Download RenalTubularTransportof AminoAcids

RenalTubularTransportof AminoAcids
John Atherton Young and Benedict Sol Freedman
Cushny in 1917 first remarked on the extensive amino acid reabsorption
which occurs in the nephron. Although many workers since then have
studied the nature and localization of the reabsorptive mechanism, progress has been slow because of the technical difficulties of micropuncture
work. The bulk of filtered amino nitrogen is reabsorbed in the proximal
tubule although the possibility of there being more distal reabsorptive (or
secretory) sites cannot be excluded. It is also uncertain whether all segments of the proximal tubule contribute equally to the reabsorptive process.
Amino acid reabsorption is an active process involving numerous illdefined steps, the first of which is binding to the brush borders. Renal amino
acid transport mechanisms are of two kinds: the high-capacity lowspecificity systems transport whole groups of amino acids-the
acidic,
basic, neutral, and imino-glycine groups-while the other, the low-capacity
high-specificity systems, transport single or perhaps pairs of amino acids
only.
Although
a great deal of information
has been
published
on the mechanisms
of amino acid transport in isolated
cells and tissues (1), rather less
work has been done on the equally important
topic
of transepithelial
transport.
Transport
across the
intestine
is technically
easy to study, so most of
our knowledge
of transepithelial
transport
comes
from studies
on that organ (2). The kidney
is
technically
much more difficult to study than the
intestine,
so our knowledge
of renal tubular transport of amino acids is scanty and comes mainly
from such indirect approaches
as clearance studies.
With the development
of sophisticated
techniques
for micropuncture
and microperfusion
of segments
of kidney tubules
and for microanalysis
of fluid
samples so obtained
(3-5) it should already have
proved possible to study renal amino acid transport at the tubule level. Yet it is only recently that
such studies have been undertaken
(6-9).
From the 1)epartment
of Physiology,
University
of Sydney,
Sydney,
N.S.W.
2006, Australia.
This review was prepared
from a paper presented
at the 5th
annual
meeting
of the Australasian
Society
for Nephrology
and
the 3rd Kanematsu
Institute
Conference
on the Kidney,
held at
Sydney
Hospital
in October
1969.
Received
Nov. 17, 1970; accepted
Nov. 25, 1970.
Why has the tubular
transport
of such compounds been so long neglected?
The reasons are
several and they are all compelling:
(a) There are a
great many different amino acids, and chemically
they are quite diverse.
(b) Since all cells must
transport
amino acids for their own metabolic
purposes
it is difficult
to distinguish
between
simple cell uptake
and trans-cellular
transport.
(c) There exists not one (or even four) distinct
amino acid transport
mechanism,
but rather
a
mosaic of neatly fitting processes,
each with its
own substrate
specificity,
affinity, and transport
rate. (d) Amino acid transport
is interlocked
with
the transport
of electrolytes
and other organic
compounds.
(e) The lack of specific chemical,
let
alone microchemical,
methods
for amino acid
determination
makes it necessary
first to separate
amino acid mixtures into their components
before
one can begin to measure them individually.
Even
then, the ninhydrin
reaction,
which one uses to
measure
amino acids, is not easy to work with.
None of these reasons need deter one from studying
amino acid transport
but, taken together, they have
discouraged
investigation.
Although
it has been 53 years since it was first
realized
that amino acids were absorbed
in the
CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 245
nephron, no review of the topic has been published.
At a time when techniques
are at last available
which permit a detailed study of the problem to be
made it seems highly
desirable
that
available
knowledge be summarized.
Historical Aspects
\hen
Ludwig,
in 1844, first proposed
his theory
of glomerular
filtration
and selective renal tubular
reabsorption
(10-12) to explain the formation
of
urine he stated that plasma-water
and salts dissolved in it would be filtered across the glomerular
capillary
walls whereas
proteins
and fats and
minerals
bound to these substances
would not.’
It was not until many years later that anyone
sought to explain the presence or absence of amino
acids in urine in terms of his theory. Indeed, before
1900, amino acids were not known to be normal
constituents
of the urine (12, 13) although
they
had been recognized
as occurring
in pathological
urine since 1810, when Wollaston
(14) described
the first recorded case of cystinuria.
Wollaston
did
not, of course, recognize the chemical nature of the
compound.
Slowly, from 1820 on, when Bracannot
first isolated
glycine
and leucine from protein
hydrolysates,
the realization
developed that amino
acids were the sole products
of protein hydrolysis
and that they occur in the free form widely
throughout
the animal kingdom
(15). Eventually,
in 1900, Pfaundller
(16) demonstrated
that small
quantities
of amino acids were present in normal
human urine.
In 1908, Sorensen
(17) published
a reliable
titrimetric
method for the determination
of total
a-amino-nitrogen,
which was soon used to determine the normal
a-amino-nitrogen
excretion
of
humans; this was found to be about 1 to 2% of the
total urinary
nitrogen
(18, 19). This finding was
confirmed
by many subsequent
workers
(20-24),
most of whom
measured
the urinary
aminonitrogen
excretion
of both normal
subjects
and
patients
suffering
from a variety
of diseases,
especially hepatic disease. An accurate
gasometric
method,
suitable
for the determination
of the
amino-nitrogen
content
of blood plasma,
was
devised by van Slyke in 1912 (25, 26), who used it
to determine
the plasma amino-nitrogen
concentration in normal human subjects.
He pointed out
that no previous
investigator
had succeeded
in
demonstrating
that free amino
acids normally
circulate
in the plasma,
although
a number
of
‘“Wir nehmen nun aber hypothetisch
Gefasswandungen
die Eigenthumlichkeit
flussigen und aufgelosten Bestandtheilen
einen Theil der Extractivstoffe
und die
gelosten Saize durch sich hindurchtreten
s#{228}mmtliche
Proteinsubstanzen,
die Fette
Verbindung
befindlichen
mineralischen
hindurchiassen”
(10).
weiter an, dass diese
besitzen,
von den
des Blutes nur Wa.sser,
freien nur im Wasser
zu lassen, w#{228}hrend
sie
und die mit beiden in
Bestandtheile
nicht
246 CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971
workers
had given amino acids parenterally
to
animals and demonstrated
that increased amounts
of amino-nitrogen
appear
in the urine
(27-31).
Similarly,
Folin and Denis (32) had fed amino
acids to cats and demonstrated
indirectly
(by
measuring
total plasma nitrogen
and subtracting
urea and protein nitrogen)
that some of the aminonitrogen had been absorbed from the intestine
and
appeared in the plasma.
By this time, Ludwig’s
mechanistic
theories
(10-12)
concerning
the formation
of urine were
beginning
to gain
ascendancy
over
those
of
Heidenhain
(33) who claimed
that
urine was
formed
by a process
of both glomerular
and
tubular
secretion.
Ludwig’s
theories,
interpreted
in the light of subsequent
knowledge,
required that
if amino acids circulated
freely in the plasma, unbound to protein, then they must be freely filtered
across the glomerulus,
and, since only a small
amount appeared in normal urine, then reabsorbed
in the nephron.
In 1913, Abel and his colleagues
(34-37), using their brilliantly
devised technique
of hemodialysis
in the living animal, demonstrated
that plasma amino acids did indeed circulate freely
in the plasma and were readily filterable
across a
semipermeable
membrane.
To
adherents
of
Ludwig’s
theories
this demonstration
made the
concept
of renal tubular
reabsorption
of amino
acids almost inescapable.
Cushny
(38) in the first
edition
(1917) of his famous
monograph
“The
Secretion of Urine” said:
“The proportion
of urea to the total
non-protein
nitrogenous
bodies is much higher in the urine than in
the blood. Thus the nitrogen
of urea makes up about
40% of the total non-protein
nitrogen
of the blood,
that of the mono-amino
acids rather less than 40%,
while in the urine the urea nitrogen
is 80% of the
total nitrogen
and that of the amino-acids
only 3%”
(footnote to p 17).
and
“The cells lining the tubule
thus absorb
from the
glomerular
filtrate a slightly alkaline fluid containing
sugar, amino-acids
and other similar food substances,
and chloride, sodium and potassium
in approximately
the proportions
in which they are present in normal
plasma, or in the artificial mixtures
which have been
introduced
for the perfusion
of surviving
organs”
(p4fl.
At the time of the second edition
of his monograph
(1926), little further
information
was to
hand, and Cushny (39) merely repeated
his earlier
remarks, commenting
that:
“.
. . it
may be assumed that there is a high threshold
for such readily available
foods as the amino-acids,
perhaps equal to that of dextrose”
(p 12).
