Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1107 Regulatory Functions of the Juxtaglomerular Apparatus BY RUISHENG LIU ACTA UNIVERSITATISUPSALIENSIS UPPSALA 2002 Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in Physiology presented at Uppsala University in 2002 ABSTRACT Liu, R. 2002. Regulatory Function of Juxtaglomerular Apparatus. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1107. 41pp. Uppsala. ISBN 91-554-5199-3 The tubuloglomerular feedback mechanism is an important regulator in the juxtaglomerular apparatus and it detects flow dependent alterations in luminal NaCl concentration ([NaCl]) at the macula densa (MD) cell site via a Na+-K+-2Cl cotransporter. Signals are sent by the MD to adjust the afferent arteriole tone and altering release of renin. This signaling mechanism is unclear but MD cell calcium concentration, release of ATP and nitric oxide (NO) might be important. In cultured rat glomerular mesangial cells the NO production was measured using confocal microscopy and calcium responses to ATP was measured with fura-2 using imaging techniques. NO from spermine-NONOate and L-arginine could resensitize, desensitized ATP receptors in a cGMP independent way. In mesangial cells from spontaneously hypertensive rats (SHR) less NO effect was found on ATP receptor de/resensitization indicating an impaired NO release or effect. The macula densa cells were studied using microperfusion techniques with confocal and video imaging systems. Changes in [Ca2+] from exposed macula densa plaques were assessed upon addition of agonists added to bath. The order of i efficacy of agonists was UTP = ATP >> 2MesATP = ADP. Dose response curve for ATP added in bath showed an EC of 50 15 µM. Macula densa cell volume and NO concentration increased considerably with increasing luminal [NaCl] indicating an important role for NO in the signaling process to counteract a vasoconstrictor response and reset the sensitivity of the tubuloglomerular feedback mechanism. In conclusion, the results showed 1). NO can increase the P2Y receptor resensitization in rat glomerular mesangial cells, acting through a cGMP-independent pathway. 2) An impaired NO generation/effect on P2Y receptors in mesangial cells from SHR rats. 3) Macula densa cells possess P2Y purinergic receptors on basolateral and that activation of these 2 receptors results in the mobilization of Ca2+. 4) Increased luminal [NaCl] delivery increased cell volume and the NO productions in the macula densa cells. Key words: Mesangial, desensitization, nitric oxide, confocal, calcium, P2Y receptor, macula densa, NaCl, luminal, microperfusion, angiotensin. Ruisheng Liu, Department of Medical Cell Biology, Division of Integrative Physiology, Uppsala University, Biomedical Center, Box 571, 75 123 Uppsala, Sweden © Ruisheng Liu 2002 ISSN 0282-7476 ISSBN 91-554-5199-3 Printed in Sweden by Universitetstryckeriet, Uppsala 2002 This thesis is based on the following studies, which will be referred to their Roman numbers: I. Nitric Oxide induces resensitization of P2Y nucleotide receptors in cultured rat mesangial cells. Liu R., Gutiérrez A., Ring A., and Persson A.E.G. J Am Soci Nephol In press 2002. II. The effects of nitric oxide on P2Y receptorresensitization in spontaneously hypertensive rat mesangial cells. Liu R., and Persson A.E.G. Submitted III. Purinergic receptor signaling at the basolateral membrane of macula densa cells. Liu R., Bell P.D., Peti-Peterdi J., Kovacs G., Johansson A., and Persson A.E.G. Submitted IV. Changes of nitric oxide concentration in macula densa cells caused by changes in luminal NaCl concentration. Liu R., Pittner J., and Persson A.E.G. Submitted Reprint was made with permission from the publisher. CONTENTS LIST OF ABBREVIATIONS……………………………………………… 1 1. INTRODUCTION……………………………………………………….. 2 1.1 Juxtaglomerular apparatus—background……………………………….. 2 1.2 Tubuloglomerular feedback……………………………………………… 3 1.3 Purinergic receptors in TGF regulation…………………………………. 3 1.4 Receptor desensitization…………..……………………………………….. 5 1.5 4,5-diaminofluorescein diacetate.