!    "     

#$ % % &&'()%

%)%  *+'(*)%)* ,%&*' * ,,%&% -..- Macula Densa Derived Nitric Oxide and Kidney Function

ANNA OLLERSTAM Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in Physiology presented at Uppsala University in 2002

ABSTRACT

Ollerstam, A. 2002. Macula Densa Derived Nitric Oxide and Kidney Function. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 1144. 47 pp. Uppsala. ISBN 91-554-5293-0.

The kidney is the major organ regulating the extracellular fluid volume and thereby the arterial blood pressure. The neuronal isoform of nitric oxide synthase (nNOS) in the kidney is predominantly located in the macula densa cells. These cells are sensors for both renin release and the tubuloglomerular feedback mechanism (TGF), which is an important regulator of the glomerular filtration rate and afferent arteriole tone. The aim of this investigation was to elucidate the function of nNOS in the macula densa cells. Acute nNOS inhibition in rats resulted in an increased TGF responsiveness and unchanged blood pressure while, after chronic inhibition, the TGF was normalised and the blood pressure was elevated. The plasma renin concentration was elevated in rats on long- term low salt diet, but was not significantly affected by chronic nNOS inhibition. On the other hand, nNOS inhibition for four days increased plasma renin concentration in rats treated with a low salt diet. The renal vasculature of rats exhibits a diminished renal blood flow and intracellular Ca2+ response to angiotensin II after one week blockade of nNOS while angiotensin II’s effect on the renal blood flow was abolished after four weeks treatment. Acute extracellular volume expansion diminish the TGF sensitivity thus assisting the elimination of excess fluid but after acute addition of nNOS inhibitor to volume expanded rats the TGF sensitivity restored. In conclusion, the results from the present study suggest an important role for nNOS in the macula densa cells in the regulation of the arterial blood pressure and the modulation of the TGF response.

Key words: Nitric oxide, rats, kidney, macula densa cells, tubuloglomerular feedback, renin, angiotensin II, blood pressure, hypertension, renal blood flow, neuronal nirtic oxide synthase.

Anna Ollerstam, Division of Integrative Physiology, Department of Medical Cell Biology, Uppsala University, Biomedical Centre, P.O. Box 571, SE-751 23 Uppsala, Sweden

 Anna Ollerstam 2002

ISSN 0282-7476 ISBN 91-554-5293-0

Printed in Sweden by Uppsala University, Tryck och medier, Uppsala 2002

2 This thesis is based on the following papers, which will be referred to in the text by their Roman numerals:

I Increased blood pressure in rats after long-term inhibition of the neuronal isoform of nitric oxide synthase. Ollerstam A, Pittner J, Persson AEG, & Thorup C. 1997 J Clin Invest. 99(9): 2212-8.

II Effects of long-term inhibition of neuronal nitric oxide synthase on blood pressure and renin release. Ollerstam A, Skøtt O, Ek J, Persson AEG & Thorup C. 2001 Acta Physiol Scand. 173: 351-8.

III Reduced rat renal vascular response to Ang II after chronic inhibition of nNOS. Ollerstam A, Salomonsson M & Persson AEG. 2002 Submitted.

IV Neuronal nitric oxide synthase inhibition restores the tubuloglomerular feedback response after volume expansion. Brown R*, Ollerstam A* & Persson AEG. 2002 *equal contribution, Manuscript

Reprints are made with permission from the publishers. Paper I is republished with permission of The American Society for Clinical Investigation, Inc.; permission conveyed through Copyright Clearance Centre, Inc. Paper II is republished with permission of Scandinavian Physiological Society.

3 TABLE OF CONTENTS

LIST OF ABBREVIATIONS...... 5

INTRODUCTION...... 6

AUTOREGULATION...... 7 Myogenic response ...... 8 Tubuloglomerular feedback...... 8 NITRIC OXIDE...... 9 RENIN-ANGIOTENSIN SYSTEM ...... 11 AIMS OF THE INVESTIGATIONS...... 13

MATERIALS AND METHODS ...... 14

ANIMALS...... 14 SURGICAL PREPARATION (STUDIES I, II, III & IV)...... 14 TAIL-CUFF BLOOD PRESSURE MEASUREMENTS (STUDIES I & II)...... 14 WHOLE KIDNEY CLEARANCE MEASUREMENTS (STUDIES I, II & IV)...... 15 STOP-FLOW PRESSURE MEASUREMENTS (STUDIES I & IV)...... 15 MEASUREMENTS OF SNGFR (STUDIES I & IV) ...... 16 ISOLATED BLOOD-PERFUSED JUXTAMEDULLARY NEPHRON PREPARATION (STUDY I) ...... 17 SAMPLING AND RENIN ASSAY (STUDY II)...... 17 RENAL BLOOD FLOW MEASUREMENTS (STUDY III) ...... 18 MEASUREMENTS OF CYTOSOLIC CALCIUM CONCENTRATION (STUDY III) ...... 18 DISSOLVING 7-NI (STUDIES I, II, III & IV)...... 18 STATISTICAL ANALYSES (STUDIES I, II, III & IV)...... 19 EXPERIMENTAL PROTOCOL ...... 19 Study I...... 19 Study II ...... 20 Study III ...... 20 Study IV ...... 21 RESULTS AND COMMENTS...... 23

STUDY I...... 23 STUDY II ...... 24 STUDY III...... 25 STUDY IV...... 26 DISCUSSION ...... 28

CONCLUSIONS ...... 36

SUMMARY IN SWEDISH/ SAMMANFATTNING PÅ SVENSKA...... 37

ACKNOWLEDGEMENT...... 40

REFERENCES...... 42

4 LIST OF ABBREVIATIONS

∆PSF Maximal TGF response 7-NI 7-Nitro indazole Ang II Angiotensin II Bw Body weight cGMP Cyclic guanosine monophosphate eNOS Endothelial nitric oxide synthase GFR Glomerular filtration rate HS High salt i.p. Intraperitoneal i.v. Intravenous IMCD Inner medullary collecting duct iNOS Inducible nitric oxide synthase JGA Juxtaglomerular apparatus L-Arg L-arginine

L-NAME Nω-nitro-L-arginine methyle ester

L-NNA Nw-Nitro-L-arginine LS Low salt MAP Mean arterial blood pressure MD Macula densa NE Norepinephrine nNOS Neuronal nitric oxide synthase NO Nitric oxide

PSF Stop flow pressure

PT Proximal tubular free-flow pressure RBF Renal blood flow SNGFR Single nephron glomerular filtration rate TGF Tubuloglomerular feedback TP Turning point PRC Plasma renin concentration VE Volume expansion

5 INTRODUCTION The fluids of the body can be considered to be divided between two compartments, the intracellular and the extracellular. The extracellular compartment can be further divided into plasma, interstitial fluid and transcellular fluid. Several systems are involved in maintaining a stable environment for the cells. The kidneys have a vital role in the regulation of extracellular, and indirectly of intracellular, fluid volume and composition. In addition to concentrating and eliminating waste products they also control the osmolality, fluid volume, acid-base status and ionic composition of the extracellular fluid and through this also the blood pressure. The kidneys also produce hormones such as renin, erythropoietin and 1.25-dihydroxy-D3.

The human kidney consists of approximately one million nephrons, which are the functional units of the kidney (Fig. 1), in which filtration, reabsorption and secretion takes place. The kidneys receive approximately 20% of the cardiac output through the renal arteries. After several divisions of the vessels blood reaches the glomerulus via the afferent arteriole. In the glomerulus approximately 20% of the blood is filtered into the tubule system through Bowman´s capsule. The blood that is not filtered exits the glomerulus via the efferent arteriole and then the kidney through the renal vein.

5 8 4 12

1. Glomerulus 2. Bowman´s capsule 7 3. Afferent arteriole 4. Efferent arteriole

3 5. Proximal tubule 6. Loop of Henle 6 7. Macula densa cells 9 8. Distal tubule 9. Collecting duct

Figure 1. Schematic drawing of the nephron.

6 The primary urine flows though the proximal tubule and down through the loop of Henle. Here, where the distal part of the tubule begins, the tubule returns back to its own glomerulus where it comes in contact with the afferent and efferent arterioles. This structure is called the juxtaglomerular apparatus (JGA) and was first described by Golgi in 1889 (Golgi, 1889). The tubular fluid flows through the distal tubule and is led to the renal pelvis through the collecting ducts. The formation of urine is the balance between glomerular filtration rate (GFR) and tubular reabsorbtion, which takes places throughout the nephron. Changes in GFR could give rise to hyper- or hypotension since the tubular system has a limited ability to reabsorb fluid and electrolytes.

Autoregulation GFR and renal blood flow (RBF) are kept rather stable to allow for precise regulation of reabsorbtion and secretion in the tubules (Fig. 2). This process is called autoregulation and is independent of nerves. Autoregulation is active within a wide rage of mean arterial blood pressure (MAP) changes and the RBF and GFR remain fairly constant even though the MAP fluctuations between 80 to 160 mmHg (Forster and Maes, 1947, Baer and Navar, 1973, Arendshorst, et al., 1975). Autoregulation is divided into two different mechanisms, the myogenic response and the tubuloglomerular feedback (TGF).

Autoregulatory

1.5 15

1.0 10 RBF (l/min) GFR (ml/min) 0.5 5

80 160 MAP (mmHg)

Figure 2. Drawing showing the autoregulation of glomerular filtration rate (GFR, ---) and renal blood flow (RBF, –––). When the mean arterial blood pressure (MAP) varies between 80 and 160 mmHg GFR and RBF remains fairly constant.

7 Myogenic response Increases in wall tension of the afferent arteriole, caused by an increased perfusion pressure, elicits an automatic contraction of the smooth muscle fibres in the vessel wall. This constriction increases the resistance, restricting the flow and pressure and thereby decreases the perfusion pressure.

Tubuloglomerular feedback The TGF response is the result of a constriction of the afferent arteriole after an increased filtration and was described as its seen today in 1964 (Thurau, 1964, Guyton, et al., 1964). In the wall of the tubulus of the JGA there are specialised cells called the macula densa (MD) cells. A regulatory function of the MD cells was first suggested by Reuter in 1925 (Ruyter, 1925). In 1939 Goormaghtigh (Goormaghtigh, 1939) postulated that the MD cells were able to sense the flow in the tubule and elicit a negative feedback response. The MD cells have a Na+/K+/2Cl--cotransport in the apical membrane (Gonzalez, et al., 1988, Schlatter, et al., 1989) and this cotransporter can be blocked with the loop diuretic furosemide. It has been shown that the TGF mechanism is blocked by loop diuretic (Wright and Schnermann, 1974, Odlind and Lonnerholm, 1982).