Between
1917 and 1938, the modern
theories of
renal function
were more adequately
formulated
and subjected
to experimental
proof. Thus gbmerular
filtration
and tubular
reabsorption
were
demonstrated
directly
in animals
by micro-
puncture
(40, 41) and were quantitated
indirectly
in man (42-44);
tubular
secretion
of exogenous
substances
was established
(45); the concepts of
glomerular
filtration
rate and of renal clearance
were formulated
(46, 47); and the concept of a
maximum
(Tm) to reabsorption
and excretion was
formulated
and established
experimentally
(4850). These developments
[reviewed fully by Smith
(51) and Pitts (52)1made it possible to re-examine
the manner
in which the kidney was thought
to
handle amino acids and to propose and test hypotheses
by analogy
with the renal handling
of
such substances
as glucose.
The study of amino acid transport
has been
greatly facilitated
in recent years by a number of
technical
advances.
With a few exceptions
(53)
there had been no specific methods
available
to
determine
the concentration
of individual
amino
acids in a mixture until 1943 to 1944, when microbiological
analyses
and paper
chromatography
were introduced.
Since then, methods
have been
developed
and improved
which
have made it
possible for individual
amino acids to be studied
rather
than
total
a-amino-nitrogen:
microbiological analyses
(54-56), specific enzymic analysis
(57), paper chromatography
(58-60), ion-exchange
column chromatography
(61), high-voltage
paper
electrophoresis
(62-65),
thin-layer
chromatography
(66), and gas chromatography
(67).
The
ready commercial
availability
of amino acids with
‘4C, H, and other radioactive
labels has also
helped to make study of amino acid transfer more
specific.
Site of Amino Acid Reabsorption
within the Nephron
It is only since the 1950’s that direct experimental
evidence
has been produced
to localize
transport
of amino acids to the proximal
tubule.
However,
clinicians,
on the basis of indirect
evidence, had long suspected that amino acids were
reabsorbed
in the same segment of the nephron as
was glucose. Thus, cases of hereditary
diseases had
been described
(associated
with the names
of
de Toni, Fanconi,
Lignac, and Debre)
in which
there appeared
to be an associated
defect in renal
tubular
reabsorption
both of amino acids and
glucose (68-74).
Hence, when Walker
(75) used
micropuncture
techniques
to localize glucose reabsorption
to the proximal tubule, the presumption
was strong that this would also be the site of amino
acid transport.
Furthermore,
there was a large,
albeit conflicting,
body of morphological
data to
suggest that there were anatomical
abnormalities
in the proximal
tubules of patients
suffering from
the Fanconi group of syndromes
(69, 76-82).
A more direct attempt
to localize amino acid
reabsorption
was made by Neame
(83, 84), who
demonstrated
that
incubated
slices of kidney
cortex could actually
concentrate
the L-forms of
histidine, proline, tyrosine, and ornithine
although,
in comparison
to other tissues such as brain and
intestine,
the concentration
gradients
established
were not very remarkable.
Nevertheless,
since
kidney cortex slices consist principally
of proximal
tubular segments, the presumption
was that amino
acid uptake
was related
to normal
proximal
tubular
amino acid reabsorption.
This and many
subsequent
studies of cortex slices suffer from the
defect that one has no knowledge
of the direction
of the amino acid transport
processes under investigation.
Thus, one cannot
distinguish
between
uptake from the luminal and from the interstitial
surfaces
of the cells, nor can one distinguish
between simple uptake and trans-cellular
reabsorption. This latter
objection
also applies
to the
histochemical
studies of Lee et al. (85, 86), who
demonstrated
that amino acid droplets
appear in
the cytoplasm
of proximal
tubular
cells after
administration
of amino acids to the rat. The
picture
emerging
from these various studies has
become even more confused since the demonstration that slices of kidney medulla and papilla can
also concentrate
amino acids (87, 88).
The technique
of stop-flow
analysis
(89) provided a tool for more direct localization
of renal
tubular
amino
acid transport.
These
studies
demonstrated
that the naturally
occurring
amino
acids were reabsorbed
in a segment of the nephron
coextensive
with
glucose
reabsorption
and
p-aminohippurate
secretion
(90-96). Although
an
attempt
to use this technique
to localize amino
acid transport
more precisely to various segments
within the proximal tubule has been made (92, 97),
it seems clear that this is not really possible with
the stop-flow
technique
(96). Figure 1 illustrates
typical amino acid stop-flow patterns
for the rat
kidney.
By injecting
so-called area-specific
toxins either
into the circulation
or retrograde
up the ureters,
Wright and Nicholson
claimed to be able to produce localized lesions in various segments
of the
nephron.
From such studies
they reported
that
amino acid reabsorption
occurred
principally
in
the more proximal parts of the proximal tubule with
subsidiary
reabsorptive
sites located
in the remainder
of the proximal
tubule and in the distal
tubule (98-100). They also claimed to have demonstrated
an amino acid secretory
site in the more
distal part of the proximal
tubule.
These studies
have not met with general acceptance,
however,
because it is difficult
to predict
just what nonspecific effects the so-called “area-specific
toxins”
might have on kidney tubule cells.
The most direct evidence
for localization
of
amino acid reabsorption
to the proximal
tubule
comes from a few recent micropuncture
and microperfusion
experiments.
Bergeron
(101) injected
radioisotopes
of leucine and lysine into the lumen
CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 247
LIECTIN0
pucr
cells has demonstrated
that these structures
can
bind amino acids specifically.
Similar findings for
D-glucose have also been reported
(105). Brush
borders occur only on proximal tubule cells; this is
strong evidence
that the proximal
tubule is the
major site of amino acid reabsorption.
PROXO-jAL
:--C
C0,
4
075
05
0?5
Active or Passive Transport
TAURINE
Cc’
6
Cc
_,,.j-GLUTAMICACID
100
no
ALAN ME
-
35
25
Cc
______,.
CREATININE
_
4
60
___
1
CUMULATIVE
2
S
URINE
MASS,
6C
0
fl
Fig. 1. Stop-flow analysis of renal tubular reabsorption of
glycine, serine, taurine, glutamic acid, and alanine in a
rat undergoing osmotic diuresis
The rat was infused intravenously at a rate of 0.39 mI/mm with
physiological bicarbonate-saline
containing, per liter, 80 g of
creatinine (as osmotic diuretic), 1.0 g of p-aminohippuric acid
(PAR)
and 0.03 mole each of L-phenylalanine, L-alanine, Lglutamic acid, taurine, and L-arginine.
After 30 mm two clearance collections (A and B) were performed and then the ureteric
catheter was obstructed for 3 mm. The obstruction was
then released and the emergent urine was collected in small
drops. Subsequently, two more clearance collections (C and D)
were performed. The urine to plasma concentration ratio (U/Pc,)
is shown to indicate the region of distal nephron water reabsorption. The PAH/creatinine clearance ratio (C,.AH/cC,)
is shown to
indicate the site of proximal tubular PAR secretion. The amino
acid/creatinine clearance ratios are so shown; it can be seen
that the four amino acids and taurine were reabsorbed in a segment of the nephron coextensive with i’.ii
secretion. LTaken
from Young and Edwards (95)1
of proximal
tubules
and, with autoradiographic
techniques,
demonstrated
uptake of radioactivity
by the cells of the entire proximal
tubule. In later
microperfusion
studies
(6, 102), labeled
amino
acids were injected
into the proximal
convoluted
tubule,
the pars recta, and the distal tubule, and
extensive
uptake
of radioactivity
in both the
proximal
convolution
and the pars recta
was
demonstrated.
The technique
of microperfusion
between
oil blocks (3) has recently
been used to
quantitate
reabsorption
of i-histidine
and glycine
from the proximal tubule (7-9). Although
all these
studies have unequivocally
identified the proximal
tubule
as the principal
site for amino acid reabsorption,
they cannot he said to have excluded
the possibility
that a small fraction of amino acid
reabsorption
occurs more distally, as is now known
to occur in the case of D-glucose (103).
Another
very recent study (104) with isolated
suspensions
of brush borders from kidney tubule
248 CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971
The fact that a great many amino acids are
present
in the urine in concentrations
less than
those in plasma
strongly
suggests
that there is
active reabsorption
of these amino acids in the
nephron.
Even when the urine/plasma
ratios for
naturally
occurring amino acid are greater than one,
the values, when corrected for distal tubular water
reabsorption,
suggest
that
their
concentrations
in the proximal
tubule would be less than those
in plasma
(106, 107). More direct evidence
of
active reabsorption
of amino acids comes from
stop-flow studies (90, 91, 94-97). In such studies,
the clearance
ratio of amino acid to inulin or
creatinine
was shown to decrease in the proximal
tubule
to values less than unity,
indicating
reabsorption
against
a concentration
gradient.
As with other tissues, renal cortex slices can be
shown to take up amino acids against a concentration
gradient,
which is suggestive
of active
transport.
As mentioned
above,
however,
the
direction of amino acid transport
cannot be ascertained from such studies.
Slices do offer one advantage:
it is possible to study the effects of metabolic inhibitors
on amino acid uptake mechanisms.
