Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1107

Regulatory Functions of theJuxtaglomerular Apparatus

BY

RUISHENG LIU

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 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+]i from exposed macula densa plaques were assessed upon addition of agonists added to bath. The order of

efficacy of agonists was UTP = ATP >> 2MesATP = ADP. Dose response curve for ATP added in bath showed an EC50 of

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 P2Y2 purinergic receptors on basolateral and that activation of these

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

1

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+]i cytosolic calcium concentration

2

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

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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

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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 (P2X1-7) and five mammalian P2Y receptors

(P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) have been cloned (Ralevic and Burnstock 1998). Activation

of both receptors increases cytosolic calcium concentration ([Ca2+]i), the difference being that

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 P2Y1

and P2Y2 receptors (Ecelbarger et al. 1994; Paulais et al. 1995; Cha et al. 1998). In proximal

tubules express the P2Y1 type (Yamada et al. 1996). In outer medullary collecting duct, P2Y1,

P2Y2 and P2Y4 receptors are expressed (Bailey et al. 2000). Mesangial cells contain P2Y2

receptors (Gutierrez et al. 1999).

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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

6

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., NO2-and NO3

-) and reactive oxygen species like superoxide (O2-.), H2O2, and

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

7

plane). This leads to considerable blurring of the images, making the fine details inside a

specimen difficult to resolve. In CLSM the specimen is illuminated in the focal plane by

using suited optics. A tiny diaphragm (slit) is placed in front of photomultiplier tubes (PMT)

to ensure that all out of focus fluorescence light does not reach the detector. By proper optical

alignment of the illumination and detection light paths only emitted light is collected from the

optical plane that is in focus. In this way, optical slices (sections) can be cut through the

specimen by adjusting the z-position of the microscope table. This distinct CLSM property is

called “optical sectioning” and results in a high axial resolution. By adjusting the size of the

slit, the thickness of the optical section can be adjusted (Figure 1).

CLSM has exhibited several advantages over conventional optical microscopy. The

most important of these stem from the fact that out-of-focus blur is essentially absent from

confocal images, giving the capability for direct non-invasive serial optical sectioning of

intact and even living specimens (Sheppard and Shotton 1997). This leads to the possibility

of generating three-dimensional images of thick transparent objects such as biological cells

and tissues. In a similar way, it allows profiling of the surfaces of 3-D objects and multi-layer

structures such as integrated circuits, again, by a non-contacting and non-destructive method

(Quesada et al. 1991; Weaver et al. 1991; Hardman and Spooner 1992; Dailey and Smith

1993; Zhu et al. 1994; Belichenko and Dahlstrom 1995; Feng et al. 1995; Huser et al. 1996;

Wedekind et al. 1996; Favard et al. 1999; Ono et al. 2001; Zucker and Price 2001).

2. AIMS OF PRESENT STUDY

The aims of the present study were: 1). To characterize the effect of NO on the

desensitization-resensitization cycle of the ATP-induce [Ca2+]i response in Sprague Dawley

(SD) and spontaneously hypertensive rats (SHR). 2). To study the existence and type of

purinergic receptors expressed by MD cells and to determine, functionally, if purinergic

8

receptors were preferentially located at the apical or basolateral surfaces of MD cells. 3). To

measure the NO concentration in MD caused by the luminal [NaCl] alterations.

3. MATERIALS AND METHODS

3.1. Isolation and culture of mesangial cells

Rat glomerular mesangial cells were cultured as described previously in our lab

(Kurtz et al. 1982; Gutierrez et al. 1999). In short, both kidneys from male SD, SHR and

WKY rat (120-170 g) were removed and decapsulated under sterile conditions, respectively.

