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      Expression Profiling of Crystal-Induced Injury in Human Kidney Epithelial Cells

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          Abstract

          Background: Deposition of crystals within tubular lumens is a feature of many kidney stone diseases, including crystals of calcium oxalate monohydrate (COM) in primary hyperoxaluria and of 2,8-dihydroxyadenine (DHA) in adenine phosphoribosyltransferase deficiency. Crystals are injurious to renal epithelial cells, but the molecular bases of cell injury have not been well characterized. Methods: We used a cDNA microarray to identify the time-dependent changes in gene expression associated with the interaction of COM or DHA crystals with primary cultures of normal human kidney cortical epithelial cells. Results: We observed gene expression changes that were common to both crystal types, as well as a number of crystal-specific responses. A subset of genes known to be aberrantly expressed in kidney tissue from stone formers also showed an altered expression in COM- or DHA-treated normal human kidney cortical epithelial cells. Conclusions: Our results show that cultured epithelial cells exposed to COM or DHA crystals demonstrate cellular responses that may be physiologically relevant, thus suggesting that this experimental system may be useful for elucidating the mechanisms of crystal-induced renal cell injury.

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          Most cited references 37

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          Gene expression levels assessed by oligonucleotide microarray analysis and quantitative real-time RT-PCR – how well do they correlate?

          Background The use of microarray technology to assess gene expression levels is now widespread in biology. The validation of microarray results using independent mRNA quantitation techniques remains a desirable element of any microarray experiment. To facilitate the comparison of microarray expression data between laboratories it is essential that validation methodologies be critically examined. We have assessed the correlation between expression scores obtained for 48 human genes using oligonucleotide microarrays and the expression levels for the same genes measured by quantitative real-time RT-PCR (qRT-PCR). Results Correlations with qRT-PCR data were obtained using microarray data that were processed using robust multi-array analysis (RMA) and the MAS 5.0 algorithm. Our results indicate that when identical transcripts are targeted by the two methods, correlations between qRT-PCR and microarray data are generally strong (r = 0.89). However, we observed poor correlations between qRT-PCR and RMA or MAS 5.0 normalized microarray data for 13% or 16% of genes, respectively. Conclusion These results highlight the complementarity of oligonucleotide microarray and qRT-PCR technologies for validation of gene expression measurements, while emphasizing the continuing requirement for caution in interpreting gene expression data.
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            Hyperuricemia and urate nephropathy in urate oxidase-deficient mice.

            Urate oxidase, or uricase (EC 1.7.3.3), is a purine metabolic enzyme that catalyzes the conversion of uric acid to allantoin in most mammals except humans and certain other primates. The loss of urate oxidase in the human during primate evolution predisposes man to hyperuricemia, a metabolic disturbance that can lead to gouty arthritis and renal stones. To create a mouse model for hyperuricemia and gout, and to address the question of whether urate oxidase is essential in lower mammalian species, we have disrupted the urate oxidase gene in the mouse by homologous recombination in embryonic stem cells. Unlike the human situation, urate oxidase deficiency in mice causes pronounced hyperuricemia and urate nephropathy. More than half of the mutant mice died before 4 weeks of age, indicating that urate oxidase is essential in mice. These mutant mice may also serve as animal models for hyperuricemia and its related nephropathy in humans.
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              Calcium oxalate crystal adherence to hyaluronan-, osteopontin-, and CD44-expressing injured/regenerating tubular epithelial cells in rat kidneys.

              Retention of crystals in the kidney is an essential early step in renal stone formation. Studies with renal tubular cells in culture indicate that hyaluronan (HA) and osteopontin (OPN) and their mutual cell surface receptor CD44 play an important role in calcium oxalate (CaOx) crystal binding during wound healing. This concept was investigated in vivo by treating rats for 1, 4, and 8 d with ethylene glycol (0.5 and 0.75%) in their drinking water to induce renal tubular cell damage and CaOx crystalluria. Tubular injury was morphologically scored on periodic acid-Schiff-stained renal tissue sections and tissue repair assessed by immunohistochemical staining for proliferating cell nuclear antigen. CaOx crystals were visualized in periodic acid-Schiff-stained sections by polarized light microscopy, and renal calcium deposits were quantified with von Kossa staining. HA was visualized with HA-binding protein and OPN and CD44 immunohistochemically with specific antibodies and quantified with an image analyzer system. Already after 1 d of treatment, both concentrations of ethylene glycol induced hyperoxaluria and CaOx crystalluria. At this point, there was neither tubular injury nor crystal retention in the kidney, and expression of HA, OPN, and CD44 was comparable to untreated controls. After 4 and 8 d of ethylene glycol, however, intratubular crystals were found adhered to injured/regenerating (proliferating cell nuclear antigen positive) tubular epithelial cells, expressing HA, OPN, and CD44 at their luminal membrane. In conclusion, the expression of HA, OPN, and CD44 by injured/regenerating tubular cells seems to play a role in retention of crystals in the rat kidney.
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                Author and article information

                Journal
                NEP
                Nephron Physiol
                10.1159/issn.1660-2137
                Nephron Physiology
                S. Karger AG
                1660-2137
                2006
                April 2006
                11 April 2006
                : 103
                : 1
                : p53-p62
                Affiliations
                aDepartment of Genetics, Rutgers University, Piscataway, N.J., bDepartment of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Mich., cDepartment of Pediatrics, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, New Brunswick, N.J., dDepartment of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, Ind., and eDepartment of Medicine, Medical University of Ohio at Toledo, Toledo, Ohio, USA
                Article
                90503 Nephron Physiol 2006;103:p53–p62
                10.1159/000090503
                16374038
                © 2006 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 4, Tables: 1, References: 57, Pages: 1
                Product
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/90503
                Categories
                Original Paper

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