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Am J Physiol Renal Physiol
278: F999–F1005, 2000.
Amino acid transport in podocytes
JOACHIM GLOY,1 STEFFEN REITINGER,1 KARL-GEORG FISCHER,1
RAINER SCHREIBER,2 ANISSA BOUCHEROT,2 KARL KUNZELMANN,2
PETER MUNDEL,3 AND HERMANN PAVENSTÄDT1
1Department of Medicine, Division of Nephrology, and 2Department of Physiology,
Albert-Ludwigs-University Freiburg, D-79106 Freiburg, Germany; and
3Department of Medicine and Department of Anatomy and Structural Biology,
Albert Einstein College of Medicine, Bronx, New York
podocytes; amino acid transport; puromycin
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
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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 g-interferon (Sigma Chemical) to enhance expression of the T-antigen. To induce differentiation, podocytes
were maintained on type I collagen at 37°C without g-interferon (nonpermissive conditions). A detailed characterization
of these cells has been published previously (25). For experi-
0363-6127/00 $5.00 Copyright r 2000 the American Physiological Society
F999
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Gloy, Joachim, Steffen Reitinger, Karl-Georg Fischer,
Rainer Schreiber, Anissa Boucherot, Karl Kunzelmann, Peter Mundel, and Hermann Pavenstädt. Amino
acid transport in podocytes. Am J Physiol Renal Physiol 278:
F999–F1005, 2000.—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 B0/1), 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 Na1 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 Na1-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.
F1000
AMINO ACID TRANSPORT IN PODOCYTES
GAATCAG, r-TGAGTTGGGGACATGAGTGA; product size:
259 bp); 4) mouse mNBAT (B0,1; 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, rAAGTCAACATCGTGAACCCC; product size: 455 bp). PCRamplification 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 6 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 B0/1, 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 5 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.
AA depolarize Vm and increase Gm in podocytes.
Podocytes had a Vm of 264 6 1 mV (n 5 169). 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 6 1 mV that was accompanied by an increase in Gm
from 1.3 6 0.3 to 1.8 6 0.3 nS (n 5 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 6 2 mV (n 5 9)
induced by 50 mM of Ala. Similar to Ala, the neutral AA
methionine (Met), leucine (Leu), phenylalanine (Phe),
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ments, 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 Na1 was
replaced by N-methyl-D-glucamine1(NMDG1). 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 1027 M
Ca21 activity, to which 100–300 mg/l nystatin were added.
The patch pipettes had an input resistance of 2–3 MV. 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 640
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 13 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-CGCCTCTGAGAAG-
AMINO ACID TRANSPORT IN PODOCYTES
F1001
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 B0/1, cationic AAT systems CAT1 and CAT3, and anionic AAT systems
EAAT2 and EAAT3 (1–11). Experiments were
performed by using RT (RT1) or no RT (RT2) in
each setup.
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.
derivate mAIB depolarize podocytes in a concentrationdependent manner. In contrast, BCH, an agonist of
system L, had no effect.
Extracellular Na1 concentration ([Na1]e) dependence
of AAT in podocytes. Figure 5A shows the effect of Ala in
the presence and absence of extracellular Na1. Deple-
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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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 6 1
mV, n 5 11). Only higher concentrations of Glu ($10
mM, n 5 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 6 4 (Phe, 50 mM) to 65 6 25%
(Ala, 1 mM). The maximal depolarization and conductance increase obtained with different AA are summarized in Table 1.
