Nephron Number, Renal Function, and Arterial Pressure in
Aged GDNF Heterozygous Mice
Luise A. Cullen-McEwen, Michelle M. Kett, John Dowling, Warwick P. Anderson, John F. Bertram
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Abstract—The loss of one allele for glial cell line– derived neurotrophic factor (GDNF) results in ⬇30% fewer but normal
sized glomeruli in young mice. Low nephron number, inherited or acquired, has been linked to increased risk of
development of hypertension and renal failure. This study examines whether GDNF heterozygous mice, with an inherent
reduction in nephron number, demonstrate a deterioration in renal structure and function and rise in arterial pressure in
later life. Fourteen-month-old male GDNF heterozygous (n⫽7) and wild-type (n⫽6) mice were anesthetized and
prepared for measurement of mean arterial pressure, glomerular filtration rate (GFR), and renal blood flow. After
measurement of renal function, kidneys were fixed for stereological determination of total glomerular number and mean
glomerular volume. Mean arterial pressure was, on average, 18 mm Hg higher in GDNF heterozygous (98⫾4 mm Hg)
than wild-type mice (80⫾2 mm Hg; P⬍0.01). However, GFR (0.656⫾0.054 versus 0.688⫾0.076 mL/min per g kidney
wt) and renal blood flow (5.29⫾0.42 versus 4.70⫾0.34 mL/min per g kidney wt) were not different between groups.
Fourteen-month-old GDNF heterozygous mice had ⬇30% fewer glomeruli than wild-type mice (9206⫾934 versus
13440⫾1275; P⬍0.01) and significantly larger glomeruli (4.51⫾0.39 versus 3.72⫾0.63⫻10⫺4mm3; P⬍0.01). Thus,
aged GDNF heterozygous mice maintained a normal GFR and renal blood flow despite reduced nephron numbers. The
elevated arterial pressure, glomerular hypertrophy, and hyperfiltration demonstrated in the GDNF heterozygous mice at
this age may indicate a compensatory mechanism whereby GFR is maintained in the presence of a reduced nephron
endowment. (Hypertension. 2003;41:335-340.)
Key Words: mice 䡲 hypertension, genetic 䡲 kidney 䡲 blood flow 䡲 arterial pressure
after birth. In contrast, GDNF, RET, and GFR␣1 heterozygous mice were both fertile and viable. Whereas the RET and
GFR␣1 heterozygotes demonstrated a normal renal phenotype, the GDNF heterozygotes showed an array of renal
phenotypes, ranging from two smaller kidneys, many with
abnormal shapes and cortical cysts, to unilateral renal
agenesis.10 –13
These results indicated that GDNF gene dosage influenced
kidney development, with the loss of one allele being sufficient to cause a significant renal phenotype. Recently we
found that the kidneys of these GDNF heterozygous mice at
30 days of age were ⬇25% smaller than their wild-type
littermates despite similar body weights.17 Furthermore, stereologic estimates of nephron number identified a 30%
decrease in nephron endowment in young heterozygous
GDNF mice compared with wild-type mice. The GDNF
heterozygous mouse thus provides a genetic model with
which to test the hypothesis that an inherent reduction in
nephron number contributes to the development of cardiovascular and renal disease that is uncomplicated by changes in
birth and body weight. In the majority of cases in humans,
I
t has been hypothesized that low nephron numbers in the
kidney may increase the risk of development of cardiovascular diseases such as hypertension and chronic renal failure
and reduce the long-term success of renal allografts.1– 4 Thus,
factors that affect nephrogenesis in the fetus may not only be
critical in kidney development but also affect subsequent
adult kidney function and underlie much subsequent renal
pathology and abnormal physiology.
Glial cell line-derived neurotrophic factor (GDNF)5–7 has
been shown to play a key role in kidney development through
actions at the RET and GFR␣1 receptor and coreceptor.8,9
Specifically, GDNF has been demonstrated to initiate budding of the ureteric duct from the Wolffian duct, branching of
the ureteric epithelium within the metanephric mesenchyme,
and the formation of new nephrons at the branch tips.5–7
Increasing the levels of exogenous GDNF in metanephric
culture medium leads to increases in both the number of
ureteric branches and number of developing nephrons.7 In the
late 1990s, knockout studies demonstrated that homozygous
null mutants for GDNF,10 –13 as well as RET14 and
GFR␣1,15,16 showed bilateral renal agenesis and died shortly
Received October 3, 2002; first decision October 21, 2002; revision accepted November 26, 2002.
