Combined inhibition of 20-hydroxyeicosatetraenoic acid formation and of epoxyeicosatrienoic acids degradation attenuates hypertension and hypertension-induced end-organ damage in Ren-2 transgenic rats.

Abstract

Recent studies have shown that the renal CYP450 (cytochrome P450) metabolites of AA (arachidonic acid), the vasoconstrictor 20-HETE (20-hydroxyeicosatetraenoic acid) and the vasodilator EETs (epoxyeicosatrienoic acids), play an important role in the pathophysiology of AngII (angiotensin II)-dependent forms of hypertension and the associated target organ damage. The present studies were performed in Ren-2 renin transgenic rats (TGR) to evaluate the effects of chronic selective inhibition of 20-HETE formation or elevation of the level of EETs, alone or in combination, on the course of hypertension and hypertension-associated end-organ damage. Both young (30 days of age) prehypertensive TGR and adult (190 days of age) TGR with established hypertension were examined. Normotensive HanSD (Hannover Sprague-Dawley) rats served as controls. The rats were treated with N-methylsulfonyl-12,12-dibromododec-11-enamide to inhibit 20-HETE formation and/or with N-cyclohexyl-N-dodecyl urea to inhibit soluble epoxide hydrolase and prevent degradation of EETs. Inhibition in TGR of 20-HETE formation combined with enhanced bioavailability of EETs attenuated the development of hypertension, cardiac hypertrophy, proteinuria, glomerular hypertrophy and sclerosis as well as renal tubulointerstitial injury. This was also associated with attenuation of the responsiveness of the systemic and renal vascular beds to AngII without modifying their responses to noradrenaline (norepinephrine). Our findings suggest that altered production and/or action of 20-HETE and EETs plays a permissive role in the development of hypertension and hypertension-associated end-organ damage in this model of AngII-dependent hypertension. This information provides a basis for a search for new therapeutic approaches for the treatment of hypertension.

Full text

Page 1

The renal circulation, tubular reabsorption and release of
renin are under multiple control by the renal nerves,
hormones and paracrine active agents (DiBona & Kopp,
1997). Therefore, removal of the neural input by renal
denervation, leading to inhibition of tubular transport
(denervation natriuresis) and, under some circumstances, to
a decrease in renin secretion and an increase in renal blood
flow may be expected to be compensated, at least in part,
by control systems working in the opposite direction. For
instance, humoral vasoconstrictor agents could help offset
the loss of vasoconstrictor tone provided by noradrenaline
released from renal nerve endings. However, it is possible
that some of the compensatory mechanisms do not come
into play immediately after denervation but require some
time to develop. For instance, the sensitivity of the tubulo-
glomerular feedback has been reported to be decreased as a
function of time when measured 60—240 min after acute
denervation. (Thorup et al.1995).
The standard technique of renal denervation necessitates
considerable dissection around the kidney, cutting all visible
nerve fibres of the renal pedicle and painting the renal
artery with phenol solution in ethanol. This procedure is
extended in time and traumatising; the conspicuous signs of
trauma are alternate periods of constriction and dilatation
of the renal artery. Therefore, the protocols in such acute
studies allowed at least one hour postdenervation before
starting measurements; by that time a new steady-state has
presumably been established. An exception to this standard
approach have been studies in which local anaesthetics were
applied to the renal pedicle for partial reversible suppression
of transmission in the renal nerves (Richardson et al. 1974;
Grady&Bullivant, 1992).
In the search for a method to enable assessment of the early
effects of renal denervation, before potential compensatory
mechanisms have come into play, we have developed a
technique enabling rapid denervation by a procedure that is
Journal of Physiology (2001), 531.2, pp.527—534
527
Early effects of renal denervation in the anaesthetised rat:
natriuresis and increased cortical blood flow
ElzbietaKompanowska-Jezierska, AgnieszkaWalkowska, EdwardJ. Johns*
andJanuszSadowski
Laboratory of Renal and Body Fluid Physiology, Medical Research Centre of the Polish
Academy of Sciences, PL 02-106 Warsaw, Poland and *Department of Physiology,
Medical School, Birmingham B15 2TT, UK
(Received25August2000;acceptedafterrevision31October2000)
1.A novel method of renal denervation was developed based on electro-coagulation of tissue
containing most of the sympathetic fibres travelling towards the kidney. Kidney tissue
noradrenaline was decreased to 4·7% of the content measured in the contralateral
innervatedkidneywhenstudied3dayspostdenervation.
2.The method was utilised in anaesthetised rats to examine the effects of denervation within
the heretofore unexplored first 75 min period postdenervation. Sodium excretion (UNaý )
increasedsignificantly(+82%, P < 0·03) overthe25—50 min afterdenervation. Inaparallel
group, with a lower baseline UNaý, there was also a significant increase in UNaý (+54%,
P < 0·03) withinthefirst25 min. Therenalperfusionpressurewasmaintainedataconstant
valueandtheglomerularfiltrationratedidnotchangeafterdenervation.
3. Renal cortical and medullary blood flows (CBF, MBF) were estimated as laser Doppler flux
andmedullarytissueionconcentrationwasestimatedaselectricaladmittance(Y).Following
denervation, in both groups CBF increased significantly within the first 25 min (+12%,
P < 0·01 and +8%, P < 0·05, respectively) while MBF did not change or decreased slightly;
Ydidnotchange.
4.The data document the development of natriuresis within the first 25—50 min after
denervation. The increase in CBF indicated that, prior to denervation, the cortical, but not
medullary, circulation was under a tonic vasoconstrictor influence of the renal nerves. Such a
dissociation of neural effects on the renal cortical vs. medullary vasculature has not been
previouslydescribed.
11612
Keywords:

