Nitric Oxide in the Kidney
ii
Colloquium Digital Library of Life Sciences The Colloquium Digital Library is an original collection of PDF e-books for researchers, instructors, and students in the biomedical life sciences. Each PDF e-book in the digital library is an accessible overview of a research area or topic, authored by a leading expert in the field. They are intended as timesaving research tools for non-specialists in related fields, and as teaching materials for upper-level courses in the biomedical life sciences. The Digital Library is organized by Lecture Series, each covering a different research area. We invite you to browse through all of our series to find e-books of interest to you: http://www.morganclaypool.com/page/LSindex Access is free for readers at institutions that license Colloquium. Please e-mail
[email protected] for more information.
iii
Colloquium Series on Integrated Systems Physiology: From Molecule to Function to Disease Editors D. Neil Granger, Louisiana State University Health Sciences Center Joey P. Granger, University of Mississippi Medical Center Physiology is a scientific discipline devoted to understanding the functions of the body. It addresses function at multiple levels, including molecular, cellular, organ, and system. An appreciation of the processes that occur at each level is necessary to understand function in health and the dysfunction associated with disease. Homeostasis and integration are fundamental principles of physiology that account for the relative constancy of organ processes and bodily function even in the face of substantial environmental changes. This constancy results from integrative, cooperative interactions of chemical and electrical signaling processes within and between cells, organs and systems. This eBook series on the broad field of physiology covers the major organ systems from an integrative perspective that addresses the molecular and cellular processes that contribute to homeostasis. Material on pathophysiology is also included throughout the eBooks. The state-of the-art treatises were produced by leading experts in the field of physiology. Each eBook includes stand-alone information and is intended to be of value to students, scientists, and clinicians in the biomedical sciences. Since physiological concepts are an ever-changing work-in-progress, each contributor will have the opportunity to make periodic updates of the covered material. Published titles (http://www.morganclaypool.com/toc/isp/1/1)
Copyright © 2015 by Morgan & Claypool Life Sciences All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means—electronic, mechanical, photocopy, recording, or any other except for brief quotations in printed reviews, without the prior permission of the publisher. Nitric Oxide in the Kidney Jennifer M. Sasser www.morganclaypool.com ISBN: 9781615046669 paperback ISBN: 9781615046676 ebook DOI: 10.4199/C00117ED1V01Y201408ISP056 A Publication in the Colloquium Series on INTEGRATED SYSTEMS PHYSIOLOGY: FROM MOLECULE TO FUNCTION TO DISEASE Lecture #56 Series Editor: D. Neil Granger, LSU Health Sciences Center, and Joey P. Granger, University of Mississippi Medical Center Series ISSN ISSN 2154-560X
print
ISSN 2154-5626
electronic
Nitric Oxide in the Kidney Jennifer M. Sasser Department of Pharmacology and Toxicology University of Mississippi Medical Center
COLLOQUIUM SERIES ON Integrated Systems Physiology: From Molecule to Function to Disease #56
vi
ABSTRACT Nitric oxide (NO) is a key regulator of various cellular signaling pathways throughout the body and plays an important role in renal function under normal physiological and pathophysiological conditions. NO plays a major role in the regulation of renal hemodynamics, tubuloglomerular feedback, sodium transport in the nephron, pressure natriuresis, and renal fibrosis and injury; moreover, a deficiency in NO is characteristic of chronic kidney disease in both human patients and in experimental animal models. The goal of this book is to highlight the actions of NO within the kidney and its effects on the regulation of renal blood flow and tubular transport.
Key Words nitric oxide, nitric oxide synthase, renal hemodynamics, renal blood flow, glomerulus, renal vasculature, sodium transport, nephron, pressure natriuresis, tubuloglomerular feedback
vii
Contents 1.
Introduction to Nitric Oxide.................................................................................1 1.1 NO Synthesis....................................................................................................... 1 1.2 Distribution of NOS Isoforms within the Kidney............................................... 3 1.3 Modulators of NO Release.................................................................................. 6
2.