From the Department of Anatomy and Cell Biology (L.A.C.-M., J.F.B.) and the Department of Physiology (M.M.K., W.P.A.), Monash University,
Victoria, Australia; and the Department of Anatomical Pathology, Alfred Hospital (J.D.), Melbourne, Australia.
Drs Cullen-McEwen and Kett contributed equally to this work.
Correspondence to John F. Bertram, PhD, Department of Anatomy and Cell Biology, PO Box 13C, Monash University, VIC, 3800, Australia. E-mail
[email protected]
© 2003 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
DOI: 10.1161/01.HYP.0000050961.70182.56
335
336
Hypertension
February 2003
cardiovascular and renal disease does not become apparent until
later in life. Thus this study examines whether GDNF heterozygous mice with a 30% reduction in nephron endowment go on to
demonstrate a deterioration in renal function, glomerular hypertrophy, and/or increases in arterial pressure later in life.
Methods
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
All experiments were approved in advance by Monash University
Departments of Physiology and Anatomy and Cell Biology Animal
Ethics Committees and were conducted in accordance with the
Australian Code of Practice for the Care and Use of Animals for
Scientific Purposes.
Our GDNF mouse colony was initially established with founders
from the laboratory of Dr Heiner Westphal (Laboratory of Mammalian Genes and Development, National Institutes of Health, Bethesda, Md).10,11 Male GDNF heterozygous mice (129Sv-C57BL/6
hybrid; 6th generation C57BL/6 back-cross) were mated with female
C57BL/6 mice. After weaning, tail tissue was obtained from all mice
for genotyping by polymerase chain reaction,10,11,17 and the mice
were coded such that the following experiments were carried out in
a blinded fashion.
Blood Pressure and Renal Function
At 14 months of age, male GDNF wild-type (14.0⫾0.3 months;
n⫽7) and heterozygous (14.1⫾0.2 months; n⫽6) mice were anesthetized (Inactin, 100 mg/kg IP; Sigma Chemical Co; and ketamine,
10 mg/kg IP; Parnell Laboratories) and placed on a heating table to
maintain body temperature at 37°C. The trachea was catheterized
(PE-90), and a stream of O2 was blown onto the end of the tube to
maintain a stable arterial pressure throughout the experiment. The
left femoral artery was catheterized (pulled SV-50) for measurement
of blood pressure and heart rate and to obtain a terminal arterial
blood sample, and the left femoral vein (pulled SV-50) was catheterized for infusion of maintenance fluids (6% BSA, 2.5 ␮L/min
during surgery). After surgery, the infusion was changed to a 1%
BSA solution containing 3H-inulin (5.58 ␮Ci/mL) and 14C-PAH (1.7
␮Ci/mL) for estimation of glomerular filtration rate (GFR) and
effective renal plasma flow by renal clearance methods, and the mice
were allowed 1 hour to equilibrate. The equilibration period was
followed by two 20-minute urine collection periods, after which an
arterial blood sample (100 ␮L) was taken. Urinary protein concentrations were measured by means of the Bradford method.18 Sodium
and potassium concentrations were analyzed with a Technicon
autoanalyzer flame photometer IV.
At the completion of the experiment, kidneys were rapidly
excised, weighed, and immersion-fixed in 2% paraformaldehyde and
2% glutaraldehyde in 0.1 mol/L phosphate buffer. The left kidneys
were then processed for embedding in glycolmethacrylate for stereologic estimation of total nephron number and mean glomerular
volume. Right kidneys were processed and embedded in paraffin,
and sections were stained with hematoxylin and eosin, periodic acid
Schiff’s reagent, and Masson’s trichrome strain for assessment of
renal pathology.
Estimating Kidney Volume
Kidney volume was estimated by means of the Cavalieri principle.19,20 Briefly, whole kidneys embedded in glycolmethacrylate
were exhaustively sectioned at 20 ␮m, and every 10th and 11th
section was collected and stained with periodic acid Schiff’s reagent.
The “10th” section of each pair was then placed on a microfiche
reader (magnification ⫻24.25), and a stereologic test grid (2⫻2cm)
was placed on the microfiche screen. Kidney volume (Vkid) was
estimated using the formula:
V kid⫽⌺P⫻a共 p兲⫻T⫻1/f
where ⌺P is the total number of points counted, a(p) is the area
associated with each grid point, T is section thickness, and 1/f is the
inverse of the section sampling fraction.
