Clinical Science and Molecular Medicine (1911) 53,9-15.
Renal handling of dibasic amino acids
and cystine in cystinuria
TOMOAKI KATO
Department of Pediatrics, School of Medicine, Nagoya University, Nagoya, Japan
(Received 21 June 1976; accepted 2 February 1977)
Summary
that observed under the endogenous condition when the filtered load was high. The
amino acid load caused only a gradual decrease in cystine reabsorption in the cystinuric
patients.
5. In the heterozygous subjects the slope of
the titration curves and the depression of the
tubular reabsorption were intermediate between
those of the control and homozygous subjects.
1. The effect of intravenous infusion of
L-lysine and L-arginine on the tubular reabsorption of dibasic amino acids and cystine
was studied in normal individuals and in
homozygous and heterozygous subjects with
cystinuria.
2. The control subjects reabsorbed almost all
filtered lysine and arginine until the filtered load
was elevated about fourfold. With further
increased loads the tubular reabsorption began
to fall and tended to approach a maximum
reabsorption rate. By contrast, the homozygous subjects could not reabsorb the elevated
amino acid beyond the endogenous capacity
until the filtered load was increased seven- to
ten-fold. When the filtered load was further
increased, tubular reabsorption proceeded at
the normal rate in the cystinuric patients.
3. These findings may be explained by a lowcapacity transport system, which acts at low
substrate concentrations, being defective in the
cystinuric subjects, while a high-capacity
transport system, which predominates at high
substrate concentrations, remains intact.
4. Lysine and arginine infusion depressed
the percentage tubular reabsorption of other
dibasic amino acids and cystine both in the
control and the cystinuricsubjects. In the control
subjects the amino acid infusion caused a
gradual linear fall in the fractional reabsorption
of the dibasic amino acids and cystine, whereas
the depressed reabsorption of the dibasic amino
acids in the cystinuric patients returned to
Key words: amino acid transport, cystine,
cystinuria.
Introduction
Cystinuria is an hereditary disorder characterized by a defective amino acid transport in
the renal tubule and the intestinal mucosa
(Crawhall & Watts, 1968). Cystine, lysine,
arginine and ornithine are excreted in large
quantities in the urine of cystinuric patients as
a result of the tubular reabsorption defect.
It seems likely that cystine not only shares a
common transport system with the dibasic
amino acids, lysine, arginine and ornithine, but
that it also has a separate transport system
from that of dibasic amino acids, as isolated
hypercystinuria, with a tubular reabsorption
defect only for cystine (Brodehl, Gellissen &
Kowalewski, 1967), and also hyperdibasicaminoaciduria, with a tubular defect for dibasic
amino acids only (Perheentupa & Visakorpi,
1965), both exist. Experiments with kidney
slices from cystinuric subjects (Fox, Thier,
Rosenberg, Kiser & Segal, 1964) have shown
a defect in the transport of the dibasic amino
acids with no corresponding abnormality in
Correspondence: Dr Tornoaki Kato, Department of
Pediatrics, School of Medicine, Nagoya University,
65 Tsurumai-cho, Showa-ku, Nagoya, Japan.
9
Tomoaki Kato
10
consent was obtained from the subjects or their
guardians after the study had been explained
to them.
A11 studies were performed in the morning
after an overnight fast. Before and during the
procedures, water was given orally to produce
a diuresis exceeding 5 mllmin. Urine was collected by spontaneous voiding. After a control
clearance period of 20 min (period l), an amino
acid solution (0.8-1-0 mllkg of 1 molil solution
of L-lysine/HCI or 0-7-0.9 ml/kg of 1 mol/l
solution of L-arginine/HCI) was infused into
an antecubital vein over 3 min. After infusion,
urine specimens were collected during three
consecutive clearance periods (periods 2, 3 and
4) of 20-30 min. Blood samples were obtained
at the midpoint of each clearance period.
Endogenous creatinine clearance was determined simultaneously for each period to
estimate the glomerular filtration rate.
Blood samples were centrifuged immediately
and serum was deproteinized by adding
sulphosalicylic acid (0.16 mol/l) at a ratio of
1:3. The supernatant was stored at -20°C.
Urine samples were kept frozen until analysed.