The mechanism of the TGF is as follows. An increase in glomerular capillary pressure causes an increased GFR. This elevation gives rise to an increased tubular flow with an increased NaCl load past the MD cells. The MD cell’s Na+/K+/2Cl--cotransporter increases its activity and the intracellular concentrations of Na+, K+ and Cl- increase. This elicits a signal originating in the MD cells and transmitted to the afferent arteriole. The transmitted signal causes the afferent arteriole to constrict, resulting in a decreased GFR. The TGF can be divided into three steps, the sensing in the MD cells, signalling from the MD cells to the afferent arteriole and the vasoconstrictor response of the afferent arteriole resulting in the decreased GFR. The sensing of tubular fluid in the MD cells occurs through the Na+/K+/2Cl-cotransporter located in the apical membrane (Gonzalez, et al., 1988, Schlatter, et al., 1989). It has been shown that increased NaCl concentration in the tubular fluid gives rise to a depolaization of the basolateral membrane of the MD cells (Schlatter, et al., 1989, Lapointe, et al., 1990) and

8 that the depolarization is necessary for a TGF response (Ren, et al., 2001). Cl- exits the cell via the basolateral Cl- channels, which in turn leads to depolarization of the MD and thereby induces the TGF response (Ren, et al., 2001).

The mediator of the signal from the MD cells to the afferent arteriole was first believed to be the renin-angiotensin system (Thurau and Schnermann, 1998, Thurau, et al., 1972). This was not found to be the case, in fact TGF and renin release are regulated in opposite directions after variations in salt concentration past the MD cells (Skott and Briggs, 1987). We now know that the mediator is most likely adenosine. In 1982, Osswald and co-workers suggested a coupling between the metabolic rate of the MD cells and the release of adenosine (Osswald, et al., 1982). The elevated transport rate in the MD cells will increase the consumption of ATP in these cells, leading to a breakdown of ATP to AMP and further on to adenosine (Thomson, et al., 2000). An increased delivery of adenosine into the renal interstitium could elevate the interstitial adenosine concentration around the afferent arteriole. We found that transgenic mice missing the adenosine A1 receptor lack TGF response (Brown, et al., 2001). This finding is entirely in line with the original suggestion made by Osswald and colleagues (Osswald, et al., 1982), where they saw adenosine as a mediator of the TGF mechanism rather than as a modulator. From a modulator one would not expect a total absence of response.

There is an important function in resetting of the TGF system. This means that the information transmitted by local factors such as interstitial pressure, prostaglandins, kinins etc. will determine what filtration rate the existing condition could allow. E.g. in dehydration the TGF system is activated to large extent even though the filtered load is lower than normal to avoid further fluid losses (Selen, et al., 1983). In the resetting process many different hormones and local factors are involved; Ang II, prostaglandins, kinin, thromboxane and others (Schnermann, et al., 1998).

Nitric oxide Nitric oxide (NO) is formed together with L-citrulline from molecular oxygen and L- arginine (L-Arg) in an enzyme-catalysed reaction. It has a number of effects in the

9 body ranging from the cardiovascular and nervous system to being involved in the host defence system. The physiological actions of NO were first shown in the vasculature by Furchgott and Zawadzki in 1980 (Furchgott and Zawadzki, 1980). It has also been found that NO is an activator of soluble guanylate cyclase, leading to formation of cyclic guanosine monophosphate (cGMP), which in turn activates protein kinase G, resulting in a decreased in cytosolic calcium in the vascular smooth muscle cells (Moncada and Higgs, 1993). NO has a high affinity for heme which is important since guanylate cyclase contains a heme group.

There are three known isoforms of NOS, the neuronal isoform (nNOS or NOS I), the inducible isoform (iNOS or NOS II) and the endothelial isoform (eNOS or NOS III). The nNOS and the eNOS are said to be constitutively expressed in the tissue and are Ca2+ dependent, while the iNOS is inducible and Ca2+ independent. However, it has been reported that the iNOS is also constitutively expressed in the kidney (Ahn, et al., 1994). All NOS isoforms are dimeric enzymes containing heme, flavin adenine dinucleotide, flavin mononucleotide and tetrahydrobiopterin as bound groups. They also contain binding sites for L-Arg, calcium-calmoduline and NADPH. In the kidney nNOS is to a large extent expressed in the MD cells (Wilcox, et al., 1992, Mundel, et al., 1992, Thorup, et al., 1993). The enzyme nNOS has also been found to be expressed in the medullary thick ascending limb (McKee, et al., 1994), inner medullary collecting duct (IMCD) (Mattson and Bellehumeur, 1996, Roczniak, et al., 1998, Roczniak, et al., 1999, Wu, et al., 1999) and in the principal cells of the cortical collecting duct (Bachmann, et al., 1995, Wang, et al., 1998).

The half-life of NO under physiological conditions is very short and NO can react with superoxide anion to produce peroxynitrite anion. It has been shown that if the superoxide anions in the JGA are removed NO:s inhibitory effect on the TGF is decreased (Wilcox and Welch, 2000). It has also been found that NO from nNOS has a diminished role in inhibiting the TGF response in spontanously hypertensive rats (Thorup and Persson, 1996), which was at least in part due to an increased level of superoxide anions (Welch, et al., 2000).

10 To study the effects of NO one can use different inhibitors of NOS. For unselective

NOS inhibition there are many different substances to choose from, such as Nw-Nitro- G L-arginine (L-NNA), Nω-nitro-L-arginine methyle ester (L-NAME) or N -mono- methyl-L-arginine. These are structural analogues of the substrate L-Arg. To increase NO release high doses of L-Arg or a NO donor e.g. SNAP can be given. Inhibition of NOS with an unselective blocker results in an increased blood pressure, due to an increase in vascular resistance. In the kidney the increased renal vascular resistance after unselective NOS inhibition leads to a decreased GFR and RBF (Baylis, et al., 1990, Tolins, et al., 1990). Chronic administration of unselective inhibitors of NOS has been found to cause a sustained elevation of arterial blood pressure and an increase in renal vascular resistance (Baylis, et al., 1992, Bouriquet and Casellas, 1995, Bank, et al., 1994, Jover, et al., 1993, Ribeiro, et al., 1992). Convincing evidence for a strong renal influence in this type of hypertension has been demonstrated. Still, the use of unselective NOS inhibitors are combined with a wide range of unselective pressor effects on peripheral vascular resistance (Vallance, et al., 1989, Johnson and Freeman, 1992), as well as unknown effects on the central nervous system (Cabrera and Bohr, 1995).

To be able to study the actions of nNOS situated in the MD cells a selective inhibitor is needed. 7-nitro indazole (7-NI) is a relatively selective inhibitor of nNOS (Moore, et al., 1993) and should therefore exert its major renal NO inhibitory effects on nNOS in the MD cells. In acute experiments 7-NI has been shown to inhibit nNOS without effecting blood pressure (Beierwaltes, 1995, Thorup, et al., 1996). Still, it is as potent as unspecific L-Arg analogues in enhancing TGF responsiveness and reducing GFR (Thorup, et al., 1996). 7-NI selectively modulates nNOS activity by binding reversibly to the heme group of nNOS and in this way interfering with tetrahydrobiopterin binding to the enzyme (Mayer, et al., 1994).

Renin-angiotensin system The existence of renin was first found in 1898 by Tigerstedt and Bergman (Tigerstedt and Bergman, 1898) and Ruyter described granular cells in the wall of the afferent

11 arteriole in 1925 (Ruyter, 1925). Fourteen years later, in 1939, it was suggested by Goormaghtigh that these cells secreted renin (Goormaghtigh, 1939). A decreased NaCl concentration in the tubular fluid passing the MD cells stimulates renin release from the granular cells in the afferent arteriole (Skott and Briggs, 1987, Schnermann, 1998, Lorenz, et al., 1991). Renin is an enzyme and not itself a vasoactive substance. It acts by splitting a plasma protein called angiotensinogen to angiotensin I. Angiotensin I has only mild effects on blood pressure but is further broken down to the potent vasoactive agent angiotensin II (Ang II) in the small vessels in the lungs by the enzyme angiotensin converting enzyme. Ang II acts through constriction of arterioles and to some extent veins. This increases the peripheral resistance and venous return. Another action of Ang II is the decreased excretion of salt and water. This is achieved in different ways, by directly effecting the kidneys or through increased aldosterone secretion. The direct effect that Ang II has on the kidney is probably mainly due to the vasoconstriction of the vessels, which decreases the blood flow through the kidney, leading to decreased filtration of fluid to the tubules. Aldosterone acts on the principal cells in the distal tubules or cortical collecting ducts and increases Na+ reabsorbtion (Guyton, 1991).

The effect of NO on renin release is still not clear. Investigators have reported that NO stimulates renin secretion, both in vivo (Naess, et al., 1993, Persson, et al., 1993) and in vitro (Fischer, et al., 1995, Schricker, et al., 1995), while others have reported an inhibitory effect of NO on renin secretion (Vidal, et al., 1988). It has also been suggested that it may be important from which side the NO is applied to the granular cells. NO from the MD cells may stimulate renin release, while NO derived from the endothelial cells in the afferent arteriole may inhibit renin release (Beierwaltes, 1995, He, et al., 1995). Kurtz and co-workers suggested that NO might stimulate or inhibit renin release from the juxtaglomerular cells by activating the phosphodiesterase III or cGMP-dependent kinase II (Gambaryan, et al., 1998, Kurtz, et al., 1998).

12 AIMS OF THE INVESTIGATIONS

The general purpose of this investigation was to elucidate the function of the nNOS in the MD cells.

I This study was designed to study the chronic and acute effects of selective inhibition of nNOS on blood pressure and renal hemodynamics in male Sprague-Dawley rats. We hypothesized that long-term (four weeks) 7-NI treatment would activate TGF, leading to a decreased GFR and thereby elevated blood pressure.

II The purpose of this examination was to study the effect of specific blockade of nNOS on blood pressure and renin release in rats treated with low, high and normal salt diets and elucidate the role of MD derived nNOS in blood pressure regulation and renin release.

III The aim of this study was to examine the short (one to two weeks) and long- term (four weeks) effects of nNOS inhibition on the Ang II and NE actions on RBF in vivo in the presence and absence of unselective NOS blockade. We also investigated the calcium response in the smooth muscle cells of isolated rat afferent arterioles in response to Ang II, NE and depolarization with KCl after one to two weeks of nNOS inhibition.

IV This study was designed to study if MD derived nNOS could reset the TGF sensitivity after extracellular volume expansion (VE). We wanted to investigate if NO is involved in the desensitisation of the TGF seen after VE.

13 MATERIALS AND METHODS For more detailed information about the different methods and materials consult the different studies.

Animals The experiments were carried out on male Sprague Dawley rats from Møllegaard Breeding Center, Copenhagen, Denmark. The local Ethics Committees for animal experiments in Uppsala or Lund approved all studies.

