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386 Original article
Development of cardiovascula
r disease due to renalinsufficiency in male sheep following fetalunilateral nephrectomyReetu R. Singha, Kate M. Dentonb, John F. Bertrama, Andrew J. Jefferiesa,Geoffrey A. Headc, Paul Lombardod, Michal Schneider-Kolskydand Karen M. Moritza,e
Background Renal insufficiency is associated with the
development of cardiovascular disease.
Objectives This study investigated whether reduced fetal
renal mass resulted in renal insufficiency, hypertension,
cardiac dysfunction and whether these changes progressed
with age.
Methods and results Fetal uninephrectomy was
performed at 100-day gestation (term, 150 days) and
studies performed in male sheep from 6 weeks to
24 months of age. Renal function declined with age in sham
animals as demonstrated by increasing plasma creatinine
levels and urinary excretion of albumin. The age-related
decline in renal function was exacerbated in animals that
had undergone fetal uninephrectomy. Evidence of renal
insufficiency was indicated from as early as 6 weeks of age
with elevations in plasma creatinine (Ptreatment < 0.001), urea
(Ptreatment < 0.001) and sodium (Ptreatment < 0.05) levels in
uninephrectomized lambs as compared with sham animals.
At 6 months, urinary albumin excretion (P < 0.001) was
increased and urinary sodium excretion (P < 0.001)
decreased in the uninephrectomized animals. By
24 months, renal function had deteriorated further
with significant progression of albuminuria
(PtreatmentTage < 0.001). Elevation of mean arterial pressure
(�15 mmHg) was associated with significantly increased
cardiac output, stroke volume and plasma volume at
6 months; arterial pressure (�27 mmHg) had increased
further in uninephrectomized animals at 24 months and was
opyright © Lippincott Williams & Wilkins. Unautho
0263-6352 � 2009 Wolters Kluwer Health | Lippincott Williams & Wilkins
driven by increased total peripheral resistance. Cardiac
functional reserve (dobutamine challenge) was reduced in
uninephrectomized animals at 6 and 24 months of age
(Ptreatment < 0.001), and this was associated with left
ventricular enlargement (P < 0.001) and reduced fractional
shortening (P < 0.01).
Conclusion Fetal uninephrectomy causing a reduction in
nephron endowment results in an accelerated age-related
decline in renal function. This is associated with an early
onset of elevated blood pressure and impairments in
cardiac structure and function. J Hypertens 27:386–396
Q 2009 Wolters Kluwer Health | Lippincott Williams &
Wilkins.
Journal of Hypertension 2009, 27:386–396
Keywords: cardiovascular, dobutamine, hypertension, nephron number,renal, uninephrectomy
Abbreviations: Ang II, angiotensin II; CO, cardiac output; HR, heart rate;MAP, mean arterial pressure; PRA, plasma renin activity; SV, stroke volume;TPR, total peripheral resistance; UNaV, urinary excretion of sodium; uni-x,unilateral nephrectomy; WP, wedge pressure
aDepartment of Anatomy and Developmental Biology, bDepartment of Physiology,Monash University, Clayton, cBaker Heart Research Institute, Melbourne,dDepartment of Medical Imaging and Radiation Sciences, Monash University,Clayton, Victoria and eSchool of Biomedical Sciences, University of Queensland,St. Lucia, Queensland, Australia
Correspondence to Karen M. Moritz, PhD, School of Biomedical Sciences,University of Queensland, St. Lucia, QLD 4057, AustraliaTel: +61 7 3365 4598; fax: +61 7 3365 1299; e-mail: k.moritz1@uq.edu.au
Received 21 August 2008 Accepted 2 October 2008
IntroductionReduced renal mass at birth has been linked to renal
insufficiency, hypertension and increased risk of cardio-
vascular disease [1–3]. In patients with unilateral renal
agenesis, in whom there is a reduced nephron endow-
ment from birth, the incidence of renal insufficiency and
proteinuria is common [4,5], and many children born with
a solitary kidney have reduced renal function and elev-
ated arterial pressure [6,7]. Increasing evidence suggests
that patients with mild-to-moderate renal dysfunction are
at a higher risk of death from associated cardiovascular
events than developing end-stage renal disease [8].
Human and animal studies have provided evidence for an
association between nephron endowment and the patho-
genesis of hypertension [9–15]. Keller et al. [10] demon-
strated a strong correlation between reduced numbers of
nephrons in hypertensive patients when compared with
age-matched normotensive controls. The caveat of these
studies being that it is not known when the reduction in
nephron number occurred and, indeed, whether the
reduced nephron number and subsequent hypertension
is more than an association. Studies on animal models
have gone some way in addressing these issues. Studies
have shown that maternal perturbations such as maternal
rized reproduction of this article is prohibited.
