development of salt-sensitive hypertension in …171674/fulltext01.pdfacta universitatis upsaliensis...

ACTA

UNIVERSITATIS

UPSALIENSIS

UPPSALA

2008

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 324

Development of Salt-SensitiveHypertension in Hydronephrosis

MATTIAS CARLSTRÖM

ISSN 1651-6206ISBN 978-91-554-7137-8urn:nbn:se:uu:diva-8586

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LIST OF PAPERS

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�

AIMS .............................................................................................................19�

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�

CONCLUSIONS ..............................................................................................62�

FUTURE PERSPECTIVES...............................................................................63�

SAMMANFATTNING PÅ SVENSKA ................................................................64�

ACKNOWLEDGEMENTS ...............................................................................66�

REFERENCES ................................................................................................70�

ABBREVIATIONS

ADMA Asymmetrical dimethylarginine

c-UNX Contralateral nephrectomy

eNOS Endothelial nitric oxide synthase

GFR Glomerular filtration rate

GU Goldblatt units

HN Hydronephrotic

HNR Hydronephrotic ratio

HS High salt

i-UNX Ipsilateral nephrectomy

L-NAME NG-nitro-L-arginine methyl ester

LS Low salt

MAP Mean arterial blood pressure

nNOS Neuronal nitric oxide synthase

NO Nitric oxide

NS Normal salt

NS2 Normal salt (second time)

PRC Plasma renin concentration

PUUO Partial unilateral ureteral obstruction

PBUO Partial bilateral ureteral obstruction

PFF Free-flow pressure

PSF Stop-flow pressure

�PSF Maximal TGF response

RBF Renal blood flow

SDMA Symmetrical dimethylarginine

SHR Spontaneous hypertensive rats

7-NI 7-Nitroindazole

TGF Tubuloglomerular feedback

UPJ Ureteropelvic junction

11

INTRODUCTION

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

buffer (1.0% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM NaF,

80mM Tris, pH 7.5) containing enzyme inhibitors (Complete Mini; 1 tab-

30

let/1.5 ml; Roche Diagnostics, Mannheim, Germany). Samples were run on

7.5% Tris-HCl gels with Tris/glycine/SDS buffer. After transfer to nitrocel-

lulose membranes, the proteins were detected with rabbit anti-nNOS (1

μg/ml; Zymed Laboratories; Invitrogen, Carlsbad, CA, USA), mouse anti-

eNOS (1 μg/ml; Zymed Laboratories) and HRP-conjugated secondary anti-

bodies (goat anti-rabbit and goat anti-mouse, 1:5000; Sigma Aldrich) by an

ECL-camera (Kodak image station 2000; New Haven, CT, USA). ß-actin

was detected with mouse anti-rat ß-actin antibody (1:10,000; Sigma Aldrich)

and secondary HRP-conjugated goat-anti mouse antibody (1:60,000; Kirke-

gaard and Perry Laboratories, Gaithersburg, MD, USA).

Histology (Studies III and IV) The animals were anaesthetized and a catheter was placed in the left carotid

artery to allow formalin (4% in PBS) infusion. The abdomen was opened

and the right renal vein was cut at the same time as the infusion started. The

infusion continued for 10 minutes and the kidneys were then removed. Sagit-

tal slices of the renal tissue were fixed in buffered formalin and embedded in

paraffin. Embedded tissue blocks were cut into 5-μm thick sections and

stained with Haematoxylin and Eosin (HE), Periodic Acid-Schiff (PAS)

stain and Picro-Sirius stain for a blind histological evaluation.

Stereology (Study III) To assess glomerular area and volume, a light microscope (Leitz DMRB,

Leica Microsystems, Wetzlar, Germany) with a CCD camera (AxioCam

color, Carl Zeiss, Oberkochen, Germany) was used. The cortical area of one

section was photographed and a computer program (Scion image, Scion

Corporation, MD, USA) was then used to determine the area of all visible

glomeruli in the cross-section. From the measured glomerular area, an ap-

proximation of the real area and volume was calculated with the equation D=

31

4/� × d, where D is the real diameter and d is the measured mean glomerular

diameter.72

Stop-flow pressure measurements (Study IV) Characteristics of the TGF were determined with stop-flow pressure tech-

niques as described earlier.73 The animals were anaesthetized, a flank inci-

sion was made and the kidney was prepared for micropuncture experiments

(Figure 9).

Figure 9. Set-up for renal micropuncture experiment.

Randomly chosen proximal tubular segments on the surface were punctured

with a sharpened glass pipette (OD 3-5 �m) filled with a lissamine green-

stained 1M NaCl solution. The pipette was connected to a servo-nulling

pressure system (World Precision Instruments, New Haven, CT, USA) to

determine the proximal tubular free-flow pressure (PFF). A second pipette

(OD 7-9 �m), filled with artificial ultrafiltrate (140 mM NaCl, 5 mM KCl, 2

mM CaCl2, 1 mM MgCl2, 4 mM NaHCO3, 7 mM urea, and 2 g/L Lissamine

green, pH 7.4) and connected to a microperfusion pump (Hampel, Frankfurt,

32

Germany), was inserted in the last accessible segment of the proximal tu-

bule. A solid wax block was placed in between with a third pipette (OD 7-9

�m). The proximal tubular stop-flow pressure (PSF) upstream to the block

was determined at different perfusion rates (0-35 nl/min) in the loop of

Henle. The maximal change in stop-flow pressure (�PSF), was used to indi-

cate the TGF reactivity and the tubular flow rate eliciting half-maximal �PSF,

(i.e. the turning point), indicated the TGF sensitivity. To create TGF-

response curves, normalized data were used in a non-linear least squares

curve-fitting program.73 The TGF-characteristics were determined in normo-

volemic animals during control conditions and after either nNOS inhibition

with 7-NI (25 mg/kg i.p.) or intratubular infusion of L-arginine (10-2 M).