Cortical tissue was cut away from the medulla and minced in isolation buffer solution (IBS)

containing 5mM KCl, 2mM CaCl2, 130mM NaCl, 10mM glucose, 20mM sucrose and 10mM

Tris, (pH 7.4, osmolality 290 mOsm). Glomeruli were isolated by sequential sieving and

collected on a 50 µm sieve. The glomeruli were then incubated with 0.1% collagenase in IBS

for 30 min at 37 C° to remove epithelial cells and obtain glomerular cores consisting mostly

of mesangium and capillary loops (Striker et al. 1980). The glomeruli suspension was

centrifuged at 2200 rpm for 7 min at room temperature. The pellet was resuspended with

10ml RPMI 1640 medium, supplemented with 18% fetal calf serum (FCS), 100 U/ml

penicillin, 100 µg/ml streptomycin, 0.25 µg/ml amphotericin B and 0.66 U/ml insulin. The

RPMI 1640 medium contained D-valine instead of L-valine. D-valine inhibits fibroblast

growth (Ausiello et al. 1980). Aliquots of the glomeruli were transferred to 25 cm2 tissue

flasks containing 6 ml supplemented RPMI 1640 medium. The flasks were incubated at 37

C° and 5% CO2 in a humidified atmosphere in a CO2-controlled incubator for 3-6 weeks. The

medium was changed every third day. Epithelial and endothelial specific staining (cytokeratin

and factor VIII, respectively) in the confluent cultures of mesangial cells were negative,

excluding any contamination.

3.2.Glomerulus preparation:

9

Female New Zealand White rabbits weighing 1.0 - 1.5 kg were killed and the left

kidney removed and cut into several 1.5 - 3 mm transversal slices. The slices were placed in

a chilled low NaCl buffer solution, containing: 35 mM NaCl, 1.3 mM CaCl2, 1 mM MgSO4,

1.6 mM K2HPO4, 5 mM glucose, 20 mM Hepes with pH adjusted to 7.4 and addition of

sucrose sufficient to achieve an osmolality at 290 mOsm. Glomeruli with attached cortical

thick ascending limb (cTAL) and containing the MD plaque were isolated by microdissection

at 4°C under a dissection microscope (Wild, Switzerland). In some experiments, the cTAL

was carefully removed leaving the MD plaque attached to the glomerulus. In other

experiments, the cTAL was left intact for microperfusion studies.

3.3. Microperfusion method:

Individual cTAL with attached glomeruli were dissected and perfused as described

previously (Kirk et al. 1985; Gonzalez et al. 1988). The cTAL was cannulated and perfused

with the low NaCl buffer solution. The preparation was bathed continuously with Ringer

solution at a rate of 6-7 ml/min. MD cells were loaded with the fluorescent probe from the

luminal side.

3.4. Confocal settings:

The image size was set to 640X480 pixels. The confocal slit was set at a width of 25

nm. Photobleaching was kept to a minimum by maintaining laser intensity at below 30% of

maximum and using a shutter so that the preparation was exposed to laser light only during

the collection of images. Data collection, with the Noran Odyssey confocal system is

controlled by a Silicon Graphics workstation. Image acquisition was limited to 30 frames/s

and, when necessary, image noise was reduced by averaging or summing 16-32 individual

images. Sampling time for each pixel was 100 ns.

10

3.5 Fluorescence loading:

3.5.1. Fura-2 and Fura Red:

The fluorescent Ca2+ indicators fura-2 was used for video imaging and Fura Red for

confocal microscopy. The fura-2 loaded cells were excited alternately at 340 nm and 380 nm,

and the emission was measured at 510 nm. The intensity of 340/380 emission ratio was used

to determine the intracellular calcium concentration after calibration in vitro. Fura Red loaded

MD-glomerular preparations were transferred to the confocal system. Fura Red was excited

at 488 nm with an argon-ion laser, while emitted fluorescence was recorded at wavelengths

of >600 nm through a 550 nm long pass barrier filter. Relative changes of [Ca2+]i were

calculated by a normalization procedure to obtain a ‘pseudo ratio’ : (F – Frest)/Frest.

3.5.2. Calcein

Calcein was excited at 488 nm with an argon-ion laser, while emitted fluorescence

was recorded at wavelengths of 510 to 530 nm. Square-shaped regions of interest (ROIs)

were set inside the cytoplasmic area of MD cells and the mean intensity within the ROIs was

recorded every 5 s. Relative changes of cell volume and NO concentration changes were

calculated by a normalization procedure to obtain a ‘pseudo ratio’ (F-Frest)/Frest.

3.5.3 DAF-2

Excitation and recording wavelength in confocal system were similar to that in calcein

measurement. Calculations were based on the following equations.

