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      Culture Methods of Glomerular Podocytes

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          Abstract

          Podocytes (glomerular visceral epithelial cells) cover the exterior surface of the glomerular capillaries and contribute to the glomerular filtration membrane. Failure of podocyte function is involved in the progression of chronic glomerular disease; accordingly, research interest into podocyte biology is driven by the need for better protection and perhaps recovery of these cells in renal diseases. This review aims at summarizing available techniques for podocyte cell cultures from both the past and present, with special attention to the currently used methods. The establishment of classical primary cultures is based on isolation of glomeruli by differential sieving. Plating of glomeruli onto a collagen surface is followed by an outgrowth of cobblestone-like cells that, after replating, differentiate into arborized, mature podocytes. Currently, the majority of research studies use immortalized podocytic cell lines most often derived from transgenic mice bearing a conditional immortalizing gene. The podocytes can also be collected and cultured from healthy or diseased animal or patient urine. The urinary podocytes obtained from subjects with active glomerulopathies display higher proliferation potential and viability in vitro, perhaps due to disease-induced transdifferentiation. Finally, a list of phenotypic markers useful for identification and characterization of the cultured podocytic elements is provided.

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          Most cited references 54

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          NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome.

          Familial idiopathic nephrotic syndromes represent a heterogeneous group of kidney disorders, and include autosomal recessive steroid-resistant nephrotic syndrome, which is characterized by early childhood onset of proteinuria, rapid progression to end-stage renal disease and focal segmental glomerulosclerosis. A causative gene for this disease, NPHS2, was mapped to 1q25-31 and we report here its identification by positional cloning. NPHS2 is almost exclusively expressed in the podocytes of fetal and mature kidney glomeruli, and encodes a new integral membrane protein, podocin, belonging to the stomatin protein family. We found ten different NPHS2 mutations, comprising nonsense, frameshift and missense mutations, to segregate with the disease, demonstrating a crucial role for podocin in the function of the glomerular filtration barrier.
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            Mutations in ACTN4, encoding alpha-actinin-4, cause familial focal segmental glomerulosclerosis.

            Focal and segmental glomerulosclerosis (FSGS) is a common, non-specific renal lesion. Although it is often secondary to other disorders, including HIV infection, obesity, hypertension and diabetes, FSGS also appears as an isolated, idiopathic condition. FSGS is characterized by increased urinary protein excretion and decreasing kidney function. Often, renal insufficiency in affected patients progresses to end-stage renal failure, a highly morbid state requiring either dialysis therapy or kidney transplantation. Here we present evidence implicating mutations in the gene encoding alpha-actinin-4 (ACTN4; ref. 2), an actin-filament crosslinking protein, as the cause of disease in three families with an autosomal dominant form of FSGS. In vitro, mutant alpha-actinin-4 binds filamentous actin (F-actin) more strongly than does wild-type alpha-actinin-4. Regulation of the actin cytoskeleton of glomerular podocytes may be altered in this group of patients. Our results have implications for understanding the role of the cytoskeleton in the pathophysiology of kidney disease and may lead to a better understanding of the genetic basis of susceptibility to kidney damage.
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              Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines.

              Mature podocytes are among the most complex differentiated cells and possess a highly branched array of foot processes that are essential to glomerular filtration in the kidney. Such differentiated podocytes are unable to replicate and culturing of primary podocytes results in rapid growth arrest. Therefore, conditionally immortalized mouse podocyte clones (MPC) were established, which are highly proliferative when cultured under permissive conditions. Nonpermissive conditions render the majority of MPC cells growth arrested within 6 days and induce many characteristics of differentiated podocytes. Both proliferating and differentiating MPC cells express the WT-1 protein and an ordered array of actin fibers and microtubules extends into the forming cellular processes during differentiation, reminiscent of podocyte processes in vivo. These cytoskeletal rearrangements and process formation are accompanied by the onset of synaptopodin synthesis, an actin-associated protein marking specifically differentiated podocytes. In addition, focal contacts are rearranged into an ordered pattern in differentiating MPC cells. Most importantly, electrophysiological studies demonstrate that differentiated MPC cells respond to the vasoactive peptide bradykinin by changes in intracellular calcium concentration. These results suggest a regulatory role of podocytes in glomerular filtration. Taken together, these studies establish that conditionally immortalized MPC cells retain a differentiation potential similar to podocytes in vivo. Therefore, the determinative steps of podocyte differentiation and process formation are studied for the first time using an inducible in vitro model.
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                Author and article information

                Journal
                KBR
                Kidney Blood Press Res
                10.1159/issn.1420-4096
                Kidney and Blood Pressure Research
                S. Karger AG
                1420-4096
                1423-0143
                2007
                June 2007
                19 April 2007
                : 30
                : 3
                : 162-174
                Affiliations
                Institutes of aMedical Biochemistry and bClinical Chemistry and Laboratory Medicine and cDepartment of Nephrology, First Faculty of Medicine, Charles University in Prague, Prague, Czech Republic
                Article
                102520 Kidney Blood Press Res 2007;30:162–174
                10.1159/000102520
                17502717
                © 2007 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: 1, Tables: 2, References: 99, Pages: 13
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