Glomerular hypertrophy in offspring of subtotally nephrectomized ewes.
ABSTRACT We have shown that fetuses whose mothers underwent subtotal nephrectomy (STNx) before pregnancy had high urine flow rates and sodium excretions, but lower hematocrits, plasma chloride, and plasma renin levels compared with controls. To see if these functional differences in utero persist after birth and are the result of altered renal development, we studied 8 lambs born to STNx mothers (STNxL) and 10 controls (ConL) in the second week of life. These lambs were of similar body weights, nose-rump lengths and abdominal girths. Their kidney weights were not different (ConL 36.1 +/- 1.9 vs. STNxL 39.8 +/- 3.3 g), nor were kidney dimensions or glomerular number (ConL 423,520 +/- 22,194 vs. STNxL 429,530 +/- 27,471 glomeruli). However, STNxL had 30% larger glomerular volumes (both mean and total, P < 0.01) and there was a positive relationship between total glomerular volume and urinary protein excretion (P < 0.05) in STNxL. Despite this change in glomerular morphology, glomerular filtration rate, tubular function, urine flow, and sodium excretion rates were not different between STNxL and ConL, nor were plasma electrolytes, osmolality, and plasma renin levels. Thus while many of the functional differences seen in late gestation were not present at 1-2 weeks after birth, the alteration in glomerular size and its relationship to protein excretion suggests that exposure to this altered intrauterine environment may predispose offspring of mothers with renal dysfunction to renal disease in adult life.
- [Show abstract] [Hide abstract]
ABSTRACT: Synthetic glucocorticoids are commonly given to pregnant women when premature delivery threatens. Antenatal administration of clinically relevant doses of betamethasone to pregnant sheep causes sex-specific compromises of renal function and increases in blood pressure in adult offspring. However, it is unclear whether such effects are present in immature lambs. Therefore, the aims of the present study were to determine whether antenatal betamethasone at 80-81 days of gestation increases blood pressure and adversely impacts renal function in adolescent ewes and rams. Prenatal steroid exposure increased blood pressure significantly in the young male (84 +/- 2 vs. 74 +/- 3 mmHg) and female sheep (88 +/- 5 vs. 79 +/- 4), but it did not alter basal glomerular filtration rate, renal blood flow (RBF), or sodium excretion in either sex. However, antenatal betamethasone exposure blocked increases in RBF (P = 0.001), and enhanced excretion of an acute Na load (P < 0.05) in response to systemic infusions of angiotensin (ANG)-(1-7) at 10 pmol.kg(-1).min(-1) in males. In females, the natriuretic response to combined ANG-(1-7), and Na load was significantly altered by prenatal betamethasone exposure. These findings indicate that blood pressure is increased in immature animals in response to antenatal steroid exposure and that sex-specific effects on renal function also exist. These changes may reflect greater risk for further loss of renal function with age.AJP Regulatory Integrative and Comparative Physiology 09/2010; 299(3):R793-803. · 3.28 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Antenatal steroid administration is associated with alterations in fetal kidney development and hypertension. However, a causal relationship between nephron deficit and hypertension has not been established. In this study, we measured nephron number, renal function, and blood pressure in sheep exposed antenataly to betamethasone. Pregnant sheep were given 2 betamethasone doses (0.17 mg/kg) or vehicle at 80 and 81 days gestational age and allowed to deliver at term. Data were obtained from a fetal cohort and 2 adult cohorts and were analyzed by analysis of variance (ANOVA) and/or 2 sample t test. Antenatal betamethasone induced a 26% reduction in the number of nephrons in both males and females in the absence of intrauterine growth restriction and/or prematurity. Adult males presented a reduction in glomerular filtration rate (GFR; 132 +/- 12.7 vs 114 +/- 7.0 mL/min, P < .05). Betamethasone administration was also associated with an increase in arterial blood pressure of similar magnitude in male (mean arterial pressure [MAP] in mm Hg; 98 +/- 2.7 vs 105 +/- 2.4) and female (96 +/- 1.9 vs 105 +/- 2.4) adult sheep and the increase in blood pressure preceded the decrease in GFR in the males. Furthermore, we found no significant association between the magnitude of the decrease in nephron number and the magnitude of the increase in arterial blood pressure. Our data thus support the conclusion that exposure to glucocorticoids at a time of rapid kidney growth is associated with an elevation in blood pressure that does not appear related solely to the reduction in nephron number.Reproductive sciences (Thousand Oaks, Calif.) 11/2009; 17(2):186-95. · 2.31 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: 1. The aim of the present study was to test the hypothesis that the renin response to mechanisms activated by haemorrhage is programmed by exposure to maternal renal dysfunction. 2. In 26-27-day-old lambs born to ewes that had reduced renal function (STNxL, n=10) and lambs born to ewes with normal renal function (ConL, n=6), 1.6 mL/kg per min of blood was removed over 10 min. 3. Under basal conditions, the STNxL group had increased mean arterial pressure (P < 0.05). In response to haemorrhage, mean arterial pressure decreased in the STNxL group (P < 0.001), but there was no significant change in the ConL group. 4. Although plasma renin level increased in both groups (P < 0.05), the peak response was reduced and delayed in the STNxL group. In contrast, the rise in arginine vasopressin (AVP) level was similar in both groups and occurred over the same time course. At 24 h, both plasma renin and AVP level were the same as those measured before haemorrhage in both groups. Kidney renin level was similar in the two groups. 5. The attenuated renin response to haemorrhage in the STNxL group might explain the inability to maintain arterial pressure after haemorrhage. The results of the present study suggest that the renin response of the postnatal kidney to reductions in blood volume can be affected by the intrauterine environment. If these changes persist into adulthood, it suggests that permanent programming has occurred. Thus, the ability of an individual to respond to acute severe reductions in blood volume might be determined during intrauterine life.Clinical and Experimental Pharmacology and Physiology 02/2011; 38(2):102-8. · 2.41 Impact Factor