Vc1 * Cc1 = Vc2 * Cc2 (I)

Where Vc1 is cell volume and Cc1 is concentration of calcein during resting condition; Vc2 and

Cc2 are changed cell volume and changed concentration of calcein, respectively. The

concentration of calcein is proportional to its fluorescent intensity, therefore, the ratio of

concentrations is equal to the ratio of intensities. Assuming that Vc1 is 1, the changed cell

11

volume could be expressed with the changed calcein intensity (Fc2) and the intensity at basal

level (Fc1).

Vc2 = Vc1* Cc1/Cc2 = Fc1/Fc2 (II)

Delta relative changes of cell volume can be calculated as:

(Vc2 – Vc1 )/ Vc1= Fc1/Fc2 – 1 (III)

The triazolofluorescein of DAF-2 (DAF-2T) is the fluorescent form after DAF-2 selectively

traps NO between two amino groups. The relative changes of the amount of DAF-2T in MD

cells can be calculated by the changed cell volume (Vd2) multiplied with changed

concentration (Cd2) of DAF-2T divided by the basal level of cell volume (Vd1) multiplied with

concentration (Cd1) of DAF-2T.

(Vd2 * Cd2) / (Vd1 * Cd1) (IV)

Since Vd2 and Vc2 are the same, and Vd1 is regarded as 1, the Cd2 /Cd1 could be expressed by

the ratio of DAF-2T intensities in changed conditions (Fd2) and in basal level (Fd1). The delta

relative changes of DAF-2T amount can be expressed:

[(Vd2*Cd2)- (Vd1*Cd1)]/(Vd1*Cd1) = Vc2*(Fd2/Fd1) -1 (V)

Equations III and V were used to calculated the changes of cell volume and DAF-2T amount

in MD cells.

4.RESULTS

4.1. Study I:

The present study evaluates the effect of NO on P2Y nucleotide receptor

resensitization in cultured rat glomerular mesangial cells. L-arginine increased and Nω-nitro-

L-arginine methyl ester (L-NAME) decreased NO production significantly (P<0.05),

measured with the NO indicator DAF-2 using confocal microscopy. Calcium responses to

ATP were measured with fura-2 and imaging techniques. Repeated stimulation with ATP

results in receptor desensitization, characterized by lower calcium peak amplitude.

12

Desensitization was induced by challenging mesangial cells with four consecutive 2 min

pulses of ATP (0.1 mM) separated by 4.5 min control perfusions. [Ca2+]i increase evoked by

second, third and fourth ATP challenges were about 40%, 26% and 18% of the first

challenge. The NO precursor, L-arginine (10 mM) and the NO donors, spermine-NONOate

(500 µM) and sodium nitroprusside (SNP, 1 mM), were added before and during the fourth

ATP challenge. Spermine-NONOate and L-arginine induced a recovery of the [Ca2+]i

response to the fourth ATP challenge (p<0.01, 0.05 respectively). The NO synthase inhibitor,

L-NAME (5 mM) applied together with ATP, was shown to enhance desensitization. 1H-

(1,2,4)oxadiazolo(4,3-α)quinoxalin-1-one (ODQ, 30 µM), an inhibitor of guanylate cyclase,

was used together with L-arginine, SNP or spermine-NONOate. There was no significant

difference with or without ODQ. Neither ODQ nor 8-Br-cGMP, an analog of cGMP, at

different concentrations showed effects on ATP stimulated [Ca2+]i. There was no elevation of

[Ca2+]i when the cells were challenged by different concentrations (1 µM, 100 µM, 1mM, 20

mM and 30 mM) of caffeine, caffeine plus ATP (0.1 mM) and 4-chloro-3-ethylphenol (100

µM , 500 µM and 1mM), a new agonist of ryanodine receptors.

The results indicate that: NO can increase the P2Y receptor resensitization in rat

glomerular mesangial cells, acting through a cGMP-independent pathway. We found no

evidence for the existence of ryanodine sensitive intracellular calcium stores in rat mesangial

cells.