………………………………………... 5 1.6 Confocal laser scanning microscopy…………………..………………… 6 2. AIMS OF THE INVESTIGATIONS……………………………………. 7 3. MATERIALS AND METHODS………………………………………… 8 3.1 Isolation and culture of mesangial cells………………………………….. 8 3.2 Glomerulus preparation………………………………………………….. 8 3.3 Microperfusion method…………………………………………………… 9 3.4 Confocal settings………………………………………………………….. 9 3.5 Fluorescence loading…………………………………………………...... 10 4. RESULTS…………………………………………………………………. 11 4.1 Study I…………………………………………………………………….. 11 4.2 Study II……………………………………………………………………. 12 4.3 Study III…………………………………………………………………… 13 4.4 Study IV……………………………………………………………………. 14 5. DISCUSSIONS…………………………………………………………….. 15 5.1 NO production in mesangial cells…………………………………………. 15 5.2 NO induces receptor desensitization in mesangial cells…………………... 16 5.3 Contribution of c-GMP pathway and ryanodine receptors………………... 17 5.4 NO on unstimulated receptors……………………………………………... 17 5.5 Purinergic receptor signaling of macula densa cells……………………… 18 5.6 Luminal NaCl and NO production in macula densa cells…………………. 19 SUMMARY…………………………………………………………………… 21 ACKNOWLEDGEMENTS………………………………………………….. 22 REFERENCES……………………………………………………………….. 25 APPENDIX: Studies I – IV LIST OF ABBREVIATIONS JGA juxtaglomerular apparatus TGF tubuloglomerular feedback [NaCl] sodium chloride concentration MD macula densa GPCRs G-protein-coupled receptors NO nitric oxide NOS nitric oxide synthase DAF-2 DA 4,5-diaminofluorescein diacetate CLSM confocal laser scanning microscopy PMT photomultiplier tubes SD sprague dawley SHR spontaneously hypertensive rats WKY Wistar Kyoto rat L-NAME Nω-nitro-L-arginine methyl ester ODQ 1H-(1,2,4)oxadiazolo(4,3-α)quinoxalin-1-one 2-MesATP 2-methylthio-ATP IBS isolation buffer solution FCS fetal calf serum cTAL cortical thick ascending limb ROIs regions of interest [Ca2+] cytosolic calcium concentration i 1 1.INTRODUCTION 1.1. Juxtaglomerular apparatus-background: The juxtaglomerular apparatus (JGA) consists of the macula densa (MD), afferent and efferent arterioles, and the mesangium. The JGA is found at the hilus of the renal glomerulus where the thick ascending limb of the loop of Henle of the same nephron changes its morphologic characteristics as it comes in contact with the vascular pole of the glomerulus. These modified cells are MD cells. The MD can sense the sodium chloride concentration ([NaCl]) and fluid load in distal tubule, then regulate the afferent arteriole tone and renin release (Schnermann et al. 1973; Bell and Navar 1982). The glomerular arterioles (afferent arteriole in particular) present a unique morphological characteristic, which consists of the appearance of cells exhibiting both smooth muscle and endocrine features. These cells synthesize, store and release renin. They are more abundant as the arterioles approach the hilus of the glomerulus. Between the two arterioles as they enter the glomerulus there is a group of cells located in a region known as the mesangium containing mesangial cells. Mesangial cells are smooth muscle-like pericytes that abut and surround the filtration capillaries within the glomerulus. Structural and functional studies suggest that mesangial cells play an important role in regulation of glomerular microcirculation and filtration rate in both a static and dynamic fashion (Deen et al. 1972; Brenner et al. 1996). In 1925, Ruyter (Ruyter 1925) described myoepitheloid cells located in the afferent arteriole just before its penetration into the glomerulus and suggested that these cells might contribute to the regulation of blood flow through the glomerulus. In the 1930’s, the JGA became the object of intensive study for two prominent investigators, Goormaghtigh (Goormaghtigh 1932) and Zimmermann (Zimmermann 1933). They both foresaw a functional role for the JGA in the regulation of glomerular blood flow. There is now much 2 agreement that the interrelationship of tubular and vascular elements in the JGA reflects a functional connection between the vascular system and the electrolyte handling tubular system of the