Statistical analysis Values are presented as means ± SEM. Single comparisons between nor-

mally distributed parameters were tested for significance with Student’s

paired or unpaired t-test. For multiple comparisons, analysis of variance

(ANOVA) followed by the Bonferroni or Fisher’s post-hoc test, were used.

Scored data for the histological evaluation were analysed by the Kruskal-

Wallis test followed by the Mann-Whitney-U-test. Statistical significance

was defined as p<0.05.

Ethics All experiments were approved by the Uppsala Ethical Committee for Ani-

mal Experiments and performed in accordance with national guidelines for

care and use of laboratory animals.

33

RESULTS

In all studies (I-IV) the animals were in good condition, and at the beginning

of the experimental protocols there was no difference in body weight be-

tween the sham operated controls and the hydronephrotic animals. All hy-

dronephrotic animals developed salt-sensitive hypertension that correlated to

their degree of hydronephrosis. No differences were determined in heart rate

between the groups.

34

Study I Long-term effects on blood pressure

Blood pressure level was higher in the obstructed animals (106±5 mmHg)

than in the controls (87±1 mmHg). Furthermore, blood pressure increased

slowly with time in the hydronephrotic animals but not in the controls (Fig-

ure 10).

Figure 10. Long term effect on mean arterial blood pressure (MAP) in hy-

dronephrotic animals and controls. * P<0.05 compared with the initial value (within same group)

# P<0.05 compared with the controls

35

Effects of different salt loads on blood pressure

All hydronephrotic groups displayed elevated blood pressures compared

with the controls on NS, LS and HS diets (Figure 11). There were no differ-

ences in blood pressure levels between the first and second NS period. Fur-

thermore, all hydronephrotic animals displayed salt-sensitive blood pressure

that became more pronounced with the degree of hydronephrosis.

Figure 11. Mean arterial blood pressure (MAP) in controls and hy-

dronephrotic animals with different hydronephrotic degrees, treated with

normal salt (NS), low salt (LS), high salt (HS) and normal salt diet once

again (NS2). * P<0.05 compared with hydronephrotic groups on same diet.

# P<0.05 compared with NS and NS2 diet within the same group

† P<0.05 compared with LS diet within the same group.

36

Typical 24-hour circadian variations in blood pressure were observed in all

groups, with a higher blood pressure during active periods (night time).

However, the circadian variation in blood pressure pattern was less pro-

nounced in obstructed animals, with a mean blood pressure increase of

9.3±1.1% in the control animals but only 7.0±0.4% in the hydronephrotic

ones (p<0.05). Fluctuations in MAP during the day and night also appeared

less pronounced with increasing degree of hydronephrosis (Figure 12).

Figure 12. 48-hour mean arterial blood pressure (MAP) recordings in repre-

sentative animals with different degrees of hydronephrosis and in sham op-

erated control. Shaded areas represent nighttime.

37

As shown in the scatter plot (Figure 13), there was a correlation between

blood pressure and the degree of hydronephrosis, expressed as HNR.


dronephrotic animals (HN) with different hydronephrotic ratios (HNR).

38

Plasma levels were higher in the hydronephrotic rats on all diets. However, it

was only found significant on the NS diet (Figure 14). In all groups given the

LS diet, PRC increased, compared with baseline level.. Animals with severe

hydronephrosis displayed lower PRC levels than both mild and moderate

groups on all diets, however, this was only significant on the LS diet.

Figure 14. Plasma renin concentration (PRC) in controls and hydronephrotic

animals with different hydronephrotic degrees, treated with normal salt (NS),

low salt (LS), high salt (HS) diet. * P<0.05 compared with hydronephrotic groups on same diet.

# P<0.05 compared with both NS and HS diets

† P<0.05 compared with those with moderate and mild hydronephrosis on same diet

39

Study II Ipsilateral nephrectomy

In the hydronephrotic animals, ipsilateral nephrectomy attenuated both hy-

pertension and salt-sensitivity, whereas, in the controls there was a transient

increase in blood pressure (Figure 15).


dronephrotic animals with different hydronephrotic degrees, treated with NS

and HS diets, before and after ipsilateral nephrectomy (i-UNX). * P<0.05 compared with controls on same diet and with the same surgical treatment.

# P<0.05 compared with NS diet within the same group and with the same surgical treatment

† P<0.05 compared with same diet within the same group before nephrectomy.

40

Before nephrectomy, PRC was elevated in the hydronephrotic group, com-

pared with the controls, but after removal of the obstructed kidney, these

differences were eliminated (Figure 16).

