Micropuncture
reabsorption
study of renal tubular
of calcium in normal rodentd’”
WILLIAM
E. LASSITER,3
CARL
W. GOTTSCHALK,4
AND MARGARET
MYLLE
Department of Medicine, University of North Carolina, School of Medicine,
Chapel Hill, North Carolina
C
ALCIUM
EXCRETION
by the mammalian
kidney has
been extensively
investigated
by clearance
techniques
with
and is known ‘to involve
glomerular
filtration,
subsequent reabsorption
of most of the filtered calcium
load (I ). Available
evidence
indicates
that calcium
reabsorption
involves active transport,
but the nature of
the reabsorptive
process and its anatomic
localization
in the tubule
remain
unresolved.
Recent
stop-flow
studies in dogs have been interpreted
as showing active
reabsorption
of calcium in distal portions of the nephron
(2, 3). No evidence of active calcium
transport
in more
proximal parts of the nephron was found in these studies,
but the stop-flow
technique
is of limited
value in the
study of proximal
tubular
function
because it is imReceived
for publication
25 October
1962.
1 This investigation
was supported
by a grant-in-aid
from the
American
Heart Association
and by Public Health
Service Grant
H-2334.
2 Reported
in part before the American
Physiological
Society,
Atlantic
City, April, I 962 (Federation hoc. 2 I : 435, I 962).
3 Established
Investigator,
American
Heart Association.
4 Career Investigator,
American
Heart Association.
possible to control the degree to which the composition
of proximal
fluid is modified
by continuing
glomerular
filtration
during
stop flow (4), or by passage through
more distal parts of the nephron
after release of the
ureteral clamp.
On the other hand, the likelihood
of proximal
calcium reabsorption
is suggested by other studies relating
calcium to sodium excretion. Beck, Levitin,
and Epstein
(5) have presented evidence that hypercalcemia
leads to
impaired
reabsorption
of sodium by the renal tubules.
Conversely,
Walser
(6) found in diuretic
dogs that
changes
in sodium
clearance
were accompanied
by
parallel changes in calcium clearance
and that, in fact,
the clearance
of free calcium
ion equaled
the sodium
clearance.
Since the bulk of sodium reabsorption
occurs
in the proximal
tubule (7, 8), and this has been shown to
involve active transport
(9, I o), the above observations
suggest that calcium also may be actively transported
in the proximal
tubule. We have investigated
the renal
tubular
reabsorption
of calcium
in normal
rodents,
using the micropuncture
technique
to sample fluid in
individual
tubules. The results indicate that, as is true
of sodium,
calcium
is actively
reabsorbed
from all
parts of the nephron,
and furthermore,
that the bulk of
calcium reabsorption
occurs in the convoluted
portion
of the proximal
tubule.
METHODS
White Rats
Normal
rats were anesthetized
by intraperitoneal
injection
of pentobarbital,
35 mg/kg body wt., the left
kidney
exposed through
an abdominal
incision,
and
100-200
PC calcium
455 administered
in a single intravenous injection
through
an indwelling
jugular
cannula. After allowing
an hour or more for isotopic
equilibration,
fluid was collected by micropuncture
from
glomeruli
and from proximal
and distal convolutions
on
5 Obtained
from
Oak Ridge
National
Laboratory,
cat. no.
Ca-45-P-3.
Specific
activity
of various
lots was between
25,000
and 42,000 me/g calcium.
Downloaded from http://ajplegacy.physiology.org/ by 10.220.33.4 on April 1, 2017
LASSITER,
WILLIAM
E.,
CARL
W.
GOTTSCHALK,
AND
MARGARET
A~YLLE.
Micropuncture
study of renal tubular
reabsorption of calcium in normal rodents. Am. J. Physiol.
204(5) : 771I g63.Anesthetized
rats and
hamsters
were
given
775.
Ca45 intravenously,
and fluid was subsequently
collected
by
micropuncture
from glomeruli
and surface
tubules
in the rats,
and from loops of Henle
in the hamsters.
In nondiuretic
animals, fluid:plasma
calcium
ratios
averaged
0.7 I in the glo2.0 in the loop of Henle;
merulus;
0.76 in the proximal
tubule;
0.47 in the distal
convolution;
and 0.9 in ureteral
urine.
In
mannitol
diuresis,
the calcium
ratio of glomerular
fluid
was
unchanged,
but ratios as low as 0.2 I were noted in the proximal
tubule.