Glomerular Hypertrophy in Offspring of
Subtotally Nephrectomized Ewes
AMANDA E. BRANDON,1AMANDA C. BOYCE,2EUGENIE R. LUMBERS,1
MONIKA A. ZIMANYI,2JOHN F. BERTRAM,2AND KAREN J. GIBSON1*
1Department of Physiology and Pharmacology, School of Medical Sciences,
University of New South Wales, Sydney, New South Wales, Australia
2Department of Anatomy and Cell Biology, Monash University, Clayton, Victoria, Australia
We have shown that fetuses whose mothers underwent subtotal
nephrectomy (STNx) before pregnancy had high urine flow rates and
sodium excretions, but lower hematocrits, plasma chloride, and plasma
renin levels compared with controls. To see if these functional differen-
ces in utero persist after birth and are the result of altered renal devel-
opment, we studied 8 lambs born to STNx mothers (STNxL) and 10 con-
trols (ConL) in the second week of life. These lambs were of similar body
weights, nose–rump lengths and abdominal girths. Their kidney weights
were not different (ConL 36.1 6 1.9 vs. STNxL 39.8 6 3.3 g), nor were
kidney dimensions or glomerular number (ConL 423,520 6 22,194 vs.
STNxL 429,530 6 27,471 glomeruli). However, STNxL had 30% larger
glomerular volumes (both mean and total, P < 0.01) and there was a
positive relationship between total glomerular volume and urinary pro-
tein excretion (P < 0.05) in STNxL. Despite this change in glomerular
morphology, glomerular filtration rate, tubular function, urine flow, and
sodium excretion rates were not different between STNxL and ConL,
nor were plasma electrolytes, osmolality, and plasma renin levels. Thus
while many of the functional differences seen in late gestation were not
present at 1–2 weeks after birth, the alteration in glomerular size and
its relationship to protein excretion suggests that exposure to this
altered intrauterine environment may predispose offspring of mothers
with renal dysfunction to renal disease in adult life.
? 2008 Wiley-Liss, Inc.
Key words: maternal renal disease; glomerular hypertrophy;
Mothers with renal disease have an increased risk of
preterm delivery and a baby that weighs below the 10th
percentile with these risks being greater with more
severe disease (Cunningham et al., 1990; Jones and
Hayslett, 1996; Jungers et al., 1997).
To date, there have been few animal studies looking
at the effects of impaired maternal renal function on
fetal renal development and function. Most have been
conducted in rodents and have used uninephrectomy to
reduce maternal renal function (Goss, 1963; McBroom
et al., 1986; Averbukh et al., 1988, 1996; Okada et al.,
1994). In only two of these studies was renal function
reduced before mating, and in these studies unilateral
nephrectomy was carried out 1 week before mating
(Averbukh et al., 1988, 1996). Pups from these unineph-
rectomized mice had lighter body weights, but their
kidney:body weight ratios were increased (Averbukh
et al., 1988). Glomerular number was also increased in
these pups (Averbukh et al., 1988). These changes in
kidney morphology persisted and were still present at
Grant sponsor: NHMRC; Grant number: 157142; Grant spon-
sor: Kidney Health Australia; Grant number: PI040506.
*Correspondence to: Karen Gibson, Dept Physiology and
Pharmacology, School of Medical Sciences, University of New
South Wales, Sydney, NSW, 2052 Australia. Fax: 61-9385-1059.
Received 1 June 2007; Accepted 26 November 2007
Published online 29 January 2008 in Wiley InterScience (www.
? 2008 WILEY-LISS, INC.
THE ANATOMICAL RECORD 291:318–324 (2008)
sexual maturity (7 weeks of age; Averbukh et al., 1996).
The increase in the offspring’s kidney weight was due to
an increase in renal tissue, as renal water content was
the same in pups from uninephrectomized mothers and
pups from mothers that had normal renal function.
That Averbukh et al. (1988, 1996) uninephrectomized
the animals before mating might partially explain why
their results differed from those of Goss (1963) and
McBroome et al. (1986). Goss (1963) uninephrectomized
female rats on day 19 of gestation (term ?22 days;
Ozanne et al., 2003) and found that, on day 21, there
was no increase in mitotic activity in the pup kidneys.
Similarly, McBroome et al. (1986) uninephrectomized
after mating, at 5–7 days gestation, and did not find any
evidence of compensatory kidney growth in the pups. It
is possible that the stress of the uninephrectomy during
pregnancy negated any growth stimulating effects on
the fetal kidney of maternal renal dysfunction, like those
seen by Averbukh et al. (1988, 1996).
To study the effects of reduced maternal renal func-
tion on development of the kidney during intrauterine
life, we subtotally nephrectomized (STNx) nonpregnant
ewes before mating (Gibson et al., 2006). We used the
pregnant ewe and her fetus, because the fetal sheep,
like the human fetus, completes nephrogenesis in utero.
We removed one kidney from the nonpregnant ewe and
partially infarcted the remaining kidney so that 30–50%
of the kidney became ischemic. Ewes were subsequently
mated, and intact pregnant ewes from the same flock
were used as controls.
