ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2008 Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 324 Development of Salt-Sensitive Hypertension in Hydronephrosis MATTIAS CARLSTRÖM ISSN 1651-6206 ISBN 978-91-554-7137-8 urn:nbn:se:uu:diva-8586
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Development of Salt-Sensitive Hypertension in Hydronephrosis · LIST OF PAPERS I. Hydronephrosis causes salt-sensitive hypertension in rats. Carlström M*, Wåhlin N*, Sällström
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ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2008
Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 324
Development of Salt-SensitiveHypertension in Hydronephrosis
I. Hydronephrosis causes salt-sensitive hypertension in rats.
Carlström M*, Wåhlin N*, Sällström J, Skøtt O, Brown R &
Persson AEG.
J. Hypertension, 24:1437-43, 2006
*Equal contribution
II. Relief of chronic partial ureteral obstruction attenuates salt-
sensitive hypertension in rats.
Carlström M, Wåhlin N, Skøtt O & Persson AEG.
Acta Physiol. 189:67-75, 2006
III. Hydronephrosis causes salt-sensitive hypertension and impaired
renal concentrating ability in mice.
Carlström M*, Sällström J*, Skøtt O, Larsson E, Wåhlin N &
Persson AEG.
Acta Physiol. 189:293-301, 2007
*Equal contribution
IV. Role of nitric oxide deficiency in the development of hyperten-
sion in hydronephrotic animals.
Carlström M, Brown R, Edlund J, Sällström J, Larsson E, Teer-
link T, Palm F, Wåhlin N & Persson AEG.
Am J Physiol Renal Physiol. 294:362–370, 2008
CONTENTS
INTRODUCTION ............................................................................................11�Kidney function and Autoregulation........................................................11�Renal hypertension...................................................................................12�Hydronephrosis ........................................................................................13�
Incidence and aetiology .......................................................................13�Diagnostics ..........................................................................................13�Hydronephrosis and changes in renal function ...................................14�Treatment.............................................................................................15�
Hydronephrosis and Hypertension ...........................................................16�Experimental findings ..............................................................................16�
MATERIAL AND METHODS..........................................................................20�Animals ....................................................................................................20�Study protocols.........................................................................................20�Creation of partial ureteral obstruction (Studies I-IV) ..............................21�Telemetry - Implantation (Studies I-IV) ...................................................23�Telemetry - Measurements (Studies I-IV) ................................................25�Classification of hydronephrosis (Studies I-IV) .......................................25�Sampling and renin assay (Studies I-IV) ..................................................27�Ipsi- and contralateral nephrectomy (Study II) .........................................28�Ureterovesicostomy (Study II)..................................................................28�Renal excretion measurements (Studies III and IV) .................................29�L-arginine, ADMA and SDMA measurements (Study IV) ......................29�Western blotting for nitric oxide synthases (Study IV).............................29�Histology (Studies III and IV) ..................................................................30�Stereology (Study III) ...............................................................................30�Stop-flow pressure measurements (Study IV)...........................................31�Statistical analysis ....................................................................................32�Ethics........................................................................................................32�
RESULTS .......................................................................................................33�Study I ......................................................................................................34�Study II.....................................................................................................39�Study III ...................................................................................................42�Study IV ...................................................................................................45�
DISCUSSION ..................................................................................................53�Relief of chronic partial ureteral obstruction............................................53�Renal excretion measurements.................................................................54�Renin Angiotensin System .......................................................................54�Histopathological changes........................................................................55�L-NAME and blood pressure ...................................................................57�L-arginine and blood pressure..................................................................57�Nitric oxide, renal excretion and salt-sensitivity......................................58�Sympathetic nervous activity, renal excretion and salt-sensitivity ..........59�L-arginine, ADMA and SDMA................................................................59�Characteristics of TGF .............................................................................60�
Kidney function and Autoregulation The kidneys play a key role in the homeostatic regulation of body fluid sta-
tus and electrolyte balance, and consequently have a dominant role in long-
term blood pressure control.1 One of the mechanisms for achieving a stable
fluid balance is renal autoregulation. Via autoregulation, the kidneys are able
to maintain constant renal blood flow (RBF) and glomerular filtration rate
(GFR) despite wide changes in the arterial perfusion pressure.2-4 The mecha-
nisms responsible for autoregulation are the myogenic response and the tu-
buloglomerular feedback (TGF) systems. Myogenic response is an inherent
ability of the afferent arterioles to respond to changes in vessel-wall tension
by contracting or relaxing. The TGF mechanism is a negative feedback sys-
tem that operates within the juxtaglomerular apparatus (Figure 1). An in-
creased flow in the thick ascending limb is sensed by the macula densa cells,
which release a mediator that is transferred to the preglomerular vessels
causing vasoconstriction. Adenosine is the mediator of the TGF, whereas,
nitric oxide (NO) and angiotensin II are modulators.5, 6
12
Figure 1. The juxtaglomerular apparatus with the macula densa cells in the
wall of the distal tubule, glomerular arterioles (afferent and efferent), and
between these structures the extraglomerular mesangial cells.
