Clinical Science and Molecular Medicine (1975) 48, 475-489.
Secretion of inorganic phosphate in the rat nephron
J.-F.BOUDRY, U. TROEHLER, M. TOUABI, H. FLEISCH
AND
J.-P. BONJOUR
Department of Pathophysiology, University of Berne, Switzerland
(Received 25 November 1974)
Summary
1. The existence of tubular secretion of inorganic
phosphate (PI) in the mammalian kidney has been
investigated by studying the renal response of rats
infused with sodium phosphate by three different
techniques.
2. Clearance studies indicate that, in anaesthetized
rats, the net tubular reabsorption decreases markedly
in response to PI infusion. In conscious rats, the
clearance of PI slightly exceeded that of inulin at high
plasma PI concentration.
3. Free-flow micropuncture in control rats showed
a net tubular reabsorption of PI along the proximal
tubule, and probably between the end of the distal
tubule and the ureteral urine. In phosphate-loaded
rats, whether receiving parathyroid hormone or not,
an apparent net secretion of PI was observed between
the end of the distal tubule and the ureteral urine. In
the phosphate-loaded group receiving parathyroid
hormone, net secretion was also observed very
early in the proximal tubule followed by a predominant reabsorption along this segment. Thus the early
proximal tubule and probably also the terminal
nephron can be the site of either net reabsorption or
net secretion.
4. Microperfusions of proximal tubules show a fall
in the specific radioactivity of the perfused radioactive PI solution, indicating entry of PI into the
lumen.
(I)
Abb~eviations:
concentrat~on;
Ph inorganic phosphate; P, plasma
TF, tubular fluid concentration; U, urinary
concentration.
Correspondence: Dr I.-P. Bonjour, Department of Pathophysiology, University of Berne, Hugelweg 2 3012 Berne
Switzerland.
"
Key words: micropuncture, renal phosphate transport, secretion.
Introduction
It is generally accepted that, in the mammalian
kidney, inorganic phosphate (PI) is filtered at the
glomerulus and reabsorbed by the tubular epithelium,
so that the amount excreted in the urine equals that
which escapes tubular reabsorption (Pitts, 1968). The
existence of tubular secretion of PlY) defined as a
unidirectional flux from peritubular space to tubular
lumen (Pitts, 1968), has been questioned in mammals
but not in other classes such as fishes (Grafflin, 1936;
Smith, 1939), amphibians (Walker & Hudson, 1937),
reptiles (Hernandez & Coulson, 1956; Clark &
Dantzler, 1972) and birds (Levinsky & Davidson,
1957), wherein it has been clearly demonstrated.
Numerous attempts with various techniques have
been made to demonstrate a secretory flux of PI in
rats (Strickler, Thompson, Klose, Giebisch, Gluck
& Vaughan, 1964), cats (Taugner, von Brunoff &
Brown, 1953), rabbits (Falbriard, Vassalli & Schaller,
1962) and dogs (pitts & Alexander, 1944; Barclay,
Cooke & Kenney, 1949; Nicholson & Shepherd,
1959; Nicholson, 1959; Carrasquer & Brodsky,
1960; Carrasquer & Brodsky, 1961; Bronner &
Thompson, 1961; Handler, 1962; Lambert, Vanderveiken, De Koster, Kahn & De Myttenaere, 1964;
Hellman, Baird & Bartter, 1964; Davis, Kedes &
Field, 1966). Results of several investigations
(Barclay et al., 1949; Nicholson, 1959; Nicholson &
Shepherd, 1959; Carrasquer & Brodsky, 1960;
Carrasquer & Brodsky, 1961; Falbriard et al., 1962;
Davis et al., 1966) could be interpreted as evidence
475
476
J.-F. Boudry et af.
for tubular secretion of PI in mammals. However, the
failure of classical experiments to demonstrate a
significant net tubular secretion in the steady-state
condition of phosphate loading (Pitts & Alexander,
1944; Handler, 1962; Strickler et al., 1964; Hellman
et al., 1964)has led many to conclude that the tubular
secretion of PI in mammals has yet to be established.
A reappraisal of this question has been presented
by Knox, Schneider, Willis, Strandhoy & Ott (1973),
who mention that several findings in dog and man
(Barclay et al., 1949; Webster, Mann & Hills, 1967;
Ginsburg, 1972) suggest strongly the existence of a
secretory pathway. The recent demonstration of
secretion in patients with X-linked hypophosphataemia (Glorieux & Scriver, 1972) has further
underlined the need to reconsider the renal handling
of PI in mammals.
In this paper we present evidence for tubular
secretion of PI in the rat. Such evidence has been
obtained by investigating the renal response to an
acute infusion of phosphate. This response was
studied by three different techniques: clearance,
free-flow micropuncture and microperfusion of the
proximal tubule in situ.
Methods
All experiments were done on male Wistar rats
weighing 170-210 g. Before the experimental day,
the animals were kept on a commercial chow
(Altromin 1314) containing 1·1% Ca and 1'0% P
(dry weight) and had free access to distilled water.
Clearance studies were performed in anaesthetized
and conscious rats. Some of the anaesthetized
animals were thyro-parathyroidectomized 48 h
before the clearance experiment.
Clearance studies
Intact rats. Animals, fasted overnight, were
anaesthetized with a tail-vein injection of pentobarbital (Nembutal) given at a dose of 30-40 mgjkg body
weight in NaCI solution (150 mmol/l) containing
pentobarbital (20 mmol/l, 5 mgjml), Rats were
placed on a heated table to maintain body temperature. One jugular vein and one carotid artery were
catheterized and a tracheotomy was performed. A
catheter was placed in the bladder through a small
skin incision and the urethra was ligated. At the
end of the surgical procedure a priming dose of
inulin (80 mg/kg body weight) in NaCl solution
(150 mmol/l) containing inulin (16 mgjml) was
administered, followed by a constant infusion of
NaCI (150 mmol/l) containing inulin (10 mgjml)
delivered into the jugular vein at 4 ml/h.
After an equilibration period of 90 min, urine was
collected for two periods (1a and 1b) of 30 min.
