Age-related changes in the renal dopaminergic system and expression of renal amino acid transporters in WKY and SHR rats.

Abstract

This study examined age-related changes in renal dopaminergic activity and expression of amino acid transporters potentially involved in renal tubular uptake of l-DOPA in Wistar Kyoto (WKY) and spontaneously hypertensive rats. Aging (from 13 to 91 weeks) was accompanied by increases in systolic blood pressure (SBP) in both WKY and SHR. The sum of urinary dopamine and DOPAC and the urinary dopamine/l-DOPA ratio were increased in aged SHR but not in aged WKY. The urinary dopamine/renal delivery of l-DOPA ratio was increased in both rat strains with aging. LAT2 abundance was increased in aged WKY and SHR. The expression of 4F2hc was markedly elevated in aged SHR but not in aged WKY. ASCT2 was upregulated in both aged WKY and SHR. Plasma aldosterone levels and urinary noradrenaline levels were increased in aged WKY and SHR though levels of both entities were more elevated in aged SHR. Activation of the renal dopaminergic system is more pronounced in aged SHR than in aged WKY and is associated with an upregulation of renal cortical ASCT2 in WKY and of LAT2/4F2hc and ASCT2 in SHR. This activation may be the consequence of a counter-regulatory mechanism for stimuli leading to sodium reabsorption.

Full text

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Age-related changes in the renal dopaminergic system and expression of renal
amino acid transporters in WKY and SHR rats
Vanda Pinto, Joa ˜o Amaral, Elisabete Silva, So ´nia Sima ˜o, Jose ´ Miguel Cabral, Joana Afonso,
Maria Paula Serra ˜o, Pedro Gomes1, Maria Joa ˜o Pinho, Patrı ´cio Soares-da-Silva*
Institute of Pharmacology & Therapeutics, Faulty of Medicine, University of Porto, 4200-319 Porto, Portugal
1. Introduction
Dopamine is a major regulator of mammalian proximal tubule
salt and water reabsorption. In the mammalian kidney, dopamine
is primarily produced in the proximal tubule (Aperia, 2000; Gomes
and Soares-da-Silva, 2008; Soares-da-Silva and Vieira-Coelho,
1998). The dopamine precursor
DOPA) is filtered at the glomerulus and is taken up by the
proximal tubule via luminal transporters and converted to
dopamine by aromatic L-amino acid decarboxylase (AADC), which
is highly expressed in the proximal tubule (Soares-da-Silva and
Fernandes, 1991). The regulation of this non-neuronal dopaminer-
gic system depends mainly on the availability of L-DOPA, on its
L-dihydroxyphenylalanine (L-
decarboxylation into dopamine and on cell outward amine transfer
mechanisms (Pestana and Soares-da-Silva, 1994; Soares-da-Silva
and Fernandes, 1991). In the kidney, dopamine is metabolized
predominantly by catechol-O-methyl-transferase (COMT) and
monoamine oxidase to 3,4-dihydroxyphenylacetic acid (DOPAC),
and to homovallinic acid (HVA) (Pestana and Soares-da-Silva,
1994; Soares-da-Silva and Fernandes, 1991). A considerable
amount of evidence favours the view that dopamine of renal
origin plays a role in the regulation of central blood volume by
reducing the tubular reabsorption of sodium as a paracrine or
autocrine substance (Jose et al., 2003). The mechanisms through
which renal dopamine is thought to produce natriuresis involve
the activation of D1-like receptors that inhibit the activity of both
apical (e.g., Na/H exchange and chloride–bicarbonate exchange