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significantly less traumatising than the standard method.
The tissue containing most of nerve fibres entering the renal
hilus was surrounded with a thin flexible wire loop with
minimal prior dissection. After baseline measurements of
renalfunction, ahighfrequencycurrentwasappliedtothe
wire and the tissue was cut by electro-coagulation; renal
haemodynamic measurements (laser Doppler) were not
interrupted and urine collection was started almost
immediately after denervation. In the present study, this
methodwasusedtoexaminetheearlyeffectsofdenervation
onrenalcirculationandrenalexcretion.
METHODS
Experiments were designed to examine renal haemodynamics,
diuresis, natriuresis, and total ion concentration in the medullary
tissuebeforeanddirectlyafter, denervationoftheleftkidneyusing
a technique newly developed developed in our laboratory. The
experimental procedures were approved by the Ethical Committee
oftheMedicalResearchCentre, PolishAcademyofSciences.
Preparations and surgical procedures
Male Wistar rats were fed a standard pellet diet (Altromin GmbH,
Lage, Germany) and had free access to water. The animals, weighing
280—340g, were anaesthetised with sodium thiopenthal (Biochemie
GmbH, Vienna, Austria, 100 mg kg¢, i.p.), which provided stable
anaesthesia for at least the three hours needed to complete an
experiment. A polyethylene tube was placed in the trachea to
ensure free airways. The rats were placed on a heated surgery table
to maintain rectal temperature at about 37°C. The femoral vein
was cannulated for fluid infusions and the femoral artery was used
for aortic blood pressure measurement and blood sampling. The left
kidney was exposed via a flank incision, immobilised in a plastic
holder and the ureter was cannulated to permit timed urine
collections.
Subsequently, preparations were made to enable rapid renal
denervation later during an experiment. Although the kidney had
been dissected free from the adjacent tissue, special care was taken
toleaveintactthefatandconnectivetissuecephaladtothejunction
of the renal artery and the aorta. A loop of stainless steel wire,
0·1 mm in diameter, was placed around this tissue. Previous
dissection studies under a dissecting microscope have consistently
shown that most nerve fibres travelling from the coeliac ganglion
toward the renal pedicle, and many others whose origin could not
be clearly defined, also converging towards the kidney, are located
in this area; we have practiced encompassing all these fibres within
the wire loop without actual dissection. Both ends of the loop were
threaded through a segment of thick-walled polyethylene tubing,
about 0·6 mm inner diameter, and left loose. In order to effect the
denervation, the loop was tightened and fixed with a bulldog
clamp; immediately thereafter 0·15 ml of 1% novocaine solution
was injected through the tubing, twice at 20 s intervals. This was
done in order to minimize the potential trauma due to subsequent
cutting of the nerves. The trauma could include a momentary
efferent and afferent renal nerve stimulation leading to disturbances
of intrarenal or systemic haemodynamics. The tissue within the
loop, including renal nerve fibres, was electro-coagulated: a 5 MHz
current generated by a neurosurgical cautery device (Famed,
Warsaw, Poland) was applied for 5−10 s until the tissue was cut
completelyandtheloopcouldberemoved.
In all experiments a screw-controlled snare was placed around the
aorta above the renal arteries. The snare was slightly tightened to
lower blood pressure distal to the constriction site by 4—8 mmHg.