A part of the NaCl solution was then replaced by
a solution containing inorganic phosphate (PI)' as its
sodium salt, at a concentration calculated to deliver
3·33 .amol/min. After a 30 min equilibration period,
urine was collected for two periods of 30 min (2a and
2b). Finally this infusion solution was replaced by a
solution containing PI at a concentration calculated
to deliver 10 .amol/min and, after equilibrating for
30 min, two urine collections of 30 min were again
performed (3a and 3b). The pH of the three infusion
solutions was 7·4. For the two phosphate solutions,
pH 7·4 was obtained by combining Na 2HP0 4 and
NaH 2P0 4 in an appropriate proportions.
Blood (0,4 ml) was sampled from the carotid
artery at the midpoint of each urine collection
period. After centrifugation of the blood and
removal of the plasma, the erythrocytes were
resuspended in a volume of NaCl (150 mmol/l) equal
to that of plasma, and the cell suspension was
reinjected into the animal immediately after the next
blood sampling in order to minimize change in blood
volume. Urine volume was determined by weighing
after collection in tared test tubes.
Thyro-parathyroidectomized rats. With this group
of animals, the experimental design was exactly the
same as with the intact rats except for the surgical
removal of the parathyroid and thyroid glands 48 h
before the clearance study. The operation was
performed under ether anaesthesia. An additional
blood sample for calcium determination was taken
48 h later at the end of the catheter implantation
procedure and just before the administration of the
priming dose of inulin. Only rats with a plasma
calcium concentration below 1-88 mmoljl were
considered as successfully thyro-parathyroidectomized and retained for the present study.
Conscious rats. Preliminary studies indicated that
non-fasted, conscious rats displayed a higher fractional excretion of PI than fasted anaesthetized
animals. Renal clearances were therefore performed
in rats placed in restrictive cages according to a
technique previously described (Bonjour, 1966;
Bonjour, Regoli, Roch-Ramel & Peters, 1968). The
animals received a constant infusion at 4 ml/h into
a tail vein. The infused solution was the same as the
Phosphate secretion in rat nephron
solution used in the anaesthetized animals during
period 3 so that PI was delivered at 10 pmol/min.
After an equilibration period of 240 min, urine was
collected for two periods of 45 min each. Blood
samples were taken from the vein of a hind limb at
the beginning and at the end of each clearance
period. At the end of the second period, rats were
anaesthetized with ether and 6-8 rnl of blood was
taken anaerobically by aortic puncture. Blood was
centrifuged at 37°C and plasma from five rats was
pooled. Three aliquots of 3 ml each were ultrafiltered
at 37°C according to the method of Toribara,
Terepka & Deway (1957), and the concentration of
PI and calcium in plasma and ultrafiltrate was
determined.
Micropuncture experiments
Rats were fasted 12-15 h before the experiment
but free access to water was allowed. The rats were
then anaesthetized as described above for the clearance studies and placed on a heated table. Surgical
preparation involved tracheotomy and cannulation
of a jugular vein for infusion, of a carotid artery for
obtaining blood samples, and of the bladder. Each
rat was then placed on the right side, and the left
kidney was exposed through a flank incision and
placed in a small plastic cup for immobilization. The
kidney was decapsulated and covered with liquid
paraffin. The ureter of the exposed kidney was then
cannulated.
As soon as the jugular vein was cannulated, the
rats received a priming dose of inulin (400 mgjkg
body weight) in 1·0 ml of NaCI (150 mmol/I), followed by a constant infusion of NaCI containing
inulin (48 mg/ml), delivered at 4 ml/h into the jugular
vein. This dose of inulin maintained a plasma concentration of about 175 mg/lOO ml. A second solution
was infused into the tail vein at 4 ml/h. This solution
contained either sodium chloride (150 mmol/l)
(group A) or sodium phosphate at a concentration
calculated to deliver 6'66 pmol/min (group Band
group C). The pH of all solutions was 7·4. In ten of
the sixteen animals receiving the phosphate solution,
parathyroid hormone (bovine parathyroid hormone,
trichloroacetic acid powder,·· Wilson Laboratories)
was added to the jugular vein infusion, to deliver
2·5 i.u.Ih (group C). At least 120 min after the
priming injection, the passage time of the tubular
fluid was determined, in order to assess roughly
whether the kidney to be punctured was in a satis-
477
factory haemodynamic condition. It was performed
according to Steinhausen's (1963) technique by
injecting instantaneously Lissamine Green at a dose
of 3·0 mg/kg body weight in 1·5 ml/kg NaCI (150
mmol/l). After the first blood sample (0'3 ml) was
taken, urine collection from the left ureter and the
bladder was started. Micropunctures were then
performed according to a technique previously
described by Roch-Ramel, Chomety & Peters
(1968). A Leitz binocular stereomicroscope was used
at a 100 x magnification. Micropuncture capillaries
had a tip external diameter of about 10 pm. They
were filled with mineral oil (Protol USP, Witco
Chemical Co., Haarlem, The Netherlands) saturated
with water and coloured with Sudan Black.
Several micropuncture specimens of 0·1-0·2 pI were
obtained from proximal and distal tubules within
collecting periods of 5-10 min. The flow of tubular
fluid distal to the puncture site was stopped by
injection of an oil droplet. A second blood sample
was taken 45-60 min after the first one and a
second ureteral and bladder urine collection was
started. Micropunctures were again performed for
another 45-60 min period, at the end of which a final
blood sample was taken. In some experiments, the
proximal or distal puncture site was either selected
after identification by intravenous injection of 0'1 ml
of Lissamine Green solution or localized later after
latex injection and dissection, according to the
technique described by Roch-Ramel et al. (1968).
Blood samples were analysed for inulin, PI and
total calcium. Plasma concentrations of these
substances at the time of a micropuncture sampling
were obtained by interpolation of their concentration
at the midpoint of each micropuncture collection
period. Experiments in which the concentration of
any of these substances in two consecutive blood
samples differed by more than 10% were discarded.
