Amino acid transport in podocytes
Joachim
Gloy1,
Steffen
Reitinger1,
Karl-Georg
Fischer1,
Rainer
Schreiber2,
Anissa
Boucherot2,
Karl
Kunzelmann2,
Peter
Mundel3, and
Hermann
Pavenstädt1
1 Department of Medicine, Division of
Nephrology, and 2 Department of Physiology,
Albert-Ludwigs-University Freiburg, D-79106 Freiburg, Germany; and
3 Department of Medicine and Department of
Anatomy and Structural Biology, Albert Einstein College of
Medicine, Bronx, New York
 |
ABSTRACT |
It has recently been shown that
formation of podocyte foot processes is dependent on a constant source
of lipids and proteins (Simons M, Saffrich R, Reiser J, and Mundel
P. J Am Soc Nephrol 10: 1633-1639, 1999). Here we
characterize amino acid transport mechanisms in differentiated cultured
podocytes and investigate whether it may be disturbed during podocyte
injury. RT-PCR studies detected mRNA for transporters of neutral amino acids (ASCT1, ASCT2, and B0/+), cationic AA (CAT1 and
CAT3), and anionic AA (EAAT2 and EAAT3). Alanine (Ala), asparagine,
cysteine (Cys), glutamine (Gln), glycine (Gly), leucine (Leu),
methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser),
threonine (Thr), glutamic acid (Glu), arginine (Arg), and histidine
(His) depolarized podocytes and increased their whole cell
conductances. Depletion of extracellular Na+ completely
inhibited the depolarization induced by Ala, Gln, Glu, Gly, Leu, and
Pro and decreased the depolarization induced by Arg and His, indicating
the presence of Na+-dependent amino acid transport.
Incubation of podocytes with 100 µg/ml puromycin aminonucleoside for
24 h significantly attenuated the effects induced by the various amino
acids by ~70%. The data indicate the existence of different amino
acid transporter systems in podocytes. Alteration of amino acid
transport may participate in podocyte injury and disturbed foot process formation.
podocytes; amino acid transport; puromycin
 |
INTRODUCTION |
THE PODOCYTE is a highly specialized cell, forming
multiple interdigitating foot processes that are interconnected by slit diaphragms and cover the exterior basement membrane surface area of the
glomerular capillary. The contractile filaments in the foot processes
of podocytes stabilize the glomerular architecture by antagonizing the
distending forces of the capillaries, and they may modulate glomerular
filtration rate by changing the ultrafiltration coefficient
Kf (19). Damage to the podocyte leads to
proteinuria, and in several proteinuric diseases the podocyte is the
target cell of injury (18). It has been suggested that the maintenance of the differentiated podocyte structure requires a complex
intracellular pumping and trafficking of proteins through systems
similar to those that operate in other highly differentiated cells such
as neurons (18, 26). Uptake of amino acids (AA) is essential for many
cellular processes like protein synthesis, hormone metabolism, regulation of cell growth, and osmotic volume changes (5, 26, 29).
Different AA transport (AAT) systems for neutral, acidic, and basic AA
have been characterized in regard to their substrate specificity,
cotransport properties, and tissue distribution, and 20 of these AAT
have been cloned already (21, 23, 24). Very recently, it has been shown
that the formation of podocyte processes is highly dependent on a
constant fresh source of lipid and proteins (30). Therefore, AAT may
play a critical role in maintaining the differentiated structure of the podocyte.
The purpose of the present study was to investigate properties of AAT
in mouse podocytes. As a first step, we characterized AAT in podocytes
and investigated mRNA expression of various AAT by means of RT-PCR. We
then studied whether podocyte injury by puromycin aminonucleoside (PA)
may be associated with a disturbed AAT. PA nephrosis is a
well-established experimental model for minimal change disease, which
is characterized by effacement of podocyte foot processes from the
glomerular basement membrane and massive proteinuria (18,
33). Although the mechanisms of podocyte injury in PA nephrosis are
presently not clear, it is likely that basic cellular functions such as
AAT are affected.
 |
METHODS |
Cell culture.