Surgical preparation (Studies I, II, III & IV) On the day of an experiment, the rats were anaesthetised by an Intraperitoneal injection of Thiopenthal sodium or Thiobutabarbital sodium (Trapanal [study I] or Inactin [studies II, III and IV], 120 mg kg-1 body weight (Bw)), which was supplemented, if required, during the experiment. The rats were placed on a servo- regulated heating pad with a rectal probe to maintain their body temperature at 37.5° C. Trachea was catheterised to allow for spontaneous breathing. Catheters were inserted into the carotid artery and the jugular vein for arterial blood pressure measurements and infusion of maintenance fluid (saline, 0.9% NaCl; 5 ml h-1kg-1 Bw). The bladder was cannulated for urine release. In the studies where TGF, RBF or single nephron GFR (SNGFR) were measured (studies I, III and IV) a subcostal flank incision was made and the left kidney was exposed. The kidney was dissected free from surrounding tissue, placed in a lucite cup and fixed with a 3% agar-agar solution. The kidney surface was covered with mineral oil to prevent drying. After an equilibration period of at least 45 min, RBF, clearance or micropuncture measurements were started. In study IV a VE of 5% h-1 kg-1 with saline were commenced after the preparation.

Tail-cuff blood pressure measurements (Studies I & II) A tail-cuff blood pressure measuring system was used to measure systolic blood pressure in a non-invasive way. The rats were trained to become accustomed to the blood pressure measurement procedure for one week before the experimental series

14 were begun. All animals were given free access to standard rat chow and ordinary tap or 7-NI in tap water.

Whole kidney clearance measurements (Studies I, II & IV) Thirty minutes after completion of surgery, infusion of 3H-inulin in normal saline into the jugular vein was commenced. In whole kidney clearance measurements an initial bolus of 5 µCi was followed by a continuous dosage of 5 µCi h-1 After an equilibration period of 45 min two 20-min urine collection periods were begun. The urine volume was determined by weight. Before and after each collection period, blood samples were taken. These samples were centrifuged and aliquots of plasma were analysed in a scintillation counter, together with aliquots of urine. Inulin clearance was then calculated as a measure of GFR. The concentrations of sodium and potassium were assayed with a flame photometer. After an experiment was completed, kidneys were removed, cleaned of any surrounding tissue and weighed.

Stop-flow pressure measurements (Studies I & IV) TGF characteristics were determined by the stop-flow technique (Fig. 3). Under a stereo microscope, randomly chosen proximal tubular segments on the kidney surface were punctured with a sharpened glass pipette filled with 1 M NaCl solution stained with Lissamine green. The pipette was connected to a servo-nulling pressure system to determine the proximal tubular free-flow pressure (PT). By injections of the stained fluid, the tubular distribution on the kidney surface was determined. In nephrons where more than three proximal segments were identified, a second pipette was inserted in the last accessible segment of the proximal tubule. This pipette was filled with an artificial ultrafiltrate (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 4 mM NaHCO3, 7 mM urea, 2 g/l Lissamine green, pH 7.4), and connected to a micro perfusion pump. Between these two pipettes a solid wax block was placed, with a third pipette. The pressure upstream to the block, the PSF, was measured at different perfusion rates (0-40 nl min-1) in the loop of Henle. The flow was increased or -1 decreased in steps of 2.5-5 nl min and the maximal feedback response ∆PSF was -1 determined as the decrease in PSF at the 40 nl min perfusion rate, compared with PSF

15 at zero perfusion. The tubular flow rate at which 50% of the maximal pressure response was obtained, called the turning point (TP), was determined; by definition TP is a measure of the TGF sensitivity.

Perfusion pipette Pressure pipette Wax block

Figure 3. Schematic drawing of the nephron with the pipettes used for tubuloglomerular feedback determination using the stop-flow technique.

Measurements of SNGFR (Studies I & IV) For measurements of SNGFR in study I, [3H]methoxy-inulin (80 µCi h-1) in normal saline was infused into the jugular vein. Proximal and early distal tubular segments were identified with a perfusion pipette with which dye was injected intratubularely. For distal collections a glass pipette filled with black stained mineral oil was used. An oil drop was injected downstream from the puncture site and all fluid proximal to the pipette was collected. The proximal collection was done in a similar way in a segment as proximal as possible. Before the collection was begun, a hole was made in a late proximal segment to avoid a stop-flow situation. Timed samples of tubular fluid were taken and analysed for 3H activity for calculations of SNGFR. The initial distal collection was performed with fluid through the MD area and hence, with TGF activated. The subsequent proximal collection would be done with TGF inactivated, since all fluid was collected proximal to the oil drop in the proximal tubule. The larger

16 the difference between proximally and distally measured SNGFR, the stronger the activation of TGF.

For measuring SNGFR in study IV [3H]methoxy-inulin was given i.v. in a bolus dose of 25 µCi, followed by a continuous i.v. infusion of 50 µCi hr-1. Randomly chosen tubular segments on the kidney surface were punctured with a sharpened glass pipette, filled with artificial ultrafiltrate stained with Lissamine green, connected to a microperfusion pump. By injecting the stained fluid, the tubular distribution on the kidney surface was defined. A pipette filled with castor oil was injected into a tubular segment. Proximal to the oil block, tubular fluid was collected for 3 minutes while perfusing the nefron distal to the oil block. The volumes of the collected samples were measured from the length in constant bore capillaries.

Isolated blood-perfused juxtamedullary nephron preparation (Study I) Experiments were conducted as described in earlier studies (Casellas and Navar, 1984, Casellas and Moore, 1990). Briefly, a double barrel cannula was introduced into the left renal artery and pressure controlled perfusion of the left kidney was started. The perfused kidney was removed, longitudinally sectioned and the papilla was reflected upwards. All major arteries supplying the rest of the kidney were ligated except for a few afferent arterioles or a small arcuate artery with its branches in the observed area. The preparation was then transferred to a microscope and diameter measurements were taken. During measurements red blood cells were added to the perfusate. At 100 mmHg perfusion pressure, as measured at the level of the renal artery, basal vascular diameter of a selected afferent arteriole was measured and recorded.

Sampling and renin assay (Study II) Plasma renin concentration (PRC) was measured by radio immunoassay of angiotensin I (using the antibody-trapping technique; (Lykkegaard and Poulsen, 1976)). In short, 10 µl plasma from each sample was serially diluted. 5 µl of each dilution of plasma was incubated in duplicates for 24 h together with a mixture of rabbit Ang I antibody and renin substrate from 24 h nephrectomized rats, from which renin had been extracted by

17 affinity chromatography. After the incubation step, the reaction was stopped by addition of 1 ml cold barbital buffer, an Ang I tracer was added and radio immunoassay was performed.

Renal blood flow measurements (Study III) A non-cannulating Transonic probe was placed around the renal vein to measure RBF. A small catheter was introduced into the lumbar artery that was closest to the left renal artery. The catheter was advanced 1-2 mm into the renal artery. The insertion of the catheter did not affect RBF. Throughout the experiment a continuos infusion of heparinized saline was administered at a rate of 5µl min-1.

Measurements of cytosolic calcium concentration (Study III) Several thin slices (thickness 0.5-1 mm) were cut from the midregion of the kidney (Kornfeld, et al., 1994). An afferent arteriole was cut as close as possible to the bifurcation at the interlobular artery, using a sharp knife blade. After dissection, a fluorophore loading procedure was carried out (Salomonsson, et al., 1991). The preparation was loaded with fura-2 acetyl-methoxy ester. Thereafter, the proximal end of the arteriole was aspirated into a holding pipette and the glomerulus was held by a second holding pipette to secure mechanical stability. The preparations were examined with an inverted microscope and the fura-2-loaded preparation was excited with UV light of wavelengths 340 and 380 nm. The fluorescence emitted from each particular area was quantified. Cytosolic calcium concentration was calculated from the fluorescence intensity ratio 340/380 after subtraction of the background.

Dissolving 7-NI (Studies I, II, III & IV) Fresh 7-NI solutions were prepared every second day. Immediately after receiving the compound, the 7-NI was weighed in portions and stored frozen (-20 oC) in an exsicator. It is of greatest importance that 7-NI is stored frozen and completely dry. Even the slightest humidity will markedly reduce the solubility of the compound in water. The requisite quantity of frozen 7-NI was then dissolved in tap water that was gradually heated up to 80 oC, in an ultrasonic-bath. The result is a yellow, clear

18 solution without any sediment. For acute i.p. administration of 7-NI it was dissolved in peanut oil heated to 80 °C.

Statistical analyses (Studies I, II, III & IV) All values are given as mean ± SE. Values from tail-cuff measurements of blood pressure were tested with analysis of variance for repeated measures, followed by the Bonferroni test for pairwise multiple comparisons. The one sample Kolmogorov- Smirnov test for normal distribution was used for all other parameters. Normally distributed parameters were tested for significance with Student´s paired or unpaired t- test and others with the Mann-Whitney U-test. The Wilcoxon´s signed rank test was used for paired observations in the calcium measurement experiments. A P value less than 0.05 was accepted for significance.

Experimental protocol Study I The rats were divided into four groups; -Control; vehicle (tap water) for four weeks -1 -7-NIa; 7-NI (25 mg kg ) i. p. acutely -1 -7-NI1w; 7-NI (2.5 mg day ) in the drinking water for one week -1 -7-NI4w; 7-NI (2.5 mg day ) in the drinking water for four weeks

In the control and 7-NI4w groups systolic blood pressure was measured using the tail- cuff blood pressure measuring system in a non-invasive way, this was done two to three times a week for five weeks. In all groups GFR, diuresis, sodium and potassium excretion were measured. The TGF mechanism was also studied in all groups using the stop-flow technique and SNGFR. To rule out the possibility that effect of 7-NI was due to inhibition of eNOS and not nNOS the isolated blood-perfused juxtamedullary nephron preparation were used. Control animals received tap water for three weeks. 7- NI animals were treated with 7-NI (10 mg kg-1 day-1) over a period of three weeks before the experiments; L-NAME animals received a similar treatment with L-NAME

19 (6 mg day-1). Following this, carbachol (10-5 M) was added to the perfusing blood. After a 15-minute period the diameter was measured again.

Study II Two separate series of experiments were performed, short-term (4 days; series 1) and long-term (4 weeks; series 2) treated rats.

Series 1 The rats were divided into six groups;

-4d-LSV; LS diet for one week -1 -1 -4d-LS7-NI; LS diet for one week and 7-NI (10 mg kg day ) in the drinking water the last four days

-4d-CV; Normal salt diet for one week -1 -1 -4d-C7-NI; Normal salt diet for one week and 7-NI (10 mg kg day ) in the drinking water the last four days

-4d-HSV; HS diet for one week -1 -1 -4d-HS7-NI; HS diet for one week and 7-NI (10 mg kg day ) in the drinking water the last four days In all groups plasma rennin concentration, GFR, diuresis, sodium and potassium excretion were measured.