Regulation of Renal Hemodynamics by Nitric Oxide........................................... 11 2.1 Overview............................................................................................................ 11 2.2 Role of NO in Renal Autoregulation................................................................. 14 2.3 Effects of NO on the Release of Renin.............................................................. 16 2.4 Effects of NO on Renal Medullary Blood Flow................................................. 17
3.
Regulation of Natriuresis by Nitric Oxide............................................................ 19 3.1 Effects of NO on Tubular Function................................................................... 19 3.2 Role of NO in Pressure Natriuresis.................................................................... 23
4.
Role of Nitric Oxide in Chronic Kidney Disease and Hypertension...................... 25 4.1 Nitric Oxide Deficiency in Chronic Kidney Disease.......................................... 25 4.2 Nitric Oxide in Diabetic Nephropathy............................................................... 25 4.3 The Kidney in Hypertension—Role of Nitric Oxide........................................ 27
5.
Summary........................................................................................................... 31
References.................................................................................................................. 33 Author Biography....................................................................................................... 47
chapter 1
Introduction to Nitric Oxide Nitric oxide (NO) is a critical factor involved in many cellular processes throughout the body. In 1980, Furchgott and Zawadski [1] found that the endothelium produces a relaxing factor, endothelial-derived relaxin factor (EDRF), in response to acetylcholine administration. EDRF was then later identified as the gaseous molecule NO. Endothelial-derived NO freely diffuses into adjacent smooth muscle cells where it activates soluble guanylate cyclase to produce guanosine 3′,5′cyclic monophosphate (cGMP), resulting in vasodilation. Since its discovery, much research has focused on the actions of NO, and the 1998 Nobel Prize in Physiology or Medicine was awarded to Drs. Robert Furchgott, Louis Ignarro, and Ferid Murad “for their discoveries concerning NO as a signaling molecule in the cardiovascular system.” NO is a paracrine factor with many physiological actions including the control of vascular tone, inhibition of vascular smooth muscle cell prolifera tion and endothelial cell apoptosis, antithrombotic actions, cell cycle regulation, neurotransmission, and inflammation. In the kidney, NO is a critical regulator of renal hemodynamics, tubuloglomerular feedback, sodium transport in the nephron, pressure natriuresis, and renal fibrosis and injury. NO plays an important role in the control of renal function and long-term regulation of blood pressure, and kidney-derived NO regulates renal function via several mechanisms including increasing renal blood flow [2], increasing glomerular filtration [3], inhibiting sodium transport along the nephron [4–6], and regulating the release of renin [7]. Perturbations in NO signaling are associated with kidney diseases and the diseases that affect the kidney including hypertension, diabetes mellitus, ischemia/reperfusion injury, and septic shock.
1.1
NO Synthesis
NO is synthesized during the conversion of L-arginine to L-citrulline by a family of NO synthase (NOS) enzymes (Figure 1). Three mammalian NOS isoforms have been identified: neuro nal NOS (NOS1, nNOS), inducible NOS (NOS2, iNOS), and endothelial NOS (NOS3, eNOS), all of which are expressed within the kidney. NO synthesis requires the cofactors NADPH, tetrahydrobiopterin (BH4), heme, flavin adenine dinucleotide (FAD), and flavin mononucleotide
Nitric Oxide in the Kidney
FIGURE 1: Nitric oxide synthase (NOS) converts oxygen and L-arginine to nitric oxide (NO) and L-citrulline. Cofactors for this reaction include calmodulin (CaM), calcium (Ca2+), tetrahydrobiopterin (BH4), flavinmononuleotide (FMN), and flavin adenine dinucleotide (FAD). NO then activates soluble guanylyl cyclase (sGC) to convert GTP to cGMP which then activates protein kinase G (PKG) and regulates ion channel activity.