Cultivation of conditionally immortalized mouse podocytes was
done as recently reported (25). In brief, podocytes were maintained in
RPMI-1640 medium (Life Technologies) supplemented with 10% FCS, 100 U/ml penicillin, and 100 mg/ml streptomycin. To propagate podocytes,
cells were cultivated at 33°C (permissive conditions), and culture
medium was supplemented with 10 U/ml mouse recombinant
-interferon
(Sigma Chemical) to enhance expression of the T-antigen. To induce
differentiation, podocytes were maintained on type I collagen at
37°C without
-interferon (nonpermissive conditions). A detailed
characterization of these cells has been published previously (25). For
experiments, cells between passage 15 and 25 were
seeded at 37°C into 6-well plates and cultured in standard RPMI
media containing 1% FCS, 100 U/ml penicillin, and 100 mg/ml streptomycin for at least 7 days until cells were differentiated.
Patch-clamp experiments
The patch-clamp method (slow whole cell configuration) used in these
experiments has been described previously (11, 14). In brief, podocytes
were mounted in a bath chamber on the stage of an inverted microscope,
kept at 37°C, and superfused with a phosphate-buffered Ringer-like
solution. In ion-replacement studies Na+ was replaced by
N-methyl-D-glucamine+(NMDG+).
Pipettes were filled with a solution containing (in mM) 95 K-gluconate,
30 KCl, 4.8 Na2HPO4, 1.2 NaH2PO4, 0.73 Ca gluconate, 1.03 MgCl2, 1 EGTA, and 5 D-glucose, pH 7.2, as well
as 10
7 M Ca2+ activity, to
which 100-300 mg/l nystatin were added. The patch pipettes had an
input resistance of 2-3 M
. A flowing (10 µl/h) KCl (2 M)
electrode was used as a reference. The data were recorded by using a
patch-clamp amplifier (Fröbe and Busche, Physiologisches Institut, Freiburg, Germany) and continuously displayed on a pen recorder. The access conductance (Ga) was monitored
in most of the experiments by the method recently described. Membrane
voltage (Vm) of the cells was recorded continuously
by using the current-clamp mode of the patch-clamp amplifier. To obtain
the whole cell conductance (Gm), the voltage of the
respective cell was clamped in the voltage clamp mode
(Vc) to Vm. Starting at this
value, the whole cell current was measured by depolarizing or
hyperpolarizing Vc in steps of 10 mV to ±40 mV.
Gm was calculated from the measured whole cell
current (I) and Ga and
Vc by using Kirchhoff's and Ohm's laws (11).
Expression of AAT mRNA in mouse podocytes.
The RNA preparation and RT- PCR were performed according to the method
recently described (12). In brief, total RNA from cultured mouse
podocytes was isolated with guanidinium/acid phenol/chloroform extraction, and the amount of RNA was measured by spectrophotometry. For first-strand synthesis, 10 ng/µl of total RNA from podocytes were
mixed in 1× RT buffer and completed with 0.5 mM dNTP, 10 µM
random hexanucleotide primer, 10 mM dithiothreitol, 0.02 U RNAse
inhibitor/ng RNA, and 100 U Moloney murine leukemia virus RT/µg RNA
(RT was omitted in some experiments to control for amplification of
contaminating DNA).
RT was performed at 42°C for 1 h, followed by a denaturation at
95°C for 5 min. PCR was performed in duplicates in a total volume
of 20 µl, each containing 4 µl of RT reaction and 12 µl of PCR
master mixture. The mixture was overlaid with mineral oil and heated
for 1 min at 94°C. The samples were kept at 80°C until 4 µl
starter mixture, containing 10 pM each of sense and antisense primer
and 1 U Taq DNA polymerase, were added. The cycle profile included denaturation of 1 min at 94°C, annealing for 1 min at 60°C, and extension for 1 min at 72°C. Thirty to thirty-five
cycles were performed to amplify AAT DNA products. The amplification products of 10 µl of each PCR reaction were separated on a 1.5% agarose gel, stained with ethidium bromide (0.5 µg/ml), and
visualized by ultraviolet irradiation.