Renal hypertension Hypertension is a common chronic disorder worldwide and secondary forms
of hypertension are found in a large proportion of the hypertensive popula-
tion. The most common cause of secondary hypertension is intrinsic renal
disease, but virtually any form of renal pathological condition may lead to
hypertension.7 The mechanism is either renovascular, occurring through the
action of vasoactive substances, or more commonly an inability to regulate
sodium excretion with resulting chronic hypervolaemia.8 Increased activity
of the renin angiotensin system has been demonstrated in renal hypertension
in both humans and in experimental animal models. There is also increasing
evidence of a close connection between increased oxidative stress and re-
duced NO availability in the development and maintenance of hypertension.9
13
Hydronephrosis
Incidence and aetiology Hydronephrosis is a common condition and the incidence of detectable uri-
nary tract dilatation in utero is reported to be 1-1.4% of all foetuses, and
confirmed postnatally in approximately 0.5-1%.10-12 Hydronephrosis is a
condition with a distended kidney or a dilatation of the renal pelvis. The
presenting symptoms in neonates and young children are palpable mass,
failure to thrive, urinary tract infections and pain in the flank.13 There are
different forms of hydronephrosis and ureteropelvic junction (UPJ) obstruc-
tion is the most common cause of antenatal and neonatal hydronephrosis. In
this condition, there is an obstruction of the urine flow from the renal pelvis
to the proximal ureter. This obstruction may lead to progressive renal dam-
age and deterioration. The aetiology of hydronephrosis due to UPJ obstruc-
tion is still obscure and can be caused by several different mechanisms, in-
cluding both intrinsic (i.e. muscle disorientation,14 excessive collagen15 or
absence of smooth muscle cells16) and extrinsic factors such as overlying
aberrant vessel,17, 18 retroperitoneal fibrosis,19, 20 pelvic or abdominal tu-
mours21-24 or neurological deficits.25 In general, the obstruction is partial,
unilateral and congenital and also more frequently observed in boys than in
girls.26
Diagnostics Hydronephrosis is often detected during an ultrasonography of the urinary
system. This procedure reveals information about the enlargement of the
kidney and the ureter related to the UPJ obstruction, however, diagnosis is
often confirmed later by other techniques. Intravenous urography is a me-
thod where contrast medium is injected intravenously and excreted by the
kidneys. X-rays are then taken to follow the excretion of the contrast me-
dium. The intravenous urography reveals information about both drainage
14
and functioning of the kidney. Historically, intravenous urography was the
predominant method of evaluating patients with possible UPJ obstruction.
However, in the evaluation of a child with a hydronephrotic kidney, diuretic
renograms have replaced intravenous urography. The benefits are that io-
dine-based intravenous contrast is not used, radiation exposure is minimal,
and renal function can be better quantified. The disadvantage of the nuclear
medicine scan is that details about renal anatomy are not obtained.