When studied in late gestation, maternal renal com-
pensatory hypertrophy was apparent because maternal
renal mass was ?75% that of intact ewes and effective
renal plasma flow (ERPF) and glomerular filtration rate
(GFR), when expressed per kg body weight, were ?55%
of the intact values (Gibson et al., 2006). Because over
50% of maternal nephrons must have been destroyed,
the remaining nephrons must have been hyperfiltering
(Gibson et al., 2006). These STNx ewes were hyperten-
sive and had changes in fluid and electrolyte balance
and proteinuria (Gibson et al., 2006).
Renal function was different in fetuses carried by
STNx ewes (STNxF). STNxF had higher urine flows and
sodium excretion rates, proximal fractional tubular so-
dium reabsorption was suppressed, and distal fractional
tubular sodium reabsorption was enhanced (Gibson
et al., 2007). They also had lower hematocrits and
plasma chloride and plasma renin levels (Gibson et al.,
2007). This finding suggests that they were volume
expanded or there was an increase in transplacental
We carried out thepresent study
whether the abnormal in utero environment of the
renally insufficient ewe altered renal development and
affected renal function in the post natal milieu. To do
this, we studied the renal morphology and function of
STNxL and ConL in the second week of life.
Surgical Preparation of Nonpregnant
These experiments were approved by the Animal Care
and Ethics Committee, UNSW. Before mating, seven
nonpregnant maiden ewes (?1 year of age) underwent
surgery for STNx as previously described by Gibson
et al. (2006). Ewes were anesthetized with an intrave-
nous (IV) injection of 1 g of thiopentone sodium (Pento-
thal, Abbott Australasia Pty Ltd, Australia), intubated
and anesthesia was maintained by 1–3% halothane
(Fluothane, Zeneca Ltd, UK) in oxygen.
Using sterile techniques, the kidneys were exposed by
means of a paravertebral incision. One kidney was
removed and weighed, and at least one branch of the re-
nal artery to the other kidney was ligated to produce a
color change over 30–50% of the kidney surface. After
surgery, intramuscular (IM) injections of procaine peni-
cillin (Ilium Propen, 600 mg, Troy Laboratories) and
oxytetracycline (Alamycin, 288 mg, Norbrook Laborato-
ries Ltd, UK) were given to the ewes. All incisions were
infiltrated with bupivicaine (Marcain 0.5%, AstraZeneca,
Australia), and ewes were given 300 mg of buprenor-
phine IM (Temgesic, Reckitt Benckiser, Australia).
After surgery, sheep were housed in metabolic cages
in a temperature controlled room (18–228C) with other
ewes. They had free access to food (1,200 g of chaff and
300 g of oats daily) and water. Once ewes had recovered
from surgery, they were returned to the farm where
they were mated (at least 2 months after surgery).
Because the STNx ewes had at least 2 months to recover
before mating, there was no sham surgery carried out in
the control animals. In late gestation, 10 control and the
7 STNx ewes were brought back to the laboratory where
they were able to spontaneously deliver. Birth weights of
the lambs were recorded.
Lamb Surgical Preparation
Four to 7 days after birth, all lambs underwent cathe-
terization. Using sterile techniques, and under halo-
thane anesthesia (1–3% in oxygen), catheters were
placed into the femoral artery and vein as well as supra-
pubically into the bladder. Lambs were given 60 mg of
penicillin and 19.2 mg of oxytetracycline IM at induction
and after surgery. All incisions were infiltrated with
0.5% bupivicaine at the end of surgery.
Catheters were flushed daily with heparinized (100 U
ml21) 0.15 M saline, and lambs were given the same
doses of antibiotics for 2 days after surgery. Experiments
were carried out 4–5 days after surgery.
The lambs were removed from their mother and
placed in a supporting sling. The bladder was opened
and allowed to drain. An IV bolus dose of lithium chlo-
ride was given (150 mmol kg21) and a continuous infu-
sion of lithium (10 mmol kg21hr21) was administered in
0.15 M saline at 0.95 ml hr21. Lithium clearance meth-
odology was used to determine proximal sodium reab-
sorption as lithium is reabsorbed with sodium and water
in the proximal tubule, but it is not reabsorbed distally
(Lumbers et al., 1988).
After an equilibration period of at least 40 min, 3 3
30 min urine collections were made with a blood sample
(5 ml) taken at the midpoint of the second and third
urine collection periods. Blood pressure and heart rate
were measured continuously using pressure transducers
(ADI) connected to a polygraph (Model 79D, Grass
LAMBS BORN TO MOTHERS WITH RENAL DYSFUNCTION
Instrument Co., Quincy, MA). These data were collected
using an IBM compatible PC and a National Instru-
ments interface card (model 371). A postmortem was
conducted immediately after the completion of the
Lambs were killed by IV injection of 2 g of pentobarbi-
tone sodium (Lethabarb, Virbac [Australia], NSW). Body
weight, nose–rump length, and abdominal girth were
measured and recorded. Both kidneys were weighed and
photographed on a 1-cm grid for length and width meas-
urements. The right kidney was bisected longitudinally,
placed in 4% paraformaldehyde in 0.5 M phosphate
buffer for approximately 1 week and then into 70% etha-
nol ready for processing for glomerular counting. The
heart was weighed and the left and right ventricular
free walls were dissected and weighed.
When the lambs had been killed, their mothers were
weighed, a blood sample taken, and then killed with an
IV injection of 5 g of pentobarbitone sodium. Their
kidney(s) were removed, photographed, and weighed.