and Na–P cotransport) and basolateral (Na–K-ATPase and NaHCO3
cotransport) transporters (Aperia et al., 1987; Felder et al., 1990;
Jose et al., 1992; Lokhandwala and Amenta, 1991). The availability
of dopamine to activate its specific receptors is determined by
factors affecting renal synthesis, mainly the amounts of L-DOPA
and sodium delivered to the kidney and the degree of degradation
of the amine (Soares-da-Silva et al., 1993).
The spontaneously hypertensive rat (SHR) is a genetic model of
hypertension characterized by the resistance to the natriuretic
effect of dopamine and D1-like receptor agonists, as a result of a
defective transduction of the D1receptor signal in renal proximal
Mechanisms of Ageing and Development 132 (2011) 298–304
A R T I C L E
I N F O
Article history:
Received 25 February 2011
Received in revised form 1 May 2011
Accepted 6 June 2011
Available online 14 June 2011
Keywords:
Aging
Hypertension
Renal dopaminergic system
Amino acid transporters
Neurohumoral activity
A B S T R A C T
This study examined age-related changes in renal dopaminergic activity and expression of amino acid
transporters potentially involved in renal tubular uptake of L-DOPA in Wistar Kyoto (WKY) and
spontaneously hypertensive rats. Aging (from 13 to 91 weeks) was accompanied by increases in systolic
blood pressure (SBP) in both WKY and SHR. The sum of urinary dopamine and DOPAC and the urinary
dopamine/L-DOPA ratio were increased in aged SHR but not in aged WKY. The urinary dopamine/renal
delivery of L-DOPA ratio was increased in both rat strains with aging. LAT2 abundance was increased in
aged WKY and SHR. The expression of 4F2hc was markedly elevated in aged SHR but not in aged WKY.
ASCT2 was upregulated in both aged WKY and SHR. Plasma aldosterone levels and urinary noradrenaline
levels were increased in aged WKY and SHR though levels of both entities were more elevated in aged
SHR. Activation of the renal dopaminergic system is more pronounced in aged SHR than in aged WKY and
is associated with an upregulation of renal cortical ASCT2 in WKY and of LAT2/4F2hc and ASCT2 in SHR.
This activation may be the consequence of a counter-regulatory mechanism for stimuli leading to sodium
reabsorption.
? 2011 Elsevier Ireland Ltd. All rights reserved.
Abbreviations: 4F2hc, 4F2 heavy chain; ANOVA, one-way analysis of variance;
COMT, catechol-O-methyl-transferase; Ccr, creatinine clearance; DOPAC, 3,4-
dihydroxyphenylacetic acid; FENa+, fractional excretion of Na+; GAPDH, glyceralde-
hyde-3-phosphate dehydrogenase; GFR, glomerular filtration rate; L-DOPA, L-
dihydroxyphenylalanine; LAT1, L-type amino acid transporter 1; LAT2, L-type amino
acid transporter 2; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel
electrophoresis; SEM, standard error of the mean; SHR, spontaneously hypertensive
rat; WKY, Wistar Kyoto rat.
* Corresponding author. Tel.: +351 22 5513642; fax: +351 22 5513643.
E-mail address: pss@med.up.pt (P. Soares-da-Silva).
1Present address: Department of Biochemistry, Faculty of Medicine, University
of Porto, 4200-319 Porto, Portugal.
Contents lists available at ScienceDirect
Mechanisms of Ageing and Development
jo ur n al ho mep ag e: www .elsevier .c om /lo cate/m ec hag ed ev
0047-6374/$ – see front matter ? 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.mad.2011.06.003