Hydronephrosis and changes in renal function Renal function is often described in terms of GFR, and it has been noted that
changes in filtration rate are closely related to changes in RBF. The func-
tional status of the hydronephrotic kidney, as measured by GFR, remains
well preserved for several years in newborns.27-29 However, a poor correla-
tion between the severity and duration of symptoms and the degree of renal
function has been demonstrated.30, 31 Several animal models have been estab-
lished for studying ureteral obstruction.32 The model of partial unilateral
ureteral obstruction (PUUO) is used to induce hydronephrosis in many ani-
mal species after birth. In experimental hydronephrosis, GFR is reported as
increased, unchanged or decreased.32 This discrepancy may depend on the
severity and duration of the obstruction and the diuretic state. However, in
general, ureteral obstruction results in decreased RBF and GFR in the ipsi-
lateral kidney, whereas, in the non-obstructed, contralateral kidney, a com-
pensatory increase occurs. In addition, the literature on electrolyte and water
excretion during PUUO is conflicting.32
15
Treatment The treatment of symptomatic hydronephrosis is surgical and uncomplicated.
The usual repair of UPJ obstruction involves removal of the obstruction and
then a reconstruction of the continuity by a pyeloplasty (Figure 2).
Figure 2. Schematic illustration of pyeloplasty in a hydronephrotic kidney.
The obstruction is removed to drain and decompress the kidney and finally
the ureter is reconnected with the pelvic region.
Patients who demonstrated with a dilated collecting system and a non-visible
ureter on intravenous urography were considered as having an obstruction
and were operated, irrespective of symptoms. With the introduction of scin-
tigraphic methods for estimating renal function, knowledge concerning kid-
ney function in the presence of outlet obstruction increased.33 As a well pre-
served renal function in hydronephrosis has been demonstrated,27-29 the man-
agement of asymptomatic ureteral obstruction has become more conserva-
tive. This worldwide policy is new and the long-term physiological
consequences are still unknown.
16
Hydronephrosis and Hypertension Most children with hydronephrosis are not hypertensive, but there are sev-
eral reports20, 34-58 on limited numbers of patients with hypertension obvi-
ously caused by hydronephrosis, as the patients became normotensive after
nephrectomy or pyeloplasty. An increased activity of the renin angiotensin
system has been demonstrated, but the participation of renin in this hyper-
tension appears to be influenced by the duration of the obstruction, the pres-
ence or absence of a contralateral normal kidney, and other intrarenal fac-
tors.51 Other investigations are unable to show any differences in blood pres-
sure in hydronephrosis.37, 59 Furthermore, in large surveys on causes of sec-
ondary hypertension, hydronephrosis does not appear overrepresented.60
However, this relationship is difficult to interpret, as the prevalence of hy-
dronephrosis among the human population is lower than that of hyperten-
sion.
Experimental findings In previous experimental studies in our laboratory in Uppsala, weanling rats
were submitted to PUUO at three weeks of age, leading to considerable hy-
dronephrosis.61-63 Experiments were performed three weeks later. Functional
parameters such as RBF and GFR under baseline conditions were at the
same level as in controls. However, during volume expansion, major
changes in blood flow and filtration occurred.
17
Figure 3. Schematic drawing of a nephron, with the pipettes used for deter-
mining TGF-characteristics. A wax block is placed in the proximal tubule.
Distal to this block, a perfusion pipette is inserted so perfusion rate can be
changed. Proximal to the wax block, the stop flow pressure (PSF) can be
measured at different perfusion rates.
With micropuncture techniques (Figure 3), volume expansion causes a para-
doxical resetting of the TGF mechanism to a much higher sensitivity and
activity in the hydronephrotic kidney, instead of making the TGF insensitive
to flow (Figure 4).61 Consequently, the single nephron GFR and the urinary
output are maintained at low levels. These effects are interpreted as possibly
serving to protect the hydronephrotic kidney from further damage caused by
elevated intrarenal pressures. However, in the contralateral kidney, desensiti-
sation of the TGF mechanism occurs, similar to in healthy animals. The
same paradoxical resetting of the TGF, towards higher sensitivity and activ-
18
ity, is seen in rats of the Milan hypertensive strain rats,64 before the animals
develop hypertension, and in spontaneous hypertensive rats (SHR).65
Figure 4. TGF-responses before (blue curve) and after volume expansion. In
control animals (green curve), there is a resetting of the TGF mechanism to
decreased sensitivity and activity. In the hydronephrotis kidney (red curve),
there is a paradoxical shift towards higher sensitivity and activity.