Figure 16. Plasma renin concentration (PRC) in controls and hydronephrotic

animals with different degrees of hydronephrosis treated with normal salt

(NS) and high salt (HS) diets, before and after ipsilateral nephrectomy

(i-UNX). GU=Goldblatt units. * P<0.05 compared with controls on same diet and with the same surgical treatment.

† P<0.05 compared with same diet within the same group before nephrectomy.

Contralateral nephrectomy

In contrast to ipsilateral nephretomy, contralateral nephrectomy augmented

the hypertension in the hydronephrotic animals (97±2 to 107±3 mmHg),

whereas, in the controls, no persistent changes occurred (87±4 to 88±4

mmHg).

41

Ureterovesicostomy

As with ipsilateral nephrectomy, ureterovesicostomy decreased blood pres-

sure in the hydronephrotic animals, and salt-sensitivity became less pro-

nounced. These changes were not observed in the sham-operated controls.

The reduction cross-sectional kidney area (1264±178 to 780±137 mm2) cor-

related with a concomitant fall in blood pressure (Figure 17).

Figure 17. Correlation between the reduction in cross-sectional area and the

change in mean arterial blood pressure (MAP) in hydronephrotic animals

after ureterovesicostomy (UV).

42

Study III Similar the rats in Study I, the hydronephrotic mice developed salt-sensitive

hypertension (Figure 18) that correlated well with their hydronephrotic de-

gree (R2 = 0.91).

Figure 18. 24-hour mean arterial blood pressure (MAP) in controls and hy-

dronephrotic animals (HN), treated with NS and HS diets. * P<0.05 compared with controls on same diet.

# P<0.05 compared with same diet within the same group.

Renal excretion measurements

Urine excretion rate was higher in the hydronephrotic animals on both NS

(77±7 μl/24h/g BW) and HS diets (103±5 μl/24h/g BW), than in the controls

(NS 58±4 μl/24h/g BW, and HS 69±12 μl/24h/g BW). This was associated

with reduced urine osmolality in the hydronephrotic animals (1043±81

mOsm) on the NS diet, compared to the controls (1539±117 mOsm), but was

not significant on the HS diet (hydronephrotic animals 1174±73 mOsm and

controls 1215±64 mOsm).

43

Plasma renin concentration

No differences in PRC were found between the hydronephrotic (1043±119

10-5 GU/ml) and controls animals (1312±316 10-5 GU/ml) on the NS diet or

the HS diet (582±77 10-5 GU/ml hydronephrotic animals, 449±49 10-5

GU/ml controls).

Histology and stereology

The parenchymal weight of the hydronephrotic kidneys decreased, whereas,

compensatory hypertrophy and glomerular volume increase observed in non-

obstructed contralateral kidneys. The obstructed animals displayed dilated

pelvic areas with flattening of the papilla, which was associated with areas of

fibrosis, inflammation and glomerular changes (Figure 19).

Figure 19. Representative photomicrographs of a control kidney (A) and a

hydronephrotic kidney (B-D). (A) The control displays normal histoarchitec-

ture with a distinct cortex, medulla and renal papilla. (B) In hydronephrosis,

44

the pelvic area is slightly dilated, with flattening of the renal papilla. (A) and

(B) are stained with Haematoxylin and Eosin (HE). (C) With Periodic Acid-

Schiff (PAS) staining, slight sclerotic glomeruli (black arrows) and infiltra-

tion of inflammatory cells (white arrow) are observed. (D) Picro-Sirius stain-

ing showed areas with subepithelial fibrosis (black arrows).

45

Study IV Renal excretion measurements

During control conditions, urine excretion rate was higher and urine-

concentrating ability reduced in the hydronephrotic groups for both diets

(Table 1.). This was in accordance with observations from Study III.

Table 1. Renal excretion data on normal and high salt diets in controls and

unilateral (PUUO) and bilateral (PBUO) hydronephrotic animals.

* p<0.05 compared with controls on same diet

† p<0.05 compared with PUUO on same diet

After L-NAME treatment (0.5mg/ml; drinking water; one week), the con-

trols had a reduced ability for excreting sodium, potassium and total os-

moles, both during NS and HS conditions. The effects of L-NAME were less

pronounced in the hydronephrotic animals and reduced excretion was only

found for sodium and potassium during NS diet.

L-arginine treatment (1 mg/ml; drinking water; 30 days) did not affect renal

excretion; however, the hydronephrotic groups tended to increase electrolyte

and water excretion, which was not observed in the controls.

46

Plasma renin analysis

During both NS and HS conditions, PRC was higher in PUUO than in con-

trols, but not in PBUO animals (Figure 20).

Figure 20. Plasma renin concentration (PRC) in controls and unilateral

(PUUO) and bilateral (PBUO) hydronephrotic animals, treated with normal

(NS) and high salt (HS) diets. * p<0.05 compared with controls on same diet.

† p<0.05 compared with PUUO on same diet.

L-arginine, ADMA and SDMA measurements

The ADMA levels were higher in both hydronephrotic groups than in con-

trols. SDMA levels were only elevated in animals with bilateral hy-

dronephrosis (Table 2). L-arginine levels were similar between the three

groups (i.e. Controls, PUUO and PBUO).