In this circumstance,
the average
proximal
ratio was
0.61, and the distal ratio 0.07. These results indicate
active
transport
of calcium
out of all major
parts of the nephron,
with
the bulk of calcium
reabsorption
occurring
in the convoluted
portion
of the proximal
tubule.
Furthermore,
the pattern
of
tubular
reabsorption
of calcium
is similar
to that of sodium,
suggesting
that the two are related.
LASSITER,
77”
TABLE
I. Fluid: plasma Cad5 concentration ratios
in (A) nondiuretic rats and in (B) rats
during mannitol diuresis
Rat
Wt.,
g
p:::% of
Tubule
A.
.
Nondzuretic
Calcium
F/P
250
.81
20
.82
26
-40
-38
280
-85
78
l69
l 79
450
320
16
.60
170
53
-75
83
90
-5’
-57
320
43
47
-83
-70
33
*45
21
l 79
30
.80
29
58
-57
-13
270
IO
.65
70
7’
030
l 73
270
I9
36
-83
-84
22
-63
l63
55
35
.86
51
5’
.62
255
20
310
l 93
‘4
67
-79
-46
44
29
.02
350
53
58
-75
-67
59
3’
-05
.06
320
48
66
62
37
l 75
-35
925
029
460
67
59
25
.61
-85
200
35
45
38
‘91
l 24
-55
250
I9
4’
68
-49
-59
-59
-46
280
3’
50
45
-63
-59
*63
300
44
57
53
44
42
.62
.62
*79
16
.62
.60
-40
-69
-95
8
340
20
92
94
48
320
20
-74
-46
.28
. 16
.
‘7
18
964
*49
‘9
83
-45
-65
44
230
330
-57
-59
3’0
54
53
54
190
30
14
35
250
4’
IO
200
110
-57
-74
-70
-69
360
.80
.68
.81
‘4
23
glom.
.80
-71
glom.
glom.
9
.62
-75
31
71
*I4
I
rats
260
260
.82
.82
.04
-77
33
55
61
300
diuresis
glom.
35
30
57
‘7
49
.81
44
48
35
44
.21
‘7
40
51
5’
-47
48
68
39
-65
.68
-45
.82
-69
-05
diuresis
rats-Contznued
50
65
54
-5’
934
-56
220
glom.
*5I
23
35
27
-07
J5
*I3
210
30
60
-48
-55
9
25
-4
190
glom.
60
l 7=
*59
77
.03
250
glom.
glom.
glom.
-70
-83
-7’
.66
4-o
.02
38
-
. II
the surface of the kidney,
as previously
described
retrograde
flow, a droplet
of
( II, I 2). To prevent
mineral oil was injected into each tubule, and the rate
of collection
adjusted to maintain
the oil at a position
just distal to the puncture
site. In this manner approximately 0.1 ~1 of tubular
fluid could be collected
in
20-60 min. Each sample was discharged
under mineral
oil into a siliconed
glass dish and then aspirated
into
calibrated,
constant-bore
Pyrex capillary tubing, between
layers of chloroform
to prevent evaporation,
and volume
estimated
by measurement
of the length of the sample
with an eyepiece micrometer.
Samples were then discharged
onto aluminum
planchets,
and after drying
overnight
at room temperature,
radioactivity
was determined
in a windowless
flow proportional
counter.
0.8
I---- l o,.a’.* . l
0
00.
t
f
0 0
0
*
----------w-w--- a--w--e
0
a0
0.- 0
0
0
0
0
00
.66
-59
-56
0
0
.o
.61
-52
-7’
0
25
50
75
0
50
100
FIG.
I.
Calcium
45 fluid:plasma
ratios in proximal
tubule and
distal
convolution
in 19 nondiuretic
rats. Solid line through
proximal
points was fitted by the method
of least squares. Broken
line indicates
average concentration
ratio in glomerular
filtrate.
Downloaded from http://ajplegacy.physiology.org/ by 10.220.33.4 on April 1, 2017
-73
54
64
.82
-46
65
59
33
48
MYLLE
T.:11.: ic$$mifj$i
Z??
B. Mannitol
B . Mannitol
300
AND
I. Continued
TABLE
Proxima1
% of
Tubule
Rat
wt.,
g
rats
GOTTSCHALK,
TUBULAR
REABSORPTION
25
"0
OF
CALCIUM
50
2.
tubule in
distribution
Ig
Similar measurements
were also made on ureteral urine,
and on plasma obtained
from the inferior
vena cava
before and after each micropuncture.