(FMN) [8]. All three of the NOS enzymes consist of an oxygenase domain that contains a heme group and binding sites for L-arginine and BH4 and a reductase domain that contains binding sites for NADPH, FAD, and FMN. Dimerization of the NOS enzyme is required for NO production [9, 10]. NOS1 and NOS3 are constitutively expressed and are calcium/calmodulin dependent; however, NOS2 is calcium independent and is induced in response to inflammatory stimuli; therefore, these enzymes are typically classified as constitutive (NOS1 and NOS3) or inducible (NOS2). However, the constitutive NOS enzymes can also be induced in cases of increased shear stress, vas cular wall stretch, or nerve injury, and the inducible NOS2 is constitutively expressed in some cell types, including within the kidney. NO activates soluble guanylyl cyclase to produce cGMP, resulting in vasorelaxation via protein kinase G and actions on ion channels. In addition to its vasodilatory effects, NO also inhibits platelet aggregation, matrix synthesis, neutrophil adhesion, and proliferation; induces apoptosis; and regulates gene expression (Figure 2). Therefore, physiological levels of NO are critical for maintaining normal kidney function and health. On the other hand, excessive production of NO, generated by NOS2 in infiltrating inflammatory cells, can be cytotoxic and contribute to the development of renal injury in cases of ischemia, immune activation, or sepsis.
Introduction to nitric oxide
FIGURE 2: Summary of the actions of Nitric Oxide (NO). Arrowhead indicates stimulation; diamond head indicates inhibition.
1.2
Distribution of NOS isoforms within the kidney
All three isoforms of NOS are constitutively expressed within the kidney. This has been confirmed by Northern blotting, RT-PCR, and in situ hybridization at the mRNA level and Western blotting and immunohistochemical localization at the protein level. The medulla has the most expression and activity of the NOS enzymes within the kidney, with ~25 times greater activity in the medulla compared to the cortex [11]. Within the medulla, the inner medullary collecting duct is the site of the greatest NOS activity, followed by the vasa recta [11]. NOS1: The macula densa is the principal site of localization of NOS1 (Figure 3 [12, 13]) [14–16]. This isoform is also expressed in the glomerular endothelium, collecting duct, renal pelvic nerves, perivascular nerves around arcuate and interlobar arteries, inner medullary thin limb of the loop of Henle, and within the vasculature [14–18]. In addition to the full-length NOS1 enzyme (NOS1α), there are additional splice variants of NOS1. Of particular interest, recent studies have begun to investigate the localization and potential function of NOS1β in the kidney [19–21]. This enzyme is a fully active enzyme but lacks the amino terminus and PDZ domain of the full-length NOS1α. While there is some expression of NOS1β in the proximal tubule of the healthy kidney cortex of
Nitric Oxide in the Kidney
FIGURE 3: A. In situ hybridization of NOS1 in the macula densa. Used with permission from Kidney International, 1998, 54: S29–S33. B. Histochemical localization of NOS1 activity in the macula densa. Used with permission from Kidney International, 1992, 42:1017–9. G = glomerulus.
the rat, expression is significantly increased in the tubules and interstitium during chronic renal disease [19]. In the rat inner medulla, both NOS1α and NOS1β are expressed; however, the mouse renal inner medullary collecting duct expresses only the β isoform of NOS1 [20, 22]. NOS2: Although traditionally thought of as an inducible isoform of NOS expressed in inflammatory cells, NOS2 is constitutively expressed in the kidney. This isoform has been localized to the medullary thick ascending limb, proximal tubule, collecting duct, arcuate and interlobar arteries, and vasa recta [23–26]. LPS treatment results in strong NOS2 mRNA expression in mesangial cells, medullary interstitial cells, and the papillary epithelium [23–25].
Introduction to nitric oxide
FIGURE 4: Localization of NOS3 in kidneys from male and female spontaneously hypertensive rats. Panels A–B: NOS3 is present in the glomeruli (G) and arterioles (A) of the renal cortex. Panels C–D: There is prominent staining of NOS3 in endothelial cells (EC) lining large cortical blood vessels. Pan els E–F: NOS3 is present in the vascularture (V) of the renal outer medulla. Panels G–H: In the inner medulla, NOS3 immunostaining is detected in the vascularture (V) and collecting duct (CD). Used with permission from the American Journal of Physiology, 2010, 298:R61–69.