Thus, Schwartzman
et al. (108) and Segal et al. (109)
were able to inhibit
the active accumulation
of
amino acids in kidney slices with dinitrophenol
and
with anaerobiosis.
Using an isolated tubule preparation,
Hillman
(110) was able to inhibit amino
acid uptake with both dinitrophenol
and cyanide.
More recently,
he (104) studied
the binding
of
proline
to isolated
proximal
tubule
cell brush
borders (which would presumably
be a step in the
reabsorptive
process)
and found
that
it was
partially
inhibited
by cyanide and dinitrophenol.
The demonstration
of active transport
does not,
of course,
exclude
the possibility
that passive
transport
also
contributes
to
reabsorption.
Schwartzman
et at. (111) have demonstrated
autoand hetero-exchange
diffusion of dibasic amino acids
in rat kidney cortex slices; thus they observed increased accumulation
of a labeled amino acid when
the slice had been preloaded
with the same amino
acid (auto exchange)
and with a different
amino
acid (hetero exchange).
Such a phenomenon
had
been noticed much earlier in the Ehrlich ascites cell
(112, 113), although none of the neutral amino acids
that underwent
exchange
diffusion in the Ehrlich
cell was able to undergo exchange diffusion in the
kidney cortex slice. This exchange diffusion of the
dibasic amino acid, surprisingly
enough, is more
susceptible
to metabolic
inhibition
than is active
transport.
However,
as with active transport,
the
direction
of the exchange diffusion cannot be determined
in the kidney slice.
Although
the above evidence points toward the
existence
of active and passive reabsorptive
processes that require a supply of oxidative
energy,
microperfusion
of tubules
has recently
provided
evidence which suggests that aerobic metabolism
is
not always an essential
prerequisite,
at least for
histidine
reabsorption
(8). In these experiments,
tubular
reabsorpiion
of i-histidine
was significantly depressed
by the inclusion
of 40 mmolar
cyanide
in the perfusion
fluid. However
a large
part of the reabsorption
remained
uninhibited,
suggesting
the presence
of some passive process,
or at least one not inhibited
by cyanide
(see
Figure 2). A similar study on glycine transport
in
the rat nephron
showed that a component
of its
absorption
was not inhibited
by either cyanide or
dinitrophenol
(9).
mulation
and amino acid reabsorption
is far from
certain. Jacquez (115) postulated
that a zwitterion
component
such as phosphatidyl
choline might
play a role in amino acid transport,
at least in the
Ehrlich ascites cell, but subsequently
Schwartzman et at. (116), in work with the kidney cortex
slice, excluded
the possibility
of phosphatidopeptides’ playing a carrier role.
The most promising
model that seems suited for
the investigation
of carrier properties
is the isolated
brush-border
preparation.
This is already
being
exploited
for study of the glucose carrier
(105),
and the first amino acid studies
have recently
been reported
(104). These membrane
fragments
were shown to be able to bind i-proline
by a
saturable
and inhibitable
process.
More work
utilizing this technique
may illuminate
this rather
difficult problem.
Carrier
The renal transport
of amino acids is influenced
by the presence of a number of other substances
such as electrolytes
and sugars in the glomerular
filtrate and the renal capillary
blood. The extent
and the nature
of the interactions
occurring
between some of these substances
and amino acids
during reabsorption
are discussed
in this section.
Our knowledge
of the nature of the carrier or
carriers involved in amino acid transport
is rather
scanty. This is largely due to the lack of a suitable
experimental
preparation
with which to work.
The kidney cortex slice technique
was exploited
for this purpose
by Elsas and Rosenberg
(114),
who investigated
the effect of puromycin,
a protein
synthesis
inhibitor,
on amino acid accumulation.
They found that this accumulation
depended
on
synthesis
of a protein with a relatively
long halflife, although
the relationship
between slice accu-
I
I
I
80
0
60
1
End ni Proximol
0
Tobole
40
z
0
2(’
100% REABSORPTION
#{149}
138
picooIe$/mjn.
z
PERFUSED
LENGTH,
mm
Fig. 2. The effect of sodium cyanide on proximal tubular reabsorption
of L-histidine
in the rat
The left kidney was prepared for tubular microperfusion (3). Two
techniques
of microperfusion
were used: microperfusion
of
short segments between oil blocks and microperfusion of whole
nephrons. Control data from 34 animals is indicated by the shading. Approximately
80 to 90% of the infused histidine load was
reabsorbed by the time the fluid reached the end of the proximal
tubule. In five animals (indicated by the filled circles) 40 mmol of
sodium cyanide per liter was incorporated in the perfusion fluid.
Reabsorption
can be seen to have been partially inhibited
although about 70% of the infused load was still reabsorbed by
the end of the proximal tubule (Freedman and Young, unpublished data, 1970)
Interactions of Amino Acids and Other
Molecules in Transport
Cations
The two major body cations, sodium and potassium, have been shown to influence
renal amino
acid transport
(117), but the nature of the interaction is poorly understood.
Because
renal and
intestinal
amino acid transport
mechanisms
are
similar in many respects
(2) it may be useful to
consider what is known of cation-amino
acid interactions in the intestine.
It has been shown quite
conclusively
that transintestinal
amino acid flux is
related to the sodium concentration
of the medium
bathing
the intestine
but is independent
of the
bulk cellular
sodium
content
(118-120).
Two
hypotheses
have been advanced
to explain
this
interaction.
The first one (119, 121, 122) is basically
an extension of Crane’s ideas about sugar transport
(123, 124). Sodium is thought
to be involved
in
forming a ternary
complex with the amino acid
and the carrier,
which
leads in some way to
transport
of both
molecules.
The
alternative
hypothesis
(125) attributes
the sodium-amino
acid relationship
to an intracellular
sodium requirement for the coupling of metabolic energy to active
transport;
in this model
the concentration
of
extracellular
sodium is important
only because it
influences
the
concentration
of intracellular
sodium. Recent data support
the first hypothesis
(118, 120) [similar
conclusions
have also been
CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 249
reached
from studies of the Ehrlich
ascites cell
(126)].
The nature
and even the existence
of such a
relationship
in the kidney is far more equivocal.
As yet, only the technique
in which kidney slices
are studied in vitro has lent itself to the investigation of this relationship.
Fox et at. (117) demonstrated
a decreased
amino acid accumulation
in
cortex slices in the presence of low Na concentrations, with complete inhibition
of the accumulation
of some amino acids in a Na-free medium.
However, lysine accumulation
could not be abolished
even in Na-free media; ouabaine,
which inhibited
the Na-sensitive
accumulation,
had no effect on
the Na-insensitive
component
of lysine accumulation.
In the same study,
these authors
also
demonstrated
the dependence
of amino
acid
accumulation
on K concentration
in the medium,
with
optimal
accumulation
occurring
over
a
narrow
range of K concentrations.
More recent
studies
with isolated
tubule
and brush-border
preparations
have shown that amino acid transport involves at least two steps, binding
to the
brush
border
and subsequent
active
transport
(104, 110). The initial binding process is much less
dependent
on Na ions than is the subsequent
transfer of the amino acid into the cell (104).
A number
of subsequent
studies have thrown
some doubt on the interpretation
of results with
cortex slices. Segal and Smith (127), working with
kidney slices of newborn rats, showed that a Nafree medium
actually
enhanced
the concentrative accumulation
of lysine. Such a paradoxical
increase
in lysine uptake
in Na-free
media was
also reported for the toad bladder (128), but there
is as yet no explanation
of this phenomenon.
Schwartzman
(111, 116) was unable
to inhibit
lysine or arginine
accumulation
with a Na-free
medium, which is at variance with the observation
that lysine transport
is inhibited
in a Na-free
medium, as was previously
reported from the same
laboratory
(117). This discrepancy
is as yet unexplained,
but could be a result of the different
experimental
conditions
used in the two series of
experiments.
The above studies have been concerned with the
dependence
of amino acid transport
on cation
concentrations.
Some recent studies suggest that
salt and water transport
may depend in part on
sugar and amino acid transport.
Thus, the human
jejunum has been shown to have a low reflection2
coefficient
for sodium
chloride,
with the consequence that a major fraction
of salt and water
absorption
by the jejunum is the result of solvent
When a membrane separates two solutions of the same solute
but of differing concentrations,
the osmotic pressure difference
actually developed across the membrane is usually less than that
which could be expected
from the osmolality gradient. The ratio
of the ob8erved osmotic pressure gradient to that which could be
expecied by calculation
is defined as the “reflection coefficient”.
250 CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971
drag consequent
on the active
absorption
of
bicarbonate,
sugars, and amino acids (129). Since
the proximal
tubule of the kidney also has a low
reflection
coefficient for sodium chloride
(130) a
similar mechanism
relating salt, water, and amino
acid transport
may operate there.
In addition
to the above interactions,
an interesting and seemingly
unrelated
phenomenon
has
been reported
by several authors
(94, 131, 132).