Plasma concentrations of potassium, sodium, and chlo-
ride were measured using a blood gas analyzer (ABL
715 Series, Radiometer Pacific Pty Ltd). Plasma and uri-
nary osmolality were determined using a Fiske One-Ten
Osmometer (Fiske Associates, Massachusetts). Sodium
and potassium concentrations in urine were determined
using a FLM3 Flame Photometer (Radiometer Pacific
Pty Ltd). Glomerular filtration rate was estimated using
the clearance of endogenous creatinine. Creatinine con-
centrations in urine and plasma were determined using
the method of Haeckel (1980) using a micro plate reader
(model 680 XR, Bio-Rad Laboratories Pty Ltd Australia)
at 510 nm. Protein concentrations in plasma, urine, and
kidney homogenate were determined by a Lowry protein
assay (Lowry et al., 1951), which was read at 655 nm on
the micro plate reader. Lithium concentrations in both
plasma and urine were measured using an atomic
absorption spectrophotometer (Varian-Techtron Pty Ltd,
Plasma renin levels were measured using methods
previously described (Lumbers and Lee Lewes, 1979), as
the rate of formation of angiotensin I (Ang I) in ng ml21
h21when 100 ml of plasma was incubated for 2 hr at pH
7.5 and 378C with excess substrate (nephrectomized
sheep plasma, NSP). Kidney renin levels were measured
using methods previously described (Boyce et al., 2005).
Briefly, 0.5 g of renal cortex was homogenized in 4 ml of
0.03 M phosphate buffer. The supernatant diluted 1:400
was incubated with NSP for 0.5 hr at pH 7.5 and 378C
and the rate of formation of Ang I in ng ml21h21mea-
sured. Ang I concentrations were measured by radio-
immunoassay (Lumbers and Lee Lewes, 1979). Kidney
renin levels were expressed relative to protein levels
(mg Ang I h21mg protein21).
Kidney Processing and Glomerular Counting
The protocol for kidney processing and glomerular
counting is detailed extensively by O’Connell et al. (2006).
The fixed tissue was cut into slices ?2 mm thick and each
slice cut into pieces ?5 mm long. Approximately 8–12
pieces were randomly selected (with a known sampling
fraction of 1/15). These pieces were embedded in glycome-
thacrylate (Technovit 7100, Heraeus Kulzer Gmbh, Ger-
many), and each block was exhaustively sectioned at 20
mm. Every 10th and 11th sections were mounted onto
slides and stained with periodic acid-Schiff reagent (PAS)
and counter stained with haematoxylin. Using the physi-
cal disector/fractionator technique, the total number of
glomeruli (nephrons; Nglom,kid) in the kidney was esti-
mated (O’Connell et al., 2006). Mean glomerular volume
and mean renal corpuscle volume were also determined
using stereological techniques (O’Connell et al., 2006). By
multiplying total glomerular number by mean glomerular
volume, we calculated the total volume of all glomeruli in
kidneys. A similar approach was used to calculate total
renal corpuscle volumes in kidneys.
Physiological and biochemical data from all three peri-
ods were averaged to obtain a single value, and all data
are expressed as mean 6 standard error mean (SEM).
In the control group, n 5 10 for the functional parame-
ters and unless stated n 5 11 for morphological data. In
the STNxL group, n 5 8 for all variables. Data from the
two experimental groups were compared using an
unpaired t-test using SPSS (SPSS/PC; SPSS Inc., Chicago,
IL). Differences were considered significant at P < 0.05.
Maternal data were obtained from 6 control and 5
STNx ewes. Plasma creatinine, an indicator of renal dys-
function, was significantly higher in the STNx ewes
than controls (Con 0.45 6 0.01, n 5 5 vs. STNx 0.81 6
0.07 mmol/L, n 5 5, P 5 0.001). At postmortem, their
body weights were not different (Con 55.8 6 2.0 kg, n 5
6 vs. STNx 60.9 6 2.7 kg, n 5 5). Total kidney weight
was less in the STNx ewes (Con 175.3 6 5.2 n 5 6 vs.
STNx 127.6 6 6.8 g, n 5 5, P < 0.001), however their
remaining kidney had hypertrophied to be on average
double the weight of the kidney that was removed at
surgery (62.5 6 3.5g, n 5 5, P < 0.002). The remaining
kidney tended to be longer (Con 8.3 6 0.4, n 5 5 vs.
STNx 9.2 6 0.2 cm, n 5 5, P 5 0.07), and it was wider
than the kidneys of control ewes (Con 4.3 6 0.2, n 5 5
vs. STNx 5.6 6 0.2 cm, n 5 5, P < 0.05).
Birth weights were similar between groups (ConL 4.0
6 0.3 vs. STNxL 4.5 6 0.4 kg). On the day of the study,
there was also no difference in body weight (ConL 6.3 6
0.4 vs. STNxL 7.1 6 0.7 kg) and their ages were compa-
rable (ConL 10 6 1 vs. STNxL 11 6 1 days old). In the
ConL, there were four singletons and seven twins and
the ratio of males to females was 6:5. The corresponding
values in the STNxL were four singletons and four twins
and a sex ratio of 2:6.
ferences in mean arterial pressure (ConL 76.1 6 1.7 vs.
There were no dif-
BRANDON ET AL.
76.0 6 2.0 mm Hg) or heart rate (ConL 232 6 9 vs.
STNxL 213 6 15 beats/min) between the two groups.
Hematocrit and plasma composition.
were no differences between the groups in any measured
variable (Table 1).