Micropuncture capillaries were closed with wax
and immediately frozen at - 20°C. For the determination of inulin and PI the samples were transferred into silicone-treated glass cups filled with
liquid paraffin, previously saturated with water.
Aliquots of the sample were then aspirated into
calibrated constriction micropipettes.
Microperfusion experiments
Rats were prepared surgically as for the micropuncture experiments. Groups 1 and 3 received a
phosphate infusion of 6·66 pmol/min into the jugular
478
J.-F. Boudry et al.
vein, the solution used being the same as the one
infused to groups Band C in the micropuncture
study. The solution was infused at 4 ml/h, Group 2
received NaCI (150 mmol/l) at 4 ml/h. No systemic
infusion of inulin was given. After an equilibration
period of 90-120 min, a Lissamine Green passage
time was determined for the reasons outlined above
and microperfusions of proximal tubules were performed according to the method described by
Sonnenberg & Deetjen (1964). Solutions were perfused at the nominal rate of 15 or 30 nljrnin with a
10 III microsyringe (Hamilton, Bonaduz) on a
Brown Melsungen pump (Perfusor). The microcapillaries used for perfusion and collection were the
same size as those used for the free-flow micropuncture experiments. After starting the perfusion,
a minimum of 3 min equilibration time was always
allowed before beginning the collection. At the end
of the microperfusion, the perfused tubule was filled
with Neoprene (Neoprene-Latex 601-A, Dolder AG,
Basel) from a micropuncture capillary with a tip
diameter of about 15 11m, and a drawing of the
kidney surface was made to facilitate identification
of the puncture sites during microdissection. At the
end of each microperfusion, arterial blood was
sampled and the plasma concentration of PI determined. The kidneys were then frozen at - 20°C. The
length of the perfused segments was measured after
maceration of the kidneys and microdissection of
the tubules (Roch-Ramel et al., 1968).
The proximal tubules were perfused with one of
the three following solutions. Group 1: in order to
reduce the net movement of sodium and water the
solution contained NaCI (110 mmol/l) and was made
isotonic by adding raffinose at a concentration of
54 mmoljl, In addition it contained Lissamine Green
4 g/I, [methoxy-3H]inulin (specific radioactivity 86·6
mCi/g, from New England Nuclear, NET-086) 0'25
mCi/ml, [3ZP]orthophosphate, as H 3P0 4 carrier-free
(from Institut fUr Reaktorforschung, Wurenlingen,
Switzerland) 0·5 mCi/ml, and non-radioactive orthophosphate (5 mmol as a mixture of Na zHP0 4 and
NaH zP0 4 giving pH 7'4). Group 2: the same
solution as in group 1, but without the addition
of non-radioactive orthophosphate. Group 3: the
same solution as in group I, but without the addition of raffinose and with 150 mmol/l NaCl.
Chemical determinations
In the clearance studies, inulin was determined by
the anthrone method (Richterich, 1971b). In the
micropuncture study, inulin in plasma and ureteral
urine was determined by the diphenylamine method
(Harrison, 1942). A blank of 10 mg/l00 ml was
subtracted from all plasma values. For the tubular
fluid aliquots the ultramicromodification of the
diphenylamine method described by Roch-Ramel et
al. (1968) was used.
In the clearance studies, PI in plasma, urine and
plasma ultrafiltrate was determined colorimetrically
as phosphomolybdate after reduction with a 10 g/
100 ml ascorbic acid solution (Bisaz, Russell &
Fleisch, 1968). In the free-flow micropuncture
experiments, concentration of PI in plasma and
ureteral urine was determined by the Malachite
Green method, whereas concentration in aliquots of
tubular fluid was determined by an ultramicroadaptation of the same method (Richterich, 1971a).
Micropuncture fluid was aspirated into a 5-10 nl
constriction micropipette. This amount was then
blown into 2 III of distilled water, in a 0·4 ml polyethylene tube, and 10 III of the Malachite Green
reagent (Richterich, 1971a) was added. The tubes
were stoppered and kept at room temperature for 30
min. Calibration curves were obtained from standards treated in the same manner. The extinction
was read in a Zeiss PMW II spectrophotometer
at 630 nm, in a microcuvette similar to that described
by Ullrich & Hampel (1962). The extinction was
constant for at least 3 h after addition of the reagent.
As for the microdetermination, known amounts of PI
added to urine samples were completely recovered.
At the dilution used there was no interference by
Lissamine Green remaining in the tubular fluid after
determination of the passage time. The Malachite
Green method is about six times more sensitive than
the phosphomolybdate technique used for the microperfusion.
In the microperfusion experiments, plasma PI was
also determined by the Malachite Green method
(Richterich, 1971a). Determinations of the PI
concentration in perfusing solutions and tubular
fluid samples were carried out with an ultramicroadaptation of P. S. Chen's phosphomolybdate
method (Bisaz et al., 1968; Chen, Toribara &
Warner, 1956). Briefly, a 20-30 nl aliquot of a
standard solution or a tubular fluid sample was
added to 5 III of HCl (375 mmol/l) and 5 pI of
molybdate reagent (ammonium heptamolybdate c-Of
mmol/l, ascorbic acid 114 mmcl/l in HCl, 1 mol/I).
The mixture was then incubated at 100°C for 10 min.
Phosphate secretion in rat nephron
After cooling, extinction was read at 820 nm in the
above-mentioned microcuvette in the Zeiss spectrophotometer. Known amounts of PI were added to
urine samples: the recovery ranged from 90 to 110%
with a mean value not statistically different from
100%.
Plasma calcium was determined by atomic
absorption spectroscopy (Perkin-Elmer model 290
B) after dilution of the samples with 0'5% LaCh.
Processing of radioactive specimens
In the microperfusion experiments, the radioactivity of 32p and pH]inulin was measured in a
Packard Tri-Carb scintillation spectrometer (model
3950). A 5-10 nl aliquot of a perfusing solution or a
tubular fluid sample was added to 10 ml of a liquidscintillation solution made from 600 ml of toluene
and 300 ml of ethylene glycol monoethylether, in
which were dissolved 80 g of naphthalene (Merck,
no. 6200) and 7·0 g of butyl-PBD (Ciba-Geigy).