Estimating Nephron Number
The above section pairs were used to estimate nephron number,
using the physical disector/fractionator combination.20 –22 Briefly,
the section pairs were projected side by side, with two microscopes
modified for projection. One microscope was fitted with a motorized
stage and the other was fitted with a rotatable stage to enable section
alignment. A grid was placed over each field of view, and points
falling on kidney tissue (Pkid), glomeruli (Pglom), and renal corpuscles
(Pcorp) were counted. Glomeruli sampled by an unbiased counting
frame in the field of view of the 10th section that were not present
in the 11th section were counted. Those sampled in the 11th section
that were not present in the 10th section were counted to double the
efficiency of the technique. This process was repeated for each
complete pair of sections. Total nephron number (Nglom,kid) was then
estimated using the following equation:
N glom,kid⫽10⫻P s/P f⫻1/ 2f a⫻Q ⫺
where 10 was the reciprocal of the section sampling fraction, Ps the
number of points overlying all kidney sections, Pf the number of
points overlying complete kidney sections, 1/2fa the fraction of the
total section area used to count glomeruli, and Q⫺ the actual number
of glomeruli counted.
Glomerular Volume
Mean glomerular volume (Vglom) was estimated by using the following formula:
V glom⫽关V glom/V kid兴/关N glom/V kid兴
where Vglom/Vkid is equivalent to Pglom/Pkid.
The total volume of all glomeruli (Vglom
estimated by using the following formula:
(total)
) in the kidney was
V glom共total 兲⫽V glom⫻N glom,kid
.
These formulas were then adjusted for estimation of mean renal
corpuscle volume (Vcorp) and total volume of all renal corpuscles in
the kidney (Vcorp (total)).
Statistics
Differences between heterozygous and wild-type mice were tested
with an unpaired Student t test. Values are presented as mean⫾SEM
except for stereological data, which are presented as mean⫾SD.
Results
At 14 months of age, there were no significant differences in
GFR, renal blood flow, renal vascular resistance, filtration
fraction, fractional sodium and potassium excretions, or
urinary protein concentration between GDNF heterozygous
and wild-type mice (Table 1). In contrast, anesthetized mean
arterial pressures were 18 mm Hg higher in GDNF heterozygotes than their wild-type littermates (P⬍0.001; Table 1).
There were no differences in body or kidney weights between
the two groups (Table 1).
Stereology
Consistent with kidney weights, stereologic estimates of
kidney volume were not significantly different between
GDNF heterozygous mice and wild-type littermates (Table
2). Total nephron number estimates confirmed the results
obtained at 30 days of age, with aged male GDNF heterozygous mice also containing ⬇30% fewer nephrons than their
wild-type littermates (P⬍0.01). At 14 months, however, the
glomeruli of GDNF heterozygous kidneys were 20% larger
(P⬍0.01) and mean renal corpuscle volume 30% larger than
Cullen-McEwen et al
TABLE 1. Characteristics of 14-Month-Old Wild-Type and
GDNF Heterozygous Mice
Parameter
Wild-Type
GDNF
Heterozygous
Weights
Body weight, g
47⫾2
46⫾1
Left kidney weight, g
0.277⫾0.023
0.259⫾0.031
Right kidney weight, g
0.283⫾0.026
0.245⫾0.012
Total kidney weight, g
0.560⫾0.048
0.503⫾0.033
Hemodynamics and renal function
MAP, mm Hg
HR, b/min
GFR, mL/min/g kidney wt
RBF, mL/min/g kidney wt
RVR, mm Hg/(ml/min/g kidney wt)
FF, %
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Fractional sodium excretion, %
Fractional potassium excretion, %
UP, g/dL
80⫾1
98⫾3*
484⫾35
460⫾22
0.688⫾0.076
0.656⫾0.054
4.70⫾0.34
5.29⫾0.42
17.33⫾1.29
19.40⫾2.33
27⫾3
24⫾3
0.18⫾0.04
0.20⫾0.04
22.6⫾5.7
19.5⫾4.8
0.565⫾0.072
0.527⫾0.060
Values are mean⫾SEM. MAP indicates mean arterial pressure; HR, heart
rate; GFR, glomerular filtration rate; RBF, renal blood flow; RVR, renal vascular
resistance; FF, filtration fraction; and UP, urinary protein. Wild-type, n⫽5 for
MAP, HR, GFR, RBF, FF, RVR, UP; and n⫽7 for age and body and kidney
weights. GDNF heterozygous n⫽6.