Primers were selected from sequences that have been deposited in the
National Institutes of Health/National Center for Biological Information (NCBI) database. The NCBI accession numbers of the respective nucleotide sequences appear first, and in some cases, second, in parentheses: 1) mouse neutral AA transporter mASCT1 (U75215; f-ACGCAGGACAGATTTTCACC, r-TGGCTTCCACCTT-CACTTCT; product size:
313 bp); 2) mouse mASCT2 (D85044; f-CCTCCAATCTGGTGTCTGCT, r-CCGTTTAGTTGTGCGATGAA; product size: 673 bp); 3) human hB
(AA308071; f-CGCCTCTGAGAAGGAATCAG, r-TGAGTTGGGGACATGAGTGA; product
size: 259 bp); 4) mouse mNBAT (B0,+; AA509386;
f-GGATGAGGACAAAGGCAAGA, r-ATGAGCAGGAACACGGAAAC; product size: 298 bp);
5) mouse insulin-activated AA transporter mIAT (L42115;
f-TCGCTATCGTCTTTGGTGTG, r-GTATTTCCCGAGGCTGATGA; product size: 206 bp);
6) mouse cationic AA transporter mCAT1 (AA061682;
f-GAAGACTCCGTTCCTGTGTTG, r-ACCTGACCCTGCTAC-GCTTT; product size: 368 bp); 7) mouse mCAT2 (L11600; f-TACGTCCAGTGTCGCAAGAG, r-CAACGTCCCTGTAAAGCCAT; product size: 397 bp); 8) mouse mCAT3 (U70859; f-ACGGCACTTGTA-GCTTGGAC, r-AATGGACACCAGGGAGTGAG; product size:
575 bp); 9) mouse excitatory AA transporter 1 (mEAAT1;
AA553011; f-TCCCATCCCAGAGTCAGAAA, r-ATGACAGCAGTGACCGTGAG; product
size: 295 bp); 10) mouse mEAAT2 (U11763;
f-AGTGCTGGAACT-TTGCCTGT, r-GGACTGCGTCTTGGTCATTT;
product size: 1719 bp); and 11) human hEAAT3 (AA084131;
f-TCCCTAAACCCAGAGAACCA, r-AAGTCAACATCGTGAACCCC; product size:
455 bp). PCR-amplification of RT reactions without RT revealed no PCR
product, thereby excluding amplification of genomic DNA. RT and PCR
amplification were repeated in the same manner by using four different
mouse podocyte RNA samples. In addition, three different mouse
glomeruli RNA samples were analyzed for the PCR products in the same
way. Isolation and preparation of glomeruli have been described in a
previous report (10).
Chemicals.
The following agents were used. Dimethylsulfoxide was from Merck
(Darmstadt, Germany). PA and all L-amino acids used were obtained from Sigma Chemical (Deisenhofen, Germany) and Calbiochem (San
Diego, CA) in the highest grade of purity available.
Statistics.
The data are given as mean values ± SE; n refers to the
number of experiments. A paired t-test was used to compare mean
values within one experimental series. A P value <0.05 was
accepted to indicate statistical significance.
 |
RESULTS |
Identification of AAT systems in mouse podocytes by RT-PCR.
Figure 1 shows ethidium
bromide-stained agarose gel electrophoreses of PCR products for
different AAT systems in mouse podocytes. In mouse podocytes positive
expression of mRNA for the neutral AAT systems ASCT1, ASCT2, IAT, and
B0/+, the cationic AAT systems CAT1 and CAT3, and the
anionic AAT systems EAAT2 and EAAT3 could be detected. mRNA for all
these AAT systems could be amplified also in isolated mouse glomeruli (n = 3, data not shown). Additionally, in mouse glomeruli mRNA for the AAT systems EAAT1 and CAT2 were detected, which could not be
amplified in podocytes.

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Fig. 1.
RT-PCR studies with primers derived from mouse DNA sequences amplified
mRNA for neutral amino acid transport (AAT) systems ASCT1, ASCT2, IAT,
and B0/+, cationic AAT systems CAT1 and CAT3, and anionic
AAT systems EAAT2 and EAAT3 (1-11). Experiments were performed by
using RT (RT+) or no RT (RT ) in each setup.
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AA depolarize Vm and increase Gm in
podocytes.
Podocytes had a Vm of
64 ± 1 mV (n = 169). Podocytes were reversibly depolarized by a large number of AA.
Addition of alanine (Ala; 5 mM) to the bath caused a rapid and
reversible depolarization of podocytes by 32 ± 1 mV that
was accompanied by an increase in Gm from 1.3 ± 0.3 to 1.8 ± 0.3 nS (n = 13). Figure
2 gives a representative original recording
for the effect of Ala on Vm and
Gm. Figure 3 shows the
concentration response curves for the depolarizing effect induced by
different AA with a maximal depolarization of 41 ± 2 mV (n = 9) induced by 50 mM of Ala. Similar to Ala, the neutral AA methionine
(Met), leucine (Leu), phenylalanine (Phe), proline (Pro), glycine
(Gly), serine (Ser), threonine (Thr), cysteine (Cys), asparagine (Asn),
and glutamine (Gln), the acidic AA glutamic acid (Glu), and the basic
AA arginine (Arg) and histidine (His) depolarized podocytes and
increased Gm in a concentration-dependent manner.
