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      High Glucose Stimulates Ca 2+ Uptake via cAMP and PLC/PKC Pathways in Primary Cultured Renal Proximal Tubule Cells

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          Abstract

          Alteration of [Ca<sup>2+</sup>]<sub>i</sub> by hyperglycemia is implicated in the pathogenesis of diabetic nephropathy. However, the effect of high glucose on Ca<sup>2+</sup> regulation in proximal tubule cells is not known. Thus, we examined the mechanisms by which high glucose regulates Ca<sup>2+</sup> uptake in primary cultured rabbit renal proximal tubule cells. Glucose increased the Ca<sup>2+</sup> uptake in a time– and dose–dependent manner. A stimulatory effect of high glucose on Ca<sup>2+</sup> uptake is predominantly observed using 25 m M glucose (high glucose) after 1 h, while 25 m M glucose did not affect cell viability and lactate dehydrogenase release. However, 25 m M mannitol and L–glucose did not affect Ca<sup>2+</sup> uptake as compared with controls. Nifedipine and methoxyverapamil (L–type Ca<sup>2+</sup> channel blockers) blocked high–glucose–induced stimulation of Ca<sup>2+</sup> uptake. High–glucose–induced stimulation of Ca<sup>2+</sup> uptake was blocked by pertussis toxin, SQ–22536 (adenylate cyclase inhibitor), myristoylated amide 14–22 (protein kinase A inhibitor), neomycin and U–73122 (phospholipase C inhibitors), and staurosporine and bisindolylmaleimide I (protein kinase C inhibitors). In addition, KN–62 (a Ca<sup>2+</sup>/calmodulin–dependent protein kinase II inhibitor) and W–7 (a Ca<sup>2+</sup>/calmodulin antagonist) blocked high–glucose–induced stimulation of Ca<sup>2+</sup> uptake. In conclusion, high glucose stimulates the Ca<sup>2+</sup> uptake through L–type Ca<sup>2+</sup> channels via G–protein–coupled adenylate cyclase/cAMP and phospholipase C/protein kinase C pathways.

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          Most cited references 6

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          Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium

          Primary cultures of rabbit-kidney epithelial cells derived from purified proximal tubules were maintained without fibroblast overgrowth in a hormone-supplemented serum-free medium (Medium RK-1). A hormone- deletion study indicated that the primary cultures derived from purified rabbit proximal tubules required all of the three supplements in Medium RK-1 (insulin, transferrin, and hydrocortisone) for optimal growth but did not grow in response to EGF and T3. In contrast, the epithelial cells in primary cultures derived from an unpurified preparation of rabbit kidney tubules and glomeruli grew in response to EGF and T3, as well as insulin, transferrin, and hydrocortisone. These observations suggest that kidney epithelial cells derived from different segments of the nephron grow differently in response to hormones and growth factors. Differentiated functions of the primary cultures derived from proximal tubules were examined. Multicellular domes were observed, indicative of transepithelial solute transport by the monolayers. The proximal tubule cultures also accumulated alpha- methylglucoside (alpha-MG) against a concentration gradient. However, little or no alpha-MG accumulation was observed in the absence of Na+. Metabolic inhibitor studies also indicated that alpha-MG uptake by the primaries is an energy-dependent process, and depends upon the activity of the Na+/K+ ATPase. Phlorizin at 0.1 mM significantly inhibited 1 mM alpha-MG uptake whereas 0.1 mM phloretin did not have a significant inhibitory effect. Similar observations have been made concerning the Na+-dependent sugar-transport system located on the lumenal side of the proximal tubule, whereas the Na+-independent sugar transporter on the peritubular side is more sensitive to inhibition by phloretin than phlorizin. The cultures also exhibited PTH-sensitive cyclic AMP synthesis and brush-border enzymes typical of proximal cells. However, the activities of the enzymes leucine aminopeptidase, alkaline phosphatase, and gamma-glutamyl-transpeptidase were lower in the cultures than in purified proximal-tubule preparations from which they are derived.
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            High glucose concentration causes a rise in [Ca2+]i of cardiac myocytes.