kidney. However, the available experimental data, although accumulating rapidly, is not sufficiently clear to give a complete and unified description of the function of the JGA. 1.2. Tubuloglomerular feedback The tubuloglomerular feedback (TGF) is a very important function of the JGA to regulate renal hemodynamics. This mechanism operates as a negative feedback loop, sensing changes in distal nephron fluid flow rate by detecting flow dependent alterations in luminal [NaCl] at the MD. Signals are then sent by the MD cells, which alter the afferent arteriole tones (Schnermann et al. 1973; Bell and Navar 1982; Persson et al. 1991) and release renin (Schnermann 1998). In this signals transferring, the first step is related to the NaCl transport by MD, which is relatively well understood now. NaCl uptake is mostly through the Na+-K+- 2Cl cotransporter, which has been shown on a functional as well as transcriptional level (Schlatter et al. 1989; Obermuller et al. 1996). Next step is rather unclear so far. The possible mediators and modulators of the information transfer between the MD and its target cells include extracellular ion concentration, ATP, angiotensin II, adenosine, arachildonic acid metabolites and nitric oxide (NO) (Salomonsson et al. 1991; Wilcox et al. 1992; Ito and Ren 1993; Thorup and Persson 1994; Braam and Koomans 1995; Briggs and Schnermann 1996; Thorup et al. 1996; Ichihara et al. 1998; Kurtz et al. 1998; Peti-Peterdi and Bell 1999; Cruz et al. 2000; Ren et al. 2000; Wagner et al. 2000; Brown et al. 2001). It has also been found that the sensitivity of the TGF can be reset by a large number of different factors (Persson et al. 1982; Persson and Wright 1982; Schnermann et al. 1998; Persson et al. 2000). 1.3. Purinergic receptors in TGF regulation 3 Purinergic receptors have been identified as playing a major role within the juxtaglomerular apparatus (Schroeder et al. 2000). These receptors are activated by extracellular purines (adenosine, ADP, and ATP) and pyrimidines (UDP and UTP) and are important signaling molecules that mediate various biological effects in the kidney. They may serve as paracrine regulators of renal microvascular resistance (Navar et al. 1996; Osswald et al. 1997), and may modulate mesangial cell contraction, alter epithelial ion transport and influence the TGF mechanism (Franco et al. 1989; Paulais et al. 1995; Inscho et al. 1998; Fernandez et al. 2000; Gutierrez et al. 2000), via cell surface receptors for purines. Recently, it has been reported that MD cells may release ATP across the basolateral membrane via maxi-chloride channels, indicating that there may be a direct role of ATP in TGF signaling (Bell et al. 2000). There are two main families of purine receptors, adenosine or P1 receptors, and P2 receptors, the latter recognizing primarily ATP, ADP, UTP, and UDP (Abbracchio and Burnstock 1994). Based on differences in molecular structure and signal transduction mechanisms, P2 receptors are divided into two families consisting of ligand-gated ion channel-receptors and G protein-coupled receptors termed P2X and P2Y receptors, respectively. Seven mammalian P2X receptors (P2X ) and five mammalian P2Y receptors 1-7 (P2Y , P2Y , P2Y , P2Y , P2Y ) have been cloned (Ralevic and Burnstock 1998). Activation 1 2 4 6 11 of both receptors increases cytosolic calcium concentration ([Ca2+]), the difference being that i P2X receptors induce Ca2+ influx while P2Y receptors result in Ca2+ mobilization. P2Y receptors have been reported to be expressed in numerous, if not all, nephron segments. The cortical thick ascending limb (cTAL) and collecting ducts express both P2Y 1 and P2Y receptors (Ecelbarger et al. 1994; Paulais et al. 1995; Cha et al. 1998). In proximal 2 tubules express the P2Y1 type (Yamada et al. 1996). In outer medullary collecting duct, P2Y1, P2Y and P2Y receptors are expressed (Bailey et al. 2000). Mesangial cells contain P2Y 2 4 2 receptors (Gutierrez et al. 1999). 