47

Table 2. Plasma levels of L-arginine, ADMA and SDMA in controls and

unilateral (PUUO) and bilateral (PBUO) hydronephrotic animals.

* p<0.05 compared with controls

† p<0.05 compared with PUUO

Effects of L-NAME on blood pressure

During both NS and HS conditions, blood pressure was higher in PBUO (NS

132±7, HS 158±8 mmHg) and PUUO (NS 114±4, HS 129±9 mmHg) ani-

mals than in the controls (NS 85±2, HS 89±2 mmHg).

On NS diet, blood pressure response to L-NAME treatment was more pro-

nounced in the controls (26±1%) than in the PUUO (18±1%) and PBUO

(16±2%) animals.

During HS conditions, this response became more pronounced in the con-

trols (34±3%), whereas, no differences were found for the PUUO (18±2%)

and PBUO (13±2%) animals, when compared with the NS diet. L-NAME

treatment increased salt-sensitivity only in controls, and differences in salt-

sensitivity were no longer present between the PBUO (18±5%), PUUO

(13±3%) and control (12±2%) animals.

48

Effects of L-arginine on blood pressure

The hydronephrotic animals displayed salt-sensitive hypertension similar to

that observed for the L-NAME treated groups (see above). In the non-treated

hydronephrotic animals a progressive blood pressure increase was observed

during the 30 days experimental period for PUUO during HS diet (+5

mmHg) and in PBUO animals during HS (+24 mmHg) and NS diet (+14

mmHg) (Figure 21; upper panel).

Chronic L-arginine supplementation (1 mg/ml) in the drinking water for 30

days reduced the blood pressure in PUUO during both NS (-11 mmHg) and

high sodium conditions (-9 mmHg). In the PBUO animals, L-arginine treat-

ment completely prevented further increase in blood pressure during NS

conditions. During HS conditions L-arginine supplementation did not pre-

vent blood pressure increase, however, the augmentation in blood pressure

(+11 mmHg) was not as pronounced as in the non-treated group (+24

mmHg). In the control animals, L-arginine supplementation had no effect on

blood pressure (Figure 21; lower panel).

49

Figure 21. Mean arterial blood pressure (MAP) in controls and unilateral

(PUUO) and bilateral (PBUO) hydronephrotic animals, treated with NS

(dashed line) or HS diets (solid line) for 30 days, with or without L-arginine

supplementation (1 mg/ml in drinking water). * p<0.05 compared with the control period, within same group and diet.

# p<0.05 decreased compared with the control period, within same group and diet.

50

Stop-flow pressure measurements

There were no differences in PFF or PSF between the controls and hy-

dronephrotic animals during control conditions, or after 7-NI or L-arginine

administration. During control conditions, the reactivity of the TGF re-

sponse, as indicated by the �PSF, was greater in the hydronephrotic group

(14.4±1.0 mmHg) than in control animals (9.4±0.8 mmHg). The turning

point was lower in the hydronephrotic animals (14.9±1.4 nl/min) than in the

controls (19.1±1.2 nl/min), indicating higher sensitivity of TGF response in

hydronephrosis. Administration of 7-NI, increased the reactivity and sensi-

tivity of the TGF response (leftward shift) in the control animals, but had no

effect in the hydronephrotic animals. L-arginine administration reduced the

reactivity and sensitivity of the TGF response (rightward shift) in the hy-

dronephrotic animals, but had no effect in the control animals (Figure 22).

Figure 22. TGF response curves in controls and hydronephrotic animals

during control conditions (Baseline) and after administration of 7-NI or L-

arginine. * p<0.05 compared with baseline values of same group

# p<0.05 compared with values of control animals under similar conditions

51

Western Blotting for NOS

There was a reduced protein expression of nNOS in the cortex and medulla

for both hydronephrotic groups. The eNOS expression was only significantly

reduced for the PBUO animals.

Histology

The hydronephrotic kidneys displayed variable degrees of dilatation of the

pelvic area with flattening of the papilla. This was associated with areas of

fibrosis, inflammation, and glomerular and tubular changes. The severity of

the histopathological changes correlated with the degree of hydronephrosis.

Fibrotic and glomerular changes were also observed in the contralateral kid-

ney in PUUO animals (Figure 23).

Figure 23. Representative photomicrographs of hydronephrotic kidneys,

with mild (A-C) and severe histopathological changes (D-F). (A) Overview

of a mild hydronephrotic kidney with mild fibrosis and tubular dilatations.

(B) Higher magnification with tubular dilatation (red arrow) and slight in-

flammation (black arrow). (C) Mild fibrosis in medulla and corticomedullary

junction (black arrows). (D) Overview showing severe hydronephrosis with

inflammation, tubular dilatation and fibrosis. (E) Detail from cortex demon-

52

strating dilated tubuli filled with PAS-positive material (white arrow), inter-

stitial fibrosis, inflammation and tubular atrophy. Destructive glomerular

changes are conspicuous with proliferate changes, necrosis, collapse and

thickening of the bowman capsule (black arrows). (F) Areas with severe

fibrosis (black arrow), atrophy and tubular dilatation (red arrow). Photomi-

crographs A, B, D and E are stained with Periodic Acid-Schiff (PAS) and C

and F with Picro-Sirius. The control kidneys displayed normal histoarchitec-

ture with distinct cortex, medulla and renal papilla, with no histopathological

changes.