*P⬍0.001.
wild-type kidneys (P⬍0.01;Table 2). Total glomerular and
renal corpuscle volumes (product of glomerular number and
glomerular/corpuscle volume) were not different between
wild-type and GDNF heterozygous kidneys.
Renal Histology and Pathology
Kidneys of 14-month-old GDNF heterozygous and wild-type
mice both showed evidence of tubulointerstitial pathology.
Most wild-type kidneys analyzed showed areas of focal
tubular vacuolation (Figure) and mild inflammation around
the renal pelvis. GDNF heterozygous kidneys also showed
tubular vacuolation; however, the extent of vacuolation was
much greater and more widespread than in wild types
(Figure). There was no evidence of degeneration within the
vacuolated cells as they displayed viable nuclei and no signs
of necrosis or apoptosis. Despite significant hypertrophy, the
glomeruli of GDNF heterozygous kidneys showed no eviTABLE 2. Stereological Estimates for the Left Kidneys of
Wild-Type and Heterozygous 14-Month-Old Mice
Wild-Type
GDNF
Heterozygous
Kidney volume, mm3
276.5⫾32.1
247.8⫾54.5
Glomerular no.
13440⫾1275
9206⫾934*
3.72⫾0.63
4.51⫾0.39*
3
Total glomerular volume, mm
4.99⫾0.87
4.15⫾0.55
Corpuscle volume, ⫻10⫺4mm3
5.69⫾0.44
7.38⫾0.59*
Total corpuscle volume, mm3
7.67⫾1.13
6.81⫾1.10
Stereological Parameter
Glomerular volume, ⫻10⫺4mm3
Values are mean⫾SD. Wild type n⫽7; GDNF heterozygous n⫽6.
*P⬍0.01.
Renal Function in Aged GDNF Heterozygous Mice
337
dence of sclerosis or hypercellularity (Figure). In addition, of
the GDNF heterozygous kidneys analyzed, one showed areas
of focal interstitial inflammation and one contained a cortical
cyst.
Discussion
A low renal nephron number (“reduced nephron endowment”) has been linked with the development of hypertension, glomerulosclerosis, renal failure, and the long-term
failure of renal allografts. There are, however, few available
animal models with a congenital nephron deficit uncomplicated by changes in birth and adult body weight with which
to examine this link. In 1996, three independent research
groups generated mice with a homozygous null mutation for
GDNF.10,12,13 The homozygous null mutants died within the
first 24 hours of postnatal life as the result of bilateral renal
agenesis. The heterozygotes, however, were indistinguishable
from wild-type littermates in terms of body weight but had
reduced renal mass. Recently, our group published a detailed
stereological analysis of glomerular number and volumes in
these GDNF heterozygous mice, studying the mice at 30 days
of age when kidney development is complete.17 We found
that glomerular number was 30% lower than wild-type
littermates, although glomerular volumes were similar at this
age.17
The GDNF heterozygous mouse thus provides a unique
animal model with which to examine whether reduced glomerular endowment at birth is a predictor of subsequent
development of cardiovascular disease such as essential
hypertension later in life. GDNF heterozygous and wild-type
mice start to die of natural causes at around 16 months and
therefore we chose to study them at 14 months, making the
assumption that this was equivalent to late middle age in
humans, when the incidence of cardiovascular diseases such
as essential hypertension rises markedly in the population.
It has been hypothesized that reductions in glomerular
number leads to hypertrophy of the remaining glomeruli with
time. This has been well documented in the commonly used
5/6 nephrectomy model. The present study appears to indicate
that such hypertrophy also occurs when glomerular numbers
are reduced genetically. We previously reported that young
GDNF heterozygous mice (30 days old) showed reduced
glomerular numbers with no change in mean glomerular
volume and thus demonstrated, overall, a reduced total
glomerular volume (product of glomerular number and volume). In the current study, we found that by 14 months of
age, glomeruli of GDNF heterozygotes were significantly
hypertrophied such that the total glomerular volume was no
longer different between wild-type and heterozygous littermates. Whole-kidney GFR, renal blood flow, and fractional
excretions were also not different between wild-type and
GDNF heterozygous littermates. Although micropuncture
analysis was not performed in this study, an index of average
single nephron function can be calculated by dividing wholekidney GFR by the number of glomeruli. With 30% fewer
nephrons and similar GFR, GDNF heterozygous mice appear
to have marked hyperfiltration with 30% greater calculated
single nephron GFR values compared with wild-type mice
(17.7⫾1.3 and 13.5⫾1.2, respectively; P⬍0.05). Such glo-
338
Hypertension
February 2003
Light micrographs of 14-month-old wild-type
and GDNF heterozygous mice kidneys. There
was no evidence of glomerular sclerosis or
hypercellularity in either wild-type (A) or GDNF
heterozygous kidneys (B). The extent of vacuolation was greater and more widespread in
GDNF heterozygous (D) kidneys than in wild
types (C). Bar, 50 ␮m.