Plasma and kidney renin levels.
levels were similar in each group of lambs (ConL 47.3 6
8.3, n 5 9 vs. STNxL 47.0 6 10.2 ng of Ang I ml21h21,
n 5 7). Kidney renin levels were also similar (ConL 7.1
6 0.9 vs. STNxL 7.2 6 1.0 mg of Ang I h21mg protein21).
(GFR) of STNxL and intact lambs were similar (Table
2). Urine flow rates, osmolality, free water clearance,
and the excretion rates of Na1, K1and osmoles were
not different between the two groups (Table 2). Although
a statistical difference was detected for urine flow rate
(ml min21kg21), this finding was due to two high out-
liers in the STNx group. In the remaining six STNxL,
urine flow ranged from 0.015 to 0.028 ml min21kg21,
which was similar to the control group (0.012 to 0.027
ml min21kg21). Tubular function was similar in the two
groups (Table 2). Thus, there were no differences in
renal function between STNxL and intact lambs.
Glomerular filtration rates
difference in abdominal girth (ConL 45.9 6 1.2 vs.
STNxL 48.1 6 1.5 cm) or nose–rump length (ConL 69.7
6 1.2 vs. STNxL 73.6 6 2.1 cm) between groups. STNxL
and ConL had similar heart and kidney weights (Table
3). The STNxL had longer right kidneys (P < 0.05); how-
ever, their width was not different. The left kidney
dimensions were similar to those of the ConL. The
length to width ratio for both the left and the right kid-
ney were similar in both groups.
At postmortem, there was no
Glomerular number and volume in the right
One animal in the control group had a glo-
merular number (Nglom,kid) that was 4 SD away from the
mean and, hence, was excluded from further analysis of
glomerular number or volume (Nglom,kid 5 701,450).
With the remaining data, glomerular number in the
STNxL was not different from ConL (Nglom,kid ConL
423,520 6 22,194, n 5 10, vs. STNxL 429,530 6 27,471
nephrons, n 5 8). Mean glomerular volume (P < 0.001;
Fig. 1) and total renal glomerular volume (P < 0.01;
Fig. 1) were significantly larger in the STNxL, as were
mean corpuscle volume (ConL 0.99 3 10236 0.06 3
1023vs. STNxL 1.24 3 10236 0.06 3 1023mm3, P <
0.001) and total renal corpuscle volume (ConL 407 6 28
vs. STNx 536 6 47 mm3, P < 0.01).
As it has been shown previously that twinning has an
effect on nephron number and corpuscle size, compari-
sons were made between the control and STNx groups
for singletons and twins separately. For offspring of sin-
gleton pregnancies, there was no difference between the
groups in glomerular number (ConL 480,299 6 12,825, n
5 3 vs. STNx 488,978 6 17,609, n 5 4); however, mean
glomerular size was greater in the STNxL (ConL 0.74 3
10236 0.06 3 1023vs. STNxL 1.12 3 10236 0.05 3
1023mm3, P < 0.01). Similarly, in offspring of twin
pregnancies, again there was no difference in glomerular
number (ConL 399,186 6 26,515, n 5 7 vs. STNxL
370,081 6 29,249, n 5 4), although there was a strong
tendency for mean glomerular volume to be increased in
the STNxL (ConL 1.04 3 10236 0.89 3 1023vs. STNxL
1.19 3 10236 1.05 3 1023mm3, P 5 0.07).
Gender is also a confounding factor. Hence, we com-
pared female offspring of control (five females, two
twins) and STNx ewes (six females, two twins). Glomer-
ular number did not differ between the groups (ConL
433,616 6 34,832 vs. STNxL 457,223 6 22,965 glomer-
uli). However, mean glomerular volume was signifi-
cantly larger in the STNxL (ConL 0.79 3 10236 0.04 3
1023vs. STNxL 1.06 3 10236 0.05 3 1023mm3, P <
0.01). We were unable to do the same comparison with
the male offspring due to low numbers in the STNxL
(n 5 2).
In both groups, there was a positive relationship
between GFR (ml/min) and glomerular number (ConL
r25 0.717, P < 0.01, n 5 9; STNxL r25 0.796, P <
0.01, n 5 8). A positive relationship was found in both
groups between glomerular number and right kidney
weight (ConL r25 0.678, P < 0.01, n 5 10; STNxL r25
0.729, P < 0.01, n 5 8). There was also a positive rela-
tionship between the excretion of protein and total
glomerular volume in the STNxL (r25 0.528, P < 0.05,
n 5 8; Fig. 2) that was not present in the ConL (r25
0.094, not significant, n 5 9; Fig. 2). There was no rela-
tionship between MAP and either glomerular number or
total glomerular volume in either group.
We have developed a model of maternal renal insuffi-
ciency in sheep, a species that completes renal organo-
genesis in utero as does the human fetus. The kidney
completes nephrogenesis at approximately 130 days ges-
tation in sheep (term ?150 days; Robillard et al., 1981)
compared with the rat and mouse, in which nephrogene-
sis continues until 8–10 days after birth (Clark and Ber-
tram, 1999). We subtotally nephrectomized nonpregnant
ewes before mating, so that there was a chronic reduc-
tion in maternal renal mass for the whole of gestation.