4.2. Study II

The first ATP-stimulated increase in [Ca2+]i was significantly higher in SHR

(1330.25± 360.31 nM) than that in WKY (974.28 ± 397.72 nM) (P< 0.05). The ratio of

second, third and fourth ATP-stimulated increase in [Ca2+]i in SHR was significant lower than

that in WKY (P< 0.05). Spermine-NONOate and L-arginine could significantly increase the

fourth ATP-stimulated [Ca2+]i in WKY(P< 0.01, 0.05 separately). In SHR, only spermine-

13

NONOate can significantly recover the fourth ATP-challenged [Ca2+]i increase. L-NAME can

greatly reduce the second, third and fourth ATP-stimulated [Ca2+]i increase in WKY (P<0.01),

but not in SHR. There was no significant difference in [Ca2+]i increase in the presence or

absence of ODQ in both WKY and SHR. The cGMP analog, 8-Br-cGMP (10-4M to 10-6M) (n

= 53), on the fourth ATP challenges did not show any significant effect on [Ca2+]i stimulated

by ATP. [Ca2+]i was not elevated when the WKY cells were challenged by caffeine at

concentrations of 1 µM, 100 µM, 1mM, 20 mM and 30 mM separately for 2 min. When the

cells were superfused with L-NAME, L-arginine or spermine-NONOate for a period of 5 min

before and during one single ATP challenge, the responses observed in these conditions were

not significant different from the response in control experiment with WKY.

In conclusion, we found 1) A higher ATP response and a faster desensitization were found in

the SHR cells compared to WKY. 2) The presence of L-NAME enhance receptor

desensitisation in WKY but not in SHR. 3) These effects are mediated through a cGMP-

independent pathway. 4) We could not prove the existence of a ryanodine receptor to release

calcium from intracellular stores in rat mesangial cells.

4.3. Study III:

Isolated glomeruli containing MD cells, with and without the cortical thick ascending

limb, were loaded with the Ca2+ sensitive indicators Fura Red (confocal microscopy) or Fura

2 (conventional video image analysis). Studies were performed on an inverted microscope in

a chamber with a flow-through perfusion system. Changes in [Ca2+]i from exposed MD

plaques were assessed upon addition of adenosine, ATP, UTP, ADP or 2-methylthio-ATP (2-

MeS-ATP). There was no change in [Ca2+]i with addition of adenosine (10-7-10-3 M). UTP

and ATP (10-4 M) caused [Ca2+]i to increase by 268 ± 40 nM; n = 21 and 295 ± 53 nM; n = 21,

respectively, while in response to 2MesATP and ADP [Ca2+]i increased by only 67 ± 13 nM;

n = 8 and 93 ± 36 nM; n = 14, respectively. Dose response curve for ATP (10-7-10-3 M) added

14

in bath showed an EC50 of 15 µM. No effect on MD [Ca2+]i was seen when ATP was added

from the lumen. ATP caused similar increases in MD [Ca2+]i in the presence or absence of

bath Ca2+ and addition of 5 mM EGTA. Suramin (an antagonist of P2X and P2Y receptors)

completely inhibited ATP-induced [Ca2+]i dynamics. ATP-Ca2+ responsiveness was also

prevented by the phospholipase C inhibitor, U-73122, but not by its inactive analog, U-

73343.

These results suggest that MD cells possess P2Y2 purinergic receptors on basolateral

but not apical membranes and that activation of these receptors results in the mobilization of

Ca2+.

4.4. Study IV

NO concentration in MD cells was measured with confocal microscopy in isolated

perfused thick ascending limb using the NO-sensitive fluorophore DAF-2 DA. Calcein was

used to measure cell volume changes. The loop perfusion fluid was a modified Ringer

solution containing 10, 35, or 135 mM NaCl with constant total osmolarity (290 mOsm),

while the bath was perfused with the 135 mM NaCl solution. The results show that MD cell

volume and NO concentration increased considerably (19 % and 28 %, respectively) with

increasing luminal [NaCl]. Similar results were got with calcium free solution both in lumen

and bath. 5 mM L-arginine increased (30 %) the NO concentration in the MD cells. 7-

nitroindazole could totally inhibit the NO production caused by L-arginine and by increased

luminal [NaCl].

In conclusion, for the first time, we could quantitatively measure MD cell volume

changes caused by the changes of luminal [NaCl]. We found that increasing the luminal

[NaCl] resulted in an increase in cell volume. We also found that NO formation in MD cells

could be measured with DAF-2 and that NO production was increased by an increase in

15

luminal [NaCl]. An increased NO production will suppress the vasoconstriction induced by

the TGF and at the same time reduce the TGF sensitivity.