Series 2 Rats were divided into three diet groups given a low-, control- or a high salt diet for four weeks. Each diet group was subdivided into two groups that were orally treated with vehicle (tap water) or 7-NI in their drinking water (Groups 4w-LSV, 4w-LS7-NI,

4w-CV 4w-C7-NI, 4w-HSV and 4w-HS7-NI). During the four weeks of treatment, the tail- cuff blood pressure measuring system was used to measure systolic blood pressure once a week. GFR and plasma renin concentration were also determined.

Study III The rats were divided into four groups;

20 -Cyoung; vehicle (tap water) for one week -1 -1 -7-NI1w; 7-NI (10 mg kg day ) in the drinking water for one to two weeks

-Cold; vehicle (tap water) for four weeks -1 -1 -7-NI4w; 7-NI (10 mg kg day ) in the drinking water for four weeks

In all groups RBF were measured following an intra renal bolus infusion of 2 ng Ang II in 10 µl saline. After a recovery period 40 ng NE was also given as a bolus of 10 µl into the left renal artery. The NE bolus injection was used as an indication of the renal vessel contractile responsiveness. A systemic i.v. infusion of the unselective NOS- blocker L-NNA (bolus dose10 mg kg-1 and continuos infusion 3 mg kg-1 h-1) was performed and the Ang II and NE bolus infusions were repeated.

Since it is difficult to dissect out afferent arterioles in rats larger than 200 g we refrained from cytosolic calcium concentration measurements in the Cold and 7-NI4w groups. Glomeruli with an attached isolated afferent arteriole were microdissected from rats in the Cyoung and 7-NI1w groups and intracellular calcium changes were investigated. In all series the preparations were exposed to Ang II (10-8 M), NE (10-6 M) and 50 mM K+. A recovery period was allowed to elapse before application of the second drug. NE, Ang II and K+ were administered in random order to avoid cross over effects.

Study IV The rats were divided into four groups; -1 -1 -EuC; infusion of maintenance fluid 0.9% NaCl; 5 ml h kg Bw -1 -1 -VEC; a VE of 50 ml h kg with 0.9% NaCl -1 -1 -VEL-NAME; a VE of 50 ml h kg with 0.9% NaCl and an intra-tubular infusion of L- NAME (10-3 M) to unspecificely block NOS -1 -1 -VE7-NI; a VE of 50 ml h kg with 0.9% NaCl and an i. p. bolus administration of 7- NI (25 mg kg-1) to block nNOS

GFR measurements were made after one hour of VE and further 15 min subsequent to a single Intraperitoneal 7-NI dose of 25 mg kg-1. To study the TGF-mechanism after

21 VE and NOS inhibition the stop-flow and SNGFR techniques were used in all groups. After approximately one hour of VE the micropuncture studies were commenced.

22 RESULTS AND COMMENTS Study I Increased blood pressure in rats after long-term inhibition of the neuronal isoform of nitric oxide synthase Unspecific chronic NOS inhibition in rats leads to elevated blood pressure associated with increased renal vascular resistance. The aim of this study was to examine the effect of chronic (four weeks) selective inhibition of nNOS with 7-NI on rats TGF- system, GFR, and conscious blood pressure. The systolic blood pressure was measured using the non-invasive tail-cuff technique after one week of 7-NI treatment (nNOS inhibition) and had increased from 129 ± 4 to 143 ± 2 mmHg (Fig. 4). GFR was unchanged after one week of nNOS inhibition compared to control while, micropuncture studies revealed a more sensitive TGF than in controls. After four weeks of 7-NI treatment the systolic blood pressure was 152 ± 4 mmHg, but the

180

160 * * *

140 A

P (mmHg) 120

100

80 -101234 Weeks

Figure 4. Systolic blood pressure (PA) measured by non-invasive tail-cuff technique during 5 weeks in control rats ({) and in rats treated with nNOS inhibitor (7-NI; O) in their drinking water. *P<0.05 vs. vehicle treated rats. Republished with permission of The American Society for Clinical Investigation, Inc., from (Ollerstam, et al., 1997); permission conveyed through Copyright Clearance Centre, Inc.

23 elevation in TGF sensitivity had disappeared and GFR was still unchanged compared to control rats. Acute i.p. administration of 7-NI to non-treated rats did not affect blood pressure but significantly decreased GFR and resulted in an increased TGF response with increases both in reactivity indicated by an increased ∆PSF and sensitivity indicated by a decreased TP compared to control. We also performed SNGFR measurements to further evaluate the TGF response. The results support the findings obtained from the stop-flow pressure measurements.

In conclusion, chronic nNOS inhibition led to increased systolic blood pressure measured with the tail-cuff technique. The results suggest that the elevated blood pressure could be caused by an initially increased TGF sensitivity seen after acute and one week nNOS inhibition. This could lead to a decreased GFR, giving fluid retention and an increased body fluid volume.

Study II Effects of long-term inhibition of neuronal nitric oxide synthase on blood pressure and renin release Decreased NaCl load past the MD cells increases the renin release (Skott and Briggs, 1987). Since the MD cells are involved in the renin release and the MD cells contains high levels of nNOS (Mundel, et al., 1992, Thorup, et al., 1993), NO produced by nNOS in MD cells might be involved in the control of renin release. We investigated the effect of short (four days) and long-term (four weeks) 7-NI treatment on blood pressure, PRC and GFR in rats on LS, normal and HS-diets. Long-term 7-NI-treated rats (the LS and C groups) showed increased systolic blood pressure compared to rats without nNOS inhibition (LS: 149±4 vs. 133±3; C: 146±4 vs. 127±4 mmHg). Systolic blood pressure in rats receiving a HS diet did not differ from that in controls. PRC was increased in animals on the long-term LS diet (251±64 mGU ml-1) compared to C and HS rats (42±8 and 39±5 mGU ml-1, respectively), while PRC was not significantly affected by the chronic 7-NI treatment. In rats treated with 7-NI for four days, no effect on blood pressure was seen, but PRC was increased in 7-NI treated LS rats

24 140 * 120

) 100 -1

80

60

40 PRC (mGU ml

20

0 LS V LS 7-NI C V C 7-NI HS V HS 7-NI

Figure 5. Plasma renin concentration (PRC) in anaesthetised rats on a low salt (LS), control (C) or a high salt (HS) diet orally treated for 4 days with vehicle (V) or nNOS inhibitor, 7-NI. *P<0.05 vs. vehicle treated rats within the diet. Republished with permission of Scandinavian Physiological Society from (Ollerstam, et al., 2001). compared to vehicle treated LS rats (Fig. 5; 107±15 vs. 56±1 mGU ml-1). The elevation of PRC in LS rats was further enhanced by 7-NI after four days of treatment, but not affected in rats treated for four weeks.

In conclusion, this suggests that short-term inhibition of nNOS stimulates renin release, but that this stimulatory effect in the long run might be depressed by the increase in blood pressure.

Study III Acute angiotensin II after chronic inhibition of neuronal nitric oxide synthase in rats This study investigated the effect of a bolus dose of Ang II after chronic (one weeks and four weeks) inhibition of nNOS on RBF and cytosolic calcium concentration in smooth muscle cells in afferent arterioles. RBF decreased after a 2 ng bolus dose of Ang II by 60±11% in the control rats vs. 23±8% in the one week 7-NI treated group (Fig. 6). The decreased sensitivity to Ang II after one week of 7-NI treatment compared to control rats persisted after a general NOS inhibition, obtained through i.v.

25 Ang II

120

100

80 * 60

40 RBF (% of Baseline) 20

0 -20 0 20 40 60 80 100 120 140 Time (s)

Figure 6. Renal blood flow in rats given as percent of baseline in response to bolus injection of 2 ng angiotensin II (ANG II) in rats receiving vehicle (O ; n=5) or 7-NI for one to two weeks ({ ; n=5) in their drinking water. *P<0.05 vs. vehicle treated rats.

L-NNA infusion. There were no differences from control in the group receiving 7-NI in their drinking water for four weeks. Ang II gave a transient increase in cytosolic calcium concentration in the smooth muscle cells in afferent arterioles from control rats whereas this response was absent in one week 7-NI-treated rats. A possible explanation for these findings could be a down-regulation of Ang II receptors or an up- regulation of another vasodilatory system such as prostaglandins.

In conclusion, the renal vasculature of rats exhibits a diminished RBF and cytosolic calcium concentration response to Ang II after one week blockade of nNOS while Ang II´s effect on RBF was restored after the four weeks treatment.

Study IV Neuronal nitric oxide synthase inhibition restores the tubuloglomerular feedback response after volume expansion It is imperative for the body to eliminate excess water and solutes entering the body to keep a constant milieu for the cells. The TGF response is attenuated during VE allowing for increased water and salt excretion. This study was

26 50

45

40

35 EUC 30 VEC 25 VEL-NAME

20 VE7-NI

Stop-flow pressure (mmHg) 15 0 10203040 Proximal tubular perfusion rate (nl/min)

Figure 7. Tubuloglomerular feedback response in euvolumic (EUC), 5% volume expanded (VEC), 5% volume expanded with intra-tubular L-NAME (VEL-NAME) and 5% volume expanded with 7-NI Intraperitoneally (VE7-NI) treated rats Curves represent the proximal tubular stop-flow pressures after late proximal perfusion of 0 to 40 nl min-1. *P<0.05 vs. vehicle treated rats. designed to investigate whether the inhibition of all NOS or nNOS re-establishes the attenuated TGF response caused by acute extracellular VE. After addition of intra-tubular L-NAME (via the perfusion fluid) the TGF sensitivity increased, indicated by an increased maximal ∆PSF response (Fig. 7) and the TGF reactivity was also elevated seen as decreased TP. ∆SNGFR increased, indicating an increased TGF. The specific nNOS inhibitor 7-NI elicited a similar increase in

TGF responsiveness seen both in the ∆PSF (Fig. 7), TP and ∆SNGFR. Acute nNOS inhibition with 7-NI after VE decreased GFR from 1.13 ± 0.14 to 0.92 ± 0.08 ml min-1 g-1 kidney wt.

In conclusion, these results suggest that a functioning NO system, especially through the nNOS, may be important in mediating normal renal responses and that an increased production of and/or sensitivity to NO during sustained VE plays an important roll in the adaptive mechanism of the TGF.

27 DISCUSSION Renal NOS is present in at least three different isoforms. In the kidney nNOS has been found to a high extent in the MD cells of the JGA (Wilcox, et al., 1992, Mundel, et al., 1992, Thorup, et al., 1993). Administration of L-Arg analogues block of all three isoforms of NOS, while 7-NI has been shown to selectively inhibit nNOS without systemic effects (Moore, et al., 1993, Thorup, et al., 1996, Ollerstam, et al., 1997). The present studies were performed to examine the effect of acute and chronic nNOS inhibition on blood pressure and kidney function in male Sprague-Dawley rats.