Later during experiments the snare could be released or tightened
as needed, and the mean blood pressure below the snare, equivalent
to renal perfusion pressure (RPP) was maintained constant. A set
of two probes used for measurement of inner medullary blood flow
(laser Doppler flux, MBF) and tissue electrical admittance (Y) was
inserted into the kidney and another laser Doppler probe, for
measurement of cortical blood flow (CBF), was placed on the
kidneysurface(seebelow:Measurementofintrarenalblood).
During surgical procedures 3% bovine serum albumin was infused
i.v. at 2·4 ml h¢ to preserve plasma volume. Directly after
insertion of the MBFÏY measuring set, this infusion was replaced
by Ringer solution, 3·2 ml h¢. In one series (protocol II, see below)
this solution contained [methoxy-ÅH] inulin given at a rate of
6 ìC h¢, for measurement of the glomerular filtration rate (GFR).
The details of clearance procedures as well as analytical techniques
for plasma and urine osmolality, sodium, and tritiated inulin have
been described previously (Kompanowska-Jezierska et al. 1994).
After completion of the experimental protocols the animals were
killedbyanintravenousoverdoseofsodiumpentobarbitone.
Experimental protocols
Protocol I (n = 11). In addition to the surgical preparations
described earlier, the urinary bladder was cannulated and left
ureteral and right kidney (bladder) urine were collected separately.
Two control 25 min bilateral urine collections were made, the left
kidney was then denervated, urine collection was resumed and
three further 25 min bilateral collection periods were made.
Throughout the experiment RPP was maintained constant at about
120 mmHg, and CBF, MBF and medullary tissue Y were
continuouslyrecorded.
Protocol II (n = 8). Experiments were conducted 2 months later
than the other studies. Urine was collected from the left kidney
only (the bladder was not cannulated). Since the rats’ baseline
aortic blood pressure was lower than in the other groups, RPP was
initially set at 110 instead of 120 mmHg so that adjustments
needed to keep it constant could still be made as described earlier.
The sequence of control and experimental urine collections was as
in protocol I. In this series, tritiated inulin was infused and GFR
wasdetermined.
Protocol III (left renal sham denervation, n = 7). The procedure
was as in Protocol I except that the wire loop normally used for
denervationwasplacedaroundthefatandconnectivetissuelocated
caudal to the aortic—renal artery junction and this tissue was later
electro-coagulated. Urine was separately collected from the left and
therightkidneyandRPPwasmaintainedat120 mmHg.
Protocol IV (n = 6). In this study, the sequential effects of
novocaine and electro-coagulation on CBF were examined. In rats
destined for denervation and later determination of renal
noradrenaline content, prior to denervation two surface laser
Doppler probes were placed non-invasively on the dorsal surface of
the left kidney: PF407 type used in all studies (optical fibre
separation 0·15 mm) and PF416 type (separation: 0·25 mm); the
latter probe provides a deeper penetration of the light beam
i.e.measures signals within the cortex at a slightly greater depth.
Initially, CBF was recorded by the two probes for about 10 min and
thereafter novocaine was applied to the tissue containing the nerve
fibres via the tubing segment housing the wire loop. Subsequently,
about 20—25 min were allowed to observe the effect of novocaine
itself on CBF before electro-coagulation was performed. This was
followed by 10—15 min observation of CBF after electro-
coagulation.
E. Kompanowska-Jezierska and others
J. Physiol. 531.2
528