Appropriate corrections were made for background,
quenching and incomplete discrimination between
the two isotopes.
479
Results
Clearance study in anaesthetized rats
In intact rats, the renal response to a moderate
phosphate load (3,33 pmol/min) consisted of a sharp
decrease in the absolute amount of PI reabsorbed
(Table 1, period 2). A threefold increase in the rate
of PI infusion led to a further decrease in the tubular
reabsorption of PI (Table 1, period 3). At this rate of
PI infusion, two animals out of six excreted more PI
than the amount filtered. In thyro-parathyroidectomized animals, a significant decrease in the absolute
amount of PI reabsorbed was also observed, but only
during the infusion of the large dose (10 pmol/min) of
PI (Table 2, period 3). In both intact and thyroparathyroidectomized groups, plasma calcium fell
proportionately to the rate of PI infusion. The
relationship between plasma phosphate and the
phosphate reabsorbed expressed per unit of glomerular filtrate is represented in Fig. 1. This representation shows clearly that an inverse relationship
exists between the two variables in both intact and
thyro-parathyroidectomized animals.
Calculation and statistical analysis
Clearance study in conscious rats
In the calculation of the ratios for urine or tubular
fluid over plasma concentration of Ph the values of
plasma PI were not corrected for incomplete
ultrafiltrability.
All experimental results are expressed as mean
values ±SEM. Significance of difference was evaluated
by paired sample analysis and the Student's t-test.
The results of the clearance experiments in conscious rats are presented in Table 3. During phosphate loading, the fraction of phosphate filtered
which was excreted in the urine remained steady at a
value of about 1·0 for the two consecutive periods of
clearance. The ratio of the concentration of phosphate in ultrafiltrate to that in plasma sampled at the
TABLE 1. Phosphate clearance in anaesthetized intact rats
flow; Clnulin = clearance of inulin; U = urine concentration; P = plasma concentration. Results for
periods 1,2 and 3 are the mean of values for two periods each oe30 min. All results are mean values±sEM (n = 6).
Significanceofdifference between period 2 or 3 and period 1 estimated by paired sample analysis: * P< O'OS;** P< 0·01 ;
*** P<O·OOI.
V
= urine
Period 1
PI infused (pmol/min)
Plasma PI (mmolJI)
V (mljmin)
Clnulln (rnl/min)
PI filtered (pmol/min)
PI excreted (pmol/min)
PI reabsorbed (pmol/min)
(U/P)P I
(U/P)lnulln
Plasma Ca (mmoIfI)
Period 2
Ii. (2-1)
Period 3
Ii. (3-1)
10·0
4·13±0·21
0·034± 0·004
1·82±0·09
7-30±0'61
6·28±0·19
I·OHO·S8
+ 2'00+ 0,28***
+0·012+0'004*
-0·40±0·22
+ 2·67 + 1,03*
+S·S7+0·2S***
-2-88±0'94*
2'13±0'16
0·021 ± O·OOS
2·22±0·44
4·63±0·49
0·71±0·07
3·92±0·44
3·33
2'63±0'09
0·033±0·004
1·83±0·21
4·79±0·20
2·73±0·20
2·06±0·24
0'I7±0'016
0·S3±0·031
+0'36±0'042***
0·89±0·06S
+0·72±0·074***
2·47±0·04
2·24±0·OS
-0·23±0·08*
1·88±0·OS
-0·S9±0·08***
+0'SO±0'16*
+0·012±0·006
-0·39±0·19
+0·16±0·S8
+2'02+0,19***
-1·86± 0'64*
J.-F. Boudry et al.
480
TABLE
2. Phosphate clearance in anaesthetized thyro-parathyroidectomized rats
Thyro-parathyroidectomy was performed48 h beforetheclearancestudy. V = urine flow;Cinull n = clearanceof inulin;
U = urine concentration; P = plasma concentration. Results for periods 1,2 and 3 are the mean of values for two
periodseach of 30min. All resultsare meanvalues± SEM (11 = 5 unlessotherwisespecified by a numberin parentheses).
Significance of difference betweenperiod 2 or 3 and period 1 estimated by paired sampleanalysis: * P< 0·005; ** P<
0'01; *** P<O·OOL
Period 1
Pi infused(zzrnoljmin)
Plasma PI (mmol/I)
V (mljrriin)
Cinuiln (mljrnin)
Pi filtered (jlmol/min)
PI excreted(jlmol/min)
P j reabsorbed (jlmol/min)
(U/P)P1
(U/P);:;;-;;;Plasma Ca (mmol/I)
3·ll±0·08
0·054± 0·012
l-60±0·13
4·90±0·29
0·37±0·06
4·53±0·32
3·33
4·16±0·16
+1'05+0'13**
0·032± 0·007 -0'022±0'006*
1·43±0·08
-0'17±0'15
5·87±0·17
+0·97+0·40
1·95±0·1O
+1'46+0'21**
3·92±0·1O
-0·61±0·25
0·33±0·03
1·60±0·04
1·38± 0'04(3)
Micropuncture studies
The results obtained in the micropuncture are
300
to;
"2
'"
g
200
"15
E
8
1"
100
~
(;
V>
.c
~
a:
+ 0·25+ 0,03**
0·08±0·013
end of the second clearance period was 0·93 ±0'01
(n = 3). Thus the fraction of filtered Pi which
was excreted in the urine [(U/P)PJ!(U/P),nulln] was
under-estimated since plasma Pi concentration was
not corrected for the apparent incomplete ultrafiltrability of Pi.
C>
Jl (2-1)
Period 2
OL ----~----:!:--------:!,....
I
3
7
5
Plasma PI{mma!1Il
FIG. 1. Net tubular reabsorption of phosphate (PI) expressed
per 100 ml of glomerular filtrate plotted against the plasma
concentration of Pi. Phosphate was infused at 0, 3'33 and
10'0 Jlmol/min in intact rats (e, n = 6) and in thyroparathyroideclomized rats (0, n = 5) under pentobarbital
anaesthesia. Thyro-parathyroidectomy was performed 48 h
before the phosphate-loading experiment.