Macula densa derived nitric oxides effects on blood pressure and TGF Long-term treatment of rats with L-Arg analogues such as L-NAME and L-NNA has developed into a model of experimental hypertension (Baylis, et al., 1992, Bouriquet and Casellas, 1995, Bank, et al., 1994, Jover, et al., 1993, Ribeiro, et al., 1992, Bank and Aynedjian, 1993, Qiu, et al., 1998, Matsuoka, et al., 1994). This type of hypertension has been shown to be associated with dramatic changes in total vascular resistance and also in renal hemodynamics, implying that renal NO production is essential for the control of body fluid homeostasis and hence the blood pressure. In study I, acute 7-NI treatment did not induce any change in blood pressure but potently enhanced the TGF responsiveness. It is likely that this TGF activation causes the reduction in total kidney GFR seen in the rats receiving 7-NI acutely. After one week of treatment TGF sensitivity was still increased, although the increase in maximal response and the reduction in TP were not as pronounced as in acutely treated animals. The blood pressure after one week of 7-NI treatment was higher however than in the controls. After four weeks of this treatment the blood pressure was further increased, but the TGF sensitivity was completely normalised and there was no difference in TGF activation compared to that in control animals. There seems to be a parallelism between the development of hypertension in spontaneously hypertensive rats and that resulting from chronic administration of 7-NI. It is therefore particularly interesting to note that results from our lab showed an impaired effect of NOS inhibition in spontaneously hypertensive rats and Milan hypertensive strain rats as compared to their normotensive control strains (Thorup and Persson, 1996).

28 A single-dose of 7-NI acutely administered to both 7-NI1w and 7-NI4w animals did not alter the TGF sensitivity, indicating that the inhibition of nNOS was complete during dietary addition of the drug. Taken together, these results indicate that disturbed NO synthesis or an increased NO breakdown (Wilcox and Welch, 2000, Thorup and Persson, 1996, Welch, et al., 2000) might be an important factor in the development of arterial hypertension.

The findings in study I demonstrate that acute selective inhibition of nNOS results in increased TGF sensitivity, as indicated both by increased maximal TGF responsiveness and by reduction of the tubular flow rate required to activate the TGF response. Measurements of SNGFR after acute administration of 7-NI showed that the TGF system was strongly activated to reduce total kidney GFR. After 1 week of treatment TGF sensitivity was still somewhat increased, but the strong effect on GFR had faded and the blood pressure was now increased. After 4 weeks of 7-NI treatment the blood pressure was even more elevated, but the TGF activity was normalized. These results might imply that the change in TGF sensitivity induced by the 7-NI treatment activates the TGF mechanism to reduce GFR, which in turn leads to volume retention. The increased extracellular volume could then elevate the blood pressure and thereby a new steady state would be reached, where a normalization of TGF occurs at the expense of an increased blood pressure. Our results do not allow us to exclude other ways in which disturbed neuronal NO synthesis might affect the blood pressure. The blood pressure of chronically 7-NI-treated animals could, for example, be affected by altered renin secretion, but in study II we showed that inhibition of nNOS for four weeks does not significantly influence the PRC.

The mechanism behind the increase in blood pressure in study II that occurs after more than two weeks of 7-NI might also be explained as follows. In the LS diet group we found an increase in renin concentration after short-term treatment with 7-NI, which would give rise to an increased production of Ang II. At this point, the elevated Ang II production is not enough to raise blood pressure as seen from the tail-cuff measurements. However, it is well known that Ang II can sensitise the TGF

29 mechanism (Schnermann and Briggs, 1989). Shifting the TGF response to the left will reduce GFR and fluid excretion rate and create a fluid retention and, as mentioned, in study I we saw a reduced GFR in acutely 7-NI treated rats. This could be responsible for the increased blood pressure that occurs after 2-4 weeks of 7-NI treatment in the low- and normal salt diets groups. The increase in blood pressure could then act to reduce the renin release. In the HS diet group neither renin production nor arterial blood pressure measured with tail-cuff method was increased.

Resetting of TGF sensitivity A number of different hormones and local factors are important for setting the level of sensitivity of the TGF. Soon after having described the TGF operation in detail, it became clear that body fluid volume changes were associated with differences in sensitivity of the TGF mechanism.

It is clear that NO produced from the nNOS located in the MD cells is important for the setting of the sensitivity of the TGF system. For some time it has been clear that there is an important function in resetting the TGF system. This means that the information transmitted by local factors like interstitial pressure, prostaglandins, kinins etc. will determine the specific filtration rate the existing condition could allow. E.g. in dehydration the TGF system is activated to large extent even though the filtered load is lower than normal to avoid further fluid losses (Selen, et al., 1983). In the resetting process many different hormones and local factors are involved; Ang II, prostaglandins, kinin, thromboxane etc. (Schnermann, et al., 1998). In animals that are saline volume expanded a resetting to a lower TGF sensitivity occurs, as described earlier by Selen et al. (Selen, et al., 1983). This indicated that a high tubular flow was needed to activate the TGF mechanism and at the same time a low response was achieved.

Resetting of the TGF to a lower level following VE, as was observed in study IV is an important mechanism because the attenuation of the TGF response allows for a greater distal delivery of fluid before a TGF-induced reduction of the GFR takes place. This adaptation of the TGF response takes part in facilitating a return of the extracellular

30 fluid volume to a euvolemic level. If no resetting of the TGF had occurred, the TGF- induced vasoconstriction of the afferent arteriole would reduce the GFR and thereby hinder an adjustment the extracellular fluid volume. Thus, an increased production of NO would lead to a shift of the TGF sensitivity to higher TP and reduced reactivity. We found that extracellular VE reduced the reactivity and sensitivity of the TGF response, seen both in the PSF and SNGFR measurements. General NOS inhibition achieved through L-NAME or nNOS inhibition with 7-NI restored the TGF reactivity and sensitivity. In study IV the experiments measuring whole kidney GFR after extracellular VE, we found that GFR was attenuated by nNOS inhibition. In study I we also reported a decreased GFR after acute nNOS inhibition. As stated above the reduction in total kidney GFR seen after 7-NI administration is probably due to the increased TGF activity. This indicates that NO derived from the MD cells is involved in the resetting of the TGF response seen after VE. The resetting could be due to an increased NO production and/or activity. The process in which NO production is increased in the MD cells in response to different stimuli needs to be further elucidated. For the time being, it is not possible to tell what mechanism(s) that induce NO production during VE. However, since nNOS is a constitutive NOS and has a calcium/calmodulin dependent NO production an increase in MD cell intracellular calcium could be one factor responsible for the increased NOS activity. It is known that VE might release bradykinin in the kidney and bradykinin release may in turn release calcium in the MD cells, as in other renal epithelial cells (Aboolian and Nord, 1988, Pidikiti, et al., 1985).

Macula densa derived nitric oxide inhibition leads to up-regulation of other vasodilatory systems?

In study I rats treated for four weeks with 7-NI showed an initial transient drop in PSF to almost the same magnitude as the sustained level in acutely treated nephrons. This might indicate that chronic nNOS inhibition, which in the kidney predominately affects MD-NOS, exposes the involvement of the other NOS isoforms. It is also conceivable that chronic nNOS inhibition unveils an unknown vasodilator that is not as fast as NO but capable of taking over the role as a modulator of the afferent

31 arteriolar tonus. The results from study III could also support the view of another vasodilator system up-regulated in this situation, counteracting the Ang II induced vasoconstriction decreasing the RBF. In study III we also found a greater increase in

MAP after L-NNA infusion in the 7-NI4w group compared to the age matched control group. This finding might indicate that some other NOS has been upregulated. This is further supported by that we, in study I, found a significantly increased nitrate/nitrite excretion in the rats receiving 7-NI for three weeks compared to control rats (Ollerstam, et al., 1997). We have also in preliminary results found that in one week nNOS inhibited rats an iNOS inhibition increased the TGF sensitivity and that L-NAME increased further. This indicates that in this situation iNOS and eNOS is upregulated.

Thus, we postulated that the blunted Ang II response in the 7-NI1w could partly be explained by an up-regulation of another, non-nNOS, NO-system. To test this hypothesis we infused systemically the non-selective NOS inhibitor L-NNA before the acute Ang II bolus in all four groups. In this regard we found a tendency to a larger response to Ang II on RBF after L-NNA in all groups but this difference only reached statistical significance in the 7-NI1w group. This finding is consistent with the results of other studies where acute administration of L-Arg analogues, that unselectively inhibits NOS, has proven to augment the renovascular response to Ang II (Kornfeld, et al., 1994, Ito, et al., 1991). MAP increased significantly in all four groups after administration of the general NOS blocker, which is also consistent with other studies in the field (Zatz and de Nucci, 1991). The increased response to Ang II was probably not due to a generally increased responsiveness of the renal vascular system, since the response to NE was not affected by the L-NNA treatment in any of the groups.

Our results in study III thus reflected a specific action of the NO blockade on the signal pathways elicited by the activation of Ang II. Despite the augmented Ang II response after L-NNA treatment, the difference between the Cyoung and 7-NI1w groups persisted. This indicates that the blunted Ang II response in the rats treated with 7-NI for one to two weeks is not due to an unspecific up-regulation of another NOS system. Furthermore, this observation supports the specificity of the 7-NI treatment, i.e. the L- NNA treatment has another site of action than 7-NI. The existence of a second

32 vasodilator involved in TGF modulation was proposed by Vallon and Thomson (Vallon and Thomson, 1995). Furthermore, the results of another study of chronic NOS inhibition by Bouriquet and Casellas (Bouriquet and Casellas, 1995) might support the idea of a second vasodilatation system in renal arterioles. They studied the autoregulatory response in rats subjected to chronic NOS inhibition by measurements of the arteriolar diameter in the isolated juxtamedullary nephron preparation and found a diminished arteriolar response to increases in renal perfusion pressure.

Macula densa derived NOS and renin release Changes in salt intake may influence renin synthesis and secretion by several independent mechanisms: a signal from the MD cells, sympathetic nerves, blood pressure, circulating hormones and local hormones. MD-mediated renin release is clearly established as an acute control mechanism for renin release following changes in salt concentration at the macula densa site (Skott and Briggs, 1987). A decrease in NaCl concentration here stimulates renin release and vice versa. As stated earlier, MD cells are rich in nNOS (Wilcox, et al., 1992, Mundel, et al., 1992, Thorup, et al., 1993). Many investigators have studied the effect of different salt diets on MD nNOS expression. In the kidney, cortical and medullar nNOS are stimulated in different situations. The medullar nNOS has been shown to increase after high sodium intake (Roczniak, et al., 1998, Mattson and Higgins, 1996). Low salt diets increase the nNOS expression in the MD cells (Singh, et al., 1996, Tojo, et al., 2000). He et al. (He, et al., 1995) demonstrated in the isolated perfused juxtaglomerular apparatus that inhibition of NOS at the MD inhibited the renin response to a low sodium concentration. Beierwaltes (Beierwaltes, 1995) showed that acute infusion of 7-NI in rats blocked the acute effect on renin release to a bolus injection of furosemide, which acutely stimulates renin release by inhibition of MD salt transport. Siragy et al. (Siragy and Carey, 1997) showed that a LS diet caused an increased cGMP concentration in renal interstitial fluid, which was blunted by treatment with 7-NI. These data suggest that the NO production is increased in rats on a low salt LS diet and is consistent with our finding that 7-NI had a greater effect in LS rats than in HS rats in study II.