Page 2

tubules (Jose et al., 2010; Sanada et al., 1999; Zeng and Jose, 2011).
It has been suggested that increased oxidative stress in renal
proximal tubules of the SHR could be a mechanism for defective
dopamine D1 receptor/G-protein coupling (White and Sidhu,
1998). Moreover, recent studies have shown the overexpression
of Na+-independent and pH-sensitive amino acid transporter LAT2
(Slc7a8) in the SHR kidney, which might contribute to enhanced L-
DOPA uptake in the proximal tubule and increased dopamine
production (Pinho et al., 2004), as an attempt to overcome the
defect in D1receptor function.
The aging kidney undergoes structural changes that result in
quantitative alterations in some renal functions, such as a decline
in renal blood flow and glomerular filtration rate (Zhou et al.,
2008). An increasing number of studies have shown that old
animals may present particular deficiencies in the renal handling
of L-DOPA, its subsequent conversion to dopamine (Armando et al.,
1995; Soares-da-Silva and Fernandes, 1991) and at the level of
receptor number or coupling to G proteins (Kansra et al., 1997). In
the presence of age-related diseases, such as heart failure and
hypertension, these changes can be aggravated (Fischer and
O’Hare, 2010).
This study was aimed at evaluating age-related changes in the
activity of the renal dopaminergic system and the regulation of the
amino acid transporters that are potentially involved in the uptake
of L-DOPA: Na+-independent LAT1 and LAT2 and Na+-dependent
ASCT2 in SHR and their normotensive Wistar Kyoto (WKY)
counterparts.
2. Materials and methods
2.1. Animal preparation and experimental design
Five-week old male WKY and SHR were obtained from Harlan-Interfauna Ibe ´rica
(Barcelona, Spain). The rats were housed under controlled conditions (12 h light/
dark cycle and room temperature at 22 ? 2 8C) and had free access to tap water and
standard rat chow (PANLAB, Barcelona, Spain). The animals were carefully maintained
and monitored until 13 or 91 weeks of age. Blood pressure (systolic and diastolic) and
heart rate were measured in conscious animals using a photoelectric tail-cuff detector
(LE 5000, Letica, Barcelona, Spain). A minimum of 5 measures were made each time and
the mean values were used for further calculations. All rat interventions were
performed in accordance with the European Directive number 86/609, and the rules of
the ‘‘Guide for the Care and Use of Laboratory Animals’’, 7th edition, 1996, Institute for
Laboratory Animal Research (ILAR), Washington, DC.
2.2. Metabolic study
Forty eight hours before experiments, 13- and 91-week old rats were placed in
metabolic cages (Tecniplast, Buguggiate, Italy) for a 24 h urine collection. The urine
samples were collected in vials containing 1 ml of 6 M HCl to prevent spontaneous
decomposition of monoamines and amine metabolites. After completion of this
protocol, rats were anesthetized with sodium pentobarbital (60 mg/kg, i.p.). The
animals were then sacrificed by exsanguination using cardiac puncture and the
blood collected into tubes containing K3EDTA for later determination of plasma
biochemical parameters. Before excising the kidneys, a cannula was inserted in the
right ventricle of the heart and animals were perfused with ice-cold saline (0.9%
NaCl) to remove all blood from the kidneys. The kidneys were then excised,
weighed, decapsulated, and the renal cortex and medulla rapidly separated by fine
dissection. Tissue pieces were immediately frozen in liquid nitrogen and stored at
?80 8C for Western blot analysis.
2.3. Plasma and urine biochemistry
The quantification of sodium and potassium in plasma and urine was performed
by ion-selective electrodes. Creatinine was measured by the Jaffe ´ method (Chromy
et al., 2008). All assays were performed by Cobas Mira Plus analyzer (ABX