DOI:10.1097/HJH.0b013e32831bc778
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Renal and cardiovascular dysfunction Singh et al. 387
dietary protein restriction or elevation in circulating stress
hormones (glucocorticoids) during pregnancy results in a
low nephron endowment at birth [13,14,16] and sub-
sequent development of hypertension in these offspring
[13,17,18]. However, these prenatal perturbations have
effects on other developing organs [19], and some
maternal perturbations such as dietary protein restriction
or global undernutrition cause fetal growth restriction
[20–22], which may also contribute to hypertension in
these models. This makes it difficult to appreciate the
importance of a reduced nephron complement from birth
observed in these models as pertaining to the develop-
ment of renal dysfunction and cardiovascular disease.
In order to investigate the effect of a congenital nephron
deficit on adult renal and cardiovascular function, our
group has established an ovine model of uninephrectomy
[23,24]. Development of the permanent (metanephric)
kidney in sheep commences on approximately the 27th
day of gestation, and nephrogenesis is complete by the
130th day (term is 150 days) [25]. This is an ideal model as
this is similar to the situation in humans in which kidney
development commences around week 6 of pregnancy
and nephrogenesis is complete prior to birth [26]. Pre-
viously, we have reported that unilateral nephrectomy
(uni-x) during metanephrogenesis (at 100 days of
gestation) resulted in a 30% reduction in nephron number
in males, and in females at 2 years of age, there was a
modest reduction in glomerular filtration rate and
elevation in arterial pressure (�7 mmHg) [11,27].
In the present study, in order to determine the onset and
progression of disease in this model of congenital
nephron deficit, we conducted renal and cardiovascular
studies on uni-x male sheep from 6 weeks to 24 months of
age. It is well recognized that progression of renal and
cardiovascular disease is exacerbated in males as com-
pared with females [28,29]. Thus, we hypothesized that
the reduction in renal function and subsequent increase
in arterial pressure will be severe in males, the age-
related decline in renal function will be markedly accel-
erated in uni-x males and cardiac structure and function
will be reduced from an early age because of the impact
of altered extracellular fluid homeostasis as a result of
fetal uninephrectomy.
Materials and methodsAnimalsAll experiments were approved by an Animal Ethics
Committee of Monash University and were performed
in accordance with the guidelines of the National Health
and Medical Research Council of Australia. Merino ewes
carrying single male fetus of known gestational age under-
went surgery at 100 days after conception. Anesthesia was
induced in ewes and fetus with sodium pentothal (1 g
intravenous) and maintained with halothane (1.5–2% in
O2). The fetal left kidney was cleared from surrounding fat
opyright © Lippincott Williams & Wilkins. Unauth
and the left renal artery, left renal vein and ureter were
ligated (uni-x group, n¼ 5), and the kidney was excised. In
five fetuses, the kidney was cleared from the surrounding
fat but was not excised (sham-operated group, n¼ 5). After
surgery, ewes were housed in pens for 2 weeks before
being returned to the farm. After birth, lambs remained
with their mothers on pasture until weaned at 18 weeks of
age. At 5 months of age, the lambs underwent surgery, and
the right carotid artery was surgically exteriorized into a
skin fold to form a carotid arterial loop [30].
Blood samplesBlood samples through venous puncture were obtained
from animals from 6 weeks of age, fortnightly, till 16
weeks of age and through carotid artery at 6 and 24
months for measurement of plasma sodium, urea, crea-
tinine and total plasma protein concentrations. All
samples were analyzed using the Beckman Synchron
CX-5 clinical system (Beckman Instruments Inc, Full-
erton, California, USA). Plasma samples were also ana-
lyzed for osmolality by freezing point depression
(Advanced Instruments, Norwood, Massachusetts,
USA) and for renin and angiotensin II levels by radio-
immunoassay (Prosearch International Pty, Malvern,
Australia) at 6 and 24 months of age.
Renal functionAt 6 and 24 months of age, animals were brought into the
laboratory, placed in individual metabolic cages and
allowed a week of acclimatization. All animals were meal
fed a diet of hay and chaff. Following this, all animals’
daily food and water intake and urine output were mon-
itored and recorded over 6 consecutive days. Twenty-
four-hour urine samples were collected and analyzed for
sodium, albumin, total protein and osmolality.
Basal mean arterial and heart rate measurementBlood pressure (BP) (systolic, diastolic) and heart rate
(HR) were determined by an indwelling carotid arterial
catheter. These measurements were acquired every 10 s
and averaged every 10 min, over a 72-h period, and
cumulative averages of these are reported as basal mean
arterial pressure (MAP) and HR.
Measurement of cardiovascular variablesA day prior to experimentation, the animals were cannu-
lated with a Swan–Ganz catheter (Baxter, Old Toongab-
bie, New South Wales, Australia) for measurement of
cardiovascular variables as previously described [31].
Cardiovascular variables [MAP, HR, cardiac output
(CO)], central venous pressure (CVP) and mean pulmon-
ary artery pressure (PAM) were measured simultaneously
and continuously over 24 h with CO readings obtained
from a CO computer (9520A; American Edwards Labora-
tories, Irvine, California, USA). Stroke volume (SV) and
total peripheral resistance (TPR) were calculated from
the measured variables.