Page 3

Measurements of intrarenal blood flow and tissue electrical
admittance
Medullary blood flow (MBF, laser Doppler flux) was measured
using a PF402 needle probe inserted into the kidney to the depth
of the inner medulla and connected to a PeriFlux 4001 flowmeter
(Perimed, Jarfalla, Sweden). Tissue electrical admittance (Y, an
index of total ion concentration in medullary interstitium) was
measured at about the same depth, between the tips of the stainless
steelcannulahousingtheopticalfibresoftheprobeandofaparallel
iridium-platinum needle, both forming an admittance cell connected
to a conductance meter measuring at a frequency of 24 kHz
(Sadowski et al. 1997). Another laser Doppler probe (PF407) was
placed on the kidney surface for measurement of superficial cortical
blood flow. The laser Doppler flux values (a product of the number
of blood cells moving and their mean velocity, within an area less
than 1 mmÅ beneath the tip of the probes) were expressed in
arbitrary perfusion units (PU) or in volts of the analogue output
(1000 PU = 10 V). Thus, only relative changes were measured but
the calibration procedure allowed the results to be compared
betweenanimals.
Noradrenaline content of renal tissue
For verification of the effectiveness of the method described above,
the noradrenaline content of renal tissue was determined 3 days
after denervation. Six rats were anaesthetised with intraperitoneal
sodium pentobarbitone, 50 mg (kg body wt)¢ with small intra-
venous supplements as required. The left kidney was exposed and a
short non-invasive experiment consisting of measurements of renal
cortical blood flow before and after denervation was performed
(Protocol IV). Left renal denervation was performed as described
above. Subsequently, ampicillin powder, about 100 mg (kg body
weight)¢, was applied directly into the abdomen and the flank
wound was closed. Ringer solution (5 ml) was administered sub-
cutaneously and, for postoperative analgesia, metamizole sodium
(20 mg kg¢) was given. On the third day postdenervation, animals
were re-anaesthetised, both kidneys were removed through a
midline abdominal incision, cut to •0·3 g pieces, frozen on dry ice,
weighed, and stored at −80 °C until analysed. The tissue was
deproteinized with a mixture of perchloric acid and reduced
glutathione, and homogenized. A part of the homogenate was used
for determination of protein content, the rest was centrifuged and
the supernatant, after appropriate dilution, was analysed for
noradrenalineusingaradioenzymaticmethod(Johnson et al.1980).
Statistics
The significance of trends was first examined by repeat
measurement analysis of variance and then differences between
means were tested by Student’s t test for dependent variables. The
standard error of mean (s.e.m.) was used as a measure of data
dispersion.
Early effects of renal denervation in the rat
J. Physiol. 531.2
529
Fig. 1. The acute effect of renal denervation (n = 11) or sham denervation (n = 7) on sodium
excretion (UNaý ) and urine flow (ý )
The data are means ± s.e.m. for the denervated (0) or contralateral (1) kidney (n = 11), and for the sham
denervated (2) or contralateral (3) kidney (n = 7). In all experiments the renal perfusion pressure was
maintained constant at about 120 mmHg. *Significantly different from the predenervation period (second
controlperiod)at P <0·05orless.