It has been found that potassium
secretion by the
distal tubule can be sharply stimulated
by infusing
dibasic amino acids. The mechanism
of this interesting occurrence is unclear.
Sugars
The presence of glucosuria with associated amino
aciduria
in the Fanconi
syndrome
(69, 71-73)
suggested either a lesion in a common reabsorptive
site or a defect in a step common to the reabsorption of both. It is interesting
to note, therefore,
that a number
of sugars have been shown to
interact
with amino
acids in renal
transport
mechanisms.
Studies in vivo by use of clearance
techniques
have demonstrated
some of these interactions.
Glucose infusions
impair the renal absorption
of
amino acids (133). Phiorizin,
a competitive
inhibitor of glucose transport,
also inhibits
amino
acid absorption
to some extent (134). Some amino
acids-lysine,
glycine, and alanine-depress
glucose reabsorption;
others, including
aspartic
acid
and leucine, have no effect (135).
Thier (136) used the in vitro slice technique
to
demonstrate
that uptake
of some neutral
amino
acids was inhibited
by glucose,
galactose,
and
fructose.
These sugars were without
effect during
anaerobiosis
or in the presence of 2,4-dinitrophenol,
and had no effect on the accumulation
of histidine
and lysine, both of which had been shown previously to be taken up both by sodium-dependent
and sodium-independent
pathways
(117). These
findings,
together
with a failure
to observe
a
hexose-induced
change of affinity of amino acids
for the transport
sites which would be expected of
competitive
inhibition,
led Thier (136) to suggest
that the basis for the interaction
was probably
a
common
dependence
on a sodium-dependent
ase
that provides
the energy for the transport
systems.
Work on the glucose-amino
acid interaction
in the intestine
has progressed
a little
further
than in the kidney,
and points
to the
relationship
being due to some common
factor,
possibly the energy source required
for the transport of both (137).
Other Interactions
A number
stances
have
of other naturally
also been shown
occurring
to interact
subwith
amino
acids during
their transport.
Thus,
the
reabsorption
of sulfate was found to be depressed
by amino
acid infusions
(138),
and stop-flow
studies have since shown that sulfate and amino
acids are reabsorbed
in the same segment
of
nephron
(93). The reabsorption
of phosphate
has
also been linked to amino acid transport.
Thus
phosphate
reabsorption
is reduced by amino acid
infusions
(133, 139). Similarly
vitamin
D deficiency has been shown in man to impair renal
amino acid reabsorption
(140). Scriver (141) used
rats
with
experimentally
induced
vitamin
D
deficiency
and obtained
evidence
to suggest that
low serum calcium concentrations
stimulated
the
release of parathormone,
which acted in some way
to impair both renal amino acid and phosphate
reabsorption.
The dependence
of amino acid reabsorption
on
hydrogen ion concentration
has been demonstrated
in the isolated perfused
rat kidney by Weiss and
his colleagues
(142). It was found that as the
urinary pH was increased
from 5 to 8, amino acid
reabsorption
decreased
for all the amino acids
studied, and a few amino acids even exhibited
net
secretion
at the higher pH values.
In another
study with the same technique
(143), they found
that as the urine pH was increased
from 5 to 8,
the secretion of methyl and ethyl esters of some of
the amino acids used in the earlier study was
greatly decreased until there was a net reabsorptive
flux at the highest pH’s. The authors interpreted
the results in terms of a pH-dependent,
nonionic
diffusion
process operating
for these substances.
However
certain reservations
about the viability
of their isolated perfused kidneys must be kept in
mind when considering
the results. For example,
the histidine/inulin
clearance in this isolated perfused kidney was never less than 0.25 at any urinary
pH, whereas in clearance experiments
in the intact
rat the clearance
ratio at similar histidine
loads
was less than 0.025 (96). In addition,
there is an
apparent
contradiction
between
results
for the
isolated
perfused
kidney
and the kidney
cortex
slice. Segal (109) demonstrated
a marked inability
of the slice to concentrate
amino acids at pH 5 to
6, in which range the perfused
kidney
was reabsorbing
amino acids maximally,
while at pH
8 to 9 the slice showed a marked increase in concentrating
ability,
a range
in which
the perfused kidney
showed
a greatly
diminished
reabsorption
to the point of a change in the net
flux to secretion.
This discrepancy
highlights
the
difficulties
in the interpretation
of data obtained
with kidney cortex slices.
Renal Handlin9 of Optical
Isomers of Amino Acids
The possibility
that the kidney might handle Dand L-isomers3 of amino acids differently
did not
occur to many early workers.
Indeed, it was not
until the studies of Gibson and Wiseman
(144),
who demonstrated
optical specificity
of intestinal
amino acid transport
systems,
that the possibility that renal amino acid reabsorption
systems
might
show such specificity
was seriously
examined.
Wohlgemuth
(31), in 1905, gave a racemic mixture of amino acids to a rabbit and observed
that
urinary
amino acids during the next 24 h were
predominantly
of the D-form. He interpreted
this
as evidence
for differential
rates of metabolism
of the two isomers and did not consider the possibility of there being differential
renal handling
of the isomers. Subsequent
studies (28, 145-1 47)
confirmed
their findings and explained
them in a
similar fashion. However,
Albanese et at. in later
experiments
with arginine
(148) discovered
that
the liver could metabolize
both isomers equally
rapidly,
so he postulated
that there might be a
differential
renal threshold
for the L- and D-forms.
This conclusion
was also drawn from many subsequent studies (149-153).
Crampton
and Smyth (57) were the first workers
specifically
to investigate
the renal handling
of
the optical isomers of natural
amino acids. They
confirmed
that the kidney of the cat did indeed
have a lower threshold
for the D- forms of histidine, alanine, and methionine
and inclined to the
view that D- amino acids were absorbed
merely
by passive diffusion.
A number
of studies since
1953 have extended
the range of observations
to
include many other animals and a wide variety of
amino acids (138, 154-157).
Webber
(158) studied
the renal tubular
reabsorption of D- and L-aspartiC acid and their influence on the excretion
of other amino acids by the
dog. He found that both forms were reabsorbed
by
‘The
nomenclature
for designating
the optical
isomers
of
amino acids has changed
somewhat
during the last 50 years.
Before the publication
of a paper by Wohl and Freudenberg
in
1923 (235) [cited by Vickery (236)1 it was the custom to prefix
amino acids with an italicized
lower case d or 1 to indicate
the
direction
of optical
rotation.
Under
this system
a number
of
amino acids occurring
in animal protein
(the “natural”
forms)
were called dextro (d) amino acids although
it was recognized
that
all the natural
amino
acids
had
the same
configuration
about the a-carbon
atom.
These
amino
acids were: alanine,
arginine,
aspartic
acid, glutamic
acid, isoleucine,
lysine,
ornit.hine, threonine,
and valine. The system
proposed
by Wohi and
Freudenberg
(235) required that all the natural amino acids be
designated
with an italicized
lower case 1 and the optical rotation
was indicated
by a + or - after the 1. For a number of reasons
the system
never worked fully and eventually
Vickery
(236), in
1947, proposed the system now in use. The natural and unnatural
forms of amino acids are referred to not as levo and dextro but as
“ell” and “dee” and they are written as small capital letters t
and 1), connected
to the proper or trivial names of the amino
acids with a hyphen.
Recently
the absolute
configuration
of
natural amino acids about the a-carbon
atom has been determined
and, conveniently,
found to correspond
to the n form
which had arbitrarily
been assigned
to it (237). It is essential
to
be aware of these changes in nomenclature
if older papers are to
be read correctly.
CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 251
a Tm-limited
transport
mechanism
and that the
Tm for D-aspartic
acid was only slightly less than
for the L isomer. Both isomers had similar inhibitory effects on i4-glutamic acid reabsorption.
It is
unfortunate,
however,
that Webber’s
analytical
technique
could not distinguish
between
the Land D-forms of aspartic
acid, thus preventing
him
from excluding
the not unlikely
possibility
of
conversion
of the D- to the L-form in the body.
Young and Edwards
(96) have shown that L-a
methyldopa
has a competitive
inhibitory
effect on
the reabsorption
of L-histidine
whereas D-methyldopa was without inhibitory
effect.
From the above it seems clear that the kidney
has a lower threshold
for the transport
of most of
the D-isomers than for their corresponding
L-forms.
However the inference drawn by most authors that
D-amino acids are merely reabsorbed
by passive
diffusion seems open to some doubt. (a) a-amino
acids can be absorbed rather more readily than one
would expect for a merely passive process. Thus,
for example, DL-methionine
was reported to have a
maximum
clearance
of only 5% of glomerular
filtration
rate (159). (b) A Tm value for D-aspartic
acid has been demonstrated
(160). (c) The ratio of
the clearance of D-amino acids to that of inulin has
been shown to increase as plasma levels of the Damino acid increase
(57), which suggests that Damino acid absorption
is a saturable
process.