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388 Journal of Hypertension 2009, Vol 27 No 2
Blood volume measurement using fluoresceinisothiocyanate-dextranBlood and plasma volume measurements were obtained
by determining dextran space using 250 kDa fluorescein
isothiocyanate-dextran (Sigma, St. Louis, Missouri, USA)
as previously described [32].
Cardiac functional reserveCardiac functional reserve was determined by evaluating
the CO response to b-adrenergic stimulation (dobutamine
challenge) as performed previously in sheep [31]. Briefly,
on the day of experimentation, basal MAP, HR, CO,
PAM, pulmonary artery wedge pressure and CVP were
measured over 1 h. To eliminate the differences in pre-
load pressures between the animals, a plasma volume
expander was infused (hemaccel; infusion rate 15 ml/min)
in 200 ml steps until the wedge pressure had reached
10 mmHg. Once the desired wedge pressure was reached,
MAP0, CO0, HR0, PAM0, wedge pressure0 and CVP0 were
measured to establish a new baseline prior to the dobu-
tamine challenge. Dobutamine (dobutamine hydrochlo-
ride, Lilly, Indianapolis, Indiana, USA) was infused in
eight incremental doses (10 min per dose; 0.5–7.0 mg/kg
per min) or until the CO had reached a plateau. Wedge
pressure was maintained at 10 mmHg for the duration of
the experiment.
Baroreflex sensitivity at 6 months of ageBasal MAPandHR were measuredfor anhouron thedayof
this experiment, and then the arterial pressure was lowered
in each animal with sodium nitroprusside (0.5–5.0 mg/h
intravenous) or raised with phenylephrine (0.4–16 mg/h)
infusion. MAP and HR were continuously recorded for
10 min per dose of each drug. Data was analyzed using
Ramp 98 (Baker Heart Research Institute, Melbourne,
Victoria, USA), a program written in Labview language
to fit a four-parameter logistic equation to the HR–MAP
curves. This involved the Marquardt–Levenberg least
squares method as described previously [33]. From the
curves, measures of gain (delta HR/delta MAP), mid-point
MAP (BP50) and HR range were obtained.
Echocardiographic measurement at 24 months of ageEchocardiographic measurements were obtained by M-
mode scanning (HDI 5000 SonoCT; Philips) in conscious
sheep as previously described [27,31]. Measurements
obtained in diastole were left ventricular diastolic
diameter (LVD), diastolic thickness of the posterior wall
(PWD) and interventricular septum (IVSd), and measure-
ments obtained in systole were left ventricular systolic
diameter (LVS), thickness of posterior wall in systole
(PWS) and interventricular septum (IVSs). Left ventri-
cular mass was determined using the following formula
and then indexed for animal body weight. Left ventri-
cular mass (Penn conversion)¼ 1.04[(LVDþPWDþIVSd)3� (LVD)3]� 13.6 g. The percentage fractional
shortening and relative wall thickness (RWT) were cal-
opyright © Lippincott Williams & Wilkins. Unautho
culated using the following formulae: % fractional
shortening¼ (LVD�LVS)/LVD�100; RWT¼ (PWSþIVSs)/LVD [27,31].
Statistical analysisAll data were analyzed to determine the effect of treat-
ment or effect of age and the interaction between treat-
ment and age by two-way repeated measures analysis of
variance (ANOVA), unless stated otherwise. Values are
presented as mean� standard deviation (SD), with the
level of significance set at 0.05 or less. Statistical analysis
was performed using SYSTAT software (SYSTAT 11 for
Windows, SPSS Science, Birmingham, UK).
ResultsBirth weights and growth measurementsAll lambs were born at 150� 1 day. Birth weight and adult
body weights at 6 and 24 months of age were similar
between sham and uni-x animals [birth weight (kg):
sham, 4.4� 0.7, uni-x, 4.3� 0.3; 6 months (kg): sham,
39� 3, uni-x, 35� 3; 24 months (kg): sham, 67� 4, uni-x,
65� 4].
Plasma analysis (6–16 weeks)Plasma sodium concentration was significantly altered in
the uni-x as compared with sham animals (Ptreatment¼0.047, Page¼ 0.02, Ptreatment�age¼ 0.001, Fig. 1a), with
levels being lower at 6 weeks of age and higher between
10 and 14 weeks of age in uni-x animals. Plasma urea
levels were elevated in uni-x animals from 6 weeks of life
as compared with sham animals (Ptreatment¼ 0.007,
Fig. 1b). Plasma creatinine concentration progressively
increased from 6 weeks of age in uni-x animals as com-
pared with sham animals (Ptreatment�age< 0.001, Fig. 1c).
Total plasma protein concentration was higher in uni-x
animals between 6–16 weeks of age (Ptreatment¼ 0.05) as
compared with sham animals.
