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      Fluorescent Labeling of Renal Cells in vivo

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          Abstract

          In vivo fluorescence imaging, using confocal or multiphoton microscopes, provides a powerful method to analyze kidney function in experimental animals. In this review, the preparation used for physiological studies in rats is described. A variety of fluorescent probes are available to study glomerular permeability, renal blood flow, peritubular capillary permeability, cell ion concentrations, tubule transport properties, and the functional status of renal cells. We have recently used micropuncture techniques and an adenovirus vector to accomplish gene transfer into kidney tubule and endothelial cells; this new methodology will allow the dynamic study of fluorescently-labeled proteins. Two examples of the use of two-photon fluorescence microscopy to study renal pathophysiology, namely polycystic kidney disease and renal ischemia, are presented. Software is available to quantify data collected from in vivo imaging experiments and to construct 3-dimensional images of renal structures. Two-photon or confocal microscopy offers many opportunities for a better understanding of kidney function in health and disease.

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          Most cited references 15

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          The role of oxidative stress-altered lipoprotein structure and function and microinflammation on cardiovascular risk in patients with minor renal dysfunction.

          Cardiovascular disease is common in patients with chronic kidney disease (CKD). As renal function fails, many patients become progressively malnourished, as evidenced by reduced levels of albumin, prealbumin, and transferrin. Malnourished patients have increased levels of C reactive protein (CRP), interleukin-6 (IL-6), and concomitant cardiovascular disease when they reach end stage. Many diseases that cause CKD, diabetes, and hypertension are also associated with cardiovascular disease. Thus the direct effect of renal failure per se directly contributing to the inflammation-malnutrition-atherosclerosis paradigm is not completely established in early stages of CKD. Some aspects of progressive renal failure, however, cause changes in plasma composition and endothelial structure and function that favor vascular injury. As renal function fails, hepatic apo A-I synthesis decreases and HDL levels fall. HDL is an important antioxidant and defends the endothelium from the effects of cytokines. Inflammation causes further structural and functional abnormalities in HDL. Apolipoprotein C III (apo C III), a competitive inhibitor of lipoprotein lipase is increased in CKD. Serum triglyceride levels increase as a result of accumulation of intermediate-density lipoprotein (IDL) comprising VLDL and chylomicron remnants. These impede vascular relaxation and are associated with cardiovascular disease. Activation of the renin angiotensin axis is a component of many renal diseases and adaptation to loss of renal mass. Angiotensin II (AngII) activates NADPH oxidases, leading to production of the superoxide anion and decreased availability of nitric oxide (NO), further impairing vascular function. H(2)O(2), produced as a consequence of superoxide dismutation, stimulates vascular cell proliferation and hypertrophy. Leukocyte-derived myeloperoxidase functions as an "NO Oxidase" in the inflamed vasculature and contributes to decreased NO bioavailability and compromised vascular reactivity. The changes in lipoprotein composition and structure as well as AngII-mediated alterations in endothelial function amplify the effect of subsequent inflammatory events.
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            Chapter 5 Fluorescent Indicators of Ion Concentrations

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              Defective proximal tubular fluid reabsorption in transgenic aquaporin-1 null mice

              To investigate the role of aquaporin-1 (AQP1) water channels in proximal tubule function, in vitro proximal tubule microperfusion and in vivo micropuncture measurements were done on AQP1 knockout mice. The knockout mice were generated by targeted gene disruption and found previously to be unable to concentrate their urine in response to water deprivation. Unanesthetized knockout mice consumed 2.8-fold more fluid than wild-type mice and had lower urine osmolality (505 ± 40 vs. 1081 ± 68 milliosmolar). Transepithelial osmotic water permeability (P f ) in isolated microperfused S2 segments of proximal tubule from AQP1 knockout [−/−] mice was 0.033 ± 0.005 cm/s (SE, n = 6 mice, 37°C), much lower than that of 0.15 ± 0.03 cm/s ( n = 8) in tubules from wild-type [+/+] mice ( P < 0.01). In the presence of isosmolar luminal perfusate and bath solutions, spontaneous fluid absorption rates (nl/min/mm tubule length) were 0.31 ± 0.12 (−/−, n = 5) and 0.64 ± 0.15 (+/+, n = 8). As determined by free-flow micropuncture, the ratios of tubular fluid-to-plasma concentrations of an impermeant marker TF/P in end proximal tubule fluid were 1.36 ± 0.05 (−/−, n = 8 mice [53 tubules]) and 1.95 ± 0.09 (+/+, n = 7 mice [40 tubules]) ( P < 0.001), corresponding to 26 ± 3% [−/−] and 48 ± 2% [+/+] absorption of the filtered fluid load. In collections of distal tubule fluid, TF/P were 2.8 ± 0.3 [−/−] and 4.4 ± 0.5 [+/+], corresponding to 62 ± 4% [−/−] and 76 ± 3% [+/+] absorption ( P < 0.02). These data indicate that AQP1 deletion in mice results in decreased transepithelial proximal tubule water permeability and defective fluid absorption. Thus, the high water permeability in proximal tubule of wild-type mice is primarily transcellular, mediated by AQP1 water channels, and required for efficient near-isosmolar fluid absorption.
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                Author and article information

                Journal
                NEP
                Nephron Physiol
                10.1159/issn.1660-2137
                Nephron Physiology
                S. Karger AG
                978-3-8055-8074-8
                978-3-318-01315-3
                1660-2137
                2006
                March 2006
                11 April 2006
                : 103
                : 2
                : p91-p96
                Affiliations
                Departments of aMedicine and bCellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Ind., USA
                Article
                90626 Nephron Physiol 2006;103:p91–p96
                10.1159/000090626
                16543774
                © 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: 2, Tables: 1, References: 33, Pages: 1
                Product
                Self URI (application/pdf): https://www.karger.com/Article/Pdf/90626
                Categories
                Microscopic Imaging

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