53

DISCUSSION

Hydronephrosis due to UPJ obstruction is a common condition in newborn

infants and the renal function in hydronephrosis, as measured by RBF and

GFR, has been demonstrated to be well preserved for a long time.27-29 The

outcome of these observations has been a worldwide trend towards more

conservative treatment of hydronephrosis. However, the long-term conse-

quences of this new treatment strategy are unknown. In the present study,

experimental hydronephrosis, induced by a PUUO at young age, caused

renal injury and salt-sensitive hypertension in both rats and mice. The hyper-

tension and salt-sensitivity correlated well with the degree of the hy-

dronephrosis and increased slowly with time. The explanation for this time

dependency is probably progressive hydronephrosis and renal pathological

changes.

Relief of chronic partial ureteral obstruction There are several case reports20, 34-58 of hypertension in hydronephrotic pa-

tients. The hypertension is obviously secondary to hydronephrosis as neph-

rectomy or pyeloplasty normalized the blood pressure. Even though the

management policy of hydronephrosis has become more conservative, symp-

tomatic patients are treated surgically. In Study II, relief from the obstruction

by unilateral nephrectomy or ureterovisicostomy attenuated salt-sensitive

hypertension in hydronephrotic animals, whereas, an augmentation occurred

after removal of the non-obstructed, functioning kidney. The reduction in

cross sectional area of the hydronephrotic kidney (i.e. the efficiency of inter-

nal urinary drainage) correlated with the concomitant drop in blood pressure.

54

Unilateral nephrectomy in the controls caused a transient blood pressure

increase, which was not seen in hydronephrotic animals after ipsilateral

nephrectomy. This could reflect that control animals had to adapt to the loss

of a normally functioning kidney, a situation that was already present in

hydronephrosis. Moreover, contralateral nephrectomy augmented salt-

sensitive hypertension in hydronephrosis. The hypertensive mechanisms are

primarily located to the diseased kidney, insofar as relief of the obstruction

attenuated blood pressure. However, as the blood pressure levels following

ipsilateral nephrectomy correlated with the previous degree of hydronephro-

sis, secondary mechanisms must be considered.

Renal excretion measurements The renal function, in terms of GFR and RBF, are well preserved for a long

time in hydronephrotic children.27-29 In animal models with PUUO, GFR

increases, remains unchanged or decreases. However, in general, ureteral

obstruction results in decreased RBF and GFR. The literature on electrolyte

and water excretion during PUUO is also conflicting.32 This discrepancy

may depend on the severity and duration of the obstruction, the diuretic state

and the compensatory ability of the contralateral kidney. In the model for

experimental hydronephrosis presented here, the results are consistent; both

rats and mice with PUUO display increased diuresis and reduced ability to

regulate electrolyte concentrations. The changes observed could possibly be

explained by reduction of the renal medulla and down regulation of aq-

uaporins-2 and sodium transporters in the obstructed kidney.74, 75

Renin Angiotensin System Many forms of secondary hypertension display changes in the activity of the

renin-angiotensin system, leading to increased concentration of circulating

angiotensin II, a powerful vasocontrictor that acts both systemically, by af-

55

fecting vascular tone, and intrarenally, by sensitising the TGF system. In

hydronephrotic patients with hypertension, increased plasma renin levels

have been demonstrated.34, 39, 41, 43-47, 49-51, 56-58, 76 However, the participation of

renin appears to be influenced by the duration of the obstruction, the pres-

ence or absence of a contralateral normal kidney and other intrarenal factors.

In the present studies, hydronephrotic rats, but not mice, displayed elevated

PRC, compared to the controls. The elevated renin concentrations were nor-

malized after removal of the hydronephrotic kidney. However, increased

PRC cannot entirely explain the hypertension, as animals with severe unilat-

eral hydronephrosis or bilateral hydronephrosis, (i.e. those with the highest

blood pressures) were the one with the lowest PRC. These data could be

interpreted as the severely hydronephrotic kidneys are damaged and cannot

respond to different salt diets in a normal way, or, that renin is down regu-

lated due to the higher blood pressure level. It has also been suggested77, 78

that the angiotensin II concentration may be 60-100 times higher in the renal

interstitium than in the plasma as a result of intrarenal formation. In this

thesis changes of intra renal renin levels were not studied and remain to be

investigated. Studies on the genetic model of rat hypertension (SHR), dem-

onstrate an important role of renin angiotensin system in the development,

but not maintenance, of hypertension. Taken together, the renin angiotensin

system is considered involved, but changes in plasma renin levels cannot

alone be responsible for hypertension in hydronephrosis.