5. DISCUSSION

5.1. NO production in mesangial cells

NO plays an important role in the physiological regulation of hemodynamics in the

kidney (Tolins et al. 1990; Stockand and Sansom 1998). Mesangial cells may be exposed to

NO through the high production of NO from MD cells. Once iNOS in mesangial cells is

induced, it produces large amounts of NO that can influence cell and tissue function and

cause damage (Palmer et al. 1988; Nathan and Xie 1994; Willmott et al. 1996). In study I, we

found that L-arginine can increase NO concentration and that the increase caused by L-

arginine can be inhibited with L-NAME. This indicates that NO is produced by unstimulated

mesangial cells and NO production can be stimulated by the addition of L-arginine. Similar

results have been reported (Kunz et al. 1994; Mohaupt et al. 1994; Datta and Lianos 1999;

Guan et al. 1999; Kihara et al. 1999) by measuring nitrite and/or nitrate, which are end

products of NO interaction with superoxide species. They found that there is a basal level of

NO production in unstimulated mesangial cells. It has also been reported that L-NMMA can

reduce the nitrite concentration in mesangial cells under basal condition (Shultz et al. 1991;

Doi et al. 2000). Unstimulated mesangial cells do not express constitutive NOS. This might

be due to effects of cytokines or endotoxin during cell culture. Furthermore, cultured

mesangial cells in fetal calf serum (FCS) free medium showed an increased nitrite production

and iNOS expression (Rodriguez-Lopez et al. 1999; Rodriguez-Lopez et al. 2000). In present

study, 18% FCS was used during culture, 9% FCS was used in subculture for 24 to 48 hours,

and the FCS-free solution used during the experiment. This also might be another way to

enhance the activity of iNOS.

16

5.2. NO induce receptor desensitization in SD mesangial cells

In study I and II, the receptors were desensitized to a large extent after three ATP

challenges. The first ATP-stimulated [Ca2+]i increase was significantly higher in SHR than

that in WKY. This may be due to the higher receptor capacity in SHR, which has also been

found by others (Bottiglieri et al. 1988; Modrall et al. 1995; Ishikawa et al. 1996; Sidhu et al.

1998; Ozono et al. 2000). It might be the results of receptor upregulation. It also might be due

to the difference between different strains of rats. The ratio of the second, third and fourth

ATP-stimulated [Ca2+]i increase was significantly lower in SHR than that in WKY. This

reflected greater receptor desensitization in SHR. In the juxtaglomerular apparatus ATP can

contract afferent arteriole by increasing [Ca2+]i in smooth muscle cells via P2X receptors

(Gutierrez et al. 1999). At the same time, ATP can stimulate NO release from the endothelial

cells in the kidney via P2Y2 receptors (Fernandez et al. 2000). If less NO was produced, a

mechanism to explain the blood pressure increase could be presented.

The NO synthase inhibitor, L-NAME, was shown to increase purine receptor

desensitization, except in the SHR rats. An increase in NO delivery to the mesangial cells by

L-arginine or NO donors resensitized the ATP receptors. Among the NO donors, the

spermine-NONOate had the greatest effect, although it was only tested at a single

concentration. This is consistent with other reports (Muhl et al. 1996; Kone and Baylis 1997).

The results showed that in SHR there is an impairment of receptor resensitisation in the

reaction to the endogenous and exogenous NO. These findings of a larger desensitization

effect and impaired receptor resensitization in the SHR would be in line with a reduced NO

concentration in these cells. A low NO concentration has been found to be caused by an

increased formation of oxygen radicals in the juxtaglomerular apparatus sensitizing the

tubuloglomerular feedback mechanism (Thorup and Persson 1996; Welch et al. 2000).

17

5.5. Contributions of c-GMP pathway and ryanodine receptors

NO has an inhibitive effect on Ca2+ release from IP3-sensitive stores (the GPCR-PLC-

IP3 cascade) through a cGMP-dependent pathway. This has been demonstrated in various

cell systems (Karaki et al. 1988; Clementi et al. 1995; Harvey and Burgess 1996; Stockand

and Sansom 1996), a mechanism operating through the activation of protein kinas G (PKG).