When we have studied nonpregnant ewes 8 weeks after
STNx surgery, GFR was half that of control ewes (Lum-
bers et al., 2007), which indicates renal dysfunction
before mating. This is, therefore, a model of human
pregnancy occurring in association with pre-existing
mild renal impairment. Using this model of pre-existing
renal disease, in the current study we found that lambs
TABLE 1. Hematocrit and plasma composition in
lambs from control (ConL) or subtotally
nephrectomized (STNxL) mothersa
Protein (mg mL21)
Creatinine (mg dL21)
Osmolality (mosm kg H2O)
30 6 2
84 6 4
0.35 6 0.02
299 6 2
141 6 1
3.9 6 0.1
111 6 1
36.0 6 0.9
33 6 2
92 6 3
0.39 6 0.04
304 6 3
142 6 1
3.9 6 0.1
112 6 1
36.8 6 1.2
aValues expressed as mean 6 SEM.
LAMBS BORN TO MOTHERS WITH RENAL DYSFUNCTION
that had been carried by STNx ewes had glomerular
hypertrophy in the second week of postnatal life. The
extent of the glomerular hypertrophy could be estimated
from the rate of excretion of protein in the urine, which
further suggests that the integrity of this glomerular
filtration barrier is altered.
In our previous study, STNxF had altered renal func-
tion including diuresis, natriuresis, and suppression of
fractional proximal sodium reabsorption (Gibson et al.,
2007). They also had low hematocrits and plasma chlo-
ride levels, and their plasma renin levels were sup-
pressed, suggesting that they were volume expanded
(Gibson et al., 2007). One aim of the current study was
to determine whether these functional differences per-
sisted after birth. One possibility was that, after birth,
because offspring of STNx ewes would no longer be
exposed to a volume load, all the altered variables might
return to normal. Indeed we found that most of the vari-
ables that were altered during fetal life in the STNx
group were not different 7–14 days after birth. This find-
ing included sodium excretion, hematocrit, and plasma
chloride and plasma renin levels. Thus, in utero, renal
function of STNxF was determined by the abnormal
maternal milieu. Ex utero, renal function of STNxL was
determined by the lamb’s fluid intake and by extrarenal
fluid losses and was similar to that of control lambs.
Because maternal uninephrectomy in mice before mat-
ing is associated with an increase in glomerular number
and size in the offspring (Averbukh et al., 1988, 1996),
we hypothesized that glomerular number would be
increased in lambs carried by STNx ewes. This was not
the case, although glomerular volume increased (Fig. 1).
It is possible that there was no increase in glomerular
number in the STNxL because the ewes were not mated
until at least 2 months after renal surgery. By contrast,
mice were mated within a week of surgery (Averbukh
et al., 1988, 1996), and the authors suggested that
following maternal unilateral nephrectomy renotrophic
hormonal factor(s) were generated that crossed the
placenta and stimulated growth of the fetal kidneys
(Averbukh et al., 1988, 1996). In our study, mating was
delayed and maternal compensatory renal hypertrophy
probably complete. Thus, any changes/effects of this hy-
pothetical renotrophic factor on fetal renal development
would not have occurred. Alternatively, different species
might respond differently. The differences between sheep
and rodents in the time at which nephrogenesis is com-
pleted may well alter the susceptibility of these species
to those factors that affect nephron number. Finally,
Averbukh et al. (1988, 1996) used different methods to
measure glomerular number. The physical dissector/frac-
tionator method that we used is highly regarded.
The most important finding in this study was the
?30% increase in glomerular size in the STNxL (Fig. 1).
Because twinning is associated with a decrease in neph-
ron endowment and an increase in renal corpuscle
volume (Mitchell et al., 2004), we made separate
TABLE 2. Renal function in lambs from control (ConL) or subtotally
nephrectomized (STNxL) mothers
Urine flow rate
(ml min21kg body weight21)
Glomerular filtration rate
(ml min21kg body weight21)
Urinary osmolality (mosm kg H2O21)
Free water clearance (ml min21kg body weight21)
Na1(mmol min21kg body weight21)
K1(mmol min21kg body weight21)
Osmoles (mosm min21kg body weight21)
Protein (mg min21kg body weight21)
0.11 6 0.02
0.017 6 0.002
0.25 6 0.07
0.034 6 0.008*
29.1 6 3.1
4.5 6 0.4
709 6 50
20.03 6 0.00
30.7 6 6.7
4.0 6 0.6
577 6 108
20.02 6 0.01
0.20 6 0.04
2.10 6 0.24
12.1 6 1.2
210 6 13
99.96 6 0.01
95.2 6 0.9
4.8 6 0.9
98.5 6 0.5
0.27 6 0.05
2.26 6 0.43
15.3 6 2.2
201 6 18
99.94 6 0.02
92.5 6 1.2
7.4 6 1.2
98.7 6 0.7
aValues expressed as mean 6 SEM. FRx, fractional reabsorption of X; FRNaP and FRNaD, frac-
tional reabsorption of Na by the proximal and distal tubules, respectively; DRNaDD, distal reab-
sorption of Na as a percentage of distal delivery.
*Difference between ConL and STNxL, P < 0.05.
TABLE 3. Heart and kidney weights and kidney
dimensions in lambs from either control (ConL) or
subtotally nephrectomized (STNxL) mothersa
Kidney dimensions (cm)
Left kidney length
Left kidney width
Right kidney length
Right kidney width
44.2 6 2.4
15.5 6 1.0
9.7 6 0.7
36.1 6 1.9
18.4 6 1.0
17.7 6 1.1
48.3 6 3.2
17.6 6 0.9
11.0 6 0.7
39.8 6 3.3
20.0 6 1.7
19.8 6 1.6
4.4 6 0.1
2.3 6 0.1
1.9 6 0.1
4.5 6 0.1
2.3 6 0.1
2.0 6 0.1
4.7 6 0.1
2.4 6 0.1
1.9 6 0.1
4.9 6 0.2*
2.4 6 0.1
2.1 6 0.1
aValues expressed as mean 6 SEM.