Period 3
Jl (3-1)
10·0
+ 3·53+ 0,16***
6·64±0·12
0·028± 0·002 -0·026±0·012
1·28±0·09
-0·32±0·13
+ 3·39+ 0,95*
8·29±0·81
6'IH 1·40
+5'81±0'57***
2·10±0·63
-2'43±0'67*
0·79±0·06
+0'71±0'07***
1'IH 0'03(4)
summarized in Table 4. The mean values of Clnuiln for
the micropunctured kidneys of the three groups were
comparable with those usually reported in similar
conditions. In each group no significant difference
was observed between the urine flow of the punctured
and contralateral kidneys. As observed in the clearance study, Pi infusion led to a decrease in plasma
calcium. For each proximal or distal micropuncture
sample, the ratios (TF/P)P I and [(TF/P)PJ!(TF/P)
Inulin], the filtered fraction of Pi remaining at the site
of the puncture, have been designated early or late on
the basis of the corresponding value for (TF/P)inuiln.
For the proximal and distal segment the separation
between early and late localization was arbitrarily
set at a (TF/P)inulln ratio of 2·0 and 6·0 respectively.
This estimate of the anatomical site allows the fate of
Pi along the nephron in one experimental condition
to be followed. However, it does not permit the
assessment of the actual differences between groups
in the tubular handling of PI at a precise anatomical
site of the nephron. Indeed, phosphate infusion
without and especially with the administration of
parathyroid hormone could have depressed the
tubular reabsorption of sodium and water along
the proximal tubule (Agus, Puschett, Senesky &
Goldberg, 1971; Agus, Gardner, Beck & Goldberg,
1973). Thus, at least in the proximal tubule, the same
value of (TF/P)inulln might well correspond to a site
farther along this segment in group B as compared
with group A and in group C as compared with
group B.
In the animals which did not receive a P, infusion
(group A) the ratio (TF/P)P I along the proximal
Phosphate secretion in rat nephron
TABLE 3. Phosphate clearance in conscious intact rats
After an equilibration period of 240 min, urine was collected
for two periods (I and 2) each of45 min. V = urine flow;
Clnulln = clearance of inulin; U = urine concentration; P =
plasma concentration. All results are mean values± SEM
(n = 5).
PI infused <JLmol/min)
Plasma PI (mmol/I)
Plasma Ca (mmol/I)
V (mljmin)
Clnulln (mljrnin)
PI excreted<JLmol/min)
(U/P)PI
(U/P)lnulln
Period I
Period 2
10·0
6·3±0·12
1·4±0·03
0·060± 0·005
1·53±0·04
9·7±0·15
10·0
6·3±0·14
1·4±0·04
0'054± 0·003
l-60±0'06
10'1±0'29
l-04±0·03
1·07±0·01
tubule was found to be significantly below 1·0. As
shown in Fig. 2(a), this ratio remained constant
from the beginning to the end of the accessible
proximal tubule and then increased progressively
along the distal tubule. In the same group, the fraction of PI filtered which remained at the site of the
481
micropuncture [(TF/P)Pd(TF/Plnulln] declined steadily over the length of the accessible proximal tubule
(Fig. 2b). This fraction then remained constant from
the end of the proximal to the end of the distal
tubule. Finally the mean fraction of PI excreted in the
urine [(U/P)Pd(U/P)lnulln] was significantly lower
than the mean fraction remaining in the late part of
the distal tubule (0'17 as compared with 0'32,
P < 0;901, see Table 4). This could indicate that in the
non-phosphate-loaded animals, net reabsorption of
PI also took place along the terminal nephron.
In the phosphate-loaded rats which did not
receive parathyroid hormone (group B), the ratio
(TF/P)P I along the accessible proximal tubule
remained constant (Fig. 3a) but at a higher level than
that observed in the animals which did not receive a
phosphate infusion. A progressive rise in the ratio
(TF/P)P I was observed along the distal tubule (Fig.
3a). In the same group, the fraction of filtered PI
which remained at the site of the micropuncture
decreased over the length of the accessible proximal
tubule (Fig. 3b), indicating a net reabsorption of PI.
Between the end of the proximal tubule and the
TABLE 4. Micropuncture study in control rats and phosphate-loaded rats
V = urine flow; Clnulln = clearance of inulin (correspondsto the micropunctured kidney);
TF = tubular fluid concentration; P = plasma concentration; U = urine concentration;
p.k, = punctured kidney; c.k, = contralateral kidney. All results are mean values±sEM
with the number of observations in parentheses. The separation between early and late
proximalor distal tubule has been arbitrarily set at a (TF/P)lnulin ratio of 2,0and 6·0 respectively. Significance of difference as compared with the corresponding value of the late distal
tubule: *** P<O·OOI.
Group A
PI infused (pmol/min)
PTH infused (i.u.jmin)
Plasma PI (mmol/I)
Plasma Ca (mmol/l)
V (mljmin per kidney) {~:~:
Clnulln (mljmin per kidney)
(TF/)P I early proximal
late proximal
early distal
late distal
(U/P)PI
(TF/P)PI
early proximal
(TF/P)lnulin
late proximal
early distal
late distal
(U/P)PI
(U/Pbulln
B
0
Group B
6·66
Group C
2·23± 0'25(7)
2'39±0'11(7)
0'031± 0'006(7)
0·037± 0'004(7)
0·89±0·10(7)
0'79± 0'02(8)
0·75± 0'07(7)
1·22±0·14(7)
2'48±0'17(7)
4·96± 0,78(7)
4·35± 0'39(6)
1·97±0·12(6)
0'033± 0'004(6)
0·039± 0,003(6)
l-03±0'12(6)
1·35±0·10(8)
1'22± 0'07(7)
2'16±0'42(5)
4·37± 1'10(5)
25·04± 4'41(6)
6·66
0·042
4·74± 0'22(10)
l-88± 0'03(10)
0'042± 0'005(10)
0·047± 0'003(10)
0·99± 0'09(10)
l-80±0'09(12)
2·01±0·12(14)
2·16±0·40(4)
3'54± 0'70(5)
19·31± 3'13(10)
0·51± 0'03(8)
0·89± 0'04(8)
1·17± 0'06(12)
0'30± 0'03(7)
0'31±0'02(7)
0'32±0'02(7)
0·50± 0'05(7)
0·48± 0'08(5)
0·45± 0'03(5)
0·74± 0'06(14)
0·43± 0'04(4)
0·43± 0'03(5)
0'17±0'02(7)*"
0·66± 0'03(6)***
0·70± 0'03(10)**·
J.-F. Boudry et of.