33 Our results do not support the hypothesis that MD-derived NO is a mandatory participant in the long-term adaptation of the renin-angiotensin system to changes in dietary salt intake. Further support for this view is the study by Zanchi et al. (Zanchi, et al., 1995) were they found that PRC increased after NOS-inhibition in rats receiving a normal salt diet. In the group receiving a LS diet, PRC was also increased, but L- NAME had no further effect. Studies with nNOS knockout mice also supports this. The nNOS knockout mice show a normal response in renin release following a low salt intake (Harding, et al., 1997). Another study (Wagner, et al., 2000) showed that neither eNOS nor nNOS is essential for up- or down-regulation of renin expression in the eNOS and nNOS deficient mice. Despite these findings, the level of renin expression during a control situation was only half of that of the wild type mice in the eNOS -/- mice, while the renin expression in the nNOS -/- mice was unaltered. In the long run NO, from MD nNOS, is probably not important for the up- or down- regulation of renin secretion. Even though, in the acute state of nNOS inhibition, it seems to be important for the increase in renin release following low salt.

In study III the Ang II induced decrease in RBF is reduced after one to two weeks of p.o. 7-NI. While after four weeks of 7-NI treatment the Ang II induced effect on the RBF is returned to normal. This is in contrast to the effect of acute blockade of nNOS seen in vivo studies (Ichihara, et al., 1998). However, the decreased renal vascular response to Ang II in the 7-NI1w group seen in vivo was confirmed in vitro in experiments where we measured cytosolic calcium concentration in smooth muscle cells of microdissected afferent arterioles. In this series of experiments we found that there was a significant cytosolic calcium concentration response to Ang II in the control group, as has previously been shown by us and by others, in intact arterioles and dispersed smooth muscle cells from afferent arterioles (Kornfeld, et al., 1994, Salomonsson, et al., 1997, Iversen and Arendshorst, 1998, Conger, et al., 1993). This response was abolished in afferent arterioles from rats in the 7-NI1w group. In contrast, the response to NE was not different between the 7-NI treated and the control groups in the present study.

Here, we present a tentative model that might explain the somewhat paradoxical finding in the present study that blockade of a NO system that normally is considered

34 to possess vasodilating properties attenuates the vasoconstrictive action of a receptor agonist. The elevated PRC in LS diet rats after four days of 7-NI treatment in study II might give rise to an increased activity of Ang II, which leads to an increased activation of the Ang II cell surface receptors (Ollerstam, et al., 2001). As mentioned, long-term occupancy can cause down-regulation of receptors. An observation in agreement with elevated Ang II levels from the present study was that the afferent arterioles microdissected from the 7-NI1w group were substantially longer than those from the age matched control group. This finding might be explained by the trophic action of increased Ang II levels (Berk, et al., 1989). In a previous study we found that after four weeks of 7-NI treatment the elevated renin levels were returned to normal, indicating that the stimulation of renin release seen after four days of nNOS blockade is, in the long run, counteracted by other mechanisms. This notion is in accordance with the present finding indicating no difference between the Ang II induced reduction in RBF between the 7-NI4w group and their age matched controls. An alternative model to explain the absent Ang II response in the 7-NI1w group is derived from the observation that the Ang II induced tachyphylaxis in isolated rabbit afferent arterioles is attenuated after stimulation of the NO system with L-Arg (Kornfeld, et al., 1997). This is, however, a controversial issue and in a study by Ito et al (Ito, et al., 1991), no effect of NOS blockade was found on Ang II induced tachyphylaxis in perfused rabbit afferent arterioles. This might imply that the tendency to tachyphylaxis induced by receptor occupancy under normal circumstances is counteracted by the presence of local stimulation with NO.

35 CONCLUSIONS Taken together, the results from the present studies support the view that NO, derived from nNOS in the MD cells, is an important modulator of TGF. We can also conclude that:

• chronic nNOS inhibition leads to increased blood pressure. Our results suggest that the elevated blood pressure could be caused by an initially increased TGF sensitivity, leading to decreased GFR and an increased body fluid volume.

• inhibition of nNOS, stimulates renin release but that this stimulatory effect in the long run might be depressed by the increase in blood pressure.

• blockade of nNOS for one week leads to a diminished RBF and afferent arteriole smooth muscle cell cytosolic calcium concentration response following a bolus dose of Ang II.

• a functioning NO system, especially through the nNOS, may be important in mediating normal renal responses and that an increased production of and/or sensitivity to NO during sustained VE plays an important roll in the adaptive mechanism of the TGF.

36 SUMMARY IN SWEDISH/ SAMMANFATTNING PÅ SVENSKA För högt blodtryck är ett av de största hälsoproblemen i västvärlden. Det är sedan lång tid välkänt att den extracellulära vätskevolymen påverkar blodtrycksnivån. Njuren spelar en stor roll för regleringen av den extracellulära vätskevolymen. Små dagliga förändringar i vätskeutsöndring påverkar den extracellulära vätskevolymen. Regleringen av njurens vätskeutsöndring är en fråga om hur balansen mellan den glomerulära filtrationshastigheten (GFR) och resorptionen i tubulus kontrolleras och regleras. Ett av de viktigaste kontrollsystemen involverat i denna reglering är den tubuloglomerulära-feedbacken (TGF). Den juxtaglomerulära-apparaten med maculadensacellerna är beläget i distala tubuli. Dessa celler är sensorerna i TGF, som påverkar tonus i den tillförande arteriolen (leder blodet till glomerulus där filtrationen sker). Maculadensacellerna känner av salthalten i tubulusvätskan och vid höga halter skickar de en signal till den tillförande arteriolen och den drar ihop sig. Detta sänker GFR och salthalten förbi maculadensacellerna blir lägre. Ett sänkt flöde till maculadensa ökar frisättningen av renin vilket ger angiotensin II (Ang II) -bildning och blodtrycket höjs. Kväveoxid (NO) har visat sig vara viktig i moduleringen av TGF-svaret och motverkar vasokonstriktionen. Den neuronala isoformen av NO- syntetas (nNOS) finns i njuren framför allt i maculadensacellerna.

Delarbete I Increased blood pressure in rats after long-term inhibition of the neuronal isoform of nitric oxide synthase Anna Ollerstam, Janos Pittner, A. Erik G. Persson & Christian Thorup. J. Clin. Invest. 1997, 99, 2212-2218.

Syftet med studien var att undersöka hur en blockad av det neuronala NO-syntetaset i maculadensa påverkade blodtrycket och den renala hemodynamiken hos råttor. För att göra detta valde vi 7-nitroindazole (7-NI) som endast blockerar nNOS och på så sätt undviker så många oselektiva effekter som möjligt. Akut, selektiv blockering av nNOS resulterade i ökad TGF-känslighet och reaktivitet. SNGFR-mätningar (GFR mätningar i ett nefron) efter akut 7-NI administration visade att TGF var aktiverad för att minska

37 totala njur-GFR. Efter en veckas kronisk behandling var TGF-känsligheten fortfarande något förhöjd men effekten på GFR var borta och blodtrycket var förhöjt. Efter fyra veckors behandling var blodtrycket ytterligare förhöjt men TGF var normaliserat. Dessa resultat indikerar att nNOS hämning påverkar TGF så att GFR reduceras vilket leder till volymretention. Den ökade extracellulära volymen höjer blodtrycket och inhiberar TGF-mekanismen. Härigenom uppnås ett nytt steady-state läge där TGF normaliseras på bekostnad av det höjda blodtrycket.

Delarbete II Effects of long-term inhibition of neuronal nitric oxide synthase on blood pressure and renin release Anna Ollerstam, Ole Skøtt, Joakim Ek, A. Erik G. Persson & Christian Thorup. Acta Physiol Scand 2001, 173, 351-358.

NO från nNOS i maculadensa i njuren kan vara involverat i reninfrisättning. I denna studie undersökte vi hur nNOS-blockad och olika saltdieter i fyra dagar och fyra veckor påverkade blodtrycket, GFR och plasmakoncentrationen av renin. Långtidsblockering av nNOS höjde blodtrycket hos råttor efter normal- och lågsaltdiet. Ökningen i blodtryck var inte kopplat till några förändringar i GFR. Reninkoncentrationen i plasma ökade efter lågsaltdiet men ändrades inte signifikant av fyra veckors nNOS-blockad. Fyra dagars 7-N- behandling ökade plasmareninkoncentrationen hos de råttor som fick lågsaltdiet. Detta pekar på att blockering av nNOS stimulerar reninfrisättningen men att denna stimulerande effekt minskas av ökningen i blodtryck i den längre 7-NI-behandlingen.

Delarbete III Acute angiotensin II after chronic inhibition of neuronal nitric oxide synthase in rats Anna Ollerstam, Max Salomonsson & A. Erik G. Persson. 2002. Manuskript.

Denna studie undersökte hur långtidsblockering av nNOS påverkade det renala blodflödet and kalciumsvaret hos de glatta muskelcellerna i de tillförande arteriolerna

38 efter aktivering med Ang II och noradrenalin (NE). Blodflödesmätningar efter Ang II och NE bolusinjektioner i njurartären före och efter systemisk oselektiv NOS-blockad gjordes i två kontrollgrupper och två grupper behandlade med 7-NI efter en respektive fyra veckor. Kalciumsvaret mätes i arterioler från kontrollråttor och råttor behandlade med 7-NI i en vecka. Det var ingen skillnad i njurblodflödessvaret efter NE mellan grupperna. Efter en veckas 7-NI behandling minskade Ang II svaret. Denna minskade känslighet för Ang II bestod efter allmän NOS blockad. Efter fyra veckors 7-NI behandling var skillnaderna i Ang II känslighet borta. Ang II gav en transient ökning i intracellulärt kalcium hos kontrollråttorna. Denna ökning var minskad hos de 7-NI behandlade råttorna. En veckas blockering av 7-NI minskade alltså känsligheten för Ang II medan svaret på NE var oförändrat.