Page 4

RESULTS
The mean tissue noradrenaline concentration measured 72 h
after left renal denervation in six rats was 3·5 ± 0·7 pmol
(mgprotein)¢ inthedenervatedkidneywhichwaslessthan
5% of the 88·0 ± 23·6 pmol (mg protein)¢ recorded in the
contralateral innervated kidney (P < 0·001, paired Student’s
t test).
Urine osmolality in these studies ranged from 950 to 1350
mosmol (kg HµO)¢ and was not significantly changed by
denervationorshamdenervation.
It can be seen in Fig. 1 that following the denervation there
was a progressive increase in UNaý of the denervated
kidney beginning in the first postdenervation period, which
became significant at 25—50 min after denervation and
further increased in the third postdenervation period. The
changes in urine flow (ý) (Fig. 1) roughly paralleled those of
UNaý. There was no change in UNaý and ý in either the
sham-denervated kidneys or in the contralateral kidneys of
the group of rats subjected to the renal denervation. Blood
pressure remained at a constant level throughout the
observation period. Figure 2 presents data from an additional
group (protocol II) in which left kidney denervation was
performed as in protocol I but in which the baseline
conditions were slightly different. Renal perfusion pressure
was set 10 mmHg below that in protocol I, and the overall
surgical trauma was slightly less (the bladder was not
cannulated). In this group, the baseline UNaý was
approximately one half of that measured in protocol I
studies, but there was a significant increase in UNaý and ý
within the first 25 min after denervation, i.e. earlier than in
the rats subjected to protocol I. It was evident that there
was no change in GFR during experiments. There was also
nochangeinbloodpressurethroughouttheperiodofstudy.
The medullary tissue admittance (Y), an index of inter-
stitial NaCl concentration, was not significantly affected
over the 75 min following renal denervation. In the group of
rats subjected to protocol I the control value was
964 ± 49 ìS, compared with 984 ± 40 ìS measured 75 min
after denervation (a 2% difference), whilst in the group
undergoing protocol II these values were 882 ± 35 and
887 ± 38 ìS, respectively (less than 1% difference). There
was also no change in medullary tissue admittance in the
animalsinwhichshamdenervationwasperformed.
Figure 3 shows the effects of denervation on CBF and MBF
in the two renal denervation groups (protocols I and II)
and in the sham-denervated group (protocol III). In both
denervated groups, CBF increased significantly (P < 0·05)
within 25 min or less and remained elevated for the
subsequent50 min ;shamdenervationhadnoeffectonCBF.
MBF was unchanged following denervation in both protocol
I and II. Indeed, MBF tended to decrease in protocol II and
at 50—75 min was even slightly but significantly (P < 0·05)
E. Kompanowska-Jezierska and others
J. Physiol. 531.2
530
Figure 2. The acute effect of left renal denervation on ipsilateral sodium excretion (UNaý ), urine
flow (ý ) and glomerular filtration rate (GFR) in rats with renal perfusion pressure maintained
constant at •110 mmHg
The data are means ± s.e.m., n = 8. *Significantly different from the predenervation period (second
controlperiod)at P< 0·05 or less.

Page 5

lower than control (Fig. 3). However, a similar tendency to
decrease over the time period of observation was also seen
in the sham denervation experiments. In all these protocols,
blood pressure remained at an unchanged value over the
periodofexperimentation.
Additional observations on the effect of inhibition of renal
nerve activity were made in studies in which application of
novocaine was followed by a 20—25 min observation of CBF
before electro-coagulation was performed in the usual way
(Table 1, Fig. 4). Novocaine caused a significant increase in
CBF recorded both by the surface laser Doppler probe, used
inallthedefinitivestudies(PF407)andbythesurfaceprobe
measuring slightly deeper in the cortex (PF416). However,
in most cases, an initial increase in CBF was seen as soon as
novocaine was applied, but then it tended to decrease after
about 10 min. The subsequent electro-coagulation caused a
furtherincreaseinCBFwhichstartedafterabout8 min and
was complete at around 18 min. The maximum rise in CBF
after electro-coagulation was significantly higher (P < 0·05)
than that achieved after application of novocaine. The mean
overall increase after novocaine plus electro-coagulation was
Early effects of renal denervation in the rat
J. Physiol. 531.2
531
Figure 3. The acute effect of left renal denervation or sham denervation on cortical (CBF) and
medullary (MBF) blood flow (laser Doppler flux)
The data points represent means of the values averaged for simultaneous 25 min urine collection periods;
symbols refer to denervation studies at renal perfusion pressure maintained constant at 120 mmHg (0),
110 mmHg (6), and sham denervation at 120 mmHg (2). *Significantly different from the predenervation
period(secondcontrolperiod)at P< 0·05 or less.
––––––––––––––––––––––––––––––––––––––––––––– –
––––––––––––––––––––––––––––––––––––––––––––– –
Table 1. Effects of application of novocaine to the tissue containing renal nerve fibres,
and of subsequent electro_coagulation of this tissue, on the cortical blood flow
––––––––––––––––––––––––––––––––––––––––––––– –
Control
––––––––––––––––––––––––––––––––––––––––––––– –
ProbePF407 537 ± 22
ProbePF416508 ± 36
––––––––––––––––––––––––––––––––––––––––––––– –
Cortical blood flow (laser Doppler flux, perfusion units) was measured on kidney surface by a standard
PF407 probe and a PF416 probe that provided slightly deeper penetration of the laser light beam.
Means ± s.e.m., n = 6. *Significantly different from control value for the same probe, †significantly
differentfromtheprecedingnovocainevalueat P<0·05orless(testedbypairedStudent’s t test).
––––––––––––––––––––––––––––––––––––––––––––– –
––––––––––––––––––––––––––––––––––––––––––––– –
Novocaine Coagulation
575 ± 14 *
546 ± 30 *
617 ± 14 †
610 ± 21 †