            Diabetes mellitus is associated with an elevation in the basal levels of cytosolic calcium ([Ca2+]i) of cardiac myocytes. This may be due in part to a glucose-induced elevation in [Ca2+]i. The present study examined this issue and explored the cellular pathways responsible for such a phenomenon. A total of 30 mM glucose, mannitol or choline chloride, but not urea, induced a time- and dose-dependent rise in the [Ca2+]i of cardiac myocytes. G protein inhibition by GDP beta S or pertussis toxin produced significant inhibition (> or = 80%) in the rise in [Ca2+]i. Incubation of cardiac myocytes in a calcium free medium or in media containing verapamil, nifedipine or amlodipine almost completely abolished the rise in [CA2+], while ryanodine produced only small reduction (10%) in the glucose-induced rise in [Ca2+]i. Rp-cAMP or H-89, inhibitors of the cAMP-protein kinase A pathway, produced a modest decrease in the rise in [Ca2+]i, while staurosporine (an inhibitor of PKC) and HOE 694 (an inhibitor of the Na(+)-H+ exchanger) had no effect on the rise in [Ca2+]i. The results indicate that the osmotic activity of glucose (cell shrinkage) activates G protein(s), most likely through a stretch receptor, which in turn stimulates calcium channels inhibitable by verapamil, nifedipine and amlodipine, thus permitting a calcium influx into the cardiac myocytes. The increased calcium entry may stimulate a calcium release from intracellular stores by a calcium-induced calcium release process. Thus, in cardiac myocytes direct activation of calcium channels, and to a small extent activation of the cAMP-protein kinase A, and calcium-induced calcium release mediate the high glucose-induced acute rise in their [Ca2+]i.
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              Protopanaxatriol Ginsenosides Inhibit Glucose Uptake in Primary Cultured Rabbit Renal Proximal Tubular Cells by Arachidonic Acid Release

              Ginsenosides are involved in protective action against renal dysfunction and the regulation of renal functions. However, the effects of ginsenosides on glucose reabsorption are not yet known in renal proximal tubular cells. The aim of this study was to examine the effects of ginsenosides, protopanaxadiol (PD) saponin and protopanaxatriol (PT) saponin, on α–methyl–D–glucopyranoside (α–MG) uptake and its mechanism of action in primary cultured rabbit renal proximal tubular cells (PTCs). The α–MG uptake was inhibited by 90% by 0.5mM phloridizin and by removal of Na + in the PTCs. These are typical characteristics described for the proximal tubule. To determine the time– and dose–dependent effects of PD and PT saponins on α–MG uptake, PTCs were incubated with different concentrations of PD and PT saponins (10–100 μg/ml) and for different time periods (from 10 min to 24 h). PT saponin (≥50 μg/ml) from 30 min inhibited α–MG uptake; however, PD saponin did not alter the α–MG uptake at any doses and time periods. In the kinetic analysis of α–MG uptake, PT saponin produced a significant decrease in V max . The PT saponin induced inhibition of α–MG uptake was blocked by mepacrine, a phospholipase A 2 inhibitor. In addition, PT saponin increased [ 3 H] arachidonic acid release by 218% of that of control, and this effect was also completely blocked by mepacrine. In conclusion, PT saponin inhibited, in part, α–MG uptake through the phospholipase A 2 signal pathway in primary cultured rabbit renal PTCs.
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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
                2001
                2001
                24 January 2001
                : 24
                : 1
                : 10-17
                Affiliations
                aDepartment of Veterinary Physiology, College of Veterinary Medicine, Hormone Research Center, Chonnam National University, Kwangju, and bCollege of Veterinary Medicine, Seoul National University, Suwon, Korea
                Article
                54200 Kidney Blood Press Res 2001;24:10–17
                10.1159/000054200
                11174001
                © 2001 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: 9, Tables: 2, References: 31, Pages: 8
                Product
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/54200
                Categories
                Original Paper

                Cardiovascular Medicine, Nephrology

                Calcium, cAMP, G protein, Protein kinase C, Glucose, Kidney

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