Delarbete IV Neuronal nitric oxide synthase inhibition restores the tubuloglomerular feedback response after volume expansion Russell Brown*, Anna Ollerstam* & A. Erik G. Persson A. 2002. Manuskript. *Delat förstanamn

Det har tidigare visats i vår grupp att förändringar i den extracellulära vätskevolymen eller i blodtrycket kan ändra känsligheten i TGF. Detta sker via ändringar i njurens interstitiella hydrostatiska och onkotiska tryck eller ändrade blodkoncentrationer av vissa hormoner. Under volymsexpansion fann vi som i tidigare studier ett inhiberat TGF-system. I detta arbete studerade vi om NO från nNOS var involverat i den känslighetsförändringen av TGF som sker efter volymsexpansion med hjälp av två olika mikropunktionstekniker. Vi fann att nNOS blockering gav tillbaka hela TGF- svaret efter extracellulär volymsexpansion. En allmän NO blockad intratubulärt gav ingen ytterligare effekt. Vi fann också en sänkt GFR efter nNOS blockaden hos de volymsexpanderade djuren. Detta visar att nNOS NO är involverat i känslighetsförändringen av TGF efter extracellulär volymsexpansion.

39 ACKNOWLEDGEMENT This thesis is of course the result of the collaboration with many people and I wish to especially thank,

My supervisor, Erik Persson, for your belief in me and introducing me to the field of renal physiology and your never failing enthusiasm concerning the project,

Christian Thorup for teaching me the micropunction technique, encouragement, understanding and so much more,

Russell Brown for fruitful co-operation, revising the English and being a good roomie,

Pernilla Lindström for co-operation, gossip, laughs, encouragement and also being a excellent roomie,

Lena Holm, Peter Hansell, Örjan Källskog and Mats Sjöquist for good discussions, encouragement in teaching and vast knowledge of physiology which you all share,

My co-authors Ole Skøtt and Max Salomonsson for being such excellent collaborators,

Angelica Fasching, Birgitta Klang, Alf Johansson, Monika Lundahl and Britta Isaksson for help with lots of different things, taking care of rats, making everything work and above all making the lab a pleasant place to be,

Gunno Nilssson and Erik Ekström for helping me with technical matters,

AnnSofie Göransson, Lena Carlsson, Karin Öberg and Agneta Bergwall for excellent help in administration,

The Animal department, especially Kattis and Susanne, for taking such excellent care of my animals and always being so helpful, The Photo department, for helping me with posters and pictures, The Cleaning staff, for keeping it nice and tidy,

40 Cissi for being a great friend, so open to discussions about everything in life and being such a party animal, Markus for always giving me a happy face and encouragement, Fredrik for coffee, candy, cakes and coca colas and all kinds of help, Micke for helping me with the receptor studies, Ruisheng for nice discussions about the macula densa cells, Antonio, Mark and Janos for good discussions, Johanna for being a happy person helping everybody around her, Louise for being a great person to share a hotel room with,

My dear, super-friends Viktoria, Låtta, Mia and Isabella for being such fantastic friends with whom you can talk about anything, science or not (mostly the last),

Cilla, Isabella, Lotta, Stefan, Viktoria, Markus, Låtta, Erik, Danne, Anders, Lina, Anna, Jonas, Fredrik, Viktoria, Patrik, Anna and all my other friends for just being my friends,

My husband Olof and daughter Saga for support, love and for helping me keeping my priorities right in life,

My family; my mother Agneta, my sisters Eva and Åsa and their families, my family in-law Lena, Sigge and Kajsa for all their support and love. My father Leif for making it possible to have a dissertation party. I especially want to thank my Morfar, Per-Ingemar, for lots of love, encouragement and support, for always believing in me. I am so sorry you could not be here today.

* * * This thesis was financially supported by funds from the Medical faculty of Lund and Uppsala Universities, the Swedish Medical Research Council, the foundations of Ingabritt and Arne Lundberg, Berth von Kantzow, Wallenberg and Wenner-Gren.

41 REFERENCES 1. Golgi C. Annotazioni intorno all´Istologia dei rein deeúomo e di altri mammiferi e sull´Istogenesi dei canalicoli oriniferi. Atti R Acad Naz Lincei Rendiconti 1889;5:334-342. 2. Forster RP, Maes JP. Effects of experimental neurogenic hypertension on renal blood flow and glomerular filtration rate in intact denervated kidneys of unanesthetized rabbits with adrenal glands demedullated. Am J Physiol 1947;150:534- 40. 3. Baer PG, Navar LG. Renal vasodilation and uncoupling of blood flow and filtration rate autoregulation. Kidney Int 1973;4:12-21. 4. Arendshorst WJ, Finn WF, Gottschalk CW. Autoregulation of blood flow in the rat kidney. Am J Physiol 1975;228:127-33. 5. Thurau K. Renal hemodynamics. Am J Med 1964;36:698-719. 6. Guyton AC, Langston JB, Navar G. Theory for renal autoregulation by feedback of the juxtaglomerular apparatus. Circ Res 1964;15:187-197. 7. Ruyter JHC. Über einen merkwurdigen Abschnitt der Vasa afferentia in der Mäuseniere. Z Wissensch Biol 1925;2:242-8. 8. Goormaghtigh N. Existence of an endocrine gland in the media of renal arterioles. Proc Soc Exp Biol Med 1939;42:688-9. 9. Gonzalez E, Salomonsson M, Muller-Suur C, Persson AE. Measurements of macula densa cell volume changes in isolated and perfused rabbit cortical thick ascending limb. I. Isosmotic and anisosmotic cell volume changes. Acta Physiol Scand 1988;133:149-57. 10. Schlatter E, Salomonsson M, Persson AE, Greger R. Macula densa cells sense luminal NaCl concentration via furosemide sensitive Na+2Cl-K+ cotransport. Pflugers Arch 1989;414:286-90. 11. Wright FS, Schnermann J. Interference with feedback control of glomerular filtration rate by furosemide, triflocin, and cyanide. J Clin Invest 1974;53:1695-708.

42 12. Odlind B, Lonnerholm G. Renal tubular secretion and effects of chlorothiazide, hydrochlorothiazide and clopamide: a study in the avian kidney. Acta Pharmacol Toxicol (Copenh) 1982;51:187-97. 13. Lapointe JY, Bell PD, Cardinal J. Direct evidence for apical Na+:2Cl-:K+ cotransport in macula densa cells. Am J Physiol 1990;258:F1466-9. 14. Ren Y, Yu H, Wang H, Carretero OA, Garvin JL. Nystatin and valinomycin induce tubuloglomerular feedback. Am J Physiol Renal Physiol 2001;281:F1102-8. 15. Thurau K, Schnermann J. The Na concentration of the macula densa cells as a factor regulating glomerular filtration rate (micropuncture studies). 1965. J Am Soc Nephrol 1998;9:925-34. 16. Thurau KW, Dahlheim H, Gruner A, Mason J, Granger P. Activation of renin in the single juxtaglomerular apparatus by sodium chloride in the tubular fluid at the macula densa. Circ Res 1972;31:Suppl 2:182-6. 17. Skott O, Briggs JP. Direct demonstration of macula densa-mediated renin secretion. Science 1987;237:1618-20. 18. Osswald H, Hermes HH, Nabakowski G. Role of adenosine in signal transmission of tubuloglomerular feedback. Kidney Int Suppl 1982;12:S136-42. 19. Thomson S, Bao D, Deng A, Vallon V. Adenosine formed by 5'- nucleotidase mediates tubuloglomerular feedback. J Clin Invest 2000;106:289-98. 20. Brown R, Ollerstam A, Johansson B, et al. Abolished tubuloglomerular feedback and increased plasma renin in adenosine A1 receptor-deficient mice. Am J Physiol Regul Integr Comp Physiol 2001;281:R1362-7. 21. Selen G, Muller-Suur R, Persson AE. Activation of the tubuloglomerular feedback mechanism in dehydrated rats. Acta Physiol Scand 1983;117:83-9. 22. Schnermann J, Traynor T, Yang T, et al. Tubuloglomerular feedback: new concepts and developments. Kidney Int Suppl 1998;67:S40-5. 23. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 1980;288:373-6. 24. Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med 1993;329:2002-12.

43 25. Ahn KY, Mohaupt MG, Madsen KM, Kone BC. In situ hybridization localization of mRNA encoding inducible nitric oxide synthase in rat kidney. Am J Physiol 1994;267:F748-57. 26. Wilcox CS, Welch WJ, Murad F, et al. Nitric oxide synthase in macula densa regulates glomerular capillary pressure. Proc Natl Acad Sci U S A 1992;89:11993-7. 27. Mundel P, Bachmann S, Bader M, et al. Expression of nitric oxide synthase in kidney macula densa cells. Kidney Int 1992;42:1017-9. 28. Thorup C, Sundler F, Ekblad E, Persson AE. Resetting of the tubuloglomerular feedback mechanism by blockade of NO- synthase. Acta Physiol Scand 1993;148:359-60. 29. McKee M, Scavone C, Nathanson JA. Nitric oxide, cGMP, and hormone regulation of active sodium transport. Proc Natl Acad Sci U S A 1994;91:12056-60. 30. Mattson DL, Bellehumeur TG. Neural nitric oxide synthase in the renal medulla and blood pressure regulation. Hypertension 1996;28:297-303. 31. Roczniak A, Zimpelmann J, Burns KD. Effect of dietary salt on neuronal nitric oxide synthase in the inner medullary collecting duct. Am J Physiol 1998;275:F46-54. 32. Roczniak A, Fryer JN, Levine DZ, Burns KD. Downregulation of neuronal nitric oxide synthase in the rat remnant kidney. J Am Soc Nephrol 1999;10:704-13. 33. Wu F, Park F, Cowley AW, Jr., Mattson DL. Quantification of nitric oxide synthase activity in microdissected segments of the rat kidney. Am J Physiol 1999;276:F874-81. 34. Bachmann S, Bosse HM, Mundel P. Topography of nitric oxide synthesis by localizing constitutive NO synthases in mammalian kidney. Am J Physiol 1995;268:F885-98. 35. Wang X, Lu M, Gao Y, Papapetropoulos A, Sessa WC, Wang W. Neuronal nitric oxide synthase is expressed in principal cell of collecting duct. Am J Physiol 1998;275:F395-9.