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
F1002
AMINO ACID TRANSPORT IN PODOCYTES
Table 1. Maximal depolarization and maximal
conductance increase obtained with
different amino acids
Maximal
Amino Depolarization,
Acid
mV
41 6 2
40 6 4
29 6 5
28 6 3
36 6 9
55 6 7
47 6 4
34 6 4
43 6 4
45 6 4
40 6 4
861
42 6 8
23 6 6
2 6 2 (NS)
65 6 25 (1 mM)
26 6 6 (10 mM)
24 6 7 (10 mM)
7 6 4 (50 mM)
32 6 8 (5 mM)
63 6 9 (5 mM)
54 6 28 (10 mM)
50 6 19 (10 mM)
53 6 16 (1 mM)
43 6 8 (50 mM)
17 6 4 (5 mM)
3 6 4 (10 mM)
26 6 4 (10 mM)
15 6 11 (50 mM)
22 6 2 (10 mM)
Km for
Depolarization,
Na1
Dependence
mM
0.2
0.5
4.0
9.0
0.7
2.5
0.3
4.0
0.3
0.7
1.2
0.1
6.0
25.0
Yes
ND
Yes
ND
Yes
Yes
ND
ND
ND
ND
Yes
No
Partly
Yes
ND
Values are means 6 SE. Km , Michaelis-Menten coefficient; Ala,
alanine; Met, methionine; Leu, leucine; Phe, phenylalanine; Pro,
proline; Gly, glycine; Ser, serine; Thr, threonine; Cys, cysteine; Asn,
asparagine; Gln, glutamine; Arg, arginine; His, histidine; Glu, glutamic acid; Asp, aspartic acid; ND, not done.
tion of extracellular Na1 by substitution of Na1 by 145
mM NMDG1 led to a transient hyperpolarization of
podocytes from 264 6 1 to 276 6 2 mV (n 5 36). In the
absence of Na1, the depolarization and the increase of
Gm induced by 5 mM Ala was completely and reversibly
inhibited (n 5 7). Figure 5B summarizes the effect of
different AA in the absence of Na1. 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 Na1 and the depolarization induced by
Pro (5 mM) was inhibited by .90% (n 5 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 [Na1]e. The conductance increase induced
by the AA Gln, Gly, Leu, Pro, Ala (5 mM each, n 5 4–7),
and Glu (25 mM, n 5 3) were significantly inhibited in
the absence of extracellular Na1 [from 65 6 21 (Leu) to
96 6 14% (Pro)].
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.
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 Na1. Note that
depolarization induced by Ala was completely and reversibly inhibited in absence of extracellular Na1. B: summary of depolarizing
effects of different AA in absence and presence of extracellular Na1
(n 5 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 Na1, whereas arginine (Arg; 10 mM) and
histidine (His; 5 mM) depolarized equally or partly. * Statistical
significance, P , 0.05.
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 5 3,
data not shown). Pretreatment of podocytes with 100
µg/ml for 24 h slightly decreased the resting Vm of
podocytes from 264 6 1 to 254 6 2 mV (n 5 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 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 5), Gly (5 mM, n 5 5), Leu (5
mM, n 5 5), Pro (5 mM, n 5 5), Arg (10 mM, n 5 5), His
(5 mM, n 5 7), and Glu (25 mM, n 5 5) were significantly inhibited.
DISCUSSION
AAT in podocytes. Uptake of AA via membrane AA
transporters is essential for many cellular functions. It
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Ala
Met
Leu
Phe
Pro
Gly
Ser
Thr
Cys
Asn
Gln
Arg
His
Glu
Asp
Maximal
Conductance
Increase, %
AMINO ACID TRANSPORT IN PODOCYTES
is achieved by either coupling the uptake of AA to the
cotransport of Na1 (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
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.
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 B0/1,
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 [Na1]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 Na1-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 Na1-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 Larginine suggests the existence of a Na1-independent
membrane transport of L-arginine. In the absence of
extracellular Na1, the depolarization induced by the
dibasic AA His was inhibited by ,50%, suggesting that
His might also be transported via the Na1-dependent,
broad-scope AA transporter B0/1, which accepts dibasic
and some neutral AA. Alternatively, His transport
might have been inhibited by NMDG1.
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 5 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 demon-
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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).
F1003
F1004
AMINO ACID TRANSPORT IN PODOCYTES
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.
This work was supported by the Forschungskommission der
Universität Freiburg.
Address for reprint requests and other correspondence: H. Pavenstädt, Medizinische Universitätsklinik, Abt. Nephrologie, Hugstetter-
str. 55, D-79106 Freiburg, Germany (E-mail: [email protected]).
Received 28 April 1999; accepted in final form 30 December 1999.
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strated 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
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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
a-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.
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