The regulation of the TGF sensitivity is intimately coupled to NO production
in the macula densa cells. Chronic, selective blockade of neuronal nitric
oxide synthase (nNOS) increases TGF sensitivity, reduces GFR and salt and
water excretion, and leads to hypertension.66 Taken together, it appears as if
the functional adaptations to PUUO could lead to development of renal hy-
pertension.
19
AIMS
The overall aim was to determine if there is a link between hydronephrosis and the development of hypertension.
Study I � To determine whether hydronephrosis and hypertension are causally
related and to evaluate the short and long term effects of hy-dronephrosis on blood pressure level.
� To elucidate the effects of different sodium diets on the blood pres-sure and the renin-angiotensin system in hydronephrotic animals.
Study II
� To examine the effects of ipsilateral nephrectomy, contralateral nephrectomy and ureterovesicostomy on blood pressure in hy-dronephrotic animals treated with different sodium diets, to deter-mine whether salt-sensitive hypertension is of renal origin and if the mechanisms are located primarily to the hydronephrotic kidney.
� To study the effects of relief from partial ureteral obstruction (ipsi-lateral nephrectomy) on the plasma renin concentrations (PRC) in hydronephrotic animals.
Study III
� To develop a model for partial ureteral obstruction in mice. � To investigate if hydronephrotic mice develop renal injury and salt-
sensitive hypertension similar to that found in rats. Study IV
� To investigate if NO deficiency is involved in the development of salt-sensitive hypertension in hydronephrosis.
20
MATERIAL AND METHODS
Animals The experiments were conducted on male Sprague-Dawley rats (Studies I, II
and IV) and C57bl/6J mice (Study III) from the M&B Research and Breed-
ing Centre (Ry, Denmark).
A ureteral obstruction was created at young age (described below) and the
animals were then left to grow with free access to standard rat chow
(TD96329) and tap water. The experiments were conducted on adult animals
treated with different sodium diets: normal salt (NS) (0.7% NaCl;
TD96329), low salt (LS) (0.012% NaCl; TD90228) or high salt (HS) (4%
NaCl; TD92034) (Harland Scandinavia, Allerød, Denmark). All animals
were allowed to recover for at least one week after any surgical procedures
and an equilibration period of 10 days was used on the different sodium diets
before any measurements were taken.
Study protocols Study I. Blood pressure in rats with PUUO was measured at different time
points during a 5-month period. The effects of different sodium loads on
blood pressure were investigated and the PRC were analysed.
21
Study II. The effects of relief from obstruction (ipsilateral nephrectomy or
ureterovesicostomy) or contralateral nephrectomy on blood pressure and
plasma renin levels were studied in hydronephrotic rats (PUUO) with salt-
sensitive hypertension.
Study III. A model for PUUO in mice was developed. Blood pressure, renal
excretion and PRC were measured during different sodium conditions, and
renal histological and stereological changes were evaluated.
Study IV. Animals with both unilateral (PUUO) and bilateral (PBUO) hy-
dronephrosis were investigated. Blood pressure and renal excretion were
measured during different sodium conditions, before and after chronic NG-
nitro-L-arginine methyl ester (L-NAME) (0.5 mg/ml in drinking water for
one week) or L-arginine treatment (1mg/ml in drinking water for 30 days).
Plasma levels of renin, L-arginine, asymmetrical dimethylarginine (ADMA)
and symmetrical dimethylarginine (SDMA) were analysed. The TGF charac-
teristics were determined before and after administration of 7-Nitroindazole
(7-NI) or L-arginine. The protein expression of NOS-isoforms in the cortex
and medulla was analysed, and the renal histological changes were evalu-
ated.
Creation of partial ureteral obstruction (Studies I-IV) A partial unilateral (PUUO – Studies I-IV) or bilateral (PBUO – Study IV)
ureteral obstruction was created to induce hydronephrosis. Male rats (~50 g)
and mice (~7 g) underwent surgical obstruction at three weeks of age in
which the left, or both, ureters were embedded in the psoas muscle by a
modified technique to that originally described by Ulm and Miller in dogs.67
Anaesthesia was induced by spontaneous inhalation of isoflurane (Forene�,
Abbot Scandinavia AB, Kista, Sweden) and continued during surgery by
22
inhalation of ~2% isoflurane in air. Gas flow rate was set to ~150 ml/min.