482
•
40
•
(0)
30
0
3
0
a:-
c:-
2
0
0
l;:
I
d:"
•
c:-
0
•
<,
0
2
00
20
•
0
t:.
10
L
/
1·0
1·0
(b)
ee
0·5
•••
• • •e •
o
000
o
0&
0
0
........
, I
2
3
6
8
10
•
(TF/P)inulin
FIG. 2. Phosphate handling by the renal tubule in control rats (e, proximal; 0, distal). (a) The ratio for tubular fluid
(TF) P.!plasma (P) PI is plotted against the same ratio (abscissa) for inulin. The ratio ureteral urine (U) PI!
plasma (P) PI (.) is presented at the right. (b) Fraction of filtered phosphate at puncture site [(TF!P)PI!(TF!P)lnulln]
is plotted against the tubular fluid (TF)!plasma (P) ratio for inulin. The fraction of PI filtered excreted in the
ureteral urine is presented at the right (.).
late distal tubule no net movement of PI took place.
However, between the late distal tubule and the
ureteral urine, an apparent net secretion of PI was
observed (Fig. 3b). Indeed the mean fraction of PI
excreted in the urine was significantly higher than
the mean fraction remaining at the late distal tubule
(0,66 compared with 0,45, P<O'OOI, see Table 4).
The addition of parathyroid hormone to the phosphate infusion (group C) brought about a further
increase in the (TF!P)P I ratio along the proximal
tubule (Table 4, Fig. 4a). As in the other two groups,
the tubular fluid concentration of PI was altered
mainly in the very early proximal tubule, and no
significant change was observed up to the latest
portion of the accessible proximal convolution.
In group C, the mean fraction of PI filtered
observed in the early proximal tubule was more than
1,0, indicating that secretion had taken place in the
earliest part of the nephron. Nevertheless, further
down the proximal tubule, reabsorption predominated but not to the extent of reducing the fraction
remaining inthe tubule to the level observed in the
late proximal tubule in the phosphate-loaded rats
which had not received the infusion of parathyroid
hormone (Table 4). Between late proximal and early
distal tubule, however, a net reabsorption supervened so that the early distal value was similar in both
phosphate-loaded groups (groups B and C). The
administration of parathyroid hormone to phosphate-loaded animals did not modify the net handling of PI along the distal tubule. As observed in
group B, an apparent net addition of PI seemed to
take place between the end of the distal tubule and
the ureteral urine.
Phosphate secretion in rat nephron
483
40
•
•
o
(0)
7
30
a:
•
•
D:'
<,
5
2
o
3
o
fI'
20
0
•
o
o
o
•
00
~-tl----~~--~·.... ----o----- ------ --,/ I
10
L
/rL--
e
\·0
a:-
.E
a:: a::~
~
t:.
~
e
e
e
e
t:.
0·5
1·0
e
e
0
e e
e e
0
0
0
•e.
0
0
0
o
0·5
•
•••
••
0
0
(b)
2
3
i
/I
4
I
6
I
8
(TFIPhnulin
FIG. 3. Phosphate handling by the renal tubule (e, proximal; 0, distal) in phosphate-loaded rats (6'66 pmol of
PI/min). Results are presented in (a) and (b) in the same way as described in Fig. 2.
Finally, the concomitant infusion of parathyroid
hormone in phosphate-loaded rats, at a dose which
had a conspicuous phosphaturic effect in nonphosphate-loaded rats (unpublished observations),
did not further significantly increase the fraction of
filtered PI excreted in the urine (Table 4).
Mlcroperfusion study
Results of the microperfusion of proximal tubules
are summarized in Table 5. With the isotonic low
sodium solution (groups 1 and 2), the amount of PI
collected exceeded the amount perfused. A net entry
of PI was present in all tubules. Since only the
nominal perfusing flow rate of the pump was
known, no attempt was made to calculate the actual
amount of PI entering the tubular lumen. In both
groups 1 and 2, the greatest fall in the specific
radioactivity of phosphate was observed with the
longest perfused tubule.
With the 150 mmol/l NaCI solution (group 3) a net
reabsorption of PI was observed in three out of the
four perfused tubules. However, the specific radioactivity of PI is also lower in the collected fluid than
in the perfusing solution. This indicates that in this
condition, too, PI has been secreted into the tubular
lumen.
Discussion
Our clearance studies in anaesthetized rats show that
net tubular reabsorption did not remain constant but
I.-F. Boudry et al,
484
40
••
7
(0)
30
ci:'
0
5
ci:'
'iL
<,
2-
••
20
a:-
t;::
!::.
•
3
00
~., • • •
••
, _4!.
•
•
"....
0
0
••
0
0
I
0
!!
Q..
,(
10
_
L
I
••
•• •
',0
•
c
•
,---••
1·0
• • • •
• ••
• • ••
!f. ]
Q.a:-
t;::,
I- u,
~t:.
••
0·5
~
a:~
ci:'1
<,
~
Q.
<,
~2.