In general, NO stimulated activation of PKG is associated with a decrease in intracellular

calcium, a mechanism consistent with decreased contractility of smooth muscle cells. The

guanylate cyclase inhibitor ODQ and the cGMP analog, 8-Br-cGMP, were used to determine

the contribution of the cGMP pathway. The results indicate that there are no significant

differences with or without ODQ and 8-Br-cGMP. NO can also play a role in controlling Ca2+

release from ryanodine-sensitive intracellular calcium stores (Galione et al. 1993). However,

we have failed to see calcium release response when challenging with caffeine, caffeine plus

ATP and 4-chloro-3-ethylphenol, a new ryanodine receptor agonist (Shoshan-Barmatz and

Ashley 1998; Westerblad et al. 1998). We found that a low dose of caffeine (100 nM) can

inhibit calcium release challenged by ATP. Similar effects have been reported in the other

kinds of cells and tissues through IP3 sensitive intracellular calcium stores (Bezprozvanny et

al. 1994; Missiaen et al. 1998). The mechanism is not clear, probably because caffeine

inhibits the binding site of IP3 receptors and deactivates the receptors (Maes et al. 1999).

5.6. NO on unstimulated receptors

It has been shown that the receptor desensitization cascade is not activated until the

receptors are stimulated by agonists (Trowbridge et al. 1993). Thus, we can exclude the

contribution of receptor desensitization cascade in only one ATP challenge. We found that

there was no significant difference in ATP-stimulated [Ca2+]i increase after a 5 min perfusion

of different NO donors and L-NAME in SD,WKY and SHR mesangial cells. The lack of

18

effects of NO before the first stimulus indicates that NO increase P2Y receptor resensitization

in rat mesangial cells.

5.7. Purinergic receptor signaling at MD cells

Confocal microscopy has superior spatial and temporal resolution compared to

conventional image analysis systems. In study III, we found no increase in [Ca2+]i during the

administration of adenosine (10-7-10-3 M). Earlier reports have found (Schnermann 1988; Franco et

al. 1989) that adenosine A1 receptor agonists enhance TGF responses when added from the lumen.

In their studies (Schnermann 1988; Franco et al. 1989), it is possible that the A1 receptor agonists

directly affected the arteriolar smooth muscle cells. As recently reported (Gutierrez et al. 1999;

Gutierrez et al. 1999), freshly dissected renal arterioles but not cultured mesangial cells respond to

adenosine with an increase in [Ca2+]i. We found that in [Ca2+]i responses, the order of efficacy of

agonists was UTP = ATP >> 2MesATP = ADP. This is consistent with P2Y receptor expression.

Since the early maximal increase in [Ca2+]i is a good indicator of release of Ca2+ from intracellular

pools (Sipma et al. 1999), the maximal increases in [Ca2+]i obtained with each agent was recorded

in the results. Phospholipase C inhibitor, U-73122, prevented increases in [Ca2+]i , but its inactive

analog, U-73343, could not. These findings strongly support our conclusion that the most

important contribution to the rapid and maximal increase in [Ca2+]i was from Ca2+ mobilization

through the PLC-IP3 pathway and not through Ca2+ entry mechanisms (Watanabe and Endoh

1999; Doi et al. 2000; Mignen and Shuttleworth 2000). It also further supports the presence of

P2Y receptors and not P2X receptors in MD cells. This later class of receptors exhibits properties

of a non-selective cation channel that promotes increases in [Ca2+]i via Ca2+ entry (McCoy et al.

1999). The order of the efficacy of the nucleotides in MD cells was essentially the same as that

found for P2Y2 or/and P2Y4 receptors (Lustig et al. 1993; Parr et al. 1994; Bowler et al. 1995;

Godecke et al. 1996; Gutierrez et al. 1999). Pharmacological characterizations of P2Y2 and P2Y4

are almost the same, except P2Y4 receptors are less sensitive to suramin (Bogdanov et al. 1998;

19

Harada et al. 2000). Our results showed that suramin could completely block the [Ca2+]i responses

induced by ATP, so we conclude the MD cells express P2Y2 receptors.