The estimated Km values for the depolarization were
calculated as follows (in mM): 0.2 Ala, 2.5 Gly, 4.0 Leu, 0.5 Met, 9.0 Phe, 0.7 Pro, 0.3 Cys, 0.3 Ser, 4.0 Thr, 0.7 Asn, 1.2 Gln, 6.0 His, 0.1 Arg, and 25.0 Glu. Compared with the neutral AA and His the depolarization induced by 10 mM Arg was relatively weak (8 ± 1 mV,
n = 11). Only higher concentrations of Glu (
10 mM, n = 5) induced a significant depolarization, whereas aspartate in a
concentration up to 10 mM did not have any effect. With all AA except
Arg and Asp a significant increase in Gm was observed, with
a peak increase ranging from 7 ± 4 (Phe, 50 mM) to 65 ± 25% (Ala,
1 mM). The maximal depolarization and conductance increase obtained
with different AA are summarized in Table
1.

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Fig. 2.
Original recording of effect of 5 mM alanine (Ala) on membrane voltage
(Vm; A) and membrane conductance
(Gm; B) of a podocyte. Addition of Ala
leads to reversible depolarization and conductance increase.
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Fig. 3.
Concentration-response curves of effect of different amino acids
(A-C) on Vm of podocytes. n,
No. of experiments.
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Figure 4 summarizes the effects of
experiments with aminoisobutyric acid (AIB), methyl-aminoisobutyric
acid (mAIB), and bicyclic amino acid 2-aminobicyclo (2,2,1 heptane)-2-carboxylic acid (BCH). Like AA, the AAT system
A-specific agonist AIB and its methyl derivate mAIB depolarize
podocytes in a concentration-dependent manner. In contrast, BCH, an
agonist of system L, had no effect.

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Fig. 4.
Effect of aminoisobutyric acid (AIB; left), methyl
aminoisobutyric acid (mAIB; middle), and bicyclic amino acid
2-aminobicyclo (2,2,1 heptane)-2-carboxylic acid (BCH; right)
on Vm of podocytes. Amino acid transport system
A-specific agonists AIB and mAIB depolarize podocyte concentration
dependently. On the contrary, BCH, an agonist of system L, has
no effect. Nos. in brackets, no. of experiments.* Statistical
significance, P < 0.05.
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Extracellular Na+
concentration
([Na+]e)
dependence of AAT in podocytes.
Figure 5A shows the effect
of Ala in the presence and absence of extracellular Na+.
Depletion of extracellular Na+ by substitution of
Na+ by 145 mM NMDG+ led to a transient
hyperpolarization of podocytes from
64 ± 1 to
76 ± 2 mV (n = 36). In the absence of Na+, the
depolarization and the increase of Gm induced by 5 mM Ala was completely and reversibly inhibited (n = 7). Figure
5B summarizes the effect of different AA in the absence of
Na+. Similar to Ala, the depolarization induced by Gln (5 mM), Gly (5 mM), Leu (5 mM), and Glu (25 mM) was abolished in the
absence of extracellular Na+ and the depolarization induced
by Pro (5 mM) was inhibited by >90% (n = 5 for all). The
depolarization induced by 5 mM His was only partly inhibited by
~50%, and the depolarization induced by 10 mM Arg was not
significantly influenced after depletion of
[Na+]e. The conductance increase
induced by the AA Gln, Gly, Leu, Pro, Ala (5 mM each, n = 4-7), and Glu (25 mM, n = 3) were significantly inhibited
in the absence of extracellular Na+ [from 65 ± 21 (Leu) to 96 ± 14% (Pro)].

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Fig. 5.
A: original recording of effects of 5 mM Ala on
Vm of single podocyte in presence (145 mM) and
absence (0 mM) of Na+. Note that depolarization induced by
Ala was completely and reversibly inhibited in absence of extracellular
Na+. B: summary of depolarizing effects of
different AA in absence and presence of extracellular Na+
(n = 3-13 experiments). Paired experiments were
performed as demonstrated in Fig. 5A. Depolarization induced by Ala (5 mM), glutamine (Gln; 5 mM), glycine (Gly; 5 mM), leucine (Leu; 5 mM),
proline (Pro; 5 mM), and glutamate (Glu; 25 mM) was abolished in
absence of extracellular Na+, whereas arginine (Arg; 10 mM)
and histidine (His; 5 mM) depolarized equally or partly.