*Difference between ConL and STNxL, P < 0.05.
BRANDON ET AL.
comparisons between the STNxL and ConL for single-
tons and twins. As the unaltered glomerular number
and increased glomerular size in STNxL was present
when we examined singleton lambs alone and a similar
trend was seen in twin lambs as well, it is reasonable to
assume that the unaltered nephron number with glo-
merular hypertrophy was caused by the altered intrau-
terine environment and that this finding was independ-
ent of any effects related to the proportion of twins in
It has also been demonstrated that developmental pro-
gramming can sometimes affect one gender more than
the other (Sugden and Holness, 2002; O’Regan et al.,
2004; Fernandez-Twinn et al., 2005). Unfortunately
there were not enough males in the STNx group for
analyses. However, there were enough females for analy-
sis with both ConL and STNxL female groups having an
even singleton/twin ratio. Female STNxL were still
found to have glomerular hypertrophy without changes
in nephron number when compared with the controls.
Again this strengthens our statement that the off-
spring’s glomerular hypertrophy was caused by the
altered intrauterine environment due to maternal STNx.
The combination of increased glomerular size but nor-
mal nephron number in the STNxL is particularly in-
triguing. There are multiple reports of offspring born
with a nephron deficit (due to maternal intervention)
that have increased glomerular volume (Woods et al.,
2001; Wintour et al., 2003), but glomerular hypertrophy
with a normal glomerular number is relatively unusual.
Glomerular hypertrophy is often an early sign of renal
disease and is particularly associated with proteinuria
(Perico et al., 2005). Although the glomerular hypertro-
phy that we have demonstrated at 2 weeks of life may
not persist into adulthood, the positive relationship
between glomerular size and urinary protein excretion
in the STNxL could indicate a predisposition to deterio-
ration of renal function with age.
Glomerulomegaly is commonly found in patients with
morbid obesity, congenital cyanotic heart disease, and
pulmonary hypertension (Faustinella et al., 1997). In
these patients, several mechanisms have been proposed,
but the cause of the glomerular hypertrophy is contro-
versial (Faustinella et al., 1997). In the current study,
we believe that the glomerular hypertrophy was a
response to hyperfiltration seen in late fetal life. When
studied chronically at ?127 days gestation, GFR was not
significantly higher in the STNxF than ConF (Gibson
et al., 2007). However, more recent studies in older
(?139 days gestation) anesthetized fetuses, show that
GFR is clearly increased in late gestation in STNxF
(ConF 4.6 6 0.5, n 5 14, vs. STNxF 7.2 6 0.70 ml/min,
n 5 16; Turner, 2007). Hence, the extra load placed on
the glomeruli in the very late stages of gestation prob-
ably caused a work-induced hypertrophy in these ani-
mals. Of interest, because GFR increased after 130 days
in STNxF, when nephrogenesis is complete, this finding
may be a possible explanation as to why we see an
increased glomerular volume without changes in neph-
ron number in STNxL.
Women who have renal disease and become pregnant
have an increased risk of fetal morbidity and mortality.
Babies born to mothers with renal disease are more
likely to be growth restricted (Cunningham et al., 1990;
Jones and Hayslett, 1996; Jungers et al., 1997). The
STNx ewes had no overt signs of renal insufficiency.
These ewes could be maintained on pasture, were fertile,
and carried apparently healthy fetuses, as there were no
effects on lamb body weights or dimensions (Table 3;
Gibson et al., 2006, 2007). Only by careful comparison of
GFRs, renal function, and urinary protein excretion was
it possible to detect any signs of renal impairment in the
STNx ewes (Gibson et al., 2006). GFR has to be <30%
for chronic renal insufficiency to become manifest.
Therefore, these animals were not a model of human
renal disease in pregnancy because they did not have
a symptomatic illness. However, they are a model for
human subclinical renal insufficiency, and our finding
that glomerular morphology is altered in lambs carried
right kidney for lambs of control (C, n 5 10) and subtotally nephrec-
tomized (S, n 5 8) mothers. *Different to C, P < 0.01, **P < 0.001.
Mean glomerular volume and total glomerular volume of the
total glomerular volume of the right kidney in lambs of control (ConL,
circles, n 5 9) or subtotal nephrectomy (STNx; ~---, n 5 8) mothers.
A positive relationship was found in lambs born to STNx mothers
(STNxL) described by the equation: EProt 5 3.9 3 total glomerular
volume 2375, r25 0.528, P < 0.05. No such relationship was found
in the ConL (r25 0.094, not significant).
The relationship between the excretion of protein (EProt) and
LAMBS BORN TO MOTHERS WITH RENAL DYSFUNCTION
by STNx ewes suggests that these offspring may be pre-
disposed to developing renal disease in adult life.
We thank Ms. Vasumathy Kumarasamy and Ms. Pam-
ela Bode for their technical assistance. E.L. was funded
by the NHMRC and E.L. and K.G. were funded by Kid-
ney Health Australia.
Averbukh Z, Bogin E, Cohn M, Goren E, Modai D, Rosenmann E,
Weissgarten J. 1988. The renotrophic factor, a persistent stimulus
that crosses the placenta in mice. J Physiol 404:31–38.
Averbukh Z, Weissgarten J, Berman S, Cohn M, Modai D. 1996.