Histopathological changes Obstructive nephropathy is not a result of simple mechanical impairment of

urine flow, but a complex condition resulting from alterations in the glome-

rular and tubular function.79 Complete unilateral ureteral obstruction causes

rapid destruction of the kidney, but is rare in clinical practice. Experimental

ureteral obstruction is an important model for studying the mechanisms of

56

renal fibrosis and inflammation, and for evaluating the impact of potential

therapeutic approaches to ameliorate renal disease. Increased activity of the

renin angiotensin system and oxidative stress with subsequent reduction in

NO availability are involved in the development of renal pathological

changes taking place in ureteral obstruction.80-84 The magnitude of renal

injuries and renal function impairment (i.e. reduction in GFR and RBF) has

been shown to be dependent on the angiotensin II type 1 receptor (AT1-R).

Inhibition of AT1-R during nephrogenesis exacerbates renal injuries of the

obstructed kidney,85, 86 but inhibition during subsequent renal maturation has

salutary effects.87

In the model for chronic PUUO presented here, the hydronephrotic kidneys

of rats and mice displayed variable degrees of dilatation of the pelvic area,

with flattening of the renal papilla. Moreover, hydronephrotic kidneys exhib-

ited areas with subepithelial fibrosis, infiltration of inflammatory cells (i.e.

plasma cells and lymphocytes), predominantly localized to the medulla and

pelvic region, and glomerular changes (i.e. sclerosis, mesangial matrix in-

crease and collapsed glomeruli). Tubular changes (i.e. hyaline material in the

lumen, atrophy and thickening of the basal membrane) and vascular changes

(i.e. hypertrophy of the media, hyperplasia in arterioles and arteries) were

only detected in animals with severe hydronephrosis. Mild fibrotic and glo-

merular changes and compensatory hyperthrophy (i.e. increased weight and

glomerular volume) were identified in the contralateral kidneys of animals

with PUUO. Based on this evidence, the model exhibited the characteristics

for obstructive nephropathy and could therefore provide useful information

for the clinician.

57

L-NAME and blood pressure NO is a potent vasodilator that possesses an important role in blood pressure

regulation. Long term blockade of the NO system with L-NAME results in

salt-sensitive hypertension.88-92 The acute blood pressure response to NOS

inhibition appears due to generalized arterial vasoconstriction, whereas, re-

nal sodium and water handling are more important for sustained blood pres-

sure elevation and salt-sensitivity. In the present study, the effects of chronic

L-NAME administration on blood pressure and salt-sensitivity were more

pronounced in controls than in hydronephrotic animals. After L-NAME ad-

ministration, blood pressure levels were still higher in the hydronephrotic

groups, but blood pressure elevation was highest in the controls. Salt-

sensitivity increased only in control animals, and after NO blockade, no dif-

ferences between the three groups (i.e. Controls, PUUO and PBUO) were

identified. This indicated that animals with hydronephrosis had reduced NO

availability, which could contribute to the development of salt-sensitive hy-

pertension.

L-arginine and blood pressure The L-arginine-NO pathway is ascribed an important role in the develop-

ment of systemic hypertension and progressive renal disease.93-95 NOS sub-

strate supplementation has beneficial effects on both blood pressure and

renal disease in clinical trials96-98 and in experimental models.99-105 In animals

with ureteral obstruction, the administration of L-arginine increases GFR

and RBF to the post-obstructed kidney, and decreases infiltration of macro-

phages of the renal parenchyma106 and tubulointerstitial fibrosis.103 In the

present study, chronic L-arginine supplementation reduced or prevented

further increase in blood pressure in hydronephrotic animals.

58

Nitric oxide, renal excretion and salt-sensitivity Reduced NO availability in hydronephrotic animals was apparent from renal

excretion measurements. Inhibition of the NO system by chronic L-NAME

administration did not change renal excretion properties in hydronephrosis,

whereas, the controls displayed a reduction in osmolar excretion. Stimulation

of the NO system by chronic L-arginine supplementation did not change

renal excretion in the controls, whereas, the hydronephrotic groups tended to

increase electrolytes and water excretion.

NO has an important role in the control of renal sodium excretion during

physiologic conditions, and plays a critical role in the adaptation to high

sodium intake.107-110 Evidently, high sodium conditions (i.e. high salt intake)

result in high NO concentrations and a large NOS expression, especially in

the renal medulla. An increased NO concentration will result in increased

sodium and water excretion and deficient NO increase during these condi-

tions will result in sodium retention and hypertension. The mechanisms for

this are unclear, but oxidative stress has been demonstrated in the patho-

physiology of salt-sensitive hypertension9, 111 and may also be involved in

hydronephrosis.

Supplementation of antioxidant vitamin-E (�-DL-tocopherol) in rat chow

(5% w/w) for three weeks lowered blood pressure in hydronephrotic animals

both during NS conditions (114±8 to 109±9 mmHg) and HS conditions

(120±6 to 104±5 mmHg), but not in control animals [(NS: 90±9 to 88±9

mmHg; HS: 92±5 to 91±5 mmHg)] (unpublished data). Vitamin E is a po-

tent, naturally occurring antioxidant that scavenges reactive oxygen species

and lipid peroxyl radicals. In a model for chronic kidney disease (5/6 neprec-

tomy), long-term vitamin E treatment reduces superoxide production and

preserves renal NO generation.112

59

Enhanced production of free radicals, especially superoxide, will reduce NO

availability with consequences, such as, increased TGF sensitivity, increased

afferent arteriolar responsiveness, reduced medullary blood flow and in-

creased tubular reabsorption.113 In the studies presented here, the suggestion

that reduced NO availability explains salt-sensitive hypertension was further

supported by reduced NOS protein expressions in the kidney cortex and

medulla of hydronephrotic animals. The same feature of reduced NO syn-

thase activity is observed in Dahl salt-sensitive rats.113

Sympathetic nervous activity, renal excretion and salt-sensitivity Increased renal sympathetic nerve activity can increase PRC, and has been