The animals were placed on a servo regulated heating pad to maintain body
temperature between 37 and 38oC. The surgical field was sterilized with 70%
ethanol and sterile NaCl and the abdominal wall was opened through a mid-
line incision. The intestines were retracted to gain access to the left ureter,
which was dissected free under the microscope (Figure 5).
Figure 5. Creation of partial unilateral ureteral obstruction (PUUO).
The underlying psoas muscle was split longitudinally to form a groove (rats:
~15 mm; mice ~5 mm) and the ureter was placed in it. The muscle edges
were approximated above the ureter with two non-absorbable 6/0 (rats) or
7/0 (mice) silk sutures (Silk®, Ethicon, Johnson & Johnson Intl, USA), and
the ureter was thus embedded in a tunnel. The abdominal incision was then
closed in one layer with interrupted sutures. Sham operations were per-
formed in the same manner, but without dissection of the ureter or the psoas
muscle. After the operation, the animals were allowed to wake up under a
heating lamp, and were not returned to their cages until fully awake.
23
Telemetry - Implantation (Studies I-IV) Rats (Studies I, II and IV): Four to six weeks after PUUO, the animals were
prepared for blood pressure measurements. Gas anaesthesia as described
above, but with a higher concentration of isoflurane (2-2.5% in air) and also
a higher gas flow rate (180-220 ml/min), was used. The skin was sterilized
and the abdominal wall was opened, as described above. The intestines were
retracted to achieve access to the deep vessels and kidneys. A macroscopic
examination of both kidneys (see below) was performed in both sham oper-
ated and obstructed animals, and subsequently a 20 mm long segment of the
abdominal aorta was dissected free from surrounding fat and connective
tissue. The blood flow was then occluded by clamping the aorta (caudal to
the left dorsal renal muscular branch and cranial to the iliac bifurcation of
the aorta). A needle (21G X 1½¨), bent 90� at the bevelled end, was used to
puncture the aorta 5mm cranial to the iliac bifurcation. The catheter of the
telemetric blood pressure device, PA-C40 (DSI™, Transoma Medical, St
Paul, MN, USA) was then inserted into the aortic lumen (Figure 6) and the
entry site was sealed by application of n-butyl-cyanoacrylate tissue adhesive
(VetbondTM, 3M Animal Care Products, St Paul, MN, USA). The clamps
were removed, the intestines were replaced in their original position: the
transmitter was placed in the peritoneal cavity on top of the intestines.
Figure 6. Implantation of the telemetric blood pressure device (PA-C40,
DSITM) in rats.
24
Mice (Study III): Six to eight weeks after PUUO, the telemetric device (PA-
C10 (DSI™, Transoma Medical, St Paul, MN, USA) was implanted. Gas
anaesthesia was used in the same way as described above, and a midline
incision was made between the lower mandible and sternum (Figure 7). The
catheter of the telemetric blood pressure device was then inserted into the
left carotid lumen and secured by 6/0 silk sutures (Silk®, Ethicon, Johnson
& Johnson Intl, USA). The entry site was sealed by application of n-butyl-
cyanoacrylate tissue adhesive (VetbondTM, 3M Animal Care Products, St
Paul, MN, USA) and the body of the transmitter was placed subcutaneously
in the right flank.
Finally, the muscle edges were sutured and the skin incision was closed.
After surgery, the animals were placed in new cages and allowed to wake up
under a heating lamp. The hydronephrotic and sham operated animals (con-
trols) were treated exactly the same. To verify that the tip of the catheter was
correctly positioned, the transducer was switched on and the recorded radio
signal (AM 550 kHz) pulsation was confirmed.
Figure 7. Implantation of the telemetric blood pressure device (PA-C10,
DSITM) in mice (left panel). Telemetric blood pressure measurement in mice
(right panel).