0
0000
0
0
0
0·5
..,:t
•
0
(b)
{ I
6
decreased markedly in response to an elevation of
plasma PI concentration, contrary to the usual
pattern described for the renal handling of PI (Pitts,
1948). This finding is in agreement with a recent and
very similar study (Frick, 1968). In addition our
results show that decrease in the net tubular reabsorption of PI also occurs in thyro-parathyroidectomized animals, though at a higher plasma concentration of PI. The increased parathyroid hormone
secretion, which probably occurs in response to the
phosphate load through the fall in plasma calcium
(Sherwood, Mayer, Ramberg, Konfeld & Potts,
1968; Reiss, Canterbury, Bercovitz & Kaplan,
1970; Fischer, Binswanger & Blum, 1973), thus seems
not to be the only factor responsible for the fall in the
8
10
net tubular reabsorption of PI. It remains to be
established whether hypocalcaemia itself plays a role
in reducing the net reabsorption of Ph as suggested
by other observations (Lavender & Pullman, 1963;
Rasmussen, Anast & Arnaud, 1967; Glorieux &
Scriver, 1972),
As seen in the micropuncture experiment, the
large decrease in the net tubular reabsorption of PI
cannot be attributed to extracellular volume expansion since animals infused with the same volume of
isotonic NaCI display a much smaller fractional
excretion of PI. The observed response must represent
a specific phenomenon as inferred by Frick (1968)
in his similar study.
In the experiment carried out in anaesthetized
Phosphate secretion in rat nephron
485
TABLE 5. Microperfusion ofproximal tubules
In groups 1 and 2 the tubules were perfused with an isotonic solution containing NaCI (110 mmol/l) and
raffinose (54 mmol/l). In group 3 the tubule perfusion was performed with an isotonic NaCI solution (150
mmol/I). In groups 1 and 3, the plasma phosphate concentration was raised by intravenous infusion of
sodium phosphate and the solution perfused in the tubules contained phosphate at a concentration very
close to that of the plasma. In group 2, the rats received an intravenous infusion of NaCl, and only carrierfree [3 2Plorthophosphate was added to the solution used for the tubular perfusion. Concentrations of
PI, inulin and specific radioactivity (S.A.) of PI are shown for the perfusing solution (0) or the collected
fluid (f). The ratio of concentrations presented in column 5 represents the fraction of perfused PI which
has been collected. A value for the ratio above or below unity means net secretion or net reabsorption
respective ly.
Group
no.
Plasma
(mmolfl)
Length of
tubule perfused
(mm)
Perfusion
flow rate
(nJ/min)
0·7
1·0
2·0
1·0
Mean
(±SEM)
4·87
(0'23)
2
Mean
(±SEM)
Mean
(± SEM)
[Pllf· [Inulinjo
lPllo' [Inulinjf
S.A·rlS.A .•
5·54
5·32
5·32
5·32
1·58
2-36
2·74
1-62
0·59
0·39
0·33
0·43
0·48
0·48
0·50
0·50
2·35
4·31
2-46
3040
0·34
0·20
0·33
0·27
4-85
0·48
0·89
1·09
0·49
0·80
0·75
0·65
0·86
15
2·0
3·0
1·5
1·6
2·77
(0'17)
3
lPllo
(mmol/l)
30
4·5
1·8
2·5
3·0
4·70
(0'86)
intact rats, some animals displayed a negative net
tubular reabsorption, but the mean P.finulin
clearance ratio was below unity. In conscious rats,
however, the mean clearance of PI was not lower than
that of inulin in the steady-state condition of PI
infusion. The ultrafiltrability of plasma PI was found
to be 0,93, a value in agreement with that published
by Strickler et af. (1964) for phosphate-loaded rats.
Taking this into account, the amount of phosphate
excreted would slightly exceed the amount filtered.
Even without considering ultrafiltrability, a clearance
ratio P.finulin of 1·0 suggests strongly that secretion
of PI has taken place. Indeed, it is difficult to conceive that, in the presence of an increased filtered
load, the reabsorptive flux of PI would be abolished.
.Our micropuncture studies in rats which did not
receive a phosphate infusion confirmed the previous
finding of others (Strickler et al., 1964; Morel,
4'85
4·85
4·85
30
Roinel & Le Grimellec, 1969; Amiel, Kuntziger &
Richet, 1970). The mean (TF/P)P I in the whole
proximal tubule was 0'77, which corresponds very
closely to the values obtained in several earlier
studies (Strickler et al., 1964; Gekle, Stroder &
Rostock, 1969; Arniel et al., 1970). Our data confirm
that the tubular fluid concentration of PI remains
constant along the accessible proximal tubule at a
value already set in the early part of this segment and
demonstrate the absence of an appreciable net
movement of PI between the end of the proximal and
the early distal tubule and also along the distal segment, as previously reported (Strickler et al., 1964:
Morel et al., 1969; Arniel et al., 1970). Finally, the
results showed that an apparent net reabsorption of
PI takes place between the end of the distal tubule
and the ureteral urine, in agreement with other recent
free-flow micropuncture data (Arniel et al., 1970;
486
J.-F. Boudry et al.
Le Grimellec, Roinel & Morel, 1973a; Le Grimellec,
Roinel & Morel, 1973b). It seems unlikely that the
reduced fraction of filtered PI found in the ureteral
urine, compared with that in the late distal tubule,
could be due to the contribution of deep nephrons
with a higher reabsorptive capacity. Thus a net
reabsorption of PI along the terminal nephron has
to be suspected in the rats without phosphate load.
The discrepancy between these various free-flow
micropuncture studies and the results of microinjection experiments (Brunette, 1972; Staum,
Hamburger & Goldberg, 1972), which failed to
demonstrate net PI reabsorption over the last portion
of the nephron, remains to be explained.
In the phosphate-loaded animal, the main difference between our micropuncture data and the work
of Strickler et al. (1964) concerns the fraction of PI
filtered that remains at the level of the distal tubule.
In our experiments less than half the amount of PI
filtered is still present in the distal tubular fluid,
whereas the mean value obtained by Strickler et al.
(1964) was 0·71 along the same segment in phosphate-loaded rats. The net entry of PI along the
terminal part of the nephron which we have observed,
was not detected by Strickler et al. (1964). However, it should be noted that two out of seven rats
studied by Strickler et al. (1964) excreted PI in an
amount equal to or larger than that filtered. The
different pattern in the tubular handling of PI
between the two studies might be related to the
experimental conditions. In the experiments of
Strickler et al. (1964), for instance, the renal response
to phosphate loading probably took place in the
presence of systemic acidosis since monosodium
phosphate was perfused in rats that had already
received NH4CI in their drinking water. As stated
by the authors, this regimen was adopted to achieve
high rates of urinary phosphate excretion on the
basis of experiments in dogs showing a pH-dependent
transient secretion of PI (Carrasquer & Brodsky,
1960, 1961).