Plasma and renal function analysis at 6 and 24 monthsof agePlasma variables at 6 and 24 months of age
Plasma sodium and plasma osmolality were similar
between the treatment groups at 6 and 24 months of
age. Plasma sodium (mmol/l): 6 months: sham, 142.4�0.7, uni-x, 144.2� 0.9; 24 months: sham, 144.8� 1.2, uni-
x, 145.5� 1.3. Plasma osmolality (mOsm/kg water): 6
months: sham, 302.6� 1.5, uni-x, 303.2� 0.7; 24 months:
sham, 304.4� 0.7, uni-x, 302.8� 0.6. Uni-x animals had
significantly elevated plasma creatinine concentrations
at both ages compared with the sham group
(Ptreatment< 0.001, Fig. 3a). Plasma creatinine levels
increased in both groups between 6 and 24 months of
age (Page< 0.001), but this increase was lesser in the uni-x
as compared with sham animals (sham, �20%, uni-x,
�11%; Ptreatment�age¼ 0.04, Fig. 3a). Uni-x animals had
significantly elevated plasma urea levels at 6 and 24
months of age as compared with the sham group
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Renal and cardiovascular dysfunction Singh et al. 389
Fig. 2
(a) Plasma angiotensin II and plasma renin activity in sham and uni-xanimals at 6 and 24 months of age. P values are from two-way repeatedmeasures analysis of variance. Values are mean�SD. Sham (n¼5/age, clear bars); uni-x (n¼5/age, dark bars). Ang II, angiotensin II; mths,months; PRA, plasma renin activity.
Fig. 1
Plasma variables measured fortnightly between 6 and 16 weeks through venous puncture in male sheep. (a) Plasma sodium concentrations, (b)plasma urea concentrations, (c) plasma creatinine levels and (d) total plasma protein levels in sham (n¼5/age, clear bars) and uni-x (n¼5/age, darkbars) sheep. P values are from two-way repeated measures analysis of variance. Values are mean�SD. wks, weeks.
(Ptreatment¼ 0.01, Fig. 3b). Plasma urea levels decreased
from 6 to 24 months of age (Page< 0.001, Fig. 3b); how-
ever, this decrease was similar between both groups
(Ptreatment�age< 0.001, Fig. 1c). Total plasma protein
concentration was higher in uni-x animals at 6 and 24
months of age as compared with the sham group
(Ptreatment¼ 0.006, Fig. 3c). Plasma protein levels
increased with age in uni-x animals (6 months, 66� 1;
24 months, 68� 0.8; Page¼ 0.006, Ptreatment�age¼ 0.02,
Fig. 3c).
Plasma renin activity was significantly lower in uni-x
animals as compared with sham animals (Ptreatment<0.001, Fig. 2b). Plasma renin activity decreased with
age (Page< 0.001, Fig. 2b) in both groups (uni-x, 46%
and sham, 46%), though the rate of decline, in absolute
terms, was less in uni-x animals (Ptreatment�age¼ 0.005).
Uni-x animals had significantly reduced plasma angio-
tensin II (Ang II) levels as compared with sham animals at
6 and 24 months of age (Ptreatment< 0.001, Fig. 2a).
Plasma Ang II level was significantly lower in both
treatment groups at 24 months of age as compared with
6 months of age (Page< 0.001, Fig. 2a); however, the rate
of decline in plasma Ang II levels was not different
between the groups (Ptreatment�age¼ 0.2).
Urinary variables at 6 and 24 months of age
Food intake was similar between the groups at both ages
studied. Cumulative water intake was similar in both
treatment groups at both ages; water intake (l/day): 6
months: sham, 2.2� 0.2, uni-x, 2.4� 0.1; 24 months:
opyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
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390 Journal of Hypertension 2009, Vol 27 No 2
Fig. 3
Plasma and urinary variables obtained at 6 and 24 months of age as indicators of basal renal function. Data presented are as cumulative averages ofmeasurements obtained over 6 days. Plasma samples (a–c, obtained by carotid arterial catheter), (d) urine output (liters in 24 h; l/day), (e) urinarysodium excretion rate, (f) urinary albumin excretion rate in uni-x and sham animals at 6 months and 24 months of age. P values are from two-wayrepeated measures analysis of variance. Values are mean�SD. Sham (n¼5/age, clear bars); uni-x (n¼5/age, dark bars). mths, months; UalbuminV,urinary albumin excretion rate; UNaV, urinary sodium excretion rate.
sham, 2.3� 0.1, uni-x, 2.3� 0.2. Urine output was also
similar between the groups at both ages (Fig. 3d). Urinary
osmolar excretion and free water clearance were similar
between the treatment groups at both ages. Urinary
osmolar excretion (mOsm/day): 6 months: sham,
3.8� 0.8, uni-x, 4.0� 0.4; 24 months: sham, 4.1� 0.6,
uni-x, 3.9� 0.3. Free water clearance was similar between
the treatment groups at 6 and 24 months of age (6 months:
sham, �2.37� 0.23, uni-x, �2.58� 0.14; 24 months:
sham, �2.55� 0.27, uni-x, �2.44� 0.14). Uni-x animals
had significantly reduced urinary sodium excretion rates
opyright © Lippincott Williams & Wilkins. Unautho
(Ptreatment¼ 0.005, Fig. 3e) as compared with sham
animals at 6 months of age. At 24 months of age, urinary
sodium excretion rates were similar between the treat-
ment groups. This was because urinary sodium excretion
was reduced in sham animals at 24 months of age
(Ptreatment�age¼ 0.047, Fig. 3e). Uni-x animals had sig-
nificantly higher urinary protein (Ptreatment< 0.001) and
albumin (Ptreatment< 0.001, Fig. 3f) excretion rates as
compared with sham animals. Urinary excretion of
protein (Page< 0.001) and albumin (Page< 0.001)
increased with age, with protein excretion increasing at
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Renal and cardiovascular dysfunction Singh et al. 391
a greater rate in uni-x animals (Ptreatment�age¼ 0.01).