25
Telemetry - Measurements (Studies I-IV) After surgery, the animals were allowed to recover for at least one week
before blood pressure recording was started. The animal cage was placed on
a receiver and the transmitter switched on (Figure 7). The signals received
were transferred to a computer where calibrated blood pressure values were
sampled by a computer-based system for data acquisition; PC-lab version
5.0.68 Data were collected for five seconds every second minute for at least
48 hours at a time. The recorded data were continuously analysed by the
computer program, as follows: by comparing the incoming signal with a
template blood pressure curve, individual heartbeats could be identified and
stored together with the distance in time between the curves. If the time in-
terval from a heartbeat to the surrounding pressure curves differed more than
20% from the average interval in the sampling period, the beat was discarded
together with its two neighbouring waves. Pulse waves that passed this test
were used for calculation of mean arterial blood pressure (MAP) and heart
rate, which was stored together with the number of accepted pulse curves
during the 5-second collecting period in question. Blood pressure data were
collected and further analysed with a computer program developed at the
department and which discarded values considered unreasonable, i.e. lower
than 50 and higher than 220 mmHg. Data were also discarded if the number
of accepted pulse curves during a 5-second sampling period was less than
eight, indicating a disturbed transmission. The evaluation of the data with
this system provided a higher level of accuracy than conventional methods,
in which a mean value is taken straight from raw data.
Classification of hydronephrosis (Studies I-IV) The kidneys were examined macroscopically before insertion of the telemet-
ric equipment (Studies I, II and IV) or once the experiments were conducted
(Study III). The obstructed kidneys were categorized as having a normal
26
appearance (i) or as having mild (ii), moderate (iii) or severe (iv) hy-
dronephrosis (Figure 8). Grossly enlarged, sacculated kidneys without any
visible parenchyma were categorized as non-functioning (v).
Figure 8. Normal kidney and hydronephrotic kidneys with mild, moderate
and severe degree of hydronephrosis.
In this thesis, only animals with unilateral hydronephrosis were used, i.e.
animals allocated to categories (i) and (v), and those with abnormalities in
the contralateral kidney were excluded. In the sham-operated animals, both
kidneys were examined, and a telemetric device was implanted only in the
absence of macroscopic changes.
After completion of all experiments, the animals were anaesthetized and the
hydronephrotic kidney was again macroscopically examined. The degree of
hydronephrosis was measured by weight in the following way: the ureter
was ligated in situ and the vessels were cut to exsanguinate the kidney,
which was then taken out and weighed. Thereafter, the kidney was sliced
and emptied of urine, and was then reweighed to calculate the parenchymal
weight: the difference between total weight and parenchymal weight was the
weight of the urine volume. To measure the degree of hydronephrosis, the
simple equation: renal pelvic volume, as measured by weight, divided by
27
renal parenchymal weight, yielding the so-called hydronephrotic ratio
(HNR) was used.
Normal kidneys have an HNR of less than 0.1 (0.06– 0.09). An HNR of 0.1–
0.3 was classified as indicating mild hydronephrosis, 0.3 and 1.0 as moderate
hydronephrosis, and above 1.0 as severe hydronephrosis. The macroscopic
examination and weight grading corresponded well.
Sampling and renin assay (Studies I-IV) Blood samples for renin analysis were taken from the tail tip (rats) or the
orbital plexus (mice) immediately after anaesthesia at the end of each diet
period. Samples were centrifuged and instantly frozen to -70°C for later
assay. The PRC was measured by radioimmunoassay (RIA) of angiotensin I
with the antibody-trapping technique.69 In short, 10 μl of plasma from each
sample was serially diluted (25, 50, 100 and 200×). From dilution of plasma,
5 μl was incubated in duplicate for 24 hours, together with a mixture of rab-
bit angiotensin I antibody and renin substrate (Angiotensinogen equivalent to
1200 ng angiotensin I ml-1) from rats nephrectomized 24 hours previously,
from which renin had been extracted by affinity chromatography. After the
incubation step, the reaction was stopped by addition of 1 ml cold barbital
buffer, an Angiotensin I tracer was added and RIA was performed. Renin
values were standardized by reference to renin standards obtained from the
Institute for Medical Research (MRC, Holly Hill, London, UK), and the
values were expressed in standard Goldblatt units (GU).
28
Ipsi- and contralateral nephrectomy (Study II) The animals were anaesthetised with isoflurane as above and placed on a
heating pad exposing their left flank. The site of the incision was shaved and
sterilized and a flank incision exposed the left kidney. The renal artery and
vein were carefully isolated and a single ligature was placed around them
and tied tightly before making a distal cut. The ureter was exposed and two
ligatures were placed close to the UPJ and cut in between. The kidney was
removed, the incision closed and HNR calculated, as described above. The
animals were allowed to recover and telemetric measurements were per-
formed.