Diagnostics, Switzerland). Creatinine clearance (in ml/min) was calculated using
the formula Ccr= (Ucreat? Vu)/(Pcreat? 24 h ? 60) where Ucreat and Vu are the
urinary creatinine concentration and urinary 24 h volume and Pcreatis the plasma
creatinine concentration. Aldosterone in plasma samples was performed by
radioimmuno assay (Diagnostic Products Corporation; Los Angeles, CA).
2.4. Assay of catecholamines
The assay of catecholamines in urine (L-DOPA, dopamine, DOPAC and
norepinephrine) and plasma samples (L-DOPA, dopamine and DOPAC) was
performed by HPLC with electrochemical detection, as previously described
(Soares-da-Silva et al., 1994, 1993). The lower limit of detection of L-DOPA,
dopamine, norepinephrine, and DOPAC ranged from 350 to 1000 fmol.
2.5. Western blotting
Renal cortices from 13- and 91-week old WKY and SHR, were lysed in RIPA
buffer containing 150 mM NaCl, 50 mM Tris–HCl, pH 7.4, 5 mM EDTA, 1% Triton
X-100, 0.5% sodium deoxycholate, 0.1% SDS, 100 mg/ml PMSF, 2 mg/ml leupeptin
and 2 mg/ml aprotinin. Protein concentration was determined using a protein
assay kit (Bio-Rad Laboratories, Hercules, CA), with bovine serum albumin as
standard. Lysates were boiled in sample buffer (35 mM Tris–HCl, pH 6.8, 4% SDS,
9.3% dithiothreitol, 0.01% bromophenol blue, 30% glycerol) at 95 8C for 5 min.
Samples containing 50–75 mg of protein, were separated by SDS-PAGE with 10%
polyacrylamide gel and then electroblotted onto nitrocellulose membranes (Bio-
Rad). Blots were blocked for 1 h with 5% non-fat dry milk in PBS (10 mmol/l
phosphate-buffered saline) at room temperature with constant shaking. Blots
were then incubated with the antibodies rabbit polyclonal anti-LAT1 (1:500;
Serotec); goat polyclonal anti-LAT2 (1:500; Santa Cruz Biotechnology); rabbit
polyclonal anti-4F2hc (1:500; Santa Cruz Biotechnology); rabbit polyclonal anti-
ASCT2 (1:500; Chemicon International); mouse monoclonal anti-b-actin
(1:20,000; Santa Cruz Biotechnology) or mouse monoclonal anti-GAPDH
(1:60,000; Santa Cruz Biotechnology) in 5% non-fat dry milk in PBS-T overnight
at 4 8C. The immunoblots were subsequently washed and incubated with
fluorescently labeled goat anti-rabbit (1:20,000; IRDyeTM 800, Rockland);
fluorescently labeled donkey anti-goat (1:10,000; IRDyeTM800, Rockland); or
the fluorescently labeled goat anti-mouse secondary antibody (1:20,000;
AlexaFluor 680, Molecular Probes) for 60 min at room temperature and
protected from light. The membrane was washed and imaged by scanning at
both 700 and 800 nm, with an Odyssey Infrared Imaging System (LI-COR
Biosciences).
2.6. Drugs
All chemicals were obtained from Sigma (St. Louis, MO) unless otherwise stated.
2.7. Data analysis
Arithmetic means are given with standard error of the mean (SEM). Statistical
analysis was performed by one-way analysis of variance (ANOVA) followed by
Newman–Keuls test. A P value less than 0.05 was assumed to denote a significant
difference.
Table 1
Cardiovascular and physiological parameters in 13- and 91-week old WKY and SHR.
Parameter
WKY
SHR
13 weeks
n = 6
91 weeks
n = 6
13 weeks
n = 6
91 weeks
n = 6
Systolic blood pressure (mm Hg)
Diastolic blood pressure (mm Hg)
Pulse pressure (mm Hg)
Creatinine clearance (ml/min)
Urinary protein excretion (mg/24 h)
FENa+(%)
Kidney weight/tibia length (% of control)
122 ? 6
97 ? 3
25 ? 2
2.64 ? 0.26
14.01 ? 0.80
0.38 ? 0.04
100 ? 2
148 ? 3*
88 ? 3
60 ? 1*
3.9 ? 0.23*
14.77 ? 1.21
0.23 ? 0.01*
114 ? 1*
191 ? 2#
165 ? 2#
26 ? 2
1.85 ? 0.15#
26.02 ? 1.60#
0.37 ? 0.04
100 ? 1
224 ? 5*,#
132 ? 3*,#
92 ? 7*,#
2.05 ? 0.18#
41.06 ? 3.38*,#
0.23 ? 0.04*
122 ? 2*,#
*Significantly different from corresponding values in 13-week old animals (P < 0.05).
#Significantly different from age-matched WKY (P < 0.05).
V. Pinto et al. / Mechanisms of Ageing and Development 132 (2011) 298–304
299