demonstrated to lead to sodium retention and salt-sensitive hypertension.114

An impairment of the renorenal reflexes contributes to hypertension in both

two-kidney one clip model, and in SHR.115, 116 Renal denervation delays the

onset of hypertension and is associated with increased diuresis and natriure-

sis. In the current model for hydronephrosis, renal denervation of the par-

tially obstructed kidney (at three and seven weeks of age) attenuated both

hypertension and salt-sensitivity (unpublished data).

L-arginine, ADMA and SDMA The limitation of L-arginine and increased levels of endogenous NOS inhibi-

tor (ADMA) are two possible causes of NO deficiency93 in renal and cardio-

vascular disease.94, 95 An association between oxidative stress and increased

ADMA levels has also been demonstrated.117 A closely related compound

SDMA does not inhibit NOS; however, as arginine, ADMA and SDMA

share a common pathway for enter into the cell, high plasma levels of

SDMA may indirectly reduce NO production by competing with arginine in

cellular uptake.118 In this study, plasma concentrations of ADMA and

60

SDMA were elevated, whereas, L-arginine levels were normal in hy-

dronephrosis. Endogenous inhibition of NOS and competition between

SDMA and arginine within the kidney may explain the reduced NO concen-

trations and thus, cause renal injury and hypertension in hydronephrosis.

Characteristics of TGF There is a connection between renal NO deficiency, enhanced TGF respon-

siveness and development of hypertension.66, 119, 120 Micropuncture experi-

ments have showed that the TGF mechanism, which is an important regula-

tor of GFR, is reset towards higher responsiveness in hydronephrosis during

volume expansion.61 This paradoxical change of the TGF is also manifested

in Milan hypertensive strain rats before they develop hypertension by vol-

ume retention and in SHR.119 The resetting of TGF sensitivity is intimately

coupled to nNOS derived NO from the macula densa cells in the juxtaglome-

rular apparatus. Increased NO availability desensitises TGF response (right-

ward shift), whereas decreased NO or increased angiotensin II increases

responsiveness (leftward shift). Chronic blockade of nNOS by 7-NI in-

creases TGF sensitivity in a similar way as in hydronephrosis, and leads to

the development of hypertension.66

Based on this, it appeared as if the functional adaptation to PUUO could lead

to the development of NO dependent hypertension. The stop-flow pressure

measurements demonstrated increased sensitivity and reactivity of TGF re-

sponse in normovolemic hydronephrotic animals. Inhibition of nNOS in-

creased responsiveness in controls, but not in hydronephrotic kidneys. How-

ever, intratubular infusion of L-arginine, attenuated TGF response in hy-

dronephrosis, whereas, no changes were observed in the controls. The

mechanisms for NO deficiency in the juxtaglomerular apparatus, with sub-

61

sequent resetting of the TGF response, are not clear, but oxidative stress in

the diseased kidney should be considered. The increased TGF responsive-

ness in SHR is similar to that seen in hydronephrosis and increased respon-

siveness can be diminished by administration of tempol (SOD-mimetic).120

62

CONCLUSIONS

Hydronephrosis, due to chronic PUUO, causes salt-sensitive hypertension in

both rats and mice. Hypertension can be markedly attenuated by relief of the

obstruction, thus, the mechanisms appear to be of renal origin and primarily

located to the diseased kidney. Hydronephrotic animals display an abnormal

renal excretion pattern with increased diuresis and impaired urine-

concentrating ability. This is associated with histopathological changes simi-

lar to that observed in obstructive nephropathy (i.e. interstitial fibrosis, in-

flammation, glomerular and tubular changes). These new findings imply that

the current conservative treatment strategy in hydronephrosis should be re-

considered in favour of treatment that is more active, in order to prevent the

development of renal injury and hypertension in later life. The mechanisms

for salt-sensitive hypertension in hydronephrosis are unclear and remain to

be further investigated. Increased renin angiotensin system activity, due to

ureteral obstruction, might be involved in the development but not necessary

the maintenance of hypertension. Reduced NO availability in hydronephrosis

may also be the result of increased oxidative stress in the diseased kidney.

Renal NO deficiency, and subsequent resetting of the TGF mechanism, ap-

pear to play an important part in contributing to the development of hyper-

tension in hydronephrosis.

63

FUTURE PERSPECTIVES

There is a causal relationship between experimental hydronephrosis and the

development of salt-sensitive hypertension. Experimental hydronephrosis

from chronic partial ureteral obstruction does not only provide useful infor-

mation for the clinicians, but offers a new model for studies of the mecha-

nisms behind salt-sensitive hypertension. Chronic kidney disease is accom-

panied by NO deficiency and oxidative stress, which contribute to the pro-

gression. It is possible that increased oxidative stress in the diseased kidney

can also explain the reduced NO availability in hydronephrosis. Further in-

vestigation into the role of oxidative stress with the described model for

PUUO in genetically modified mice (e.g. SOD-transgenic and SOD-knock-

out) could be beneficial.