4 1.4. Receptor desensitization Receptor response to G-protein-coupled receptors (GPCRs) agonists are usually rapidly attenuated (Freedman and Lefkowitz 1996). The mechanisms that attenuate signaling by GPCRs are of considerable interest from several viewpoints. In the healthy organism they govern the ability of cells to respond to hormones and neurotransmitters regulating intercellular signaling. Agonist removal from the extracellular fluid, receptor desensitization and receptor endocytosis, prevent uncontrolled stimulation of cells. On the other hand, receptor resensitization is also crucial, because it allows cells to maintain their ability to respond to agonists over time. Receptor desensitization, which occurs during short-term (seconds to minutes) exposure of cells to agonists, is mediated by: 1) phosphorylation, which causes uncoupling of activated receptors from G-proteins, a process that effectively terminates the signal, 2) receptor endocytosis which depletes the plasma membrane of high- affinity receptors. This receptor internalization is the first step of receptor recycling, which is a requisite for resensitization of the response. Receptor down-regulation is a loss of receptors from a cell that results from long-term (hours to days) continuous exposure to agonists (Casey 1995; Bohm et al. 1996). These regulatory mechanisms are also important from a therapeutic viewpoint (Weisman et al. 1998; Boeynaems et al. 2000). Over half of all medicines used today exert their effects through signaling pathways that involve G-proteins. Desensitization of P2Y receptors has been demonstrated in different cell lines and preparations (Weisman et al. 1998; Clarke et al. 1999). Elucidation of the mechanisms involved in P2Y receptor de- and resensitization, as well as identification of the specific enzymes that take part, will be important for the understanding of the physiological role of extracellular nucleotides and will be crucial for any possible use in therapy. 1.5. 4,5-diaminofluorescein diacetate 5 4,5-diaminofluorescein diacetate (DAF-2 DA) is a newly developed indicator for real time NO measurement with a detection limit of 5 nM (Kojima et al. 1998; Nakatsubo et al. 1998; Nagano 1999; Nagata et al. 1999). DAF-2 selectively traps NO between two amino groups, and yields triazolofluorescein (DAF-2T), which emits green fluorescence when excited at 490-495 nm. DAF-2T is not formed in the absence of NO. Stable forms of NO (e.g., NO -and NO -) and reactive oxygen species like superoxide (O -.), H O , and 2 3 2 2 2 peroxynitrite (ONOO-) do not react with DAF-2 to yield a fluorescent product (Kojima et al. 1998). The fluorescence intensity is dependent on the amount of NO trapped by DAF-2. DAF-2 has been used as a specific NO indicator in different cells and tissues (Hanke and Campbell 2000; Kimura et al. 2001; Prabhakar 2001; Rhinehart and Pallone 2001). 1.6. Confocal laser scanning microscopy The concept of confocal microscopy, first developed by Minsky, was patented in 1957. The first purely analogue mechanical confocal microscope was designed and produced by Eggar and Petran a decade later. It was not until the late seventies, with the advent of affordable computers and lasers, and the development of digital image processing software, that the first single-beam confocal laser scanning microscopes were developed in a number of laboratories and applied to biological and materials specimens. The first commercial confocal laser scanning microscopy (CLSM) systems were produced in the late eighties. During the last decade the availability of CLSM systems of ever-increasing power and sophistication has revolutionized the science of microscopy as applied to cell and developmental biology, physiology, cytogenetics, diagnostic pathology, and the material sciences (Plesch and Klingbeil 1988; Carlsson and Liljeborg 1989; Fabian et al. 1990; Goldstein et al. 1990). The principle of confocal microscopy and of CLSM in particular, is based upon a simple optical principle. In conventional fluorescence microscopy the image quality suffers from fluorescence emission from parts of the specimen outside the plane of focus (or focal 6