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merular hypertrophy and hyperfiltration is often a predictor of
glomerular damage and progression to glomerular sclerosis.23
However, GDNF heterozygous mice showed no evidence of
glomerular sclerosis or increased proteinuria at 14 months of
age. GDNF heterozygous mice did demonstrate greater proximal tubule vacuolation at 14 months of age compared with
wild-type mice. The significance of these changes on tubular
reabsorption in the GDNF mice are as yet unclear; however,
the fractional excretion of sodium and potassium were not
different between wild-type and GDNF heterozygous
littermates.
Finally, we found that these old GDNF heterozygous mice
had mean arterial pressures that were 18 mm Hg higher than
their wild-type littermates. At this time, we are not able to say
when arterial pressure became elevated. Gerlai et al,24 however, reported that the mean arterial pressure of 4- to
7-month-old GDNF heterozygous and wild-type mice were
not significantly different, suggesting the elevation in pressure observed in the present study occurs subsequent to this.
Interestingly, this group also showed plasma creatinine levels
of the GDNF heterozygous mice to be 10 times higher than
wild-type mice, suggesting that GFR tended to be decreased
in younger normotensive GDNF heterozygous mice. These
findings are compatible with Brenner’s hypothesis2 that a
reduced filtration surface area leads to the development of
glomerular hypertrophy and hypertension in order to maintain
adequate renal function. These findings suggest that the
GDNF heterozygous mice may prove to be a useful model of
essential hypertension. While anesthesia with Inactin and ketamine has been found to have only mild effects on arterial
pressure,25 conscious, sequential blood pressure recordings and
renal functions at different ages during the lifespan of the GDNF
mice are required to document the time course and accurately
determine the degree of the elevation in arterial pressure and
changes in glomerular structure and renal function.
A number of experimental models of reduced nephron
number have been previously used to study the association
between nephron number and blood pressure, each with
significant limitations. Rats born to pregnant dams fed a low
protein diet do show reduced nephron number; however, they
have other major phenotype differences from control mice
such as reduced body weight, which confounds simple
interpretation of the results.26 –28 With 5/6 nephrectomy, the
extent of nephron reduction is much more severe than would
be expected in the natural genetic variation in humans. Mice
overexpressing human insulin-like growth factor also have
reduced nephron endowment with glomerular numbers 20%
lower than control mice,29 but at this time there have been no
reports on renal function or blood pressure of these mice.
GDNF mice have the advantage that their body weights are
similar to their wild-type control mice from birth; indeed the
control mice have the added advantage of being littermates,
and the gene manipulated has a specific role in nephrogenesis
itself. GDNF has been shown to be a potent survival factor for
a variety of neuronal populations in vitro and in vivo, and
some of these populations are reduced in the GDNF null
mutant mice.12,13 However, apart from a reduction in the
number of A␤-caliber sensory nerve endings in the adult but
not neonatal whisker follicle30 and an impairment in learning
the position of a hidden platform in a water maze task,24 the
GDNF heterozygous mouse appears to have normalfunctioning dopaminergic, noradrenergic, and motor systems.12,13,24 Recently, Shen et al31 reported hypoganglionosis
of the gastrointestinal tract of GDNF heterozygous mice with
up to 1 in 5 GDNF heterozygous mice dying before weaning
because of complications resulting from enteric aganglionosis. It is important to note that GDNF heterozygous mice with
hypoganglionosis were asymptomatic and thus, given the
similar body weights and fecal pellets of GDNF heterozygous
and wild-type littermates in the current study, it appears
unlikely that enteric hypoganglionosis contributed to the
Cullen-McEwen et al
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higher blood pressure seen in these 14-month-old GDNF
heterozygous mice. Of course, one cannot exclude the possibility that undetected phenotypes might also affect the cardiovascular outcomes in these mice.