64

SAMMANFATTNING PÅ SVENSKA

Hydronefros är ett ganska vanligt förekommande fenomen hos nyfödda barn

med en incidens på cirka 0.5-1%. Vanligtvis är orsaken en förträngning i

övergången mellan uretär och njurbäcken. Under det senaste årtiondet har

behandlingspolicyn för asymptomatisk hydronefros blivit alltmer konserva-

tiv, men de långsiktiga fysiologiska konsekvenserna av detta är inte klarlag-

da. Det är väl känt att det finns en tydlig koppling mellan en förändrad njur-

funktion och utvecklingen av sekundär hypertension. I denna avhandling

undersökte vi om det finns en koppling mellan hydronefros och hyperten-

sion. Hydronefros inducerades genom att skapa en partiell förträngning av

uretären i tre veckor gamla råttor eller möss. I de vuxna djuren mättes blod-

trycket med hjälp av telemetri och njurfunktionen studerades. Båda djursla-

gen utvecklade saltkänslig hypertension och histopatologiska förändringar

såsom fibros, inflammation och glomerulära och tubulära förändringar. De

observerade förändringarna korrelerade väl med hydronefrosgraden (mild,

måttlig eller kraftig hydronefros). De hydronefrotiska djuren hade ett föränd-

rat renalt utsöndringsmönster i form av en ökad diures och en reducerad

förmåga att koncentrera urinen. Mekanismerna för hypertensionen anses

huvudsakligen vara lokaliserade till den hydronefrotiska njuren eftersom

avlägsnande av förträngningen via nefrektomi eller pyeloplastik reducerade

såväl blodtrycket som saltkänsligheten. Obstruktion av uretären kan leda till

en ökad aktivitet av renin-angiotensin systemet, vilket skulle kunna vara

involverat i utvecklingen av hypertensionen. Eftersom djuren med kraftigast

hydronefros och högst blodtryck inte hade ökade reninnivåer behöver dock

inte förändringar i detta system vara involverade i upprätthållandet av hyper-

tensionen. De hydronefrotiska djuren hade en reducerad kväveoxidtillgäng-

65

lighet, vilket kan bero på en ökad oxidativ stress i den skadade njuren. Brist

på kväveoxid i njuren och ökad känslighet av den tubuloglomerulära åter-

kopplingsmekanismen verkar spela en betydande roll i utvecklingen av hy-

pertensionen. De nya fynden indikerar att dagens konservativa behandlings-

strategi vid hydronefros borde förändras för att förhindra utvecklingen av

njurskador och saltkänslig hypertension senare i livet.

66

ACKNOWLEDGEMENTS

This study was carried out at the Department of Medical Cell Biology, Divi-

sion of Integrative Physiology, Faculty of Medicine, Uppsala University,

Sweden. The work was financially supported by the Swedish Research

Council, the Wallenberg Foundation, Wallenberg consortium North, the

Swedish Heart and Lung Foundation, the Ingabritt and Arne Lundberg

Foundation, Her Royal Highness the Crown Princess Lovisa’s Fund for Sci-

entific Research, and the Gillbergska Foundation.

I would like to express my deep and sincere gratitude to all those who have

contributed to this thesis by helping and encouraging me. In particular, I

would like to mention:

Erik Persson, my supervisor and colleague, who introduced me into the

field of renal research and who has encouraged and supported me throughout

these years. We have had a lot of inspiring discussions and interesting con-

ferences together. With your help, I have learnt how to design my own pro-

jects and the importance of individual and critical thinking.

Nils Wåhlin, my co-supervisor and colleague, for guidance and introduction

to animal studies of hydronephrosis. For support, fruitful discussions and

drafting of the manuscripts.

Russell Brown, my co-supervisor and colleague for introducing and helping

me with the micropuncture experiments, and for being Lasslo down under.

67

Johan Sällström, my friend and colleague, for all the good moments we

have had together, inside and outside the lab. For being patient and always

helpful when data support was needed. No one can work like you Living-

stone.

Enyin Lai and Zufu Ma, for good collaboration during days, nights and

weekends in the lab, listening to Chinese music.

Andreas Patzak and the German team, for good collaboration, fruitful dis-

cussions and nice times in Uppsala and Berlin.

Erik Larsson, for interesting discussions and for helping me with the histo-

logical evaluations

Ulrika Larsson, for helping me with the histological preparations

Peter Thorén, AstraZeneca, Mölndal, Sweden, is acknowledged for help

with the development of the telemetric analysis program.

Ole Skøtt, Department of Physiology and Pharmacology, University of

Southern Denmark, Odense, Denmark, for help with the analysis of the

plasma renin concentration.

Joel Petersson, for these 10 years together at BMC. You are a good friend

and a good chef.

Angelica, Britta and Annika for all your knowledge and help in the lab.

All the colleagues at the Department of Medical Biology, Uppsala Uni-

versity: Lina, Fredrik, Olof, Louise, Mia, Johanna, PO, Andrei, Jenny, Ma-

68

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

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