Although urinary albumin excretion increased at a lesser
rate, in percentage terms, with age in uni-x animals as
compared with sham animals (Ptreatment�age¼ 0.001,
Fig. 3f), urinary albumin excretion was more than
100% greater in uni-x animals at both ages. Urinary
protein concentration (g/day): 6 months: sham, 0.35�0.02, uni-x, 0.44� 0.01; 24 months: sham, 0.46� 0.03,
uni-x, 0.67� 0.03 (Ptreatment< 0.001, Page< 0.001,
Ptreatment�age¼ 0.01).
Chronic measurement of mean arterial pressure andheart rate obtained over 72 hUni-x animals had significantly elevated MAP at both
ages as compared with sham animals [MAP (mmHg): 6
months: sham, 76� 4, uni-x, 92� 2; 24 months: sham,
78� 4, uni-x, 105� 3; Ptreatment< 0.001, Page< 0.001].
Uni-x animals had a greater increase in MAP with age
(Ptreatment�age¼ 0.04). HR was similar between uni-x and
sham animals at 6 months of age [HR (beats/min): 6
months: sham, 75� 7, uni-x, 74� 4] and increased
similarly in both groups with age [HR (beats/min):
24 months: sham, 82� 4, uni-x, 80� 3; Page< 0.01,
Ptreatment�age¼ 0.2).
Cardiovascular variables measured over 24 hCardiovascular measurements made over 24 h in all
animals are shown in Fig. 4. Similar to measurements
obtained over 72 h, MAP was significantly elevated in
opyright © Lippincott Williams & Wilkins. Unauth
Fig. 4
Cardiovascular variables measured continuously over 24 h and blood volumepressure, (b) cardiac output, (c) total peripheral resistance, (d) heart rate, (e) stor uni-x (n¼5/age, dark bars) male sheep at 6 and 24 months of age. P valumean�SD. CO, cardiac output; HR, heart rate; MAP, mean arterial pressure
uni-x animals at 6 and 24 months of age as compared with
sham animals (Ptreatment< 0.001, Fig. 4a). HR was similar
between the treatment groups at both ages but was higher
at 24 months of age as compared with 6 months of age in
both groups (Page¼ 0.003, Fig. 4d). CO was higher at 6
months of age in uni-x animals, but by 24 months of age,
CO was similar in both groups because of an increase in
the sham group (Ptreatment�age¼ 0.02, Fig. 4b). SV was
elevated in the uni-x group at 6 months of age but was
similar in both groups at 24 months of age because of a
significant decrease in SV in uni-x animals with age
(Page�treatment¼ 0.009, Fig. 4e). TPR was similar between
the treatment groups at 6 months of age, increasing with
age in uni-x animals but decreasing with age in the sham
group (Ptreatment�age< 0.001, Fig. 4c). Uni-x animals had
significantly higher CVP as compared with sham animals
at 6 and 24 months of age [CVP (mmHg): 6 months: sham,
1.8� 0.8, uni-x, 5.2� 0.8; 24 months: sham, 3.3� 1.7,
uni-x, 5.0� 1.2; Ptreatment< 0.001, Page¼ 0.3,
Ptreatment�age¼ 0.1]. PAM was higher in uni-x animals
at 6 months of age as compared with the sham group but
was similar between the treatment groups at 24 months of
age [PAM (mmHg): 6 months: sham, 11.0� 1.1, uni-x,
14.0� 0.7; 24 months: sham, 13.0� 2.2, uni-x, 13.8� 1.3;
Ptreatment¼ 0.008, Page¼ 0.2, Ptreatment�age¼ 0.09].
Plasma and blood volume measurementsUni-x animals had significantly elevated blood volume at
6 months of age as compared with the sham group, but
orized reproduction of this article is prohibited.
in uni-x and sham animals at 6 and 24 months of age. (a) Mean arterialroke volume and (f) blood volume in sham operated (n¼5/age, clear bars)es are from two-way repeated measures analysis of variance. Values are; mths, months; SV, stroke volume; TPR, total peripheral resistance.