The addition of parathyroid hormone to the
phosphate infusion brought about a net entry of PI
into the early proximal tubule. This effect, however,
did not increase further the urinary excretion of PI in
the phosphate-loaded rats since it was compensated
before the accessible early distal tubule by a predominant reabsorption. Although our study was not
designed to localize the site of action of parathyroid
hormone, it is noteworthy that the decrease in the
net reabsorption of PI along the proximal tubule in
response to the hormone agrees with previous
micropuncture studies in rats (Gekle, 1971) and dogs
(Agus et al., 1971, 1973). It also fits the increase in
the tubular reabsorption observed in the proximal
tubule after parathyroidectomy in rats (Carone,
1964; Gekle, 1971; Frick, 1972) and dogs (Beck &
Goldberg, 1973). Earlier stop-flow studies in dogs
(Lambert et al., 1964; Samiy, Hirsch & Ramsay,
1965) and very recent microinjection experiments in
rats (Brunette, 1972) also suggest a proximal site for
the action of parathyroid hormone on the tubular
handling of PI.
In our experiments, a net secretion of PI appeared
to take place between the late distal tubule and the
ureteral urine in both phosphate-loaded groups. The
question arises whether this finding can be explained
on the basis of a functional heterogeneity between
superficial and deep (mid-cortical and juxtamedullary) nephrons. As calculated by Amiel et al.
(1970) on the basis of the nephron population fraction (De Rouffignac & Morel, 1967), and the mean
individual glomerular filtration rate (Baines & De
Rouffignac, 1969), the superficial nephrons in the rat
would be responsible for about 55% of the total
glomerular filtration rate. If one assumes that no
net movement of PI occurs between the late distal
tubule of the superficial nephrons and the ureteral
urine, the fraction of PI filtered that is excreted in the
final urine by the deep nephrons should be 1·0 and
0·9 for the phosphate-loaded groups with and without administration of parathyroid hormone respectively. Thus, in the former case, the absence of a
secretory flux along the deep nephron or the terminal
part of the superficial nephrons would require that the
reabsorptive flux of PI is completely abolished along
the entire length of the deep nephrons. This seems to
be a quite untenable hypothesis.
Our micropuncture experiments in control and
phosphate-loaded rats demonstrate the role played
by the early proximal tubule and probably by the
terminal nephron, in the renal handling of PI. The
relative importance of these two tubular segments
with regard to the final regulation of PI excretion
requires to be assessed. Our results show that
alteration in the reabsorption of PI along the proximal tubule can be fully or partly compensated before
the tubular fluid enters the last portion of the
nephron. They also suggest that the terminal nephron
is not inert in the regulation of PI excretion, but
might be the critical segment where a specific and
precise regulating mechanism operates to achieve a
Phosphate secretion in rat nephron
final urinary output of PI adjusted to the needs of the
organism.
The micro perfusion experiments provide evidence
supporting the idea that PI can enter into the tubular
lumen at least at the level of the proximal epithelium.
Such entry can be observed at endogenous or
elevated plasma concentrations of PI. It also occurs
whether the net flux of PI is directed into or out of
the tubular lumen. Thus the fall in the specific radioactivity that we observed cannot be attributed
merely to a phenomenon of exchange diffusion
between tubular and cellular Pl. Evidence for an
entry of PI into the proximal tubule has also been
obtained by Murayama, Morel & Le Grimellec
(1972), with an isotonic NaCI solution containing a
low PI concentration (560 /lmol!!). However, the
authors ascribe this entry to an initial passive leak at
or near the perfusion site. In our experiments there is
a significance positive correlation (r = +0'81,
P < 0'05, n = 8) between the length of the tubules
perfused with the isotonic low-sodium solution
(groups 1 and 2) and the decrease in the specific
radioactivity ratio of Pl. This finding would speak
against an artifact caused by 'permeability damage'
due to the presence of the microperfusion pipette.
In summary, the results we have obtained in
clearance studies, free-flow micropuncture and
micro perfusion of proximal tubule all support the
concept of a secretion of PI in the rat kidney.
Recently, clearcut evidence of the renal secretion
of PI in man has been published (G1orieux & Scriver,
1972). The authors showed that in mutant hemizygotes with X-linked hypophosphataemia, a component which probably stimulates the reabsorptive
flux was lacking. Consequently a secretory flux was
revealed by the presence of net secretion during
phosphate infusion. Now that a secretory mechanism
has been demonstrated in the mammalian kidney its
importance in the overall regulation of PI excretion
and the factors which might modulate it remain to be
established. The use of techniques such as the microperfusion of isolated tubules from different parts of
the nephron should provide answers to these
fundamental questions.
Acknowledgments
Part of this work was presented at the Seventh
Meeting of the European Society for Clinical Investigation [abstract published in European Journal of
Clinical Investigation (1973), 3, 216] and at the
487
annual meeting of the Swiss Society of Nephrology,
Lausanne, 1973 [abstract published in Kidney International (1974), 5, 309]. This work has been supported by the Swiss National Research Foundation
(3.326.70 Sr) and by the US National Institutes of
Health (AM 07266).
We are especially grateful to Dr F. Roch-Rame1
and Dr J. Diezi for their help and advice in setting
up our micropuncture unit. We are indebted to Dr
M. Abramow, Dr J. Diezi and Dr F. Roch-Ramel
for stimulating discussion of the present investigation. We wish also to thank Miss S. Beck, Miss U.
Largiader, Mrs C. Marti, Miss T. Rolli and Miss
U. Zwicky for their skilful technical assistance, Mrs
B. Gyger for her secretarial help and Miss T. Rolli
for the photographic work performed. We are indebted to Dr Ch. Dowse for reading the manuscript.
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