Methods
Amino acids in urine and serum were deterThe subjects were four homozygous cystinuric
mined with an automatic amino acid analyser
patients, six parents of the patients, and seven
(Spackman, Stein & Moore, 1958). Urinary
normal healthy volunteer subjects, ranging
and serum creatinine were measured by the
in age from 6 to 45 years. The cystinuric patients
method of Bonsnes & Taussky (1945).
excreted an excess of cystine, lysine, arginine
The creatinine clearance rate (Cc,) was
and ornithine in the urine (Table 1) and had
calculated and, from serum amino acid conformed renal cystine calculi. One patient ((3) centration (PA*),the filtered load of amino
still had a stone in the right kidney at the time
acid (FAA)was estimated as: F A A = Pan x Ccr.
of the study. The control subjects excreted
The net tubular reabsorption rate of amino
normal amino acids and the heterozygous
acid (RAA) was calculated as: RAA= F A A cystinuric patients excreted moderately inUVAA(UVAAbeing the excretion rate of amino
creased amounts of dibasic amino acids, as was
acid). The percentage tubular reabsorption of
confirmed semiquantitatively by two-dimenamino acid (TAA) was calculated as: T A A =
sional thin-layer chromatography. Informed
100 * RAAIFAA.
the uptake of cystine. Studies on rat (Rosenberg, Downing & Segal, 1962) and human
(Fox et al., 1964) renal cortex slices have shown
that the dibasic amino acids have a common
transport system that is not shared by cystine.
Furthermore, the transport systems for lysine
and cystine in rat kidney are not the same (Segal
& Smith, 1969).
There is evidence of two different systems in
membrane transport of the dibasic amino acids
by human kidney cortex slices (Rosenberg,
Albrecht & Segal, 1967), skin fibroblasts
(Groth & Rosenberg, 1972) and erythrocytes
(Gardner & Levy, 1972). One is active at low
substrate concentrations and the other at high
substrate concentrations. Recently, cystine has
also been shown to have two transport systems
(Kaye & Nadler, 1976).
I have studied the effect of intravenous
infusion of L-lysine and L-arginine on the tubular reabsorption of the dibasic amino acids and
cystine to clarify the mechanisms of renal
transport in cystinuria.
TABLE
1. Urinary excretion of cystine and dibasic amino acids in the cystinuric subjects
For normal subjects (n = 10) mean results+ SD are shown.
Urinary excretion (mg/g of creatinine)
Subject
c1
c2
c3
c4
Normal
Age
(years)
Sex
6
11
10
15
M
M
M
F
Cystine
775
233
710
614
11*1+0-7
-
Lysine
Arginine
Ornithine
1320
1880
4390
1160
33.5k4.8
1030
612
2060
627
2*85+056
453
393
861
231
1.67+0-32
Renal handling in cystinuria
Results
The results of the renal handling of the dibasic
amino acids and cystine before and after
L-lysine and L-arginine infusion are summarized
in a Table which has been deposited as Clinical
Science and Molecular Medicine Table 7712
with the Librarian, the Royal Society of
Medicine (1 Wimpole Street, London WIM
8AE), who will issue copies on request. The
cystinuric patients had a normal creatinine
clearance with no decrease of the clearance
during the elevated diuresis. Lysine infusion
caused an increase in the filtered load of arginine, and arginine infusion was accompanied by
an increase of filtered lysine and ornithine in all
three groups, i.e. normal subjects, homozygous
cystinuric and heterozygous cystinuric patients.
There was no change in the filtered load of
cystine after lysine or arginine infusion in all
subjects except one cystinuric patient (C3), who
showed a decrease of filtered cystine after the
amino acid loading.
The data concerning the effect of increasing
the filtered load of lysine and arginine by infusion on the reabsorption rate of the infused
amino acid are also presented in the Table
7712 deposited as detailed above. The control
subjects could reabsorb almost all the filtered
amino acid in clearance period 1, whereas,
when the filtered load was increased, the
percentage of the fractional reabsorption
gradually fell (periods 2, 3 and 4). In the cysti-
nuric patients, three patients (Cl, C3 and C4)
could hardly reabsorb the fiItered amino acid
when the filtered load was low. The fractional
reabsorption rate of lysine by patient C3 and
that of arginine by patients C1, C3 and C4 gave
a negative value in period 3 or 4. In patient C2,
however, there was a small increase in the fractional reabsorption with low filtered loads. When
the filtered load was completely elevated
(period 2), all the patients (except C1, in whom
the filtered load of arginine did not increase
enough to elevate the reabsorption rate) could
reabsorb the filtered amino acid almost to the
same degree as the control subjects.