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392 Journal of Hypertension 2009, Vol 27 No 2
Fig. 5
Cardiovascular response to dobutamine challenge in sham operatedand uni-x male sheep at 6 and 24 months of age following fetaluninephrectomy at 100-day gestation. (a) Maximal change in cardiacoutput from the baseline (functional reserve), (b) maximal change instroke volume and (c) maximal change in heart rate over the course ofdobutamine infusion is represented. Sham (6 months), closed squarewith solid line; uni-x (6 months), closed square with broken line. Sham(24 months), open square with solid line; uni-x (24 months), opensquare with broken line. P values are from two-way repeated measuresanalysis of variance. Values are mean�SD. CO, cardiac output; HR,heart rate; SV, stroke volume.
blood volume was similar between the treatment groups
at 24 months of age because of an increase in blood
volume in sham animals (Fig. 4f, Ptreatment�age¼ 0.001).
Similarly, plasma volume was elevated in uni-x animals at
6 months of age but was similar to sham animals at 24
months of age because of an increase in plasma volume
with age in the sham group [plasma volume (ml/kg): 6
months: sham, 32.3� 1.4, uni-x, 40.5� 1.9; 24 months:
sham, 40.6� 1.5, uni-x, 42.5� 2.0; Ptreatment< 0.001,
Page< 0.001, Ptreatment�age< 0.001]. Hematocrit was
similar in each group at each age.
Cardiac functional reserve in response to dobutamineVariables corrected for preload
Baseline values for MAP, CO, TPR, HR, SV and PAM
measured over 1 h were similar to those obtained over 24 h
at both ages studied. Wedge pressure was significantly
elevated in uni-x animals at both ages as compared with the
sham group [wedge pressure (mmHg): 6 months: sham,
5.9� 0.9, uni-x, 8.1� 0.8; 24 months: sham, 6.8� 1.0, uni-
x, 8.2� 0.8; Ptreatment< 0.001, Page¼ 0.3, Ptreatment�age¼0.3]. The total volume of hemaccel required to maintain a
wedge pressure of 10 mmHg was significantly lower in uni-
x animals as compared with sham animals at 6 months of
age but was similar between the treatment groups at 24
months of age, with both groups requiring lower volumes
to achieve and maintain a wedge pressure of 10 mmHg at
24 months of age (6 months: sham, 804� 134, uni-x,
604� 9 ml; 24 months: sham, 550� 100, uni-x,
420� 130 ml; Ptreatment¼ 0.031, Page¼ 0.01).
Cardiovascular response to dobutamine infusion
Cardiovascular response to dobutamine was analyzed
using two-way repeated ANOVA to examine the effect
of uni-x and age over graded doses of dobutamine.
Cardiac functional reserve (COmax-0 ml/kg per min) was
significantly reduced in uni-x animals as compared with
sham animals (Ptreatment< 0.001, Fig. 5a); the reduction
in cardiac reserve did not progress between 6 and 24
months of age (Page¼ 0.1). The increase in HR in
response to dobutamine challenge was reduced in uni-
x as compared with sham animals (Ptreatment¼ 0.003,
Fig. 5c). The expected increase in SV seen in sham
animals was absent in uni-x animals at both 6 and
24 months of age, with SV in fact decreasing
(Ptreatment< 0.001, Page¼ 0.001, Fig. 5b). The fall in SV
observed at 6 months of age in uni-x animals had become
greater by 24 months of age as compared with the sham
group (Ptreatment�age¼ 0.007).
Baroreflex sensitivity determined at 6 months of ageBaroreflex function was only determined at 6 months of
age, and data between the two groups was analyzed
using a two-tailed Student’s t-test. Data are presented
in Table 1. Basal MAP was significantly elevated in the
uni-x group as compared with the sham group, and HR
was similar between the groups. The baroreflex curve was
opyright © Lippincott Williams & Wilkins. Unautho
significantly shifted to the right (higher pressure) in uni-x
animals as compared with sham animals at 6 months of
age. However, gain and HR range were similar between
the groups (Table 1).
Echocardiography at 24 months of ageMeasurements for left ventricular dimension are shown
in Table 2. Left ventricular mass was significantly
increased in uni-x animals at 24 months of age as com-
pared with the sham group. Left ventricular diameter
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Renal and cardiovascular dysfunction Singh et al. 393
Table 1 Baroreflex measurements at 6 months of age
6 months Sham, n¼5 uni-x, n¼5
Basal MAP (mmHg) 75.8�4.7 90.8�2.5MMM
Basal HR (beats/min) 83.0�11.0 77.2�12.4BP-50 75.3�3.0 81.5�2.0MMM
Gain (beats/min per mmHg) �4.4�2.5 �4.8�4.2HR range (beats/min) 46.5�8.7 53.7�11.4
Data shown for basal mean arterial pressure, basal heart rate, blood pressure atmidpoint (BP-50), gain and heart rate range at 6 months of age. Values aremean�SD. P values are from two-tailed, Student’s t-test. BP, blood pressure; HR,heart rate; MAP, mean arterial pressure. MMMP<0.001.
(during diastole and systole), posterior wall thickness
(during diastole and systole) and interventricular septal
diameters (during systole) were all significantly increased
in uni-x animals as compared with sham animals at 24
months of age. RWT and fractional shortening were also
significantly greater in uni-x animals as compared with
sham animals at 24 months of age (Table 2).