(d)
By analogy with the intestine
one would expect at
least D-methionine
to be actively reabsorbed,
because it has been demonstrated
that rat intestinal
mucosa can move this amino acid against a concentration
gradient (161).
Embryonic and Postnatal Development of
Renal Amino Acid Transport
It was known as early as 1911 that infants excreted relatively
more amino acids in the urine
than adults (162). This has been amply confirmed
by many other workers
(163-1 72). An especially
complete study was that of Brodehl and Gellissen
(173), who measured
endogenous
clearances
of 17
amino acids as well as glomerular
filtration
rate in
infants and older children and calculated
the fractional reabsorption
of each amino acid studied. In
infants they found a decreased efficiency of absorption of threonine,
serine, alanine, valine, phenylalanine, glycine, histidine,
cystine, lysine, arginine,
ornithine,
and proline. However,
methionine,
leucine, isoleucine,
and tyrosine
were absorbed
as
efficiently as in older children. The authors suggest
that these results are best explained by postulating
a degree of glomerulo-tubular
imbalance
in the
infant-i.e.,
the amount of tubular tissue had not
developed sufficiently
to keep pace with developing
glomerular
filtration.
This view is supported
by the
finding of higher values and greater heterogeneity
for the ratio of glomerular
surface area to proximal
252 CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971
tubular volume in infants than in children or adults
(174). However,
the demonstration
that
some
amino acids are more involved than others in this
general
decrease
in reabsorption
points
to the
implication
of other factors, such as the maturation
of specific transport
systems.
Webber (175) has observed a similarly increased
amino acid excretion
in immature
rats, which occurred in spite of reduced
filtered loads. He accounted for this in the rat in the same terms as did
Brodehl
and Gellissen
(173) for the human
(see
above).
Recently,
Cooke
and Young (1971, in
preparation)
studied amino acid transport
in the
embryonic
and postembryonic
chick. They found
that the fractional
reabsorption
of the basic amino
acids, arginine and lysine, and the neutral amino
acid, phenylalanine,
was at least as complete in the
20-day-old
embryo as in older birds. On the other
hand, the fractional
absorption
of alanine
and
glycine was less in the embryo
than in hatched
birds. The authors
felt that their data suggested
that the basic transport
system for amino acids may
have developed earlier than that for neutral amino
acids.
Because of the difficulty
of working with embryos,
a number
of developmental
studies
on
amino acid transport
have been carried out with
kidney cortex slices. Webber has observed a more
rapid initial uptake
of amino acids in mature
as
compared with newborn rat kidney tissue, although
the final concentration
ratios attained
were always
lower in the mature tissue (176, 177). He suggested
that, despite the greater concentrative
ability of
newborn kidney tissue, the finding of slower initial
uptake
could possibly
account
for the decreased
reabsorptive
capacity
of the immature
whole
kidney. However a later study by Segal and Smith
(127), who used higher substrate
concentrations,
has failed to demonstrate
a lower initial uptake
rate for lysine in the immature
kidney.
Renal Amino Acid Transport Groups
Although
all amino acids have in common the
general configuration
around the a-carbon
atomi.e., a hydrogen
atom, an amino group, a carboxyl
group, and an R-group--the
differences
in the Rgroup among individual
amino acids allow for a
useful classification
to be made on the basis of
structure
alone. Such a classification,
as shown
below, provides
a framework
for studying
transport grouping, without prejudging
function.
Monoaminomonocarboxylic
acids with aliphatic
chains (glycine, alanine, serine, and threonine).
side
Monoaminomonocarboxylic
acids with branched
chains (valine, leucine, and isoleucine).
side
Aromatic
Heterocydic
amino
acids (phenyinlanine
amino acids
Monoaminodicarboxylic
aspartic acid).
(tryptophan
acids
and tyrosine).
and histidine).
(glutamic
acid
and
Diaminomonocarboxylic
ornithine).
Diaminodicarboxylic
acids
arginine,
and
-9
amino
(cystine).
acids
(cysteine,
C
cystine,
and
Amide-group amino acids (glutainine and asparagine).
Imino acids (proline and hydroxyproline).
On the basis of their great variety in structure,
it
would not seem likely that one transport
mechanism could serve for the reabsorption
of all of these
amino acids. Indeed, evidence obtained
from competition
studies in vivo and in vitro, as well as
“experiments
of nature” involving genetic defects,
suggests the existence of at least four amino acid
transport
systems
in mammalian
kidney.4 These
four systems will be called, for convenience,
the
neutral,
basic, acidic, and iminoglycine
systems.
The amino acids transported
by each system are
enumerated
below and the experimental
evidence
for the existence of each system will be discussed.
Neutral system
(a) Aliphatie
and branched
chain amino
(b) Heterocyclic
amino acids
(c) Aromatic amino acids
(d) Amide group amino acids
(e) Methionine
and cysteine
#{149}
Basic system
(a) Diaminomonocarboxylic
acids
(b) Diaminodicarboxylic
acids (possibly)
#{149}
Acidic system
(a) Monoaminodicarboxylic
acids
#{149}
Iminoglycine
system
(a) Imino acids
(b) Glycine
‘In
addition to the four major transport
systems, there is
some evidence to suggest that there may be a fifth system for
of -alanine,
fl-aminoisobutyric
acid,
The system has been demonstrated
in Ehrlich
(238) and in mouse (239) and human (240) kidney.
of taurine in rat kidney has also been demonstrated
analysis
[See Figure
1 and
I
(96)1.
,..-7
C,
.6
‘
.4
1
Cp
10
“cr6
-,
.4
CUIAIJLATIVE
Fig. 3A. Stop-flow
of L-a-methyldOpa
analysis
URNE
.5
l.A5S
of renal
tubular
reabsorption
in a rat undergoing osmotic diuresis
The animal was infused intravenously at a rate of 0.39 mI/mm
with a physiological salt solution containing, per liter, 80 g of
creatinine, 1.0 g of p-aminohippuric
acid, and 1 mmol of L-methyldopa-2-’4C. The experimental protocol was the same as that
used in Fig. 1. The experiment demonstrates proximal tubular
reabsorption of the synthetic a-methyl amino acid. [Taken from
Young and Edwards (96)]
acic1,
Neutral system. Genetic
evidence
for the existence of a separate
transport
mechanism
for the
neutral amino acids has come from studies of the
rare, recessively
inherited
phenotype
known as
Hartnup
disease
(106, 157, 178-183).
Cusworth
and Dent (180) measured
amino acid clearances
in
this disease, and found that the clearances
of all
the neutral amino acids were greatly increased,
except that of glycine, which was only slightly
affected, and proline, which was not affected at all.
Various authors have demonstrated
competition
for reabsorption
between
pairs of neutral
amino
acids (6, 184). More comprehensive
studies have
shown the effect of infusion of single neutral amino
acids on the excretion
(185-187) and the clearance
(160, 188) of the other neutral amino acids. The
evidence suggests that all the neutral amino acids
the transport
j
-a
acids
Sulfur-containing
methionine).
(lysine,
and taurine.
ascites
cells
Reabsorption
by stop-flow
CUMULATIVE
URINE MASS
Fig. 38. The
effect of L-methyldopa
on proximal
tubular
reabsorption
of L-histidine,
as illustrated
by stop.flow
analysis experiments (for details see Figure 1)
A stop-flow pattern in a control rat (broken lines) shows extensive reabsorption of L-histidine in the proximal tubule. In a rat
infused with L-methyldOpa (solid lines) the proximal reabsorptive trough for L-histidine is abolished despite the much lower
plasma histidine
concentration.
[Taken from Young and
Edwards (96)]
are transported
by a common system. In addition,
two synthetic
amino acids, i.-a-methyldihydroxyphenylalanine
(a-methyldopa)
and a-aminoisobutyric acid, have been shown to be transported
by this neutral system, competing
with the natural
amino acids for transport
(96, 189, 190).
Figure
3A shows a stop-flow pattern demonstrating
proximal tubular
reabsorption
of methyldopa,
while
Figure 3B illustrates
its inhibitory
effect on tubular
histidine reabsorption.
CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 253
The studies of Oxender and Christensen
(191) on
neutral amino acid uptake by Ehrlich ascites cells
suggested the presence of two transport
mechanisms
within the neutral
system-an
“A” or alaninepref erring system, and an “L” or leucine-pref erring
system.
The overlap between
these two systems
was so extensive
that nearly all the neutral amino
acids were transported
by both systems, but with
differing affinities for each. These findings were of
particular
interest,
as the authors
were able to
interpret
earlier renal competition
data (184, 188)
in terms of an A and L dichotomy
of neutral amino
acid transport
in the kidney. It should be pointed
out however that not all authors believe that the
evidence
supports
the existence
of the A and L
systems in the Ehrlich cell (192) or in the intestine
(118).