The procedure, for the contralateral nephrectomy, was the same as for the
ipsilateral nephrectomy, except that the contralateral kidney was removed.
Furthermore, the protocol for the telemetric measurements was identical to
that in the ipsilateral nephrectomy group. In the control animals, a left side
nephrectomy was performed.
Ureterovesicostomy (Study II) The same kind of anaesthesia was used and the abdomen was opened
through a sterile midline incision and the ipsilateral kidney, ureter and blad-
der were exposed and visually examined. To validate the outcome of the
ureterovesicostomy, the cross-sectional area of the kidney (length x width
mm2) was measured, before and after this procedure. A sterilized plastic
catheter was inserted through a punctured hole within a purse-string suture in
the bladder, and the purse-string was drawn up tightly and tied. Two sutures
were then placed around the ureter distal to the pelvic area, the catheter was
allowed to enter the exposed pelvic region through a punctured hole and
finally secured. In control animals, the ureter was visually examined but not
29
catheterised. The incision was closed and the animals were allowed to re-
cover before telemetric measurements were performed.
In animals with an increased cross-sectional area at the time of euthanasia,
the ureterovesicostomy was considered unsuccessful and the data were dis-
carded.
Renal excretion measurements (Studies III and IV) Animals were housed individually for 24 hours in metabolism cages. Urine
production and water consumption were determined gravimetrically. Sodium
and potassium concentrations were measured by flame photometry (FLM3;
Radiometer, Copenhagen, Denmark) and osmolality by depression of the
freezing point (Fiske®Micro-sample Osmometer, Model 210; FiskeAssoci-
ates, Norwood, MA, USA).
L-arginine, ADMA and SDMA measurements (Study IV) From anaesthetised controls, PUUO and PBUO animals, 1 ml blood was
withdrawn of from the carotid artery and plasma concentrations of L-
arginine, ADMA, and SDMA were determined simultaneously by high-
performance liquid chromatography, as described previously70 but with mod-
ified chromatographic separation conditions.71
Western blotting for nitric oxide synthases (Study IV) The animals were anaesthetized and a catheter was placed in the left carotid
artery. The infusion of cold phosphate buffered saline (PBS) was started
once the right renal vein was cut and the kidneys were explanted for Western
blotting. Renal cortex and medulla were separated and homogenized in lysis
lou, Nina, Sara, Åsa, Olerud, Ulrika, Joey, Gustav, Micke, Magnus and Shel-
ler for making the department a more enjoyable place. The professors: Lena
Holm, Peter Hansell, Mats Sjöquist, Leif Jansson, Ulf Eriksson and Örjan
Källskog for a friendly and inspiring atmosphere. Göran for being a fantastic
inst.tekniker and Gunno for helping out with different technical problems
and for sharing many pigs together. Agneta, Karin and Marianne for the
administrative help.
All my other friends and colleagues at BMC, who are struggling with the
projects.
The animal department for taking good care of my rats and mice.
Apotekarsocieteten and Anna-maria Lundins Stiftelse for giving me the
opportunity to present my findings at conferences.
All my friends in Stockholm, Göteborg, Malmö, Köpenhamn, Helsingfors
and Uppsala, for the nice times we have had throughout these years. Your
friendship has really contributed to the completion of my thesis. Thank You!
My “Norwegian family”: Erling, Bergliot, Siri & Øivind with relatives and
friends for all the nice moments we have shared during these years and for
always being so nice and friendly to me. Thanks to you I have learnt to ap-
preciate some important things in life i.e fiskekaker, brunost, hjemmlaged
pizza alt. Peppes pizza deal, att gå på tur, and most importantly att ikke tap i
femkamp.
Kristin, just for being the one you are…
69
Last, but by no means least, my caring Family, all my relatives and friends
in Blekinge. Mamma, Pappa, Andreas, Anna och mina underbara brorsdött-
rar Emmy och Minna! Tack för allt stöd, ni har verkligen ställt upp genom
åren!
70
REFERENCES
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