DiscussionIn the present study, we show that male sheep born with a
congenital nephron deficit as a result of fetal uninephrect-
omy exhibit an early onset of renal dysfunction and
elevated arterial pressure. Both age-related decline in
renal function and elevation in arterial pressure were
accelerated in uni-x animals. In addition, this study shows
that uni-x male sheep had significant cardiovascular
pathophysiology as indicated by reduced cardiac func-
tional reserve from as early as 6 months of age. This
reduction in cardiac reserve in uni-x animals persisted
with ageing and was accompanied by significant left
ventricular hypertrophy and reduced cardiac contractility
at 24 months of age.
Following uninephrectomy, each nephron in the remain-
ing kidney must process a greater volume of plasma to
maintain extracellular fluid volume homeostasis. To
opyright © Lippincott Williams & Wilkins. Unauth
Table 2 Echocardiography data in male sheep at 24 months of age
Males at 24 months Sham, n¼5 Uni-x, n¼5
Body weight (kg) 67.0�3.46 65.2�3.56LVD (cm) 3.47�0.18 4.36�0.15MMM
PWD (cm) 1.00�0.18 1.43�0.24M
IVSd (cm) 0.95�0.22 1.03�0.12LVS (cm) 2.28�0.1 3.35�0.06MMM
PWS (cm) 1.42�0.3 2.83�0.06MMM
IVSs (cm) 1.39�0.25 1.87�0.1MM
LV mass (g) 98.21�10.3 195.86�14.7MMM
LV mass indexed (g/kg) 1.48�0.23 3.01�0.28MMM
% FS 34.4�4.96 23.2�2MM
RWT (cm) 0.81�0.07 1.08�0.03MM
Values are mean�SD. P values are from two-tailed Student’s t-test. % FS,percentage fractional shortening; IVSd, interventricular septum in diastole; IVSs,interventricular septum in systole; LV, left ventricular; LVD, left ventricular diameterin diastole; LV mass indexed, left ventricular mass corrected for body weight; LVS,left ventricular diameter in systole; PWD, posterior wall thickness in diastole; PWS,posterior wall thickness in systole; RWT, relative wall thickness. MP<0.05.MMP<0.01. MMMP<0.001.
achieve this, each nephron undergoes hypertrophy, and
the hormonal and autoregulatory mechanisms regulating
renal function are likely reset. The adaptations that the
kidney undergoes in response to uninephrectomy are
different in neonates and adults [34,35]. At birth, renal
function is immature, with the infant having a relatively
poor capacity to regulate sodium and water [35]. In the
postnatal period, the nephron undergoes rapid growth
and maturation, particularly in terms of upregulation of
tubular transport mechanisms [35]. An acceleration of this
maturation process has been reported following neonatal
uninephrectomy [34,36]. Our studies demonstrated
increased plasma sodium, creatinine and urea levels in
conscious uni-x lambs. This suggests that the uni-x
kidneys have altered renal function from early in life,
which is supported by the fact that urinary sodium
excretion was significantly reduced in uni-x animals at
6 months of age despite similar sodium intake. Thus, at
6 months of age, the elevated blood volume and increase
in arterial pressure was the result of sodium retention by
the remaining kidney in uni-x animals. In addition, at
6 months of age, early signs of renal damage are evident
with significant proteinuria and albuminuria, indepen-
dent risk factors for cardiovascular disease [37]. This set
of data provides one of the strongest supports for the
Brenner hypothesis that proposes that a congenital
nephron deficit predisposes to the development of
hypertension [38].
In this study, increases in plasma creatinine and albumi-
nuria between 6 and 24 months of age indicate evidence
of an age-related decline in renal function in sham
animals. This age-related decline in renal function was
exacerbated in animals that had undergone fetal unine-
phrectomy. Renal function has been shown to decline
with age [39] among healthy humans, and this decline in
function may be attributed to a loss of functional
nephrons with ageing [40]. Removal of functioning
nephrons in renal ablation models results in increased
glomerular hydrostatic pressure and hypertrophy [2].
However, these compensatory hemodynamic and hyper-
trophic adaptations over time result in glomerular injury
and loss that precede end-stage renal failure [41]. It is
plausible that the aforementioned compensatory adap-
tations in response to a nephron deficit progressed more
rapidly in uni-x animals. Whether there is a progressive
loss of nephrons in this model with ageing needs to be
determined as it may potentially explain the early onset
and quite rapid decline in renal function observed in
uni-x animals.
The elevation in arterial pressure in uni-x animals at 6
months of age was driven by an increase in CO associated
with an elevated blood volume. However, by 24 months
of age, the elevation in arterial pressure in uni-x animals
was maintained by increased TPR rather than CO. Eich
and colleagues [42,43] have also shown an elevated
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394 Journal of Hypertension 2009, Vol 27 No 2
cardiac index in young hypertensive patients, but upon
follow-up, the CO had normalized although TPR
had increased. The reasons for this switch from an
elevated CO to elevated peripheral resistance in chronic
hypertension are not clear but may be a result of total
body autoregulation [44] and enhanced sympathetic tone
[45]. Sympathetic hyperactivity is observed in the initial
stages of hypertension in humans [46] and has been
shown to be associated with a reduced baroreflex gain
[47]. Our examination of baroreflex function at 6 months
of age showed that the baroreflex curve in uni-x animals
was shifted to the right (higher pressure setting), but
baroreflex gain was similar to that of control animals. Our
findings are consistent with those of Ligtenberg et al. [48]
who have also reported normal baroreflex gain and a
rightward shift in the baroreflex curve in patients with
chronic renal failure.