Page 3

3. Results
3.1. Blood pressure data and renal function
As expected, the systolic and diastolic blood pressures (SBP and
DBP) determined by the tail-cuff method were significantly higher
in both 13- and 91-week old SHR than in age-matched WKY (Table
1). Moreover, aging was accompanied by increases in SBP in both
WKY and SHR. DBP remained unaltered in aged WKY, but a
significant decrease was observed in aged versus young SHR (Table
1). No difference in pulse pressure (defined as SBP minus DBP) was
found between young WKY and SHR. Pulse pressure increased with
age in both WKY and SHR but at 91 weeks of age SHR had higher
pulse pressure than age-matched WKY (Table 1). Creatinine
clearance (Ccr) levels were decreased in SHR in comparison to
age-matched WKY (Table 1). Urinary protein excretion was
significantly higher in SHR than in age-matched WKY at 13 and
91 weeks of age and increased significantly with age (Table 1).
Moreover, evaluation of FENa+ in WKY and SHR showed a
significant decrease in this parameter at the age of 91 weeks in
both rat strains (Table 1). Kidney/tibia length ratios were assessed
for WKY and SHR (Table 1). Aging was associated with increases in
kidney/tibia length ratio in both WKY and SHR. However, increases
in kidney size were more marked in SHR (Table 1).
3.2. Activity of the renal dopaminergic system
In the present study, the urinary excretion of dopamine and its
metabolite DOPAC was evaluated in 13- and 91-week old WKY and
SHR (Fig. 1). No changes were found in urinary L-DOPA indexed to
urinary creatinine in aged WKY. However, L-DOPA excretion was
decreased in aged SHR, though the difference did not reach
statistical significance (Fig. 1A). Aging was accompanied by slight
increases in urinary dopamine in WKY and SHR, though not
statistically significant. However, urinary dopamine in 91-week
old SHR was higher than that in age-matched WKY (Fig. 1B).
Urinary DOPAC was significantly increased in aged SHR but not in
aged WKY rats (Fig. 1C). More complete information on the L-DOPA
renal turnover is obtained when the sum of urinary dopamine and
DOPAC is considered. The sum of urinary dopamine and DOPAC
was found to be increased in aged SHR but not in aged WKY
(Fig. 1D). Furthermore, urinary dopamine + DOPAC was markedly
increased in aged SHR in comparison to age-matched WKY
(Fig. 1D).
The enhanced urinary excretion of dopamine and DOPAC in the
SHR may reflect their enhanced ability to synthesize dopamine.
The urinary dopamine/L-DOPA ratio (a measure of renal L-DOPA
utilization and of renal dopamine-synthesis efficiency) in 91-week
old SHR was markedly higher than in young SHR (Fig. 2A). The
dopamine/L-DOPA ratio was also greater in 91-week old SHR than
in age-matched WKY (Fig. 2A). No differences were detected with
aging in the WKY (Fig. 2A). On the other hand, the ratio between
urinary dopamine and the renal delivery of L-DOPA (another index
of renal dopamine production) was greater in aged WKY and SHR
than in young animals (Fig. 2B). However, the dopamine/renal
delivery of L-DOPA ratio was significantly increased in aged SHR
when compared to age-matched WKY (Fig. 2B). The renal delivery
of L-DOPA, which considers L-DOPA plasma levels and creatinine
clearance (plasma L-DOPA ? creatinine clearance), decreased with
age in WKY and SHR, though the difference did not reach statistical
significance in WKY (Fig. 2C). Moreover, the renal delivery of L-
DOPA in 91-week old SHR was significantly lower than in age-
matched WKY (Fig. 2C). As depicted in Table 2, aging was
accompanied by decreases in plasma levels of L-DOPA, dopamine
and DOPAC in both WKY and SHR.
3.3. Renal expression of LAT1, LAT2, 4F2hc and ASCT2
Age-related changes in the amino acid transporters that are
potentially involved in the uptake of L-DOPA were evaluated in the
renal cortex of 13- and 91-week old WKY and SHR. As depicted in
Fig. 3A LAT1 expression levels were downregulated in 91-week old
WKY and SHR rats when compared to young animals. On the other
hand, LAT2 abundance was significantly upregulated in 91-week
old WKY and SHR, as compared to young animals (Fig. 3B). Aging
had no effect on 4F2hc protein abundance in WKY, whereas 4F2hc
WKYWKYSHRSHR
00
1010
2020
3030
*
*
13 week
91 week
s
s
#
#
Urinary L-DOPA
(nmol/mg creatinine)
WKY
SHR
SHR
0.0
0.0
0.5
0.5
1.0
1.0
1.5
1.5
2.0
2.0
2.5
2.5
#
#
#
#
13 weeks
91 weeks
91 weeks
Urinary dopamine
(nmol/mg creatinine)
WKY
SHR
SHR
0
0
2
2
4
4
6
6
*#*#
13 week
91 week
s
s
s
##
Urinary DOPAC
(nmol/mg creatinine)
WKYWKYSHRSHR
00
22
44
66
88
13 weeks
91 weeks91 weeks
*#*#
##
Urinary dopamine+DOPAC
(nmol/mg creatinine)
AA
BB
CC
DD
13 wee ks
91 wee ks
Urinary L-DOPA
(nmol/mg creatinine)
WKY
13 weeks
Urinary dopamine
(nmol/mg creatinine)
WKY
13 week
91 week
s
Urinary DOPAC
(nmol/mg creatinine)
13 weeks
Urinary dopamine+DOPAC
(nmol/mg creatinine)
Fig. 1. Urinary excretion of L-DOPA (A), dopamine (B), DOPAC (C), and sum of urinary dopamine and DOPAC (D) indexed to urinary creatinine in 13- and 91-week old WKY and
SHR. Each bar represents the mean ? SEM of 6 rats. Significantly different from corresponding values in 13-week old animals (*P < 0.05) and significantly different from age-
matched WKY (#P < 0.05) using the Newman–Keuls test.
V. Pinto et al. / Mechanisms of Ageing and Development 132 (2011) 298–304
300