Page 6

approximately 20% with the PF407 probe and 15% with
thePF416probe.
DISCUSSION
The effectiveness of our method of renal denervation was
validated by determination of tissue noradrenaline content.
The degree of depletion, 5% of the concentration
measured in the contralateral innervated kidney, compares
satisfactorily with the data reported by authors using a
standard method of renal denervation. A precise
comparison would be difficult because, over the years,
methods used for noradrenaline determination have varied
and the results have been expressed differently: either per
gram of tissue or per gram of tissue protein. In two more
recent studies using the radioenzymatic method, as in our
study, noradrenaline concentration after denervation
equalled less than 10% (Fernandez-Repollet et al. 1985) and
less than 2% (Greenberg et al. 1993) of the content
measuredintheinnervatedkidney.
Acute denervation effects on renal excretion and GFR
Denervationnatriuresisis
reproducible phenomenon, dependent on exclusion of the
neural stimulatory influence on tubular reabsorption and
oneofthemajorgoalsofthepresentstudywastoestablish
its earliest point of onset. Therefore, we attempted to
awelldescribed and
avoid circumstances which could alter UNaý independent
of changes in renal sympathetic nerve activity. Even modest
changes in RPP can affect UNaý and it is noteworthy that
in our studies at RPP of 120 mmHg (protocol I) baseline
UNaý was roughly double that measured at 110 mmHg
(protocol II). To take into account this phenomenon, we
maintainedRPPprecisely
experiments.
constantthroughout
The data showed that the natriuresis began within the first
25 min and was fully expressed by 50 min after denervation,
which was a time frame that had not been accessible to study
using the more conventional methods of renal denervation.
The different consecutive phases of classical denervation,
such as severing nerve fibres in the hilus, stripping the
adventitia off the renal artery and painting the vessel with
phenol solution, result in alternate periods of constriction
and dilatation of the renal artery which were of variable
duration. It is most likely that together these manoeuvres
would be associated with intrarenal vasoconstriction and
vasodilatation, andfluctuationsofGFR.
Here the excretion of sodium and water increased
progressively throughout the 75 min observation period. It
was of interest that there was no decrease in UNaý on the
contralateral side, as reported by a number of workers and
described as a postdenervation reno—renal reflex enhance-
E. Kompanowska-Jezierska and others
J. Physiol. 531.2
532
Figure 4. A record of the effects of novocaine application and subsequent electro-coagulation of
the tissue encompassing most renal nerves on the cortical blood flow
Cortical Blood Flow (CBF, laser Doppler flux in perfusion units) was measured by two surface probes: the
standardPF407probeandthePF416probecharacterizedbyaslightlydeepertissuepenetration.