Self-absorption
corrections were not necessary because of the small size of
the samples. After the initial equilibration
period, plasma
radioactivity
declined
slowly in logarithmic
fashion,
hr. Similar
with a half-disappearance
time of 134-2
studies were performed in normally hydrated, nondiuretic
animals,
and in rats undergoing
osmotic diuresis inat
duced by the intravenous
infusion of 20 % mannitol
5 ml/hr.
At the end of each experiment
the kidney
was removed and macerated,
and puncture
sites were
localized by microdissection
(I I ). As in previous studies,
the proximal
tubule is considered
to extend from the
glomerulus
to the beginning
of the thin descending
limb of the loop of Henle; the distal convolution,
from
the macula densa to the point at which two or more
convolutions
join to form a collecting
duct. Most of the
rats were males of the Wistar strain, but several females,
both Wistar
and Sprague-Dawley,
were also used.
Weights varied from I I o to 460 g, with most animals
being in the 2oo-300-g
range.
Hamsters
Golden hamsters, 80-1 I o g in weight, were prepared
in similar fashion, except that the upper part of the
left ureter was opened to expose the tip of the renal
papilla.
Fifty to one hundred
microcuries
of Ca45 was
injected
intravenously,
and after equilibration,
fluid
was collected
by micropuncture
from loops of Henle
and from the orifices of terminal collecting ducts. Analyses
were performed
as noted above.
RESULTS
Nondiuretic
Animals
Micropuncture
studies were performed
in 19 nondiuretic
rats and 4 hamsters. Fluid :plasma
Ca45 concentration
ratios in the rat tubules
are recorded
in
I A. Localization
of puncture
sites is indicated
Table
as per cent of total length of tubule or convolution.
Proximal tubule. Fluid was collected from 4 glomeruli
rats. The
and 30 proximal
tubules in I 7 nondiuretic
average fluid : plasma calcium
concentration
ratio was
and 0.76 in the proximal
0.7 I in the four glomeruli
convolution.
As is shown graphically
in Fig. I, the
calcium
concentration
ratio remained
nearly constant
along the entire length of the convolution.
The equation
of the straight line fitted to the points by the method of
x. Although
the
least squares is y = 0.71 + 0.0012
calcium ratio appeared
to rise slightly along the tubule,
the increase is small and of questionable
significance.
The difference
between
the average calcium
ratios in
the first and last halves of the proximal
convolution,
0.74 and 0.79, respectively,
is not statistically
significant (P > 0.10).
Loop of H&e.
Samples were collected
from
nine
loops of Henle at the tip of the renal papilla
in four
hamsters. The average fluid:plasma
calcium
ratio of
these samples was 2.0 (range 1.3-2.4).
Distal convolution. Tubular
fluid was collected from 27
distal convolutions
in 14 nondiuretic
rats. The average
fluid:plasma
calcium
ratio of these samples was 0.47
As shown in Fig. I, the calcium
(range o. I 3-0.89).
concentration
ratio varied
widely
among
individual
tubules, but in nearly all the samples it was lower than
the concentration
in proximal
tubules
or glomerular
filtrate.
Ureteral urine. The average Ca45 concentration
in 66
rats
samples of ureteral
urine from the 19 nondiuretic
was 0.9 times the concentration
in plasma. The U/P
urine
calcium
ratio in 14 samples of collecting-duct
from 4 nondiuretic
hamsters averaged I .2.
Osmotic Diuresis
Brisk osmotic diuresis was induced
in 19 rats by
intravenous
infusion of 20 % mannitol
solution at the
rate of 5.0 ml/hr. Micropuncture
results are summarized
IB.
The average fluid : plasma calcium
conin Table
centration
ratio was 0.69 in 5 glomeruli,
and 0.61 in
54 samples of proximal
fluid. The lowest fluid: plasma
and as is shown graphically
in
ratio observed was 0.21,
Fig. 2, the ratio in 24 of the 5 4 tubu les sampled was
less than 0.60. In contrast,
all but I of 30 proximal
tubules in the nondiuretic
rats had ratios greater than
The calcium
concentration
in fluid from distal convolutions in the diuretic animals was very low, averaging
only 0.07 X plasma concentration
in 11 collections.