Basic system. It is generally
agreed that the
diaminomonocarboxylic
acids-lysine,
arginine,
and ornithine
(commonly
referred to as the basic
amino acids)-are
reabsorbed
by a common transport system. This has been demonstrated
unequivocally by competition
studies in man, dog, and
rat (6, 90, 97, 184-187,
193-1 95). However,
the
position
of cystine in this transport
group has
proved
an enigma,
and the question
is still not
completely
resolved.
The first evidence
supporting
the grouping
of
cystine and thc basic amino acids in a common
system came from a study of the hereditary
disease, classical cystinuria.
This disease is marked
by large increases in the excretion
of cystine and
the basic amino acids (196, 197). Thus it was postulated
that cystinuria
represented
a genetically
determined
defect of the common transport
system
for these amino acids. That these amino acids
share a common system was further supported
by
clearance
and competition
studies in man (194)
and dog (94, 160).
However
in the last few years much evidence,
gathered
mainly from in vivo and in vitro experiments in the rat, indicates
that, at least in that
animal,
cystine is not transported
by the same
mechanism
as the three basic amino acids (109,
111, 127, 195, 198, 199). Similarly,
recent in vivo
and in vitro studies
of cystinuria
have thrown
doubt on the concept of a single transport
defect
for cystine and the basic amino acids. Crawhall
(200) reported
that while cystine
clearances
in
cystinurics
were invariably
greater than the gbmerular
filtration
rate (GFR), indicating
net secretion, the clearance
of the basic amino acids was
less than
GFR,
indicating
net
reabsorption.
Fox et al. (117) studied
uptake
of amino acids
into kidney cortex from cystinuric
patients
and
found no impairment
in cystine
uptake,
while
lysine uptake was reduced by half. Thus, it would
appear
that
in the human
cystinuric
kidney,
cystine is handled differently
from the basic amino
acids, suggesting separate transport
mechanisms.
254 CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971
Direct
evidence
for the presence
of separate
transport
systems for cystine and the basic amino
acids has come from the chance discoveries
of
some rare genetically
determined
aminoacidurias.
Brodehi (01) reported
an aminoaciduria
marked
by excessive cystine clearance
in the presence of
normal basic amino acid clearance.
Such isolated
cystinuria
is also found normally
in the Kenyan
blotched
genet (202, 203). Following
this line of
reasoning, one might also predict the occurrence
of
a genetic aminoaciduria
involving
only the three
basic amino acids, but not cystine. In fact such an
aminoaciduria
has recently
been discovered
and
investigated
(204).
It appears then, at least in man and the rat, that
cystine and the basic amino acids are transported
by separate
mechanisms.
The question
of what
causes competition
for reabsorption
between cystine and the basic amino acids in man and dog, as
well as the reason for impaired handling of cystine
and the basic amino acids in cystinuria,
still remains unanswered.
One possibility
might be that
the basic amino acids and cysteine (the intracellular form of cystine) share a common effiux pathway from the tubule cells. This appears to be the
case in the rat (199). If such a mechanism
were
defective in cystinuria,
there could be a “pile-up”
of intracellular
cysteine, resulting in the failure of
reabsorbed
cystine to be reduced
to cysteine.
A
mass action effect could then prevent cystine movement across the membrane.
Acidic system. The results of competition
studies
in the dog suggested
that the two monoaminodicarboxylic
acids-glutamic
acid and aspartic
acid-were
transported
together
by a common
system (94, 185-188).
In these experiments,
jul usion of one amino acid caused decreased
reabsorption of the other. However,
as the infusion of one
of these amino acids caused a marked increase in
the plasma
concentration
of the other (188), it
was not certain whether the decreased reabsorption
was the result of competition
for transport,
or was
merely a consequence
of increased
filtered load.
Webber (158) was able to resolve this question by
determining
the transport
characteristics
of each
of the two acidic amino acids individually.
He
found a transport
maximum
(Tm) for both of
these amino acids [in marked contradistinction
to
Gerok and Gayer (97, 193), who were unable to
show a Tm for glutamic
acid], which enabled him
to show that the increased
plasma concentration
caused by infusion
was not sufficient
to cause
saturation
of the transport
mechanism,
and thus
the decreased
reabsorption
observed
was a direct
consequence
of competition
for a common transport system.
A different technique,
which avoided the problem of increased
plasma concentrations,
was used
to examine the acidic transport
system in the rat.
By microinjection
of the acidic amino acids into
single nephrons,
Bergeron and Morel demonstrated
Tm values for both aspartic
and glutamic
acid
(102). They also showed marked mutual competition for transport
between
the two acidic amino
acids when they were injected
into the nephron
simultaneously.
A variety of neutral amino acids
caused no inhibition
of acidic amino acid transport.
These data suggest strongly
that glutamic
and
aspartic acids are reabsorbed
by a single system in
the rat kidney,
a system which is quite distinct
from that available for neutral amino acids.
Iminoglycine
system. The investigation
of some
metabolic
disorders has led workers to propose the
existence
of a common transport
system for the
imino acids, proline and hydroxyproline,
and the
neutral
amino acid, glycine. In Hartnup
disease,
where there is a defect in the reabsorption
of all
other neutral amino acids, glycine reabsorption
is
only slightly
affected,
and reabsorption
of the
imino acids not at all (180, 205). Scriver el al. (206)
made a study of a disease characterized
by high
plasma proline concentrations
and increased excretion of the imino acids and glycine. They found
that only when the proline level exceeded its Tm
was there an increased excretion of the imino acidglycine
group,
and this phenomenon
could be
mimicked
in normal humans by artificially
raising
the plasma
proline
levels. In addition
to this
disease, the authors cited other cases of convulsive
disorders
accompanied
by hyperexcretion
of the
imino acids and glycine in which there were normal
plasma
amino acid concentrations.
These cases
seemed to have impairment
of a transport
system
common to the imino acids and glycine.
Although the above studies indicate that there is
a common transport
system for the imino acids and
glycine, this of course does not preclude
the possibility of there being additional
systems capable
of transporting
one or other of these amino acids
individually.
In fact there is a good deal of evidence
pointing
to the existence
of multiple
transport
mediations
within the iminoglycine
system. Scriver
et al. (206, 207), from competition
studies in humans,
demonstrated
that
some neutral
amino
acids that had no effect on the reabsorption
of
imino acids nevertheless
had slight effects on glycine reabsorption.
From this they suggested
that
the imino acids and glycine might have separate
transport
mediations.
However
in similar studies
in the dog, Webber (188) demonstrated
inhibition
of the reabsorption
of both the imino acids and
glycine by the same neutral
amino acids that
Scriver et al. (206, 207) had used in their study.
Although
this discrepancy
may be due to a species
difference,
a more probable
explanation
would be
that the levels of the neutral amino acids achieved
in Scriver’s
study
were not sufficient
to cause
inhibition
of the reabsorption
of the imino acids or
glycine (indeed they were not sufficient
to cause
inhibition
of the reabsorption
of other neutral
amino acids). Wilson and Scriver (195) used slices
of rat kidney cortex to examine the affinity constants for transport
and inhibition
of the imino
acids and glycine. On the basis of variation
and
diversity of these constants,
the authors concluded
that, rather than a single common transport
system for this group, the system must be subdivided,
supplemented,
or both. These authors had reached
much the same conclusion previously
(208), believing that there were discrete catalytic
sites for the
individual
amino acids within this system. Genetic
evidence for the existence
of multiple
mediations
within the iminoglycine
system
has come from
studies
of both heterozygotes
and homozygotes
with an iminoglycinuric
trait (209). Scriver (209)
rejected
the possibility
of altered
affinity
of a
common
transport
system.
Rather,
he proposed
that a high-capacity
transport
system, common to
all these compounds,
was defective
and that the
residual
reabsorption
observed
was due to two
other low-capacity
systems,
one of these being
only for the two imino acids. These conclusions,
with suitable
modifications,
are compatible
with
preliminary
findings from isolated
rabbit
tubule
preparations.
Thus, Hillman and Rosenberg
(104)
demonstrated
three distinct transport
systems for
proline: one shared with alanine, a second shared
with glycine, and a third only with hydroxyproline.
This tubule
preparation,
together
with isolated
brush
border
preparations,
seems to offer the
greatest promise for the elucidation
of the properties of these multiple transport
mediations.
Intergroup
interactions.
Although
the evidence
for the existence
of the four transport
groups is
fairly clear, some explanation
for the large number
of intergroup
interactions
that have been reported
is necessary.
For example,
in competition
studies
(160, 188) in dogs it was found that the basic
amino acids inhibited
transport
of some neutral
amino acids and vice versa, and also that some
neutral
amino acids inhibited
transport
of the
imino acids.
Two possible theories
could account
for these
interactions.