Nitric Oxide in the Kidney
NOS3: Within the kidney, NOS3 expression is not only found in the endothelium, but also in epithelial cells along the renal nephron. NOS3 has been localized to the glomerular endothelium, preglomerular vasculature, afferent and efferent arterioles (with greater staining observed in the efferent arteriole compared to the afferent arteriole), medullary vasa recta, proximal tubules, thick ascending limb, and collecting duct (Figure 4 [28]) [14, 27].
1.3
Modulators of NO Release
NO/cGMP signal transduction may be affected by changes in NOS enzyme expression, by alter ations in substrate or cofactor availability, inhibition of NOS activity by endogenous inhibitors, by posttranslational modifications of the enzymes, or by the quenching of bioactive NO by reactive oxygen species, among other mechanisms (Figure 5). Substrate Availability: Arginine is the rate-limiting substrate required for the production of NO. In states of substrate deficiency, NOS is no longer able to produce NO but may instead produce oxygen-free radicals [29]. Arginine is synthesized within the proximal tubule of the kidney [30], and although plasma arginine levels are not changed in the setting of chronic kidney disease, there is reduced arginine production by the kidney in human and severe experimental CKD [31, 32]. Furthermore, there may be an intracellular arginine deficiency due to impaired uptake of arginine
FIGURE 5: Schematic of possible mechanisms that regulate NO bioavailability. NOS, nitric oxide synthase; ADMA, asymmetric dimethylarginine; ROS, reactive oxygen species; NO2/NO3, NO oxidation products; BH4, tetrahydrobiopetrin. Arrowhead indicates stimulation, diamond head indicates inhibition.
Introduction to nitric oxide
FIGURE 6: Schematic of pathways affecting arginine levels in the kidney. (1) argininosuccinate lyase; (2) arginine transporters; (3) arginine decarboxylase; (4) L-arginine:glycine amidinotransferase; (5) arginine-tRNA synthetase; (6) arginase; (7) nitric oxide synthase; (8) ornithine decarboxylase; (9) ornithine aminotransferase; (10) ornithine transcarbamylase. Used with permission from Kidney International, 2002, 61:876–881.
from the plasma into the cell due to increased inhibitors of transport as well as a reduction in the number of cationic acid transporters available [33–36]. Arginine levels are also regulated by enzymes that consume arginine; arginase consumes ar ginine to produce urea and ornithine. The kidney has high expression of arginase II, and both arginase I and II are expressed in the vasculature; therefore, arginase competes with NOS for arginine within the kidney and can limit the amount of arginine available for NO production. Inhibition of arginases can restore NO production and reduce renal injury in experimental hypertension and CKD [37–40]. These pathways are illustrated in Figure 6 [36]. Inhibitors of NOS: Endogenous inhibitors of NOS, such as NG-monomethyl-L-arginine (LNMMA) and asymmetric dimethyl arginine (ADMA), reduce the production of NO. These are arginine analogs that act as competitive, reversible inhibitors of the NOS enzymes. Studies have shown that ADMA levels increase in several disease states including chronic kidney disease, diabetes, and hypertension, and ADMA is a major risk factor for cardiovascular disease [41]. Formed by methylation of arginine resides on proteins and normal protein breakdown, ADMA is enzymatically degraded by dimethylarginine dimethylaminohydrolase (DDAH, Figure 7 [41]). The DDAH enzymes are highly expressed in the kidney (as well as the liver and vascular endothelium) [42]; therefore, the expression/activity of DDAH is an important regulator of renal NO production via
Nitric Oxide in the Kidney
FIGURE 7: Protein arginine methyltransferases (PRMTs) methylate proteins, and asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are then generated during protein turnover. Most ADMA is degraded by dimethylarginine dimethylaminohydrolase (DDAH), with the remainder of the ADMA and most of the SDMA excreted by the kidney. Used with permission from Kidney International, 2006, 70:2053–5.