Interestingly, an acquired nephron deficit after surgical
reduction of renal mass does not always lead to hypertension,
even when the reduction is greater than the 30% congenital
loss demonstrated in GDNF heterozygous mice. A 50%
reduction in glomerular number after unilateral nephrectomy
in rats does not lead to the development of hypertension
unless the nephrectomy is performed just after birth.32,33 In
humans, adult unilateral nephrectomy does not lead to increased prevalence of hypertension in kidney donors unless
these donors have underlying conditions such as obesity or
diabetes.34,35 However, there does appear to be an increased
risk of hypertension for patients who had a kidney removed
as children because of Wilms tumor.36,37 Even more dramatic
losses in renal mass such as those seen with surgical fivesixths nephrectomy does not always lead to hypertension in
rats unless generated by unilateral nephrectomy plus infarction of two thirds of the other kidney.38 Of course, with these
surgical models of renal and glomerular deficit, there is an
acute and quite dramatic physiological reaction to the loss of
renal mass involving the renal sympathetic nervous system
and various hormonal systems leading to immediate (minutes) doubling of sodium and potassium excretion and followed by (hours-days) marked elevations in GFR, renal blood
flow, and cardiac output, falls in renal vascular resistance,
and compensatory growth of the remaining renal tissue in the
following days to weeks.38 – 43 Such dramatic changes cannot
be compared with the situation of inherent nephron deficit in
which, one could argue, there is no acute physiological
reaction but rather a slow adaptive response to the growing
needs of the animal.
In summary, mice heterozygous for the GDNF gene that
have 30% fewer nephrons than wild types show elevated
arterial pressure, normal GFR, and thus hyperfiltration in old
age. Unlike the situation at 1 month, glomeruli of 14-monthold GDNF heterozygous mice are hypertrophied; however,
this occurs without evidence of glomerular pathology. Thus,
the results found in this low nephron-number mouse, uncomplicated by changes in body weight, are in accord with the
hypothesis of Brenner et al2 that a reduction in nephron
number from birth leads to the development of hypertension
and hyperfiltration.
Perspectives
Several animal models exist to examine the link between
acquired reductions in nephron number and the subsequent
development of cardiovascular and renal diseases; however,
there are few animal models of congenital nephron deficit. Our
current results suggest that the GDNF heterozygous mouse, with
an inherent 30% reduction in nephron number, may provide a
useful animal model to study the role of nephron endowment in
the pathogenesis of essential hypertension.
Acknowledgments
This work was supported by the Australian Research Council. Luise
Cullen-McEwen was funded by a Biomedical Research Scholarship
Renal Function in Aged GDNF Heterozygous Mice
339
from the Australian Kidney Foundation. Michelle Kett was funded
by a National Health and Medical Research Council Project Grant
(124404). The authors would like to acknowledge the assistance of
Katrina Worthy.
References
1. Mackenzie HS, Lawler EV, Brenner BM. Congenital oligonephropathy:
the fetal flaw in essential hypertension? Kidney Int Suppl. 1996;55:
S30 –S34.
2. Brenner BM, Garcia DL, Anderson S. Glomeruli and blood pressure: less
of one, more the other? Am J Hypertens. 1988;1:335–347.
3. Brenner BM, Milford EL. Nephron underdosing: a programmed cause of
chronic renal allograft failure. Am J Kidney Dis. 1993;21:66 –72.
4. Brenner BM, Chertow GM. Congenital oligonephropathy and the
etiology of adult hypertension and progressive renal injury. Am J Kidney
Dis. 1994;23:171–175.
5. Sainio K, Suvanto P, Davies J, Wartiovaara J, Wartiovaara K, Saarma M,
Arumae U, Meng X, Lindahl M, Pachnis V, Sariola H. Glial-cell-linederived neurotrophic factor is required for bud initiation from ureteric
epithelium. Development. 1997;124:4077– 4087.
6. Pepicelli CV, Kispert A, Rowitch DH, McMahon AP. GDNF induces
branching and increased cell proliferation in the ureter of the mouse. Dev
Biol. 1997;192:193–198.
7. Towers PR, Woolf AS, Hardman P. Glial cell line-derived neurotrophic
factor stimulates ureteric bud outgrowth and enhances survival of ureteric
bud cells in vitro. Exp Nephrol. 1998;6:337–351.