Page 7

ment of the innervated kidney function (DiBona & Kopp,
1997). One possible explanation may be that there is a delay
in the reflex enhancement of tubular sodium reabsorption in
the innervated kidney and that the effect did not become
apparentoverthetimecourseofourexperiments.
Since the denervation natriuresis was not associated with
any alteration in GFR, the early progressive increase in
sodium and water excretion was most likely due to a
progressive postdenervation decrease in tubular sodium
reabsorption. This postdenervation decrease in sodium
reabsorption in the proximal tubule has been documented
by many groups of workers (DiBona & Kopp, 1997). This
observation would also accord with early morphological
data indicating the presence of nerve endings in contact
with the tubular cells of the thick ascending limb of Henle’s
loop. Moreover, there is functional evidence from tubule
microperfusion experiments of an increase in sodium
transport in this segment after renal nerve stimulation
(DiBona & Sawin, 1982) and a decrease in sodium transport
following renal denervation (Bencsath et al. 1985). The
action of the renal nerves on the thick ascending limb of
Henle’s loop could manifest itself as a decrease in electrolyte
concentration in the medullary interstitium and, indeed,
one study reported a postdenervation decrease in sodium
and total solute concentration in tissue slices of the renal
medulla (Kurkus et al. 1980). Therefore, in the present
study we measured electrical admittance of medullary
tissue (Y) as an index of local ion concentration and NaCl
transport in medullary loops (Sadowski et al. 1997). We
found no change in medullary tissue Y, indicating no
distinct effect of renal denervation (protocols I and II) or
sham denervation on salt concentration in the medullary
interstitium. The reasons underlying this may be straight-
forward. The denervation would have reduced proximal
tubular reabsorption and thereby increased delivery of fluid
to the loop. Because the transport in this segment is clearly
flow dependent, it is possible that any intrinsic decrease in
NaCl transport in the ascending limb of Henle’s loop
resulting from denervation would be, at least in part, offset
by an increase in transport related to a greater inflow to
thissegment.
Acute effects of denervation on renal cortical and
medullary blood flow (CBF, MBF)
The evidence from a range of previous investigations has
shown that in conscious animals renal denervation does not
cause any increase in total renal blood flow, which was
probably due to low baseline renal sympathetic nerve activity.
In animals that were anaesthetised andÏor subjected to a
surgical procedure, a modest increase or no change in blood
flow has been reported. The actual response would depend,
presumably, on the animal species and various features of
the experimental conditions which determined the level of
predenervationrenalnerveactivity(DiBona&Kopp, 1997).
Theclassicalmethodologymakesuninterruptedmeasurement
ofrenalcirculationbeforeandafterdenervationimpracticable
because of the mechanical interventions and disturbances.
In this study both CBF (which can be taken as a reliable
index of the total renal blood flow) and MBF were measured
continuously and the data before and immediately after
denervation could be compared directly. The clear post-
denervation increase in CBF (but not in MBF) observed
under our experimental conditions suggested that the cortical
circulation was under a tonic vasoconstrictor influence of the
renal sympathetic nerves. There seems to be no doubt that
the increase in renal cortical circulation after the electro-
coagulationprocedurewasaconsequenceoftheinterruption
of the renal sympathetic nerves. This interpretation was
further supported by the studies in which novocaine
application and electro-coagulation were separated in time.
The CBF responses to pharmacological blockade and to
surgical denervation were similar although the former were
less pronounced and transient. In agreement with earlier
studies (Richardson et al. 1974; Grady & Bullivant, 1992),
the local anaesthetic must have partially interrupted neural
traffic to the kidney. As the action of novocaine is short
lasting, this would explain why the increase in CBF began
to subside after about 10 min. The subsequent electro-
coagulation caused a further significant and lasting increase
in CBF (Table 1, Fig. 4). Indirectly, the clear increase in
CBF after application of the novocaine to the area
encompassed by the wire loop indicated that the tissue
which was electro-coagulated contained most of renal nerve
fibres. A second fibreoptic laser Doppler probe (PF416) was
used in this study, which provided a greater penetration of
the laser beam than that obtained with the standard PF407
probeusedintheotherexperimentalseries. Theincreasesin
CBF measured with the two probes after novocaine
application were comparable, which suggests that the increase
in CBF was not confined to the most superficial layer of the
cortex.
It was interesting that MBF did not increase after
denervation, indeed, a trend for a reduction and, ultimately,
a slight significant decrease was observed in one group of
renal-denervated rats (Fig. 3). One possible explanation
might be that the decrease in MBF could have resulted from
a decrease in RPP. It is recognised that within the medulla
autoregulation of the renal blood flow is not as efficient as in
the cortex (Pallone et al. 1990). Accordingly, it could be
argued that even a moderate decrease in RPP could have
caused a depression of medullary circulation and offset any
increase in MBF in response to renal denervation. However,
thiswouldnotapplyinthepresentexperimentsasRPPwas
not allowed to vary by more than 1—2 mmHg. A more
likely reason for a decrease in MBF would be some
deterioration of medullary circulation with time, which was
presumably more pronounced in the group of rats whose
RPP was maintained at a lower level (110 vs. 120 mmHg). It
was apparent that the deterioration in the medullary
circulation was not alleviated by renal denervation as the
decreasing trend was similar to that observed in the sham-
denervatedkidney(Fig. 3).
Early effects of renal denervation in the rat
J. Physiol. 531.2
533