The one theory
envisages
only a
limited number of transport
systems, which have
very wide and overlapping
substrate
specificities.
The other envisages
a heterogeneity
of transport
systems of two basic types-namely,
high-capacity
low-specificity
systems,
which
would
transport
groups of amino acids, and low-capacity
highspecificity
systems,
which would transport
perhaps only one or two amino acids. The existence of
two general types of genetically
determined
aminoacidurias,
one in which
the defect
covers
a
whole transport
group-e.g.,
classical
cystinuria
and Hartnup
disease-and
the other,
in which
there is defective
transport
of only one or two
amino
acids-e.g.,
isolated
hypercystinuriawould support the latter theory. Also, the investigation
by Scriver
(209) of the iminoglycinuric
CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 255
trait provided
evidence
that supports
the argument that there are two different types of system
for the transport
of the imino acids and glycine.
Although
only a small amount
of experimental
work has been directly involved with examining the
possibilities
of two types of transport
system,
a
number of unrelated
observations
lend support to
this theory.
Brown (210) reported
that cycloleucine, a neutral amino acid that is not metabolized,
when administered
to man in large doses, caused a
very large increase in the excretion
of the basic
amino acids and cystine, with no change in their
plasma levels. Christensen
and Cullen (189) demonstrated a similar large increase in the excretion
of
the basic amino acids and cystine,
as well as
marked effects on neutral
amino acids, after administration
to rats of large doses of a-aminoisobutyric
acid (AIB),
another neutral amino acid
that is not metabolized.
Moreover,
after the plasma
AIB levels had dropped
from their very high initial
levels, the hyperexcretion
of the basic amino acids
and cystine disappeared,
leaving a residual hyperexcretion of the neutral amino acids. These results
could possibly be accounted
for on the basis of the
in vitro results of Segal et al. (109), who found that
AIB,
cycloleucine,
some basic, and some neutral
amino acids could be metabolized
by a high-capacity system. More complete studies by these authors showed that at least lysine and glycine were
both transported
by two systems-one
with a low
capacity
and high affinity, and the other with a
high capacity
and low affinity. Both lysine and
glycine uptake by this latter system were inhibited
by AIB, suggesting
that this system may transport
both neutral and basic amino acids. A similar nonspecific low-affinity
system, known as the “lysineaccepting
system,”
that transports
both neutral
and basic amino acids, has been reported
in the
Ehrlich ascites cell (211). The existence of such a
system could explain the interaction
between the
neutral and basic amino acids in Webber’s studies
(160, 188) and might also explain the inhibition
of
the reabsorption
of the basic amino acids and cystine by high levels of cycloleucine
in Brown’s
study (210). Similarly,
the results of Christensen
and Cullen (189) (see above) could mean that AlE
in high concentrations
inhibited
the common highcapacity
system shared by the neutral
and the
basic amino acids, while in lower concentrations,
it inhibited
only the low-capacity
neutral system.
Reabsorption Kinetics and Transport Maxima
Information
about
reabsorption
kinetics
has
been
obtained
by studying
the clearance
of
amino acids both at endogenous
plasma concentrations
and after intravenous
infusions of amino
acids so as to produce a wide range of exogenous
plasma amino acid concentrations.
Endogenous
amino acid clearance. Prior to the
256 CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971
development
of chromatographic
separation
techniques,
the plasma
concentrations
of individual
amino acids could not be measured,
and early studies were concerned only with the clearance of total
a-amino
nitrogen.
Such measurements
indicate
that a-amino
nitrogen
reabsorption
is 98 to 99%
complete
in normal man (180, 212). Apart from
man, endogenous
a-amino
nitrogen
clearance
has
been studied only in two species-the
dog (98) and
the alligator (sic) (213).
Although reabsorption
of total a-amino nitrogen
is 99% complete in the human, the percentage
reabsorption
of individual
amino acids varies widely.
Cusworth and Dent (180) found that the percentage
reabsorption
of histidine
(90 to 95%) was constantly lower than that of other amino acids.
Thus
glycine reabsorption
ranged from 95 to 98% and
serine was about 98%; for most of the other amino
acids, percentage
reabsorption
was 99%. The relatively low histidine percentage
reabsorption
could
be explained
by its having a low affinity for the
neutral
transport
system. This possibility
is supported by findings in the dog (160, 188). In these
studies, histidine
reabsorption
was more inhibited
by alanine,
glycine,
and phenylalanine
infusion
than was the reabsorption
of any other neutral
amino acid. Also, histidine,
as an inhibitor,
was
the least effective of all the neutral
amino acids
studied. To a lesser extent, the same is also true of
glycine. Table 1 shows a more complete
list of
individual
endogenous
amino acid clearances
in
humans,
as reported
by a number
of authors.
In
the lowest row are shown mean values
± SD for
nine subjects
studied
with ion-exchange
chromatography.
It can be seen that only glycine and
histidine
clearances
were appreciably
greater than
1 mI/mn,
which indicates
substantially
less than
99% reabsorption.
In the dog (98) the percentage
reabsorption
of nearly every amino acid exceeded
99%. Nevertheless,
the percentages
of histidine
and glycine reabsorbed
were only 97.6 and 98.4%,
respectively,
which shows the same general trend
as in man, allowing for the greater overall efficiency
of reabsorption
in the dog.
Although
clearance data provide the best means
for comparing
the renal handling of various amino
acids, some information
can be obtained
by examination
of urine/plasma
concentration
ratios
(u/p) for the various amino acids. Evered
(107)
compared
the u/p ratios of individual
amino acids
for various species. As could be expected,
the u/p
ratios of histidine
and glycine in man were much
larger than those of the other amino acids. The
u/p ratios for glycine were relatively
large also in
the rat, sheep, and cow. However,
the u/p ratio
for histidine
was not enhanced
in rabbit, cat, rat,
sheep, or cow-indicating
a possible species difference in the transport
of this amino acid, at least.
Exogenous
amino acid clearances-the
titration
curve and Tm. To gain a more complete
picture
about the kinetics of the renal handling
of amino
acids, it is necessary
to study excretion
and reabsorption patterns,
not only at endogenous
plasma
amino acid concentrations,
but also at exogenous
levels. Excretion
curves obtained
in this way are
referred to as titration
curves by analogy with similar curves obtained
during the study of the renal
handling of glucose (48, 49). In these experiments,
glucose filtered load was plotted
against
glucose
reabsorption
and the point beyond
which reabsorption
became constant
was referred to as the
transport
maximum
(Tm).
Such curves
of reabsorption
vs. load were termed “titration
curves”
since it may be considered
that the tubules have
been “titrated”
to a saturation
value of transport
by raising the load.
Using a similar technique,
Pitts (214, 215) infused a mixture of amino acids into dogs and obtained a titration
curve for total a-amino nitrogen
that had a demonstrable
Tm. In man, the curve
does not show a definite Tm, although a divergence
in the percentage
of a-amino
nitrogen reabsorbed
was found as the filtered load increased (216). This
has been taken as indicating
that reabsorption
was
approaching
a Tm value. The titration
curves for
dog and man are shown in Figures 4 and 5, respectively.
The apparent
simplicity
of the a-amino nitrogen
titration
curves disappears
when one comes to consider the renal handling
of the amino acids individually.
Many
authors
have obtained
such
“titration
curves” for individual
amino acids; in
some cases, they observed a true Tm phenomenon,
in others there was no tendency toward a Tm value
whatsoever,
and in yet others there was a progressive decline in percentage
amino acid reabsorption
without,
however,
any tendency
to reach a maximum reabsorptive
rate. Table 2 summarizes
the
available
data for a large number of amino acids
whose titration
curves have been studied.
From
an inspection
of the table it can be seen that there
are numerous
discrepancies
among the various reports. There seems to be general agreement
that a
transport
maximum
phenomenon
can be observed
for L-arglnine5
(in dog and rat),
L-alanine (in cat
and dog), L-aspartic
acid (in dog and rat) and
L-proline and L-hydroxyprohne
(in man). The discrepancies
among the various studies may partly
result from difficulties
in determining
individual
amino acids. However,
even when one considers
only those studies in which column chromatog-
‘It is interesting
to note that Gerok and Gayer (92) found
that the argilline
Tm in the dog could be abolished
by the
simultaneous
infusion
of either
were inclined
to attribute
concentration
of arginine
histidine
or glutamic
this to a metabolic
within
renal
tubular
acid.
lowering
cells.
They
of the
Whatever
the explanation,
if the phenomenon
is widespread,
it may well
account for the discrepancies
seen among the various papers in
which amino acid Tm’s have been studied.
C
E
0
C
C
LU
0
I-
z
0
z
AMINONITROGEN FILTERED, (rrmol/rnIE)
Fig. 4. Total a-amino-nitrogen
plotted against the filtered
load of amino-nitrogen
dog undergoing
infusion
reabsorption
intravenous
and excretion
in a
with gycine
[Taken from Pitts (52,214)]
if
o E
-_____
---#{149}------
-
-.