This must be considered
only an approximate
estimate
of the calcium concentration
in these tubules, because
the level of radioactivity
in the samples was too low to
permit adequate
counting
statistics. The calcium
concentration
in ureteral urine of the d iuretic animals was
also very low, averaging
0.04 X plasma concentration
in 26 samples.
Downloaded from http://ajplegacy.physiology.org/ by 10.220.33.4 on April 1, 2017
Calcium
45 fluid : plasma ratios in proximal
rats during
mannitol
diuresis.
Shaded area indicates
of values in nondiuretic
animals.
FIG.
773
LASSITER,
774
TABLE
2. cO??@riSOn
and stable Ca in rats
Ofurinary
excretion of Ca45
Calcium
U/P Ratio
Chemical
Cad5
r
I
1
0.41
2
I
0.41
o. 18
0.42
I .06
o-33
0.42
2
0.19
Source
230-g
260-g
220-g
Male,
nondiuretic
Male,
nondiuretic
Female,
saline diuresis
360-g
Male,
saline
diuresis
Urine
Urine
Urine
Urine
Urine
Urine
.04
0.32
I
o. 18
0.17
Spec$c Activity Studies
DISCUSSION
Approximately
70 % of plasma calcium was found in
this study to be filtrable
at the glomerulus.
The degree
of protein binding of calcium in rat plasma has not been
but our data are compatible
previously
investigated,
with studies in dogs (I 6) and humans
(I 7), which indicate that at physiological
temperature
and pH, 6070% of plasma calcium
is ultrafiltrable.
Our results
further
demonstrate
that although
there is net transtubular
loss of calcium out of all parts of the nephron,
the bulk of calcium reabsorption
normally
occurs in the
convoluted
portion of the proximal
tubule. The average
calcium concentrations
at various points along the rat
nephron,
expressed as a fraction
of the concentration
in glomerular
filtrate, are recorded
in the first column
of Table
3. Average
concentration
ratios for inulinC1400H
at the same levels, obtained
in a comparable
group of animals (8), are noted in the second column.
As is shown in the third column of the table, the calcium
ratio divided
by the inulin ratio at any point in the
AND
MYLLE
nephron is a measure of the quantity
of calcium reaching
that point
relative
to the amount
filtered
at the
glomerulus.
Thus it is seen that net reabsorption
of
calcium
equivalent
to approximately
two-thirds
of the
filtered load occurs in the proximal
convolution,
20-25 %
in the loop of Henle, and I o % in the distal convolution,
with most of the small amount remaining
at the end of
the distal convolution
being reabsorbed
in the collecting
ducts. This demonstration
that the bulk of calcium
occurs in the proximal
convolution
is
reabsorption
consistent with other observations
on the function of this
part of the nephron,
which has been previously
shown
to be the major site of reabsorption
of water, sodium,
chloride,
and bicarbonate
(7, 8, I 8).
Calcium loss in the loop of Henle appears to take place
primarily
from the ascending
limb.
Although
one
cannot, of course, make precise quantitative
comparisons
between fluid from long loops of juxtamedullary
nephrons
in the hamster and surface tubules in the rat, the high
calcium
concentration
found at the bends of these
loops points to the ascending limb as the important
site
of calcium reabsorption
in the loop of Henle. Thus the
major site of calcium loss in the loop appears to coincide
with the locus of sodium transport
(8).
Although
in nondiuretic
animals
no decrease in
calcium concentration
occurs in the proximal
convolution, the results in mannitol
diuresis indicate
active
transport
of calcium in this segment. If water reabsorption is restricted
by the presence of nonreabsorbable
solute, the tubular
concentration
of calcium
falls. *4s
noted above, under these conditions
we have observed
calcium concentrations
in the proximal
tubule as low
Since the lumen of
as 0.2 I X plasma concentration.
the proximal
tubule has been shown to be negatively
charged with respect to its exterior (I g, ao), calcium ion
under these conditions
is being reabsorbed
against both
an electrical and a chemical gradient,
and hence by an
active process. Similar observations
have been made with
respect to sodium in the proximal
tubule (9, I 0). In
nondiuretic
animals,
sodium with its attendant
anions
forms the bulk of osmotically
active solute in proximal
fluid, and as sodium is reabsorbed
no measurable
conTABLE
3. Average concentration ratios, tubular fluid:
glomerular jltra te, in nondiuretic rats
Source
Early
proximal
Late proximal
Early
distal
Late distal
Ureteral
urine
Calcium
Inulin
Calcium
I .o
x
100
100
37
3
5
I .I
0.7
0.6
I-3
14
6;:
0.2
TABLE 4. Comparison of average calcium and sodium
concentration ratios in nondiuretic rodents
Source
Proximal
convolution,
rats
Loop of Henle,
hamsters
Distal
convolution,
rats
Ureteral
urine,
rats
Calcium
Fluid/Glom.