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A possible dissociation of the effects of renal sympathetic
nerveactivityontherenalcorticalandmedullarycirculations
had been proposed previously but the findings of earlier
studies were questioned because of an inadequacy of the
methods used for the separate measurement of blood flow in
the two kidney zones (DiBona & Kopp, 1997). More recently,
both afferent and efferent arterioles of juxtamedullary
glomeruli in the hydronephrotic kidney preparation (a non-
filtering kidney with reduced blood flow) were found to
constrict after renal nerve stimulation (Chen & Fleming,
1993), compatible with a role for renal nerves in the control
of medullary circulation. On the other hand, laser Doppler
measurements of cortical and papillary blood flow in
anaesthetised rats showed that the blood flow responses to
renal nerve stimulation were much smaller in the papilla
where a 4% reduction was seen only with stimulation at the
highest frequency rate of 5 Hz (Rudenstam et al. 1995).
These data are consistent with the present denervation
studies which suggested that the medullary circulation in
anaesthetised rats was not under the tonic influence of the
renalnerves. Overall, thereisincreasingevidencethatcontrol
mechanisms of the medullary and cortical circulations may
differsignificantly.
Insummary, wehavedevelopedanovelapproachforarapid
renal denervation in the rat, which is less invasive but as
effective as the classical approach, as demonstrated by the
marked reduction in tissue noradrenaline level. Owing to
the reduced invasiveness of our procedure, measurements of
renal haemodynamics and renal excretion were not
interrupted at the moment of denervation and protocols
could be designed whereby an experimental intervention
and measurement (including suitable controls) could be
made before and then after denervation, within the same
experiment in one animal. It was found that the responses
of the cortical and medullary circulation were dissociated:
CBF increased almost immediately after denervation whilst
MBFdidnot. Inaddition, wedemonstratedthatanatriuresis
developed within about 0·5 h after denervation, without
changesinGFR.
Bencsath, P., Szenasi, G. & Takacs, L. (1985). Water and electrolyte
transport in Henle’s loop and distal tubule after renal
sympathectomy in the rat. American Journal of Physiology 249,
F308—314.
Chen, J. & Fleming, J. T. (1993). Juxtamedullary afferent and
efferent arterioles constrict to renal nerve stimulation. Kidney
International 44, 684—691.
DiBona, G. F. & Kopp, U. (1997). Neural control of renal function.
Physiological Reviews 77, 75—197.
DiBona, G. F. &Sawin, L. L. (1982). Effectofrenalnervestimulation
on NaCl and HµO transport in Henle’s loop of the rats. American
Journal of Physiology 243, F576—580.
Fernandez-Repollet, E., Silva-Netto, C. R., Colindres, R. E. &
Gottschalk, C. W. (1985). Role of renal nerves in maintaining
sodium balance in unrestrained conscious rats. American Journal of
Physiology 249, F819—826.
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during normal activity in conscious unrestrained rats. American
Journal of Physiology 262, R926—932.
Greenberg, S. G., Enders, C. & Osborn, J. L. (1993). Renal nerves
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(1994). Role of vasopressin V2 receptors in modulation of the renal
cortico-papillaryNaClgradient. Pfl?ugers Archiv 428, 410—414.
Kurkus, J., Sadowski, J., Gellert, R. & Krus, S. (1980). Influence
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Zanchetti, A. (1974). Mechanisms of renal release of renin by
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stimulation in vivo. Effects on regional renal haemodynamics in the
Wistar rat, studied by laser-Doppler technique. Acta Physiologica
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Sadowski, J., Kompanowska-Jezierska, E., Dobrowolski, L.,
Walkowska, A. & Badzynska, B. (1997). Simultaneous recording
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Acta Physiologica
Acknowledgements
The study was supported in part by a Polish—British joint project
grant (Royal Society, UK). We are greatly indebted to Dr J?ozef
Langfort for help in catecholamine determination in the renal
tissue.
Corresponding author
E. Kompanowska-Jezierska: Laboratory of Renal Physiology,
Medical Research Centre, Polish Academy of Sciences, Pawinskiego
5, 02-106Warsaw, Poland.
Email:renal@cmdik.pan.pl
E. Kompanowska-Jezierska and others
J. Physiol. 531.2
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