Development of cardiovascular disease due to renal insufficiency in male sheep following fetal...

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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-Kolskyd
and 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: [email protected]

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

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DOI:10.1097/HJH.0b013e32831bc778

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http://dx.doi.org/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.

orized reproduction of this article is prohibited.

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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-


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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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


opyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

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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


(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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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


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


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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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


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


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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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


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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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


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.


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