8. Treanor JJ, Goodman L, de Sauvage F, Stone DM, Poulsen KT, Beck CD,
Gray C, Armanini MP, Pollock RA, Hefti F, Phillips HS, Goddard A,
Moore MW, Buj-Bello A, Davies AM, Asai N, Takahashi M, Vandlen R,
Henderson CE, Rosenthal A. Characterization of a multicomponent
receptor for GDNF. Nature. 1996;382:80 – 83.
9. Vega QC, Worby CA, Lechner MS, Dixon JE, Dressler GR. Glial cell
line-derived neurotrophic factor activates the receptor tyrosine kinase
RET and promotes kidney morphogenesis. Proc Natl Acad Sci U S A.
1996 93;10657–10661.
10. Pichel JG, Shen L, Sheng HZ, Granholm AC, Drago J, Grinberg A, Lee
EJ, Huang SP, Saarma M, Hoffer BJ, Sariola H, Westphal H. Defects in
enteric innervation and kidney development in mice lacking GDNF.
Nature. 1996;382:73–76.
11. Pichel JG, Shen L, Sheng HZ, Granholm AC, Drago J, Grinberg A, Lee
EJ, Huang SP, Saarma M, Hoffer BJ, Sariola H, Westphal H. GDNF is
required for kidney development and enteric innervation. Cold Spring
Harb Symp Quant Biol. 1996;61:445– 457.
12. Moore MW, Klein RD, Farinas I, Sauer H, Armanini M, Phillips H,
Reichardt LF, Ryan AM, Carver-Moore K, Rosenthal A. Renal and
neuronal abnormalities in mice lacking GDNF. Nature. 1996;382:76 –79.
13. Sanchez MP, Silos-Santiago I, Frisen J, He B, Lira SA, Barbacid M.
Renal agenesis and the absence of enteric neurons in mice lacking GDNF.
Nature. 1996;382:70 –73.
14. Schuchardt A, D’Agati V, Larsson-Blomberg L, Costantini F, Pachnis V.
Defects in the kidney and enteric nervous system of mice lacking the
tyrosine kinase receptor Ret. Nature. 1994;367:380 –383.
15. Enomoto H, Araki T, Jackman A, Heuckeroth RO, Snider WD, Johnson
EM Jr, Milbrandt J. GFR alpha1-deficient mice have deficits in the
enteric nervous system and kidneys. Neuron. 1998;21:317–324.
16. Cacalano G, Farinas I, Wang LC, Hagler K, Forgie A, Moore M,
Armanini M, Phillips H, Ryan AM, Reichardt LF, Hynes M, Davies A,
Rosenthal A. GFRalpha1 is an essential receptor component for GDNF in
the developing nervous system and kidney. Neuron. 1998;21:53– 62.
17. Cullen-McEwen LA, Drago J, Bertram JF. Nephron endowment in glial
cell line-derived neurotrophic factor (GDNF) heterozygous mice. Kidney
Int. 2001;60:31–36.
18. Bradford MM. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding. Anal Biochem. 1976;72:248 –254.
19. Pakkenberg B, Gundersen HJ. Total number of neurons and glial cells in
human brain nuclei estimated by the dissector and the fractionator. J
Microsc. 1988;150:1–20.
20. Kett MM, Alcorn D, Bertram JF, Anderson WP. Glomerular dimensions
in spontaneously hypertensive rats: effects of AT1 antagonism.
J Hypertens. 1996;14:107–113.
21. Bertram JF. Analyzing renal glomeruli with the new stereology. Int Rev
Cytol. 1995;161:111–172.
340
Hypertension
February 2003
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
22. Nyengaard JR, Bendtsen TF. Glomerular number and size in relation to
age, kidney weight, and body surface in normal man. Anat Rec. 1992;
232:194 –201.
23. Hostetter TH, Olson JL, Rennke HG, Venkatachalam MA, Brenner BM.
Hyperfiltration in remnant nephrons: a potentially adverse response to
renal ablation. J Am Soc Nephrol. 2001;12:1315–1325.
24. Gerlai R, McNamara A, Choi-Lundberg DL, Armanini M, Ross J,
Powell-Braxton L, Phillips HS. Impaired water maze learning performance without altered dopaminergic function in mice heterozygous for
the GDNF mutation. Eur J Neurosci. 2001;14:1153–1163.
25. Lorenz JN. A practical guide to evaluating cardiovascular, renal, and
pulmonary function in mice. Am J Physiol. 2002;282:R1565–R1582.