* Statistical significance, P < 0.05.
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Pretreatment with PA inhibits AAT in podocytes.
Addition of 100 µg/ml PA to the bath solution did not
significantly change resting Vm during 5-10
min (n = 3, data not shown). Pretreatment of podocytes with 100 µg/ml for 24 h slightly decreased the resting Vm
of podocytes from
64 ± 1 to
54 ± 2 mV (n = 24).
Figure 6 shows an original experiment of
the effect of 5 mM Ala on Vm and
Gm in a PA-treated podocyte. After 24-h incubation with PA the depolarization and the increase of Gm
induced by Ala were almost completely inhibited (n = 5). Figure
7 summarizes the effects of different AA on
Vm and Gm in PA-treated
podocytes. Similar to Ala, the depolarization and the
Gm increase induced by Gln (5 mM, n = 5),
Gly (5 mM, n = 5), Leu (5 mM, n = 5), Pro (5 mM,
n = 5), Arg (10 mM, n = 5), His (5 mM, n = 7),
and Glu (25 mM, n = 5) were significantly inhibited.

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Fig. 6.
Influence of 5 mM Ala on Vm (A) and
Gm (B) of podocyte after preincubation for
24 h with 100 µg/ml puromycin aminonucleoside (PA). Note that
Ala-induced depolarization and its effect on Gm
were strongly attenuated compared with control cells (see Fig 2).
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Fig. 7.
Summary of inhibitory effects of an incubation of podocytes with PA
(100 µg/ml for 24 h) on amino acid-induced depolarization (A)
and conductance increase (B). Note that amino acid-induced
depolarization and increase of whole cell conductance were
significantly inhibited.
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 |
DISCUSSION |
AAT in podocytes.
Uptake of AA via membrane AA transporters is essential for many
cellular functions. It is achieved by either coupling the uptake of AA
to the cotransport of Na+ (secondary active transport) or
by the negative cell membrane potential that is used as a driving force
(26). Under physiological conditions plasma concentration of all free
AA is ~2.5 mM, and a daily load of ~450 mmol of AA passes the
glomerular filtration barrier. During proteinuric states, however, this
amount may be strongly increased and not only tubular cells but also
podocytes are faced with much higher concentrations of AA due to
hydrolysis of oligopeptides within the Bowman's space (29).
Disturbance of AAT has been assumed in podocyte damage in cystinosis,
suggesting that AAT might play a role in the maintenance of podocyte
function (32). Here, we demonstrate active AAT in mouse podocytes and mRNA expression for several AA uptake transporters such as the neutral
AAT systems ASCT1, ASCT2, IAT, and B0/+, the cationic AAT
systems CAT1 and CAT3, and the anionic AAT systems EAAT2 and EAAT3. All
AAT detected in cultured podocytes could also be identified in isolated
mouse glomerula, suggesting that these systems are also present in vivo.
Patch-clamp studies showed that neutral AA and L-glutamate
led to a concentration- and
[Na+]e-dependent depolarization and
conductance increase in podocytes, with Km values
very similar to rat kidney proximal tubule cells (16, 28).
Depolarization was also induced by the specific substrates AIB and
mAIB, indicating that mouse podocytes also possess the widely
distributed AAT system A for uptake of small neutral AA, the
cDNA code of which has not yet been cloned (17). System A AAT
has been reported to be involved in cell volume and osmolyte regulation
(4, 6, 17), which may be essential for podocyte function during
physiological states and proteinuric diseases. Podocytes also express
Na+-dependent neutral AA transporters ASCT1 and ASCT2,
which are distributed in a wide variety of cell types and are
structurally related to glutamate transporters (5, 26). ASCT1
transports Ala, Ser, Thr, Cys, and Val, whereas ASCT2 has a broader
substrate selectivity; i.e., it also accepts AA with longer side chains such as Glu and Met (3, 17). The presence of the cationic AAT systems
CAT1, CAT2, and CAT3 allows the Na+-independent uptake of
basic and dibasic AA (Arg, Lys, Orn, and Hist) (8). Podocytes seem to
express CAT1 and CAT3 but not CAT2. However, expression of all three
CAT transporters was detected in glomerula, indicating that CAT2 is
expressed in other glomerular cells. In this regard it has been shown
that within rat glomerula CAT2 is expressed in parietal cells of
Bowman's capsule (2).