Page 4

expression levels were significantly increased at 91 weeks of age in
SHR when compared to 13-week old SHR (Fig. 3C). ASCT2 was
increased at 91 weeks of age in both WKY and SHR when compared
with 13-week old animals (Fig. 3D).
3.4. Neurohumoral parameters
Aging was accompanied by increases in plasma aldosterone
levels in both WKY and SHR. However, at 91 weeks of age SHR had
increased circulating aldosterone levels when compared to age-
matched WKY (Fig. 4A). Plasma aldosterone levels were accompa-
nied by decreases in the UNa+/K+ratio in both WKY and SHR
though the mineralocorticoid response to endogenous aldosterone
was markedly greater in 91-week old SHR than in age-matched
WKY rats (Fig. 4B). Similar to plasma aldosterone levels, urinary
excretion of noradrenaline was also greater in aged WKY and SHR
than in young animals, but levels in 91-week old SHR were higher
than in age-matched WKY rats (Fig. 4C).
4. Discussion
The present study evaluated age-related changes in dopami-
nergic activity and amino acid transporter expression in WKY and
SHR. The results indicate that aged SHR have increased renal
cortical LAT2/4F2hc and ASCT2 abundance and increased efficien-
cy in the formation of renal dopamine. Moreover, activation of the
renal dopaminergic system is accompanied by an increase in the
activity of the sympathetic and renin–angiotensin–aldosterone
systems.
There are conflicting results concerning the effect of age on
renal dopamine production. Although several studies have shown
an association between age and deficiencies in the renal handling
of L-DOPA (Armando et al., 1995; Kansra et al., 1997; Soares-da-
Silva and Fernandes, 1991; Vieira-Coelho et al., 1999), other
authors have reported no alterations in renal dopamine production
with aging (Komori et al., 1997; Lehmann et al., 1985; Nicolau
et al., 1985). In the present study, no significant changes with aging
were found in urinary excretion of L-DOPA, dopamine and DOPAC
or in the urinary dopamine/L-DOPA ratio in WKY. However, results
indicated that young WKY had increased renal delivery of L-DOPA
when compared to aged rats though the urinary excretion of L-
DOPA was the same. This may have led to the accumulation of
plasma levels of
L-DOPA in young WKY. Since the urinary
dopamine/L-DOPA ratio only takes into account levels of urinary
dopamine and L-DOPA no differences were found between young
and aged WKY, regarding the ability to form dopamine. However, a
significant increase in the dopamine/renal delivery of L-DOPA ratio
in aged WKY indicates that the ability to produce dopamine may be
increased in aged WKY.
In comparison to aged WKY, aged SHR had increased urinary
excretion of dopamine and DOPAC. Additionally, urinary dopa-
mine/L-DOPA and urinary dopamine/renal delivery of L-DOPA
ratios were increased in aged SHR, indicating that aged SHR may
have an enhanced ability to produce dopamine than aged WKY.
Previous reports by other authors have shown that dopamine
production and excretion in the SHR were normal or increased
when compared with those in WKY, though most studies were
conducted in young animals (Herlitz et al., 1982; Kuchel et al.,
1987; Racz et al., 1985; Yoshimura et al., 1990). Our group reported
that SHR maintained on a normal-salt diet had, at 4 weeks of age
but not at 12 weeks, overexpression of LAT2 and increased tubular
uptake of L-DOPA (Pinho et al., 2007). Accordingly, the enhanced
ability to take up
L-DOPA in the pre-hypertensive SHR was
suggested to take place as an attempt to overcome the deficient
dopamine-mediated natriuresis generally observed in this genetic
AAA
WKYWKYWKY SHRSHRSHR
0.00.00.0
0.20.20.2
0.40.40.4
0.6 0.6 0.6
0.80.80.8
*#*# *#
13 week
91 week91 week91 week


s
sss
###
Urinary dopamine/L-DOPA
(ratio)
BBB
WKY
SHRSHRSHR
0.00.00.0
0.10.10.1
0.20.20.2
0.30.30.3
0.40.4 0.4
0.50.50.5
*# *# *#
13 week
91 week91 week91 week


s
sss
Dopamine/
renal delivery of L-DOPA
(nmol/min)
***
CCC
WKY
SHRSHRSHR
000
555
101010
151515
*#*#*#
13 weeks
91 weeks91 weeks 91 weeks
Renal delivery of L-DOPA
(nmol/min)
13 week13 week

ss
Urinary dopamine/L-DOPA
(ratio)
Urinary dopamine/L-DOPA
(ratio)
WKY
13 week13 week

ss
Dopamine/
renal delivery of L-DOPA
(nmol/min)
WKY
Dopamine/
renal delivery of L-DOPA
(nmol/min)
WKY
13 weeks13 weeks
Renal delivery of L-DOPA
(nmol/min)
WKY
Renal delivery of L-DOPA
(nmol/min)
Fig. 2. Urinary dopamine/L-DOPA ratios (A), dopamine/renal delivery of L-DOPA
ratios (B) and renal delivery of L-DOPA (C) in 13- and 91-week old WKY and SHR.
Each bar represents the mean ? SEM of 6 rats. Significantly different from
corresponding values in 13-week old animals (*P <0.05) and significantly different
from age-matched WKY (#P < 0.05) using the Newman–Keuls test.
Table 2
Plasma levels of L-DOPA, dopamine and DOPAC in 13- and 91-week old WKY and SHR.
Parameter
WKY
SHR
13 weeks
n = 6
91 weeks
n = 6
13 weeks
n = 6
91 weeks
n = 6
L-DOPA (pmol/ml)
Dopamine (pmol/ml)
DOPAC (pmol/ml)
4.07 ? 0.31
10.67 ? 1.02
4.62 ? 0.58
2.00 ? 0.21*
0.56 ? 0.09*
1.75 ? 0.41*
5.33 ? 0.36#
15.44 ? 2.97
5.01 ? 0.60
2.89 ? 0.21*
0.42 ? 0.07*
2.90 ? 0.63*
*Significantly different from corresponding values in 13-week old animals (P < 0.05).
#Significantly different from age-matched WKY (P < 0.05).
V. Pinto et al. / Mechanisms of Ageing and Development 132 (2011) 298–304
301

