolonged rounds of treatment are required for eradication. Understanding parasite biology is crucial to unravelling the complex processes involved in hos

Both NHERF3 and NHERF2 are necessary for multiple aspects of acute regulation of NHE3 by elevated Ca2+, cGMP, and lysophosphatidic acid.

The intestinal epithelial brush border Na+/H+ exchanger NHE3 accounts for a large component of intestinal Na absorption. NHE3 is regulated during digestion by signaling complexes on its COOH terminus that include the four multi-PDZ domain-containing NHERF family proteins. All bind to NHE3 and take part in different aspects of NHE3 regulation. Because the roles of each NHERF appear to vary on the basis of the cell model or intestinal segment studied and because of our recent finding that a NHERF3-NHERF2 heterodimer appears important for NHE3 regulation in Caco-2 cells, we examined the role of N

Effects of aberrant Pax6 gene dosage on mouse corneal pathophysiology and corneal epithelial homeostasis.

BACKGROUND: Altered dosage of the transcription factor PAX6 causes multiple human eye pathophysiologies. PAX6⁺/⁻ heterozygotes suffer from aniridia and aniridia-related keratopathy (ARK), a corneal deterioration that probably involves a limbal epithelial stem cell (LESC) deficiency. Heterozygous Pax6(+/Sey-Neu) (Pax6⁺/⁻) mice recapitulate the human disease and are a good model of ARK. Corneal pathologies also occur in other mouse Pax6 mutants and in PAX77(Tg/-) transgenics, which over-express Pax6 and model human PAX6 duplication. METHODOLOGY/PRINCIPAL FINDINGS: We used electron microscopy to

Npt2a and Npt2c in mice play distinct and synergistic roles in inorganic phosphate metabolism and skeletal development.

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare autosomal recessively inherited disorder, characterized by hypophosphatemia, short stature, rickets and/or osteomalacia, and secondary absorptive hypercalciuria. HHRH is caused by a defect in the sodium-dependent phosphate transporter (NaPi-IIc/Npt2c/NPT2c), which was thought to have only a minor role in renal phosphate (P(i)) reabsorption in adult mice. In fact, mice that are null for Npt2c (Npt2c(-/-)) show no evidence for renal phosphate wasting when maintained on a diet with a normal phosphate content. To obtain insig

Crb1 is a determinant of retinal apical Müller glia cell features.

Mutations in the human Crumbs homologue-1 (CRB1) gene cause retinal blinding diseases, such as Leber congenital amaurosis and retinitis pigmentosa. In the previous studies we have shown that Crb1 resides in retinal Müller glia cells and that loss of Crb1 results in retinal degeneration (particularly in the inferior temporal quadrant of the mouse eye). Degeneration is increased by exposure to white light. Here, we studied the role of light and aging to gain a better understanding of the factors involved in the progress of retinal disease. Our data reveal that light is neither sufficient nor req

Menkes Copper ATPase (Atp7a) is a novel metal-responsive gene in rat duodenum, and immunoreactive protein is present on brush-border and basolateral membrane domains.

We previously noted strong induction of genes related to intestinal copper homeostasis (Menkes Copper ATPase (Atp7a) and metallothionein) in the duodenal epithelium of iron-deficient rats across several stages of postnatal development (Collins, J. F., Franck, C. A., Kowdley, K. V., and Ghishan, F. K. (2005) Am. J. Physiol., 288, G964-G971). We now report significant copper loading in the livers and intestines of iron-deficient rats. These findings are consistent with the hypothesis that there is increased intestinal copper transport during iron deficiency. We additionally found that hepatic At

Improved diabetic syndrome in C57BL/KsJ-db/db mice by oral administration of the Na(+)-glucose cotransporter inhibitor T-1095.

1. The therapeutic effects of an orally active inhibitor of Na(+)-glucose cotransporter (SGLT), T-1095 (a derivative of phlorizin; 3-(benzo[b]furan-5-yl)-2',6'-dihydroxy-4'-methylpropiophenone 2'-O-(6-O-methoxycarbonyl-beta-D-glycopyranoside)) were examined in C57BL/KsJ-db/db (db/db) mice, a genetic animal model of obese type 2 diabetes. 2. The higher renal SGLT activity in db/db mice than normoglycaemic C57BL/KsJ-db/+m (db/+m) mice may support the rationale for using an SGLT inhibitor in the treatment regimen for type 2 diabetes. Both T-1095 and its metabolite, T-1095A, which had approximatel

Cryptosporidium parvum induces host cell actin accumulation at the host-parasite interface.

Cryptosporidium parvum is an intracellular protozoan parasite that causes a severe diarrheal illness in humans and animals. Previous ultrastructural studies have shown that Cryptosporidium resides in a unique intracellular compartment in the apical region of the host cell. The mechanisms by which Cryptosporidium invades host intestinal epithelial cells and establishes this compartment are poorly understood. The parasite is separated from the host cell by a unique electron-dense structure of unknown composition. We have used indirect immunofluorescence microscopy and confocal laser scanning mic

Nephritogenicity and immunocytochemical localization of the 70-kilodalton glycoprotein subunit (gp70) of Heymann antigen.

Heymann nephritis (HN) is an experimentally induced glomerulonephropathy of the rat characterized by subepithelial immune deposits and proteinuria. Immunization with a complex multimeric glycoprotein, gp600, comprising four subunits gp330, gp140, gp110, and gp70 has been shown to induce the complete form of the disease including proteinuria. Examination of three different batches of heterologous anti-gp600 antisera by immunoblot technique showed that the reactivity toward gp70 was dominant and common to all three antisera. gp70 was isolated from Triton X-100-solubilized Fx1A by lectin Lens cul

Clinical significance of enzymatic deficiencies in the gastrointestinal tract with particular reference to lactase deficiency.

The study of deficiencies of small intestinal brush-border hydrolases increased our knowledge about the specific functions of hydrolases in the digestion of smaller molecules on the microvillus surface of the absorptive cells. The sucrase-isomaltase (SI) complex has been shown to be synthesized as a precursor (pro-sucrase-isomaltase) which is then incorporated into the membrane. The hydrophobic N-terminal end of the molecule is anchored in the lipid bilayer. In SI deficiency the molecular base of the disease is still not clear. Absence of SI activity could be due to complete lack of precursor

The functional implications of common genetic variation in CYP3A5 and ABCB1 in human proximal tubule cells.

BACKGROUND: Calcineurin inhibitors (CNIs) are the primary immunosuppressive drugs used in solid organ transplantation but are associated wit … genotype and PTC biomarkers, 11 subclones were selected. In vitro PTC morphology with brush border microvilli was confirmed. CYP3A5*1 carriers had increased 1-OH/4-OH MDZ formation versus homozygous *3 carriers (mean: 2.36 (95% CI:1.11-3.40) vs 0.88 (95% CI:0.48-1.27); p < 0.05). P-gp activity was confirmed by calcein accumulation (mean 38.6%; 95% CI:32.8-4 …