Perhaps the most interesting finding of this study is the
significant reduction in cardiac functional reserve in uni-x
animals from as early as 6 months of age. This defect in
cardiac reserve in uni-x animals appeared to be almost
solely due to a failure to increase SV at both ages. Lele et al.[49] have shown reduced peak cardiac performance in
patients with hypertrophic cardiomyopathy that is related
to a failure to augment SV. Echocardiographic measure-
ments at 24 months of age showed that uni-x males had
significant left ventricular hypertrophy accompanied with
reduced fractional shortening indicating reduced cardiac
contractility [50]. This reduction in cardiac contractility
may be responsible for the failure to augment SV in the
uni-x group at 24 months of age. However, it should be
noted that cardiac reserve was similarly reduced in uni-x
animals at both ages. Although not investigated in this
study, we speculate that the onset of left ventricular
hypertrophy may have occurred earlier in uni-x males.
An increase in cardiac load as a result of elevated venous
return (preload) and elevated BP are common precursors to
the onset of ventricular hypertrophy [51]. Certainly, the
incidence of left ventricular hypertrophy has been
reported to be higher in children with chronic renal insuf-
ficiency [52], and Mitsnefes et al. [53] have reported the
development of left ventricular diastolic dysfunction in the
early stages of mild-to-moderate renal failure in children.
The early onset of renal impairment, elevated BP and
blood volume [54] may have triggered the development of
ventricular hypertrophy earlier in uni-x animals and may
explain the significantly reduced cardiac reserve at 6
months of age. Although cardiac function was not
examined, echocardiography revealed no incidence of
ventricular hypertrophy in our previous uninephrecto-
mized cohort of females at 2 years of age [27]. Preservation
of myocardial mass has been reported to be better in aging
women than men [55], and certain forms of familial hyper-
trophic cardiomyopathies are also reported to be more
severe in men than women [56]. It is interesting that fetal
uni-x resulted in such significant cardiac impairment from
opyright © Lippincott Williams & Wilkins. Unautho
an early age. It is possible that these changes in cardiac
function are not simply compensatory responses due to
postnatal deficiencies in renal function but may be the
result of changes in fetal cardiovascular hemodynamics,
induced by the nephron deficit, in the uni-x fetus that may
have impinged upon normal heart development.
Previously, we have reported that fetal uninephrectomy in
female sheep also results in elevated arterial pressure and
reduced glomerular filtration rate from 6 months of age
[11,27]. Although not directly comparable, the degree of
elevation in arterial pressure observed in males in the
present study was far greater at both ages than the previous
female cohort (6 months, 15 vs. 7 mmHg; 24 months, 27 vs.
5 mmHg) [11,27]. Our findings in uni-x males are consist-
ent with those of Woods et al. [15] who also observed a
greater degree of hypertension in male rats than in females
following uninephrectomy in the neonatal period. This
data also parallels human data showing that females are
protected from both functional and structural damage with
advancing age as compared with males [57].
In conclusion, although chronic renal insufficiency has
long been recognized a major independent risk factor for
cardiovascular disease, it is only more recently that
patients with mild-to-moderate renal dysfunction have
been shown to also have an increased risk of developing
cardiovascular disease [58]. This study shows that the
onset of renal disease occurs at an early age in animals
born with a nephron deficit, and the decline in renal
function with age is very rapid in the uninephrectomized
male sheep. This early impairment in renal function
results in elevated blood volume and venous return,
which may be the precursor to the onset of hypertension
in uni-x animals in this model. However, the chronic
elevation in arterial pressure with age is maintained by an
elevated peripheral resistance, similar to observations in
humans [42]. Most interestingly, uni-x animals exhibited
a significant reduction in cardiac reserve from an early
age, and this reduction in function appears to be associ-
ated with significant left ventricular hypertrophy and
reduced cardiac contractility resulting from elevated pre-
load (elevated venous return) and afterload on the heart.
This study highlights the need to monitor children born
with a suspected low nephron endowment for early signs
of renal and cardiovascular disease.
AcknowledgementThis project was supported by a National Heart Founda-
tion Grant (G 05M 2110) and Monash University Research
Fund. R.R.S. was supported by an Australian Postgraduate
Award, and K.M.M. was supported by an NH&MRC
Career Development Award. The authors would like to
thank Mr Alex Satragno and Mr Alan McDonald for
assistance in surgical preparation of the animals.
There are no conflicts of interest.
rized reproduction of this article is prohibited.
C
Renal and cardiovascular dysfunction Singh et al. 395
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