44 36. Wilcox CS, Welch WJ. Interaction between nitric oxide and oxygen radicals in regulation of tubuloglomerular feedback. Acta Physiol Scand 2000;168:119-24. 37. Thorup C, Persson AE. Impaired effect of nitric oxide synthesis inhibition on tubuloglomerular feedback in hypertensive rats. Am J Physiol 1996;271:F246-52. 38. Welch WJ, Tojo A, Wilcox CS. Roles of NO and oxygen radicals in tubuloglomerular feedback in SHR. Am J Physiol Renal Physiol 2000;278:F769-F776. 39. Baylis C, Harton P, Engels K. Endothelial derived relaxing factor controls renal hemodynamics in the normal rat kidney. J Am Soc Nephrol 1990;1:875-81. 40. Tolins JP, Palmer RM, Moncada S, Raij L. Role of endothelium-derived relaxing factor in regulation of renal hemodynamic responses. Am J Physiol 1990;258:H655-62. 41. Baylis C, Mitruka B, Deng A. Chronic blockade of nitric oxide synthesis in the rat produces systemic hypertension and glomerular damage. J Clin Invest 1992;90:278-81. 42. Bouriquet N, Casellas D. Chronic L-NAME hypertension in rats and autoregulation of juxtamedullary preglomerular vessels. Am J Physiol 1995;269:F190- 7. 43. Bank N, Aynedjian HS, Khan GA. Mechanism of vasoconstriction induced by chronic inhibition of nitric oxide in rats. Hypertension 1994;24:322-8. 44. Jover B, Herizi A, Ventre F, Dupont M, Mimran A. Sodium and angiotensin in hypertension induced by long-term nitric oxide blockade. Hypertension 1993;21:944-8. 45. Ribeiro MO, Antunes E, de Nucci G, Lovisolo SM, Zatz R. Chronic inhibition of nitric oxide synthesis. A new model of arterial hypertension. Hypertension 1992;20:298-303. 46. Vallance P, Collier J, Moncada S. Effects of endothelium-derived nitric oxide on peripheral arteriolar tone in man. Lancet 1989;2:997-1000. 47. Johnson RA, Freeman RH. Sustained hypertension in the rat induced by chronic blockade of nitric oxide production. Am J Hypertens 1992;5:919-22.

45 48. Cabrera C, Bohr D. The role of nitric oxide in the central control of blood pressure. Biochem Biophys Res Commun 1995;206:77-81. 49. Moore PK, Babbedge RC, Wallace P, Gaffen ZA, Hart SL. 7-Nitro indazole, an inhibitor of nitric oxide synthase, exhibits anti-nociceptive activity in the mouse without increasing blood pressure. Br J Pharmacol 1993;108:296-7. 50. Beierwaltes WH. Selective neuronal nitric oxide synthase inhibition blocks furosemide-stimulated renin secretion in vivo. Am J Physiol 1995;269:F134-9. 51. Thorup C, Erik A, Persson G. Macula densa derived nitric oxide in regulation of glomerular capillary pressure. Kidney Int 1996;49:430-6. 52. Mayer B, Klatt P, Werner ER, Schmidt K. Molecular mechanisms of inhibition of porcine brain nitric oxide synthase by the antinociceptive drug 7-nitro- indazole. Neuropharmacology 1994;33:1253-9. 53. Tigerstedt R, Bergman PG. Niere und Kreislauf. Skand Arch Physiol 1898;8:223-71. 54. Schnermann J. Juxtaglomerular cell complex in the regulation of renal salt excretion. Am J Physiol 1998;274:R263-79. 55. Lorenz JN, Weihprecht H, Schnermann J, Skott O, Briggs JP. Renin release from isolated juxtaglomerular apparatus depends on macula densa chloride transport. Am J Physiol 1991;260:F486-93. 56. Guyton AC. Textbook of medical physiology. 8th ed. Philadelphia: W. B. Saunders Company, 1991. 57. Naess PA, Christensen G, Kirkeboen KA, Kiil F. Effect on renin release of inhibiting renal nitric oxide synthesis in anaesthetized dogs. Acta Physiol Scand 1993;148:137-42. 58. Persson PB, Baumann JE, Ehmke H, Hackenthal E, Kirchheim HR, Nafz B. Endothelium-derived NO stimulates pressure-dependent renin release in conscious dogs. Am J Physiol 1993;264:F943-7. 59. Fischer E, Schnermann J, Briggs JP, Kriz W, Ronco PM, Bachmann S. Ontogeny of NO synthase and renin in juxtaglomerular apparatus of rat kidneys. Am J Physiol 1995;268:F1164-76.

46 60. Schricker K, Hamann M, Kurtz A. Nitric oxide and prostaglandins are involved in the macula densa control of the renin system. Am J Physiol 1995;269:F825-30. 61. Vidal MJ, Romero JC, Vanhoutte PM. Endothelium-derived relaxing factor inhibits renin release. Eur J Pharmacol 1988;149:401-2. 62. He XR, Greenberg SG, Briggs JP, Schnermann JB. Effect of nitric oxide on renin secretion. II. Studies in the perfused juxtaglomerular apparatus. Am J Physiol 1995;268:F953-9. 63. Gambaryan S, Wagner C, Smolenski A, et al. Endogenous or overexpressed cGMP-dependent protein kinases inhibit cAMP- dependent renin release from rat isolated perfused kidney, microdissected glomeruli, and isolated juxtaglomerular cells. Proc Natl Acad Sci U S A 1998;95:9003-8. 64. Kurtz A, Gotz KH, Hamann M, Wagner C. Stimulation of renin secretion by nitric oxide is mediated by phosphodiesterase 3. Proc Natl Acad Sci U S A 1998;95:4743-7. 65. Casellas D, Navar LG. In vitro perfusion of juxtamedullary nephrons in rats. Am J Physiol 1984;246:F349-58. 66. Casellas D, Moore LC. Autoregulation and tubuloglomerular feedback in juxtamedullary glomerular arterioles. Am J Physiol 1990;258:F660-9. 67. Lykkegaard S, Poulsen K. Ultramicroassay for plasma renin concentration using the antibody trapping technique. Anal Biochem 1976;75:250-9. 68. Kornfeld M, Gutierrez AM, Gonzalez E, Salomonsson M, Persson AE. Cell calcium concentration in glomerular afferent and efferent arterioles under the action of noradrenaline and angiotensin II. Acta Physiol Scand 1994;151:99-105. 69. Salomonsson M, Gonzalez E, Westerlund P, Persson AE. Intracellular cytosolic free calcium concentration in the macula densa and in ascending limb cells at different luminal concentrations of sodium chloride and with added furosemide. Acta Physiol Scand 1991;142:283-90. 70. Ollerstam A, Pittner J, Persson AE, Thorup C. Increased blood pressure in rats after long-term inhibition of the neuronal isoform of nitric oxide synthase. J Clin Invest 1997;99:2212-8.

47 71. Ollerstam A, Skøtt O, Ek J, Persson AEG, Thorup C. Effects of long-term inhibitiom of neuronal nitric oxide synthase on blood pressure and renin release. Acta Physiol Scand 2001;173:351-358. 72. Bank N, Aynedjian HS. Role of EDRF (nitric oxide) in diabetic renal hyperfiltration. Kidney Int 1993;43:1306-12. 73. Qiu C, Muchant D, Beierwaltes WH, Racusen L, Baylis C. Evolution of chronic nitric oxide inhibition hypertension: relationship to renal function. Hypertension 1998;31:21-6. 74. Matsuoka H, Nishida H, Nomura G, Van Vliet BN, Toshima H. Hypertension induced by nitric oxide synthesis inhibition is renal nerve dependent. Hypertension 1994;23:971-5. 75. Schnermann J, Briggs JP. Single nephron comparison of the effect of loop of Henle flow on filtration rate and pressure in control and angiotensin II-infused rats. Miner Electrolyte Metab 1989;15:103-7. 76. Aboolian A, Nord EP. Bradykinin increases cytosolic free [Ca2+] in proximal tubule cells. Am J Physiol 1988;255:F486-93. 77. Pidikiti N, Gamero D, Gamero J, Hassid A. Bradykinin-evoked modulation of cytosolic Ca2+ concentrations in cultured renal epithelial (MDCK) cells. Biochem Biophys Res Commun 1985;130:807-13. 78. Ito S, Johnson CS, Carretero OA. Modulation of angiotensin II-induced vasoconstriction by endothelium-derived relaxing factor in the isolated microperfused rabbit afferent arteriole. J Clin Invest 1991;87:1656-63. 79. Zatz R, de Nucci G. Effects of acute nitric oxide inhibition on rat glomerular microcirculation. Am J Physiol 1991;261:F360-3. 80. Vallon V, Thomson S. Inhibition of local nitric oxide synthase increases homeostatic efficiency of tubuloglomerular feedback. Am J Physiol 1995;269:F892-9. 81. Mattson DL, Higgins DJ. Influence of dietary sodium intake on renal medullary nitric oxide synthase. Hypertension 1996;27:688-92. 82. Singh I, Grams M, Wang WH, et al. Coordinate regulation of renal expression of nitric oxide synthase, renin, and angiotensinogen mRNA by dietary salt. Am J Physiol 1996;270:F1027-37.

48 83. Tojo A, Kimoto M, Wilcox CS. Renal expression of constitutive NOS and DDAH: separate effects of salt intake and angiotensin. Kidney Int 2000;58:2075-83. 84. Siragy HM, Carey RM. The subtype 2 (AT2) angiotensin receptor mediates renal production of nitric oxide in conscious rats. J Clin Invest 1997;100:264-9. 85. Zanchi A, Schaad NC, Osterheld MC, et al. Effects of chronic NO synthase inhibition in rats on renin-angiotensin system and sympathetic nervous system. Am J Physiol 1995;268:H2267-73. 86. Harding P, Sigmon DH, Alfie ME, et al. Cyclooxygenase-2 mediates increased renal renin content induced by low-sodium diet. Hypertension 1997;29:297- 302. 87. Wagner C, Godecke A, Ford M, Schnermann J, Schrader J, Kurtz A. Regulation of renin gene expression in kidneys of eNOS- and nNOS-deficient mice. Pflugers Arch 2000;439:567-72. 88. Ichihara A, Inscho EW, Imig JD, Navar LG. Neuronal nitric oxide synthase modulates rat renal microvascular function. Am J Physiol 1998;274:F516-24. 89. Salomonsson M, Kornfeld M, Gutierrez AM, Magnusson M, Persson AE. Effects of stimulation and inhibition of protein kinase C on the cytosolic calcium concentration in rabbit afferent arterioles. Acta Physiol Scand 1997;161:271-9. 90. Iversen BM, Arendshorst WJ. ANG II and vasopressin stimulate calcium entry in dispersed smooth muscle cells of preglomerular arterioles. Am J Physiol 1998;274:F498-508. 91. Conger JD, Falk SA, Robinette JB. Angiotensin II-induced changes in smooth muscle calcium in rat renal arterioles. J Am Soc Nephrol 1993;3:1792-803. 92. Berk BC, Vekshtein V, Gordon HM, Tsuda T. Angiotensin II-stimulated protein synthesis in cultured vascular smooth muscle cells. Hypertension 1989;13:305- 14. 93. Kornfeld M, Gutierrez AM, Persson AE, Salomonsson M. Angiotensin II induces a tachyphylactic calcium response in the rabbit afferent arteriole. Acta Physiol Scand 1997;160:165-73.

49