The reabsorption titration curves of lysine
and arginine (Fig. 1) show that in the control
subjects almost 100% of the filtered amino
acid was reabsorbed, up to a load of 100
pmol min-I 1.73 m-2 for lysine and 40 pmol
rnin-' 1-73 m-* for arginine. With higher
filtered loads the reabsorption rate began to
fall, suggesting that it was approaching a
maximum rate. The cystinuric patients, on the
other hand, exhibited no ability to reabsorb the
filtered amino acid beyond the existing endogenous capacity until this load increased sevento ten-fold. When the filtered amino acid was
further increased, the reabsorption proceeded at
the normal rate in the cystinuric patients.
Parents of the patients showed a titration curve
intermediate between those of the normal and
homozygous subjects.
t
m
p:
I
._
E
5
5
11
200
P
e
C
;
c
100
n
p!
b
3
n
0
Filtered lwd ( p o l mi"-' 1.73 rn-')
FIG. 1. Titration curves of (a) lysine and (b) arginine reabsorption in three control subjects (0),
and
in four homozygous (0) and two heterozygous(A) subjects with cystinuria.
Tomoaki Kato
100
200
300
400
500 0
100
Flltered lood (pnol rnin-' 1.73 rn-')
200
300
400
FIG.2. Percentage tubular reabsorptionof(a) arginine and (b) ornithinebefore and after lysine infusion
in three control subjects (O),and in four homozygous ( 0 ) and two heterozygous (A) subjects with
cystinuria.
-s
h
.-5
h
50
-b
2
3
0
100
400 0
100
Filtered load (prnol min-' 1;/3rr1-~)
300
200
300
400
FIQ.3. Percentage tubular reabsorptionof (a) lysine and (b) ornithinebefore and after arginine infusion
in three control subjects (O),and in four homozygous ( 0 ) and two heterozygous (A) subjects with
cystinuria.
Lysine and arginine infusion depressed the
percentage tubular reabsorption of other dibasic
amino acids and cystine both in the control
and cystinuric subjects (Figs. 2-4). In the control
subjects, lysine infusion resulted in a gradual
linear decrease in the fractional reabsorption
of arginine and ornithine, and the infusion of
arginine caused a similar decrease in the reabsorption of lysine and ornithine (Fig. 2 and
Fig. 3). An increase of filtered lysine in the
cystinuric patients also diminished the fractional
reabsorption of arginine and ornithine with
low filtered loads. Further increase of the lysine
load, however, caused a return of the diminished
reabsorption to the endogenous level (Fig. 2).
Similar findings were obtained by arginine
infusion (Fig. 3).
The results of cystine rqabsorption in the
cystinuric patients after lysine and arginine
infusion differed from that of dibasic amino
acid reabsorption. An increase of the filtered
amino acid caused only a gradual decrease in
cystine reabsorption and no return to the endogenous reabsorption (Fig. 4). In the heterozygous subjects the tubular reabsorption of the
dibasic amino acids and cystine after amino
Renal handling in cystinuria
I
0
I
too
I
I
200
300
I
I
I
400
500 0
Filtered-lood (pmol rnin-' 1.73m-')
13
100
I
I
I
200
300
400
FIG.4. Percentage tubular reabsorptionof cystine before and after (a) infusion of lysine and (b) infusion
of arginine in three control subjects (0), and in three homozygous (e)and two heterozygous (A)
subjects with cystinuria.
acid load was midway between that of the control subjects and homozygous patients (Figs.
2-4).