Interrelationship between renal mass and renotropin activity in
consecutive generations of uninephrectomized mice. Am J Neph-
Boyce AC, Gibson KJ, Wintour EM, Koukoulas I, Lumbers ER.
2005. Effects of 7-day amino acid infusion on renal growth, func-
tion, and renin-angiotensin system in fetal sheep. Am J Physiol
Regul Integr Comp Physiol 289:R1099–R1106.
Clark AT, Bertram JF. 1999. Molecular regulation of nephron
endowment. Am J Physiol 276:F485–F497.
Cunningham FG, Cox SM, Harstad TW, Mason RA, Pritchard JA.
1990. Chronic renal disease and pregnancy outcome. Am J Obstet
Faustinella F, Uzoh C, Sheikh-Hamad D, Truong LD, Olivero JJ.
1997. Glomerulomegaly and proteinuria in a patient with idio-
pathic pulmonary hypertension. J Am Soc Nephrol 8:1966–1970.
Fernandez-Twinn DS, Wayman A, Ekizoglou S, Martin MS, Hales
CN, Ozanne SE. 2005. Maternal protein restriction leads to hy-
perinsulinemia and reduced insulin-signaling protein expression
in 21-mo-old female rat offspring. Am J Physiol Regul Integr
Comp Physiol 288:R368–R373.
Gibson KJ, Thomson CL, Boyce AC, Karime BM, Lumbers ER.
2006. Effects of a reduction in maternal renal mass on pregnancy
and cardiovascular and renal function of the pregnant ewe. Am J
Physiol Renal Fluid Electrolyte Physiol 290:F1153–F1162.
Gibson KJ, Boyce AC, Karime BM, Lumbers ER. 2007. Maternal re-
nal insufficiency alters plasma composition and renal function in
the fetal sheep. Am J Physiol Regul Integr Comp Physiol 292:
Goss RJ. 1963. Effects of maternal nephrectomy on foetal kidneys.
Haeckel R. 1980. Simplified determinations of the ‘‘true’’ creatinine
concentration in serum and urine. J Clin Chem Clin Biochem
Jones DC, Hayslett JP. 1996. Outcome of pregnancy in women with
moderate or severe renal insufficiency. N Engl J Med 335:226–232.
Jungers P, Chauveau D, Choukroun G, Moynot A, Skhiri H, Houil-
lier P, Forget D, Grunfeld JP. 1997. Pregnancy in women with
impaired renal function. Clin Nephrol 47:281–288.
Lowry OH, Rosebrough NJ, Farr LA, Randall RJ. 1951. Protein
measurement with the Folin reagent. J Biol Chem 193:265–275.
Lumbers E, Lee Lewes J. 1979. The actions of vasoactive drugs on
fetal and maternal plasma renin activity. Biol Neonate 35:23–32.
Lumbers ER, Hill KJ, Bennett VJ. 1988. Proximal and distal tubu-
lar activity in chronically catheterized fetal sheep compared with
the adult. Can J Physiol Pharmacol 66:697–702.
McBroom MJ, Al-Zaid NS, Dlouha H. 1986. Kidney growth and col-
lagen content in rat pups from uninephrectomized mothers. Biol
Mitchell EK, Louey S, Cock ML, Harding R, Black MJ. 2004. Neph-
ron endowment and filtration surface area in the kidney after
growth restriction of fetal sheep. Pediatr Res 55:769–773.
O’Connell AE, Boyce AC, Kumarasamy V, Douglas-Denton R,
Bertram JF, Gibson KJ. 2006. Long-term effects of a midgesta-
tional asphyxial episode in the ovine fetus. Anat Rec 288A:1112–
O’Regan D, Kenyon CJ, Seckl JR, Holmes MC. 2004. Glucocorticoid
exposure in late gestation in the rat permanently programs gen-
der-specific differences in adult cardiovascular and metabolic
physiology. Am J Physiol Endocrinol Metab 287:E863–E870.
Okada T, Yamagishi T, Morikawa Y. 1994. Morphometry of the
kidney in rat pups from uninephrectomized mothers. Anat Rec
Ozanne SE, Olsen GS, Hansen LL, Tingey KJ, Nave BT, Wang CL,
Hartil K, Petry CJ, Buckley AJ, Mosthaf-Seedorf L. 2003. Early
growth restriction leads to down regulation of protein kinase C
zeta and insulin resistance in skeletal muscle. J Endocrinol
Perico N, Codreanu I, Schieppati A, Remuzzi G. 2005. The future of
renoprotection. Kidney Int Suppl:S95–101.
Robillard JE, Weismann DN, Herin P. 1981. Ontogeny of single glo-
merular perfusion rate in fetal and newborn lambs. Pediatr Res
Sugden MC, Holness MJ. 2002. Gender-specific programming of
insulin secretion and action. Journal of Endocrinology 175:757–
Turner AJ. 2007. Control of renal hemodynamics in the developing
kidney: implications for fetal programming. Sydney: The Univer-
sity of New South Wales, Department of Physiology.
Wintour EM, Moritz KM, Johnson K, Ricardo S, Samuel CS, Dodic M.
2003. Reduced nephron number in adult sheep, hypertensive as
a result of prenatal glucocorticoid treatment. J Physiol 549:929–
Woods LL, Ingelfinger JR, Nyengaard JR, Rasch R. 2001. Maternal
protein restriction suppresses the newborn renin-angiotensin
system and programs adult hypertension in rats. Pediatr Res
BRANDON ET AL.