5.8. Luminal NaCl and NO production in MD

The fluorophore calcein was used to measure volume in MD cells. Calcein

concentration is not significantly related to changes in measure volume in MD cells

intracellular calcium and pH (Breuer et al. 1995; Crowe et al. 1995). Changes in cell volume

are expected to be reflected by changes in the fluorescence intensity, with decreased intensity

during cell swelling and increased intensity during cell shrinkage (Mandeville et al. 1995;

Sonnentag et al. 2000). In study IV, total osmolarity was held constant. When the luminal

[NaCl] solution was decreased, the MD cell volume was reduced, and when the luminal

[NaCl] was increased, the cell volume was also increased.

To measure NO we used DAF-2, a single-wavelength measurement probe, which is

affected by cell volume. Therefore, the amount of DAF-2T had to be corrected for upon

changes in cell volume. After volume correction the concentration of DAF-2T could be

determined in MD cells. The results showed that when the luminal [NaCl] was increased

from 10 mM or 35 mM to 135 mM, keeping total osmolarity constant, the DAF-2T amount

was increased significantly. When the luminal [NaCl] was decreased the amount of DAF-2T

decreased significantly. This increase in DAF-2T could be inhibited by treatment with L-

NAME or 7-nitroindazole. It is known that the fluorescence intensity of DAF-2T is pH

dependent (Kojima et al. 1998; Nagano 1999). The fluorescence of DAF-2T is comparatively

stable above pH 7, but its fluorescence dramatically decreases below a pH of 7. It has been

reported that increased luminal [NaCl] elevats pH, while decreased luminal [NaCl] lowers the

pH in MD cells through apical Na:H exchangers (Fowler et al. 1995; Peti-Peterdi and Bell

1998). In present study, when luminal [NaCl] increased, which also increase intracellular pH

in MD cells, the influence of pH value on the DAF-2T intensity should be minor. While,

20

when luminal [NaCl] decreased, the pH in MD cells will decrease, which could possibly

influence the intensity of DAF-2T significantly. As discussed above, the amount of DAF-2T

during luminal [NaCl] increase reflected the NO production in MD cells. So, the NO

production in MD cells increased significantly, following increase luminal [NaCl]. There are

rather contradictory reports regarding the distal [NaCl] and the NO production in MD cells

(Shultz and Tolins 1993; Bosse et al. 1995; Deng et al. 1995; Singh et al. 1996; Thorup and

Persson 1996; Wilcox and Welch 1996; Ichihara et al. 1998; Turkstra et al. 1998; Braam

1999). Decreased luminal [NaCl] would lower the pH in MD cells (Fowler et al. 1995),

which could greatly decrease the amount of DAF-2T (Kojima et al. 1998; Nagano 1999). The

significant decrease of DAF-2T fluorescence in this study is probably caused by the

intracellular pH fall in MD cells. With a fall in pH, it is difficult to evaluate the actual NO

productions.

Constitutive NOS is dependant on intracellular calcium and calmodulin (Sasaki et al.

2000). Our data showed that with use of Ca2+ free solution, the changes in NO intensity were

not significantly different from those with the normal Ca2+ solution. Intracellular calcium

changes, caused by alterations in luminal [NaCl] in the MD cells, would be abolished if

calcium free solutions were used in bath and lumen (Salomonsson et al. 1991; Peti-Peterdi

and Bell 1999). However, it has been reported that increased shear stress on endothelial cells

can increase NO release in an non-calcium-dependent way (Fleming et al. 1998; Fulton et al.

1999). A similar mechanism might exist in the MD cells. In addition, changes in [Ca2+]i

caused by alterations of the luminal [NaCl] was only 20 to 40 nM (Salomonsson et al. 1991;

Peti-Peterdi and Bell 1999). Such a small change might not be enough to overwhelm other

influential factors. Furthermore, the local NO production could also be regulated below the

level of NOS activity. The release of NO from the MD cells upon increased [NaCl] could,

however, be an important mechanism to counteract the TGF mediated vasoconstriction in the

afferent arteriole and also to reset the sensitivity of the TGF mechanism to a lower level.

21

SUMMARY:

• NO can increase the P2Y receptor resensitization in rat glomerular mesangial cells,

through a cGMP-independent pathway.

• We found no evidence for the existence of ryanodine sensitive intracellular calcium

stores in rat mesangial cells.

• A higher ATP response and a faster desensitization were found in the SHR cells

compared to WKY.