Page 5

model of hypertension (Jose et al., 2002; Pinho et al., 2007).
Moreover, at 4 and 12 weeks of age no differences in the urinary
excretion of dopamine or DOPAC, or in plasma aldosterone levels
were found between age-matched WKY and SHR (Pinho et al.,
2007).
The renal cortical abundance of Na+-independent LAT1 and
LAT2, 4F2hc and Na+-dependent ASCT2, amino acid transporters
potentially involved in renal tubular uptake of
evaluated in 13- and 91-week old WKY and SHR rats. The system L-
type amino acid transporters is a major route for providing living
cells with neutral amino acids including several essential amino
acids that cells are unable to synthesize such as leucine, isoleucine,
valine, phenylalanine, tryptophan, methionine and histidine
(Christensen, 1990; Silbernagl, 1979). Although the transport of
leucine by LAT1 in pig LLC-PK1 renal cells has been previously
described (Soares-da-Silva and Serrao, 2004), LAT1 has a very
limited tissue distribution in the kidney (Pinho et al., 2007). Global
gene expression monitoring by cDNA microarrays showed a
decline in the expression of y+LAT1 and B0AT1 with age in the renal
cortex (Melk et al., 2005). Similarly, in the present study aging was
accompanied by decreases in LAT1 abundance in WKY and SHR.
LAT2 is a major Na+-independent amino acid transporter expressed
mainly in transporting epithelia, such as in the kidney and
intestine (Broer, 2008), and its functionality is dependent on the
abundance of 4F2hc (Pineda et al., 1999). The heterodimerization
L-DOPA, was
of LAT2 with 4F2hc is necessary for the transporter to reach the cell
surface (Nakamura et al., 1999). Therefore, increases in 4F2hc and
LAT2 abundance may translate in increases in LAT2 functionality in
aged SHR. On the other hand, the abundance of 4F2hc does not vary
with age in WKY, which would limit the translocation of LAT2 to the
cell surface. At the apical membrane of renal proximal tubule cells
only Na+-dependent amino acid transporters ASCT2 and B0AT1 are
capable of transporting amino acids with similar characteristics to
substrates transported through system L. Analogous to the LAT2
abundance profile, ASCT2 was found to be upregulated in aged WKY
and SHR. Overall, these results suggest that activation of the renal
dopaminergic system is accompanied by increases in LAT2/4F2hc
functionality and ASCT2 overexpression in aged SHR. In contrast,
LAT2/4F2hc functionality may not have a role in L-DOPA uptake in
the renal cortex of aged WKY.
Plasma aldosterone and renal noradrenaline levels were
higher in aged SHR than in aged WKY, indicating a marked
neurohumoral activation in aged SHR. The result of these
hemodynamic and neurohumoral alterations was an increase in
renal sodium transport (as indicated by a decrease in urinary
UNa+/K+ratio), proteinuria and reductions of the renal delivery
of L-DOPA in aged SHR. Another indication of aldosterone actions
is the marked increases in kidney size in aged SHR rats.
Aldosterone directly modulates renal cell proliferation and
differentiation via stimulation of rapidly activated protein
Fig. 3. Expression of LAT1 (A), LAT2 (B), 4F2hc (C) and ASCT2 (D) in the renal cortex of 13- and 91-week old WKY and SHR. Representative immunoblots are depicted on top of
the bar graphs. Values are normalized to the level of GAPDH expression in each condition and expressed as % of 13 week-old rats. Each bar represents the mean ? SEM (n = 4
per group). Significantly different from values in 13-week old animals (*P <0.05) using the Newman–Keuls test.
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302