I .o
2.8
o-7
I.3
Sodium
Fluid/Plasma
Calcium Ratio
______.___
Sodium Ratio
I .o
I .o
I *9
o-7
o-3
I.5
I .o
4.3
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To test the validity of the radioisotope
measurements
as a true index of calcium
excretion,
simultaneous
determinations
of Ca45 and stable calcium
were performed on urine and plasma obtained
from two nondiuretic rats and from two in which the rates of urine formation and calcium excretion were increased by infusion
of 5 % NaCl, in order to shorten urine-collection
times
and to reduce dead-space errors. Collections
were begun
I hr after administration
of Ca45, thus duplicating
conditions in the micropuncture
experiments.
Stable cal(13)
or by
cium was determined
by flame photometry
the microspectrophotometric
method of Webster
( I 4).
Urine: plasma concentration
ratios for Ca45 and stable
calcium are shown in Table 2. Although
there was considerable variation
among different animals in the ratio
of urine to plasma calcium,
in each instance the ratio
computed
from radioisotope
measurements
was identical to that calculated
from the stable calcium determinations.
These results in rats are in agreement
with
similar observations
in dogs reported
several years ago
bv Govaerts
(I 5), and they indicate
that under
the
conditions of these experiments
the radioisotope
measurements provide a reliable index of calcium excretion.
GOTTSCHALK,
TUBULAR
REABSORPTION
OF
CALCIUM
775
individual
renal
tubules
in nondiuretic
rodents.
In
Table 4 average calcium and sodium concentrations
in
the various segments of the nephron
are expressed as a
fraction
of the concentrations
in glomerular
filtrate.
The sodium values are derived from previously reported
ratios are qualitatively
data (I o, 2 I). The calcium
similar to the sodium ratios at every level except in the
final urine,
and the ratios are in close quantitative
agreement
in the proximal
and distal convolutions.
The
relatively
high concentration
of calcium
in the final
urine is probably
due to the presence of large amounts
of anions which complex
calcium,
such as phosphate,
citrate,
and sulfate, in the very concentrated
urine of
these nondiuretic
animals, as suggested by Walser. The
apparent
disagreement
between the calcium and sodium
ratios in the loop of Henle is of much smaller magnitude,
and may be due to the small number of determinations
and
on which the camp arison 1s based. Both calcium
sodium concentra tions in loop fl uid should vary with the
osmolal .ity of the fluid , and the two groups of hamsters
may not have been entirely comparable
in this respect.
Although
definitive
proof is lacking,
our results are
consistent
with
Walser’s
speculative
hypothesis
that
sodium and calcium ions are in competition
for a common binding
site at the cell membrane.
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Downloaded from http://ajplegacy.physiology.org/ by 10.220.33.4 on April 1, 2017
centration
change occurs because of the simultaneous
reabsorption
of water. On the other hand, when water
reabsorption
from the tubule is limited,
as in mannitol
diuresis,
the sodium
concentration
of proximal
fluid
falls, and active transport
can be demon .strated.
found in nond iuretic
The low calci urn concentration
animals l alon .g the entire length of the distal convol ution,
active
in the face of continuing
water loss, indicates
transport
of calcium
out of this segment,
as well as
from the ascending limb of the loop of Henle. This is
further supported by the very low calcium concentration
found in the distal convolution
during mannitol
diuresis.
Further reabsorption
of calcium occurs from the collecting ducts, but it is not possible from our data to determine whether or not this involves active transport.
Since
at this locu
however,
sodium is actively transported
1s actively
we feel that it is probable
that calcium
transported
here as well.
It is apparent
from the foregoing discussion that the
pattern
of tubular
reabsorption
of calcium
bears many
(6) has
similarities
to sodiu m rea bsorp tion. Walser
of free
observed in di uretic dogs that the clearance
calcium
iou eq uals the sodium cl earance in states of
urine flow and sodium
excretion,
provided
varying
urine flow exceeds 2.5 ml/min.
Our results indicate that
this relationship
al so holds tru e at the various levels in