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      Different Effects of Growth Factors on Human Renal Early Distal Tubular Cells in vitro

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          Abstract

          Background: In the kidney, recovery from tubular damage requires regenerative mechanisms leading to re-epithelialization of the injured tubules. Current evidence supports the para- or autocrine role of growth factors in repair and regeneration of ischemic or nephrotoxic experimental acute renal failure. Methods: We evaluated the effects of EGF, HGF, IGF-1, and bFGF on human renal thick ascending limb and distal convoluted cells (TALDC) in vitro. TALDC were isolated by immunomagnetic separation and cultured. Signal transduction of the growth factors was evaluated by Western blot of ERK1/2 MAP-K phosphorylation. Cell proliferation was measured by MTT assay and a fluorometric assay. Results: A significant, dose- and time-dependent phosphorylation of ERK1/2 could be detected exclusively after stimulation with EGF. No other growth factor induced a significant MAPK phosphorylation. In the same manner, proliferation assays showed a significant growth-promoting effect of EGF. Neither HGF, nor IGF-1 or bFGF showed a stimulative effect on TALDC proliferation. Conclusion: The present study highlights the effects of growth factors on cultured TALDC and supports the hypothesis that in vivo EGF plays a para- or autocrine role during renal repair mechanisms.

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          Most cited references 19

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          Epidermal growth factor enhances renal tubule cell regeneration and repair and accelerates the recovery of renal function in postischemic acute renal failure.

          To determine the timing and location of renal cell regeneration after ischemic injury to the kidney and to assess whether exogenous epidermal growth factor (EGF) enhances this regenerative repair process to accelerate recovery of renal function, experiments were undertaken in rats undergoing 30 min of bilateral renal artery clamp ischemia followed by reperfusion for varying time intervals. Renal cell regeneration, as reflected by incorporation of radiolabeled thymidine within the kidney, began between 24 to 48 h and reached a peak at 72 h after renal ischemia. As demonstrated by histoautoradiography, renal thymidine incorporation was essentially confined to tubule cells. Morphometric analysis of histoautoradiograph sections of renal tissue demonstrated that the majority of labeled cells were found in renal cortex, but some labeled cells were also located in the inner stripe of the outer medulla, suggesting that injury to medullary thick ascending limbs also occurs in this ischemic model. Exogenous EGF administration produced increases in renal thymidine incorporation compared with non-treated animals at 24, 48, and 72 h after ischemic injury. This accelerated DNA replicative process was associated with significantly lower peak blood urea nitrogen (BUN) and serum creatinine levels, averaging 63 +/- 20 and 3.1 +/- 0.4 mg/dl in EGF-treated ischemic rats compared with 149 +/- 20 and 5.1 +/- 0.1 mg/dl, respectively, in nontreated ischemic rats, and was also associated with a return to near normal BUN and serum creatinine levels in EGF-treated animals approximately 4 d earlier than that observed in nontreated animals. This report is the first demonstration that EGF accelerates the repair process of a visceral organ after an injurious insult.
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            Isolation of proximal and distal tubule cells from human kidney by immunomagnetic separation. Technical note.

            After collagenase digestion and Percoll density gradient centrifugation of human renal tissue, tubular epithelial cells of the proximal and the distal segments were isolated with an immunomagnetic method using MACS microbeads. To enrich proximal tubular (PT) cells we used a monoclonal antibody (mAb) against aminopeptidase M (APM, CD 13), specific of the proximal tubule. Distal tubular (DT) cells were isolated through a mAb recognizing Tamm-Horsfall glycoprotein (THG), a specific antigen for the thick ascending limb and the early distal convoluted tubule. Cells of the proximal primary isolate were histochemically strongly positive for aminopeptidase M (98.6%), however, cells of the distal portion were negative (98.7%). Ultrastructural analysis of PTC primary isolates revealed highly preserved brush border microvilli, well-developed endocytosis apparati and numerous mitochondria, whereas DTC primary isolates showed smaller cells with basolateral invaginations and less apical microvilli. Characterization by immunofluorescence indicated the coexpression of cytokeratin and vimentin, whereas staining for desmin, smooth muscle actin, a fibroblast-specific marker and von Willebrand factor was negative. Cultured PT and DT cells displayed different adenylate cyclase responsiveness to hormonal stimulation. PTH (10(-6) M) increased cAMP production in distal cells up to 32.8-fold of the basal level and in proximal only up to 3.5-fold (10(-8) M, DT 14.4x and PT 2.25x). Calcitonin stimulated adenylate cyclase in DT in a dose dependent fashion (10(-6) M, 4.3x; 10(-8) M, 2.25x), whereas only a low calcitonin response was found in PT cells (10(-6) M, 1.6x; 10(-8) M, 1.4x). AVP (10(-6) M) activated the distal cAMP-production only up to 1.9x of the basal level, but the proximal cAMP-production was negligible (only 1.3x the basal level). The data of this study indicate the proximal and distal tubule origin of the cultured cells that were isolated according to their segment-specific antigens.
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              Epidermal growth factor and the kidney.

              Current information indicates that the mammalian kidney is a significant site of EGF synthesis, second only to the salivary gland in the rodent and probably exceeding most other tissues in the human species. The prepro EGF mRNA is localized to the cells of the TALH and the DCT. The EGF mRNA transcript in kidney is similar to that in salivary gland; the molecular mass of the prepro EGF protein in kidney approximates 130,000 kDa. Several EGF peptides are excreted in urine, including 6000-molecular weight peptides (composed of EGF 1-53, 1-52, 1-51, and 1-50) and a 30,000-molecular weight species with an aminoterminus portion corresponding to amino acids 829-848 of the prepro molecule. It has been suggested that prepro EGF could be a membrane protein since it contains an internal hydrophobic domain (amino acids 1039-1058) adjacent to the EGF sequence (amino acids 976-1029). The 30,000-molecular weight urinary product appears to represent a protein derived from amino acids 829 to approximately 1029 of prepro EGF, adjacent (distal) to the hydrophobic domain. Moreover, immunoelectron microscopy localizes the EGF immunoreactivity to the apical plasma membrane of the TALH and DCT cells. The molecular form of this apically localized, EGF immunoreactivity is not yet clear. Proximal, distal, and TALH cells of the renal tubules and renal medullary interstitial cells appear to have EGF receptors and respond to EGF with increased DNA synthesis and mitogenesis. Also, there is a relatively late increase in prepro EGF mRNA levels in TALH and DCT cells during the process of renal hypertrophy. Limited evidence suggests a role of EGF on tubular function mediated via basal EGF receptors. EGF peptides processed intracellularly or by membrane localized peptidases appear to be continuously excreted and secreted into urine from the apical membrane surface of the TALH and DCT cells. This urinary EGF is constantly bathing urinary tract epithelial surfaces and could play a role in maintaining surface integrity. A similar role for salivary gland EGF in saliva has been proposed for the gastrointestinal tract. It also is possible that prepro EGF is anchored in the apical membrane, where it could function as a receptor, and a role for renal tubular EGF in regulation of membrane transport events has been proposed.
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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
                2006
                November 2006
                17 November 2006
                : 29
                : 4
                : 225-230
                Affiliations
                Department of Internal Medicine III, Division of Nephrology, J.W. Goethe University, Frankfurt am Main, Germany
                Article
                95737 Kidney Blood Press Res 2006;29:225–230
                10.1159/000095737
                16960461
                © 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: 3, References: 26, Pages: 6
                Product
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/95737
                Categories
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