Page 6

kinase cascades as part of normal kidney development (Thomas
et al., 2010). The renal dopaminergic and renin–angiotensin–
aldosterone systems (RAAS) control renal electrolyte balance
through various receptor mediated pathways with counter-
regulatory interactions. In order to conserve sodium during low
sodium intake, the RAAS is upregulated in order to produce
angiotensin II (Ang II). Stimulation of the principal membrane
bound cell surface receptor for Ang II, the AT1R, leads to sodium
reabsorption. In order to eliminate sodium during high sodium
intake the local renal production of dopamine is increased
leading to inhibition of sodium reabsorption (Felder and Jose,
2006). The natriuretic renal dopaminergic system opposes the
anti-natriuretic activity of the RAAS by downregulating the
AT1R, upregulating the AT2R and inhibiting ROS generation. Each
of the individual dopamine receptors has been shown to oppose
the activity of the AT1R, with the D1R, D3R, and D5R physically
interacting with the AT1R (Gildea, 2009). Taken together, it is
suggested that the renal dopaminergic system might be a
compensatory mechanism activated by stimuli that lead to
sodium reabsorption in aged WKY and SHR. However, this
counter-regulatory mechanism is considerably more enhanced
in aged SHR. A similar mechanism has been shown in patients
with heart failure. Stimuli leading to activation of anti-
natriuretic systems and sodium retention are accompanied by
activation of the renal dopaminergic system characterized by an
increase in the renal utilization of filtered L-DOPA (Alvelos et al.,
2004; Ferreira et al., 2001, 2002).
The specific effects of aldosterone on the expression of 4F2hc
and LAT2 have recently been explored by our group (Pinho et al.,
2009). Eight-week old Wistar rats were submitted to high salt
intake (1% NaCl in their drinking water) and treated chronically
with aldosterone and/or spironolactone, a mineralocorticoid
receptor (MR) antagonist. Treatment with aldosterone signifi-
cantly increased LAT2 mRNA expression via the MR (abolished
by spironolactone), though protein levels remained unchanged.
On the other hand, aldosterone treated rats had decreased 4F2hc
protein expression in a spironolactone-independent manner.
These effects of aldosterone were accompanied by decreases in
urinary dopamine and DOPAC in a spironolactone-sensitive
manner (Pinho et al., 2009).
Studies have shown that cardiac function and coronary
hemodynamics progressively deteriorate with aging in both SHR
and WKY and that very old WKY tend to develop a significant
degree of isolated systolic hypertension (Susic et al., 1998,
2001). In the present study SBP was found to be increased in
aged WKY and SHR, displaying the same trend as the plasma
aldosterone levels. Pulse pressure has been reported to increase
significantly with age in SHR but not in WKY (Chamiot-Clerc
et al., 2001). However, these studies were conducted in rats
between 3 and 78 weeks of age. The findings show that aged
SHR has in fact an intense dopaminergic response but SBP and
pulse pressure values remain increased. The cause for this
outcome is possibly related to the defective transduction of the
D1receptor signal in renal proximal tubules usually attributed
to this strain (Jose et al., 2010). On the other hand, the activation
of the renal dopaminergic system is not as effective in aged WKY
and SBP and pulse pressure are increased in these animals.
In conclusion, aging in WKY and SHR is accompanied by
increases in renal cortical ASCT2 abundance in the former and in
increases in LAT2/4F2hc and ASCT2 abundances in the latter.
Moreover, the dopaminergic response is more enhanced in aged
SHR than in aged WKY and this is probably a result of a
compensatory mechanism activated by stimuli leading to sodium
reabsorption.

WKY
WKY
SHR
SHR
0
0
500
500
1000
1000
1500
1500
2000
2000
2500
2500
13 week
91 week
s
s
s
*
*
*#
*#
Plasma aldosterone
(pmol/L)
#
#
WKY
SHR SHR
0.0 0.0
0.5 0.5
1.01.0
1.51.5
2.0 2.0
13 week s
91 week s
**
*#*#
Urinary Na+/K+
(ratio)
AA
BB
WKY
SHRSHR
0.00.0
0.50.5
1.01.0
1.51.5
2.02.0
13 wee ks
91 wee ks
**
*#*#
Urinary noradrenaline
(nmol/mg creatinine)
CC
13 week
91 week
s
Plasma aldosterone
(pmol/L)
WKY
13 week s
91 week s
Urinary Na+/K+
(ratio)
WKY
13 wee ks
91 wee ks
Urinary noradrenaline
(nmol/mg creatinine)
Fig. 4. Plasma levels (pmol/l) of aldosterone (A) changes in urinary Na+/K+ratio (B) and urinary noradrenaline levels (nmol/mg creatinine) in 13- and 91-week old WKY and
SHR. Each column represents the mean ? SEM of 6 rats. Significantly different from corresponding values in 13-week old animals (*P <0.05) and significantly different from age-
matched WKY (#P < 0.05) using the Newman–Keuls test.
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