• The presence of L-NAME enhances receptor desensitization in WKY but not in SHR.

These effects are mediated through a cGMP-independent pathway.

• We found that MD cells possess P2Y2 purinergic receptors on the basolateral but not

apical membranes and that activation of these receptors results in mobilization of

Ca2+.

• We could quantitatively measure the MD cell volume changes with calcein and NO

formation in MD cells with DAF-2, caused by the changes of luminal [NaCl], using

confocal microscopy.

• Increasing the luminal [NaCl] resulted in an increase in MD cell volume and NO

production. An increase in NO would oppose vasoconstriction and reset the TGF

mechanism to a lower level.

22

Acknowledgements:

These studies were carried out at the Department of Physiology and Medical Cell Biology,

Division of Integrative Physiology, Uppsala University, Sweden. I would like to express my

sincere gratitude to all who have helped me in different ways to make this work possible.

I would like to express my special thanks to:

My supervisor, Professor Erik Persson, for your support and encouragement all the time.

Thanks for introducing me into this interesting area with delicate equipment and excellent

research conditions and for always shaving firm confidence in me. Without your continuous

encouragment, it would be impossible for me to finish this today.

My co-supervisors, Associate professor Peter Hansell, and Göran Sperber, for valuable

discussion, encouragement and help in different ways.

Professor Darwin Bell, for leading me into this fascinating world of microperfusion. Thanks

for sharing your skillful technique and knowledge. Thanks for your great hospitality in

Birmingham -- delicious crayfish, nice Tai food and strong “light beer”... Thanks for your

valuable contributions on my manuscript and your prompt and optimistic reply about

revisions, even though your “next week” sometimes meant next month or even longer.

Dr. Avi Ring, for sharing your broad knowledge and great ideas in science. Thanks for your

guidance and help, and for almost always being available for discussion and questions. I have

learned a lot through your critical views on my research.

23

I also want to thank Alf Johansson, for your invaluable and kind help when it was most

needed.

Dr. Janos Peti-Peterdi, and Dr. Gergly Kovacs Birmingham, USA, for sharing your skillful

techniques; for your hospitality; and for making my stay in Birmingham so wonderful.

Dr. Werner Koopman Nigmengen, the Netherlands. For valuable and kind assistance in UV

laser confocal measurement.

Dr. Yilin Ren Detroit, USA, for fruitful discussions on arteriole microperfusion and great

hospitality.

Cissi, for kindly inviting us to spend an impressive Christmas Eve with your family. Thank

you for playing Majone with us and the very good times.

Associate professor Lena Holm, Professor Mats Wolgast, Professor Mats Sjöquist,

Associate professor Örjan Källslog, Dr. Per Liss, and Dr. Magnus Roos for valuable

discussions and encouragement.

Antonio, for collaborations and kind help. Mia, for optimistic help in Swedish and

supervisor-like encouragement. Pernilla, for interesting discussions and nice collaborations

in teaching. Janos, for skillful problem-solving in lab for whatever. Anna, for good ideas and

discussions. Russell, for nice Swedish-English translation. Fredrik, for fruitful discussions

of computer games. Åsa, for kindly providing neuroblastumor cells. Victoria, Johanna,

Markus, Anca, for good times and nice talks. Angelica, Gunno and Erik for technical

support and help.

24

Thanks to all my Chinese friends in Uppsala for very good times and great friendship,

especially: Yu Jiang, Yan Hongmei, Li Zhanchun, Zhao Lijun, Su Cha, Lin Jianman,

Zhou Yinghua, Wang Shu, Feng Jiayang, Xie Rujia, Liu Xiaoli, Wang Guimin, Zhang

Lihong, Liu Chengjie, Guo Gengsheng, Song Qiao, Zou Xiang, Lu Linge, Guo Qi, Wang

Pei and Wu Ping.

My parents, sisters and brother, for their love and encouragement.

My son, Yixiao, I am proud of everything he has done.

My daughter, Yimeng, her coming like a dream in my life.

My beloved wife, Ying, for her love, support and care.

This study was financially supported by the Swedish Medical Research Council (project no.

K99-14X-03522-28D), the Wallenberg Foundation, and the Ingabritt and Arne Lundberg

Foundation.

25

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