26. Woods LL, Ingelfinger JR, Nyengaard JR, Rasch R. Maternal protein
restriction suppresses the newborn renin-angiotensin system and
programs adult hypertension in rats. Pediatr Res. 2001;49:460 – 467.
27. Moore VM, Cockington RA, Ryan P, Robinson JS. The relationship
between birth weight and blood pressure amplifies from childhood to
adulthood. J Hypertens. 1999;17:883– 888.
28. Zimanyi MA, Bertram JF, Black MJ. Nephron number in offspring of rats
fed a low protein diet during pregnancy. Image Anal Stereol. 2000;19:
219 –222.
29. Doublier S, Amri K, Seurin D, Moreau E, Merlet-Benichou C, Striker
GE, Gilbert T. Overexpression of human insulin-like growth factor
binding protein-1 in the mouse leads to nephron deficit. Pediatr Res.
2001;49:660 – 666.
30. Fundin BT, Mikaels A, Westphal H, Ernfors P. A rapid and dynamic
regulation of GDNF-family ligands and receptors correlate with the
developmental dependency of cutaneous sensory innervation. Development. 1999;126:2597–2610.
31. Shen L, Pichel JG, Mayeli T, Sariola H, Lu B, Westphal H. GDNF
haploinsufficiency causes Hirschsprung-like intestinal obstruction and
early-onset lethality in mice. Am J Hum Genet. 2002;70:435– 447.
32. Woods LL, Weeks DA, Rasch R. Hypertension after neonatal uninephrectomy in rats precedes glomerular damage. Hypertension. 2001;38:
337–342.
33. Woods LL. Neonatal uninephrectomy causes hypertension in adult rats.
Am J Physiol. 1999;276:R974 –R978.
34. Praga M, Hernandez E, Herrero JC, Morales E, Revilla Y, Diaz-Gonzalez
R, Rodicio JL. Influence of obesity on the appearance of proteinuria and
renal insufficiency after unilateral nephrectomy. Kidney Int. 2000;58:
2111–2118.
35. Toronyi E, Alfoldy F, Jaray J, Remport A, Hidvegi M, Dabasi G, Telkes
G, Offenbacher E, Perner F. Evaluation of the state of health of living
related kidney transplantation donors. Transpl Int. 1998;11(suppl
1):S57–S59.
36. Barrera M, Roy LP, Stevens M. Long-term follow-up after unilateral
nephrectomy and radiotherapy for Wilms’ tumour. Pediatr Nephrol.
1989;3:430 – 432.
37. Makipernaa A, Koskimies O, Jaaskelainen J, Teppo AM, Siimes MA.
Renal growth and function 11–28 years after treatment of Wilms’ tumour.
Eur J Pediatr. 1991;150:444 – 447.
38. Griffin KA, Picken M, Bidani AK. Method of renal mass reduction is a
critical modulator of subsequent hypertension and glomerular injury.
J Am Soc Nephrol. 1994;4:2023–2031.
39. Valentin JP. Plasma concentration of atrial natriuretic peptide after acute
reduction in functioning renal mass in the rat. Can J Physiol Pharmacol.
1997;75:153–157.
40. Lopez-Novoa JM, Ramos B, Martin-Oar JE, Hernando L. Functional
compensatory changes after unilateral nephrectomy in rats: general and
intrarenal hemodynamic alterations. Ren Physiol. 1982;5:76 – 84.
41. Shirley DG, Walter SJ. Acute and chronic changes in renal function
following unilateral nephrectomy. Kidney Int. 1991;40:62– 68.
42. Valentin JP, Ribstein J, Mimran A. Influence of dopamine and angiotensin II blockade on the acute response to unilateral nephrectomy in rats.
J Cardiovasc Pharmacol. 1994;23:246 –251.
43. Furukawa K, Ninomiya I, Shimizu J, Wada T, Matsuura Y. Renal sympathetic nerve activity and the weight of the remaining kidney in unilateral nephrectomized rats. J Auton Nerv Syst. 1997;63:91–100.
Nephron Number, Renal Function, and Arterial Pressure in Aged GDNF Heterozygous
Mice
Luise A. Cullen-McEwen, Michelle M. Kett, John Dowling, Warwick P. Anderson and John F.
Bertram
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Hypertension. 2003;41:335-340; originally published online January 13, 2003;
doi: 10.1161/01.HYP.0000050961.70182.56
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