the control of ADMA levels. Oxidative stress can also influence ADMA levels by reducing the activity of DDAH, providing yet another mechanism by which production of ROS will reduce NO bioavailability [43]. Phosphorylation: The activity of NOS1 and NOS3 is primarily regulated by posttranslational mech anisms, and these include phosphorylation of specific serine and threonine residues on the enzymes [44–46]. NOS1 is phosphorylated at serines 837 and 1451 resulting in inhibition of activity and stimulation of activity, respectively [45–49]. While other sites of phosphorylation of NOS3 have been identified, the most well-characterized sites of phosphorylation are serine 1177 and threonine 495. Phosphorylation of serine 1177 and serine 633 on NOS3 is stimulatory, and phosphorylation at threonine 495 inhibits activity of the enzyme [50]. Recent studies have demonstrated that phosphorylation of both NOS1 and NOS3 is detectable in the kidney; therefore, the phosphorylation state of these enzymes is likely an important regulator of renal NO production [51–53]. Oxidative Stress: NO rapidly reacts with superoxide to form the unstable intermediate peroxynitrite (ONOO-) [54]. Not only does this reaction quench the bioactivity of NO, but peroxynitrite is also a potent oxidant that reacts with various biologic molecules by mechanisms including oxidation of thiols and zinc fingers and nitration of tyrosine residues on proteins. Recent studies have shown that the NOS enzymes are also potential sources of superoxide, especially in the settings of hypertension
Introduction to nitric oxide
and diabetes. When there are low levels of the NOS substrate L-arginine or the cofactor tetrahydrobiopterin, the enzyme becomes “uncoupled.” In the uncoupled state, electrons are diverted from the substrate L-arginine to molecular oxygen, and the enzyme produces superoxide rather than nitric oxide [29]. NOS3 uncoupling has also been observed when the interaction of NOS3 and the molecular chaperone heat shock protein 90 is disrupted [55]. Furthermore, it has been hypothesized that the phosphorylation state of NOS3 determines whether the enzyme will produce NO or superoxide. In vitro studies predict that dephosphorylation at the threonine 497 residue acts as a switch mechanism to promote the generation of superoxide instead of NO [56]. Hypoxia: In epithelial cells of the proximal tubule, NO production is stimulated by hypoxia, and increased NO production may contribute to ischemia reperfusion injury in the kidney [57]. Studies using mice deficient for the specific NOS isoforms revealed that NO derived from NOS2 mediates tubular injury following hypoxia [58]. NO signaling can be induced by hypoxia inducible factor (HIF), and this is an important regulator of NOS activity especially in the kidney as even the healthy kidney has low oxygen levels [59]. Prolonged hypoxia increases the expression of NOS1 protein and mRNA, and chronic hypoxia also increases NOS3 gene expression [60]. Salt: Because NO plays a critical role in the regulation of sodium balance, dietary salt intake regulates NOS expression and NO production in the kidney. When rats are maintained on a high salt diet, the expression of all three NOS enzymes in the renal medulla is increased [61], and high salt intake increases the excretion of NO metabolites (NO2 and NO3) in the urine [62]. In the macula densa, however, salt intake and NOS expression are inversely related: NOS1 is decreased in response to high salt intake and increased during sodium restriction [15, 63, 64]. Salt restriction also increases renal cortical microvascular NOS3 protein expression and nitrite production from kidney cortex slices [64]. • • • •
47
Author Biography Jennifer M. Sasser, Ph.D., is an Assistant Professor of Pharmacology and Toxicology at the University of Mississippi Medical Center in Jackson, MS. She received her BChE degree in Chemical Engineering from the Georgia Institute of Technology in Atlanta, GA, and her Ph.D. in Biomed ical Science from the Medical College of Georgia in Augusta, GA, where she studied under the guidance of Jennifer S. Pollock, Ph.D. She then completed her postdoctoral training in the labo ratory of Chris Baylis, Ph.D., in the Department of Physiology and Functional Genomics at the University of Florida College of Medicine in Gainesville, FL. Dr. Sasser’s current work is focused on the role of nitric oxide deficiency in the progression of hypertension, kidney disease, and preeclampsia. She has been funded by the American Heart Association, the PhRMA Foundation, the American Society of Nephrology, and the National Institutes of Health, and she is an active member of the American Physiological Society, the American Heart Association, and the American Society of Nephrology.