-.
.
_______
___________
-
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0
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-
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15
amino -N (mg/mm/I,
Fig. 5. Total a.amino-nitrogen
reabsorption
‘0
25
73m2)
and excretion
(ordinate)
plotted
as a function
of filtered
load of aamino-nitrogen
(abscissa),
in human
subjects
undergoing infusion with casein hydrolysate
Top: Filtration. Middle: reabsorption.
difterent symbols are for different
Stalder et al. (216)]
Bottom: excretion. The
individuals
[Taken from
raphy was used for amino acid analysis,
discrepancies still exist. Thus, Gerok and Gayer (193)
failed to observe a Tm for L-glutamic
acid in the
dog, whereas
Webber
(158) observed
one. It is
possible that Gerok and Gayer may have failed
to increase the plasma concentration
sufficiently,
although
one cannot readily calculate
the filtered
load from their data. Other discrepancies
may be
due to interspecies
differences.
The shape of the “titration curve.” The titration
curve of total a-amino nitrogen reabsorption
after
glycine infusion is shown in Figure 4 [(taken from
Pitts (52, 214)]; the titration
curve of arginine reabsorption
obtained
in the chromatographic
study
of Gerok and Gayer (193) is shown in Figure 6.
In both cases, the reabsorption
curve keeps pace
initially
with filtration
and, as the Tm is approached,
the curve bends gradually.
This region
of bending
in the titration
curve, is known
as
CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 257
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CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971 259
Table 2. Transport Maxima in Renal Reabsorption of Amino AcidsN
Amino
acids
DL-Alanine
Tm demonstrated
dog’ (215)
cat’ (57)
L-A Ianine
Tm possibIV
No Tm
dog’ (233)
cat’ (156)
dog’ (138)
D-Alanine
dog’ (138)
cat’ (156)
L.Arginine
dog’ (215)
dog’ (230)
dog’ (231)
rJL#{149}Aspartic
acid
L-Aspartic acid
D-Aspartic
Glycine
L-Glutamic
acid
acid
dog’ (138)
dog4 (97,193)
rat5 (6)
dog’ (23(3)
dog4 (155)
rat5 (102)
dog4 (158)
dog’ (214)
dog’ (738)
dog’ (233)
dog’
dog’ (215)
dog’ (230)
dog4 (158)
rat5(102)
L-Histidine
DL#{149}Isoleucine
L-Leucine
L#{149}
Lysine
DL-Methionine
L.Methionine
DL-Phenylalanine
L-Proline
L-Hydroxyproline
DL-Threonine
L-Tryptophan
OL-Valine
(230)
rat4 (96)
man4 (217)
dog4 (97, 193)
cat’ (156)
rat’ (6)
dog4 (97, 193)
dog’ (231)
dog4 (97, 193)
dog’ (184)
dog’ (232)
dog’ (232)
dog’ (230)
dog’ (230)
dog’ (231)
rat5(6)
rat’ (6)
dog’ (184)
dog’ (159)
dog4 (97, 193)
dog’ (231)
cat’ (57)
dog’ (234)
rat’(6)
man4 (206)
man4 (209)
dog’ (234)
dog’ (184)
dog’ (184)
dog’ (232)
Superscriptsindicatethe method of amino acidanalysisused: 1 = totala-amino nitrogen;2 = microbiological assay;3 = enzymic
assay; 4 = paper or column chromatographic separation,followedby use of the ninhydrin reaction; 5 = radioisotopic
labeling.
Numbers
in parentheses are reference numbers.
In the case of the glucose titration
curve,
the splay is slight, and has been attributed
to
variations
in the ratio of filtered load to tubule size
in individual
nephrons
[“glomerulotubular
balance” (51)]; this concept is adequate
to explain
the small degree of splay in the glucose titration
curve. However,
the much wider splay of amino
acid titration
curves can obviously not be fully explained on the same basis. Thus, for amino acids,
some further explanation
of the splay is needed. One
possibility
is the influence of the equilibrium
constant of the transport
reaction, on the shape of the
titration
curve. In Figure 7 [taken from Sugita
(217)], the shape of the titration
curve has been
shown for equilibrium
constants
varying between
zero and infinity. As the equilibrium
constant
ap“splay.”
260 CLINICAL CHEMISTRY, Vol. 17, No. 4, 1971
infinity,
there is very little splay in the
titration
curve. Indeed,
in the case of glucose,
which exhibits only minor degrees of splay, it has
been shown by micropun cture studies (218) that the
Michaelis-Menten
constant
(Km) of membrane
transfer
approached
zero, indicative
of an equilibrium constant
approaching
infinity.
The very
wide splay in the titration
curve of amino acids
could well be due to a relatively
low equilibrium
constant for membrane
transfer. Such a possibility
is supported
by the finding of high Km (indicating
low equilibrium
constant)
for amino acid accumulation systems in the kidney cortex slices (84, 109,
219).
Bergeron and Morel (6) have recently published
titration
curves for neutral
amino acids obtained
proaches
techniques
are essentially
different.
In clearance
experiments,
the load of amino acid is increased by
increasing
the amino acid concentration
of both
0
plasma
and glomerular
filtrate,
whereas
in the
micropuncture
experiments
only the concentration
.0
of intratubular
fluid is raised. This would mean
that
the
transtubular
amino acid concentration
a
gradient
is far greater in the micropuncture
experiments
than in the clearance
experiments
and
could conceivably
enhance
the operation
of any
passive,
gradient-dependent,
reabsorptive
process. The possibility
of the existence
of passive
processes
in amino acid reabsorption
has been
Amount
filtered
suggested
by a number
of recent mieropuncture
-NH2-NI.’n/lOOnN
GF
studies. Freedman
and Young (8) showed that a
Fig. 6. Renal tubular reabsorption of L-arginine with in#{149} large component
of amino acid reabsorption
could
creasing filtered load
not be inhibited
by very high concentrations
(40
A dog was infused intravenouslywith L-arginine;
reabsorption
mmol) of cyanide
in the tubule perfusion
fluid.
has been calculated by subtracting the amount excretedfrom
Similar results were obtained
by Silbernagel
and
the calculated filtered load (broken line). A definite Tm is shown
although splay is rather wide. i-arginine was measured by the
Deetjen
(9) who were unable to inhibit
glycine
ninhydrin color reaction after separation by column chromatog.
reabsorption
with dinitrophenol
or cyanide (partial
raphy. [Taken from Gerok and Gayer (193)]
inhibition
was possible,
1)eetjen
P., personal
communication,
1970).
Validity of the concept of Tm. Numerous
workers have re-examined
the manner in which the kidney handles glucose and have demonstrated
that
Tm
the so-called
Tm for glucose reabsorption
varies
according
to the GFR (220-223).
Furthermore,
0
many workers have shown that saline loading and
N,
plasma volume
expansion
can influence
the Tm
value for reabsorption
of substances
such as gluKrO
cose, phosphate,
magnesium,
and p-aminohippuric
acid
(224-229).
Although
the influence
of
.Zm
Fill. Load
these factors on amino acid reabsorption
has not yet
Fig. 7. The influence of the equilibrium constant for
been studied,
it seems likely that similar anomamino acid reabsorption
on the shape of the titration
alies
would
be
found. These observations
call into
curve
question the whole concept of Tm.
‘flQ
“N
0
As the equilibrium
constant
(K)
approaches infinity
the splay
becomes progressively narrower. (Note that K is the reciprocal of
the Michaelis-Menten constant, K.’.) Glucose, which has been
shown to have a high equilibrium constant (218) has, indeed, a
narrow splay (49) whereas many amino acids which have low
equilibrium constants (84, 109, 219) show wide splay or no Tm at
all. [Taken from Sugita et. at. (217)]
by microinjection
of individual
nephrons.
They
plotted their data on log-log paper. However, when
their curves are replotted
on linear axes the shapes
of the titration
curves are found to be very similar
to the slowly rising curve of an active transport
process with a low equilibrium
constant.
Thus, for
these amino acids at least, it is possible that the
splay in the titration
curve reflected a low equilibrium constant. On the other hand, when the basic
amino acids were studied in this way, Bergeron
and Morel (6) obtained
titration
curves with clear
Tm’s and some degree of splay, indicating
higher
equilibrium
constants
for these amino acids. Of
course, one must be wary of extrapolating
conclusions
from
these
micropuncture
results
to
clearance experiments,
as the conditions of the two
We thank A. Cameron for his help in the preparation
of the
bibliography,
K. Davies
for her careful
preparation
of the
typescript,
and F. D. Weber for his help. We thank especially
Mrs. H. Cooke for her discussions and criticisms of the manuscript. We also thank the National Health and Medical Research
Council of Australia for the award of a stipend to one of us
(B.S.F.) and for support of the authors’ experimental work.
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