Discussion
Studies in vivo with rats (Bergeron & Morel,
1969) and cockerels (Boorman, 1971) have
shown that a maximum tubular reabsorption
for lysine and arginine can be obtained by
increasing the filtered load of the amino acids,
and that infusion of one of the dibasic amino
acids inhibits the renal reabsorption of the
others. In addition, an infusion of the dibasic
amino acids can also depress cystine reabsorption in rat kidney (Webber, Brown & Pitts,
1961). The results in my control subjects were
consistent with these findings. With high filtered
loads the tubular reabsorption rate in the control subjects tended to approach a maximum,
and lysine and arginine infusion caused a gradual fall in the percentage tubular reabsorption
of other dibasic amino acids and cystine.
The cyshnuric subjects had no ability to
reabsorb the filtered amino acid beyond the
endogenous capacity with low filtered loads,
whereas the control subjects could reabsorb
almost all the filtered load under these conditions. However, when the filtered load was
fully increased, the cystinuric patients could
reabsorb as much load as the control subjects.
This is reminiscent of the experiments in vitro
of Groth & Rosenberg (1972), implying two
separate transport mechanisms for dibasic
amino acids in cultured human fibroblasts.
A high-affinity system is preferentially responsible for transport at low substrate concentrations, and a low-affinity transport system is
active at high substrate concentrations. If the
same transport systems are postulated to exist
in the renal tubules in viuo, the findings observed in the present study can be explained
without difficulty. That is, a transport system
which acts at low substrate concentrations is
defective in the cystinuric patients, whereas a
second transport system acting at high substrate concentrations remains intact in these
patients. A negative tubular reabsorption
found with low filtered loads is probably related
to the impairment of the low-concentration
system.
In agreement with the findings of Lester &
Cusworth (1973), that the clearance of cystine,
arginine and ornithine in control and cystinuric
subjects was elevated by the infusion of lysine,
the administration of lysine and arginine in this
study inhibited the tubular reabsorption of
other dibasic amino acids and cystine both in
the control and cystinuric subjects. In the cystinuric subjects amino acid infusion caused a
further depression of the fractional reabsorption
of the dibasic amino acids at low filtered loads,
and with increased loads the depressedreabsorption returned to that observed in the endo-
14
Tomoaki Kato
genous condition. This is in contrast to findings
in iminoglycinuric subjects studied by Scriver
(1968), where proline infusion caused no
additional inhibition of glycine reabsorption.
In the present study the amino acid load
caused only a gradual fall in cystine reabsorption, and no return to the endogenous reabsorption was observed in the cystinuricpatients.
There is no reasonable explanation for these
inhibitory effects of infused amino acid on the
tubular reabsorption. How these competitive
inhibitions occur in such a defective transport
site is not yet clear.
Scriver & Wilson (1967) postulated two
transport systems for proline, hydroxyproline
and glycine from studies in vivo with iminoglycinuricsubjects (Scriver, 1968). Such patients
had a residual tubular function to reabsorb
the imino acids and glycine under endogenous
conditions, but could not reabsorb the filtered
imino acid beyond the endogenous capacity
when the imino acid was highly increased by
intravenous load. They proposed two types of
transport systems for imino acids and glycine,
i.e. a low-capacity, high-affinity transport
system acting at low substrate concentrations,
and a high-capacity, low-affinity system active
at high substrate concentrations. In iminoglycinuria the high-capacity transport system is
defective but the low-capacitysystem is normal.
This is contrary to the present findings, but the
differences may be explained by endogenous
renal clearance studies (Frimpter, Horwith,
Furth, Fellows & Thompson, 1962; Crawhall,
Scowen, Thompson & Watts, 1967; Scriver,
1968; Rosenberg, Durant & EIsas, 1968)
which have shown that patients with cystinuria
have a severe transport defect under endogenous
conditions as compared with iminoglycinuric
subjects. As the low-capacity transport system
is used at endogenous renal amino acid concentrations, a defect in this system will lead to a
severe impairment of tubular reabsorption in
the endogenous condition, as seen in cystinuria.
On the other hand, a defect in the high-capacity
transport system, as seen in iminoglycinuria,
will cause only a slight impairment of transport
in that condition because this system is used
mainly at high amino acid concentrations.
However, it remains surprising that an amino
acid i s normally reabsorbed at the impaired
transport site, when the load of the compound
is raised.
Acknowledgments
The author thanks Professor Sakae Suzuki for
advice and encouragement and Dr Junichi
Sugiura for helpful discussion. The study was
supported in part by a grant from ChubuRosai Hospital.
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