Interestingly, CAT3 has been suggested to be brain specific (8) with a
Km for Arg that is similar to the
Km observed in podocytes in this study (0.1 mM).
CAT3 mRNA has been demonstrated in rat neurons but not in glial or
brain endothelial cells (15).
The relatively small depolarization induced by L-arginine
suggests the existence of a Na+-independent membrane
transport of L-arginine. In the absence of extracellular
Na+, the depolarization induced by the dibasic AA His was
inhibited by ~50%, suggesting that His might also be transported via
the Na+-dependent, broad-scope AA transporter
B0/+, which accepts dibasic and some neutral AA.
Alternatively, His transport might have been inhibited by
NMDG+.
The examination of acidic AAT was limited due to the solubility of
glutamate and aspartate at a pH of 7.4. In higher concentrations (Km = 25 mM) glutamate also depolarized podocytes,
indicating that glutamate uptake might occur via the anionic AAT EAAT2
and EAAT3. EAAT2 has been assumed to be specifically expressed in the
brain, where it has been demonstrated in astrocytes (17). EAAT3
expression has been demonstrated in neurons, but it is also expressed
in different peripheral cells, such as in epithelial cells of the
intestine (17). As demonstrated in the present study there is a strong
concentration-dependent depolarization in podocytes induced by AA,
reflecting secondary active AAT for most neutral AA with
Km values ranging from 0.1 to 10 mM. Thus it is
apparent that a relatively small increase in AA concentration within
the Bowman's space during proteinuric states or a protein-rich diet
would lead to a relatively strong increase in depolarization, due to
increased uptake of AA and Na+ in podocytes.
PA nephrosis (PAN) is an experimental rat model of human minimal-change
disease (33). Both diseases are characterized by nephrotic range
proteinuria and podocyte foot process effacement as the morphological
hallmark (18). The precise mechanisms underlying podocyte damage in PAN
are not well known, but the foot process effacement is associated with
a disaggregation and rearrangement of actin filaments and induction of
-actinin (31, 34). After 24-h treatment with PA
Vm was only slightly decreased, indicating that PA
did not markedly alter resting ion currents in podocytes. However,
after PA treatment, AA-induced depolarization and conductance increase
were markedly inhibited, suggesting that PAN-induced injury of
podocytes is associated with a decrease in AAT.
Altered AA transport by PA may induce podocyte injury by several
distinct mechanisms. For example, inhibition of cysteine transport by
PA may lead, via reduction of intracellular glutathione levels (7), to
an imbalance of antioxidant defense mechanisms in podocytes. Oxidative
stress has been assumed to play a major role in aminonucleoside
nephrosis, (9) and a disturbance of intrinsic antioxidant defense
mechanisms in PAN participates in podocyte injury (13).
Alternatively, PA-induced disturbance of Arg uptake may change the
Arg-dependent synthesis of nitric oxide and other important second
messengers. The highest amount of intracellular Arg within the
glomerulus has been localized in podocytes (1). This may play a
critical role in podocyte function because dietary intervention with
L-arginine improves proteinuria and may reduce podocyte
damage during proteinuric states like PAN (27).
In conclusion, we have shown that differentiated podocytes express
distinct functional transporters for AA uptake. AAT in podocytes was
inhibited by PA, suggesting that it is altered during podocyte injury
in this model of proteinuric disease. These findings suggest that
normal function of AA transporters may play a role in maintaining the
differentiated cytoarchitecture of podocytes.
 |
ACKNOWLEDGEMENTS |
We thank Temel Kilic, Charlotte Hupfer, and Monika von Hofer for
excellent technical assistance. We also thank Bernd Friedrich and
Wilfried Benz from ASTRA GMBH, Hamburg, Germany, for financial support.
 |
FOOTNOTES |
This work was supported by the Forschungskommission der
Universität Freiburg.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. Pavenstädt, Medizinische Universitätsklinik, Abt.
Nephrologie, Hugstetterstr. 55, D-79106 Freiburg, Germany (E-mail:
paven{at}mm41.ukl.uni-freiburg.de).
Received 28 April 1999; accepted in final form 30 December 1999.
 |
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