Naunyn-Schmiedeberg's Arch. Pharmacol.

304, 277-281 (1978)

Naunyn-Schmiedeberg's
Archives o f
Pharmacology
9 by Springer-Verlag 1978
Renal Handling of Oxalate
A Micropuncture Study in the Rat*
R. Ha u t ma n n 1 a nd H. Os s wal d 2
Abteilung Urologic, Medizinische Fakulfftt der RWTH Aachen, Goethestrasse 27/29, D-5100 Aachen
2 Abteilung Pharmakologie, Medizinische Fakult/it der RWTH Aachen, Melatener Strasse 213, D-5100 Aachen,
Federal Republic of Germany
Summa r y. Cl ear ance a n d mi c r o p u n c t u r e exper i ment s
wer e p e r f o r me d i n r at s t o s t udy t he r enal ha ndl i ng o f
oxal at e. The 14C- oxal at e t o 3H- i nul i n cl ear ance r at i o
(Co~/Ci,) was 1.36 _+ 0. 04 a nd was l ower ed by p r o b -
eneci d ( 2 0 0 mg / k g ) t o 1.11 0.03 ( S. E. , n = 6, P
< 0. 005). An a t t e mp t was ma d e t o l ocal i ze t he a s s ume d
secr et i on o f oxal at e in t hr ee di f f er ent mi c r o p u n c t u r e
pr ot ocol s . I n free f l ow mi c r o p u n c t u r e exper i ment s
si ngl e n e p h r o n cl ear ance o f oxal at e was n o t di f f er ent
whe n obt a i ne d f r o m pr oxi ma l or di st al t ubul a r punc t u-
re sites. The f r act i onal del i ver y o f oxal at e a ve r a ge d 0. 84
0.03 r egar dl ess o f t he punc t ur e site f r o m mi d-
pr oxi ma l t o l at e distal. Thi s f i ndi ng i ndi cat es a net
out f l ux o f oxal at e i n an ear l y pr oxi ma l l oop si nce
oxal at e is f r eel y ul t r af i l t er abl e. I n mi c r ope r f us i on ex-
per i ment s t he me a n r e c ove r y o f oxal at e r a nge d f r o m
7 9 - 9 0 %. The out f l ux o f oxal at e cor r el at ed l i near l y
wi t h t he t ubul a r l oa d (r = 0.95). The resul t s suggest
t ha t no net secr et i on occur s i n super f i ci al n e p h r o n
s egment s accessi bl e f or mi c r opunc t ur e . Si nce whol e
ki dne y cl ear ances o f oxal at e al ways exceeded gl ome r u-
l ar f i l t r at i on rat e, it is c onc l ude d t ha t net a ddi t i on o f
oxal at e i nt o t he t ubul a r f l ui d can oc c ur at sites b e y o n d
t he super f i ci al l at e di st al t ubul es or is due t o hi gher
del i ver y o f oxal at e by deep cor t i cal ne phr ons .
Key wor ds : Oxal at e excr et i on - Pr obe ne c i d - Mi c r o-
punc t ur e - Mi c r ope r f us i on - Ra t ki dney.
Introduction
Ca l c i um oxal at e st ones compr i s e s ome 60 % o f t he r enal
cal cul i e nc ount e r e d ( Wi l l i ams, 1974). On t he ot he r
Send offprhlt requests to author at the above address
* This work was generously supported by the Martin Brinkmann
Foundation, Bremen, West Germany
hand, a r evi ew o f t he per t i nent oxal at e r es ear ch i ndi -
cat es t ha t our f und o f knowl e dge does n o t par al l el t he
r el evance o f ur ol i t hi asi s and, i ndeed, rel at i vel y little is
k n o wn a b o u t t he r enal ha ndl i ng o f oxal at e.
Ther e is wi de a gr e e me nt i n t ha t oxal at e is freel y
ul t r af i l t er abl e a n d is excr et ed i nt o t he ur i ne 1 . 2 - 1 . 6 -
f ol d i n excess t o i nul i n ( Cat t el l et al., 1962; Wi l l i ams et
al., 1971, 1975).
Recent l y, our g r o u p ( Ha u t ma n n et al., 1976,
Schmi t z et al., 1977) r e por t e d a 2.5 f ol d hi gher ur i na r y
excr et i on o f oxal at e c o mp a r e d t o i nul i n i n ma n a nd dog
when b o t h s ubs t ances wer e i nj ect ed i ns t a nt a ne ous l y
i nt o t he r enal ar t er y. Since pr obe ne c i d a nd hi gh
i nf usi ons o f p a r a - a mi n o hi ppur a t e ( PAH) depr essed
t he oxal at e t o i nul i n excr et i on r at i o t o 1.7, t ubul a r
secr et i on was as s umed. I n t he pr es ent s t udy mi c r opunc -
t ur e exper i ment s were pe r f or me d i n or de r t o el uci dat e
t he excr et i on me c ha ni s m, exi st i ng a pa r t f r o m gl omer u-
l ar f i l t r at i on, a nd t o l ocal i ze it wi t hi n t he ne phr on.
Methods
Experiments were performed in 21 male Sprague-Dawley rats (mean
body weight 210 g). The animals were fed an Altromin :~ standard diet
and were deprived of food 14 h prior to the experiment, while having
free access to tap water. After anesthesia with thiobutobarbital
(Inactin ~ , 100 mg/kg i.p.), the rats were placed on a heated table to
maintain their body temperature at 37 ~ C.
Clearance experiments were performed on 6 rats. Saline solution
(0.85 % NaC1) was infused via a jugular vein catheter at a rate of
3 ml/h. The carotid artery was cannulated for withdrawal of blood
samples and monitoring of blood pressure. The urine was collected
from the bladder by a catheter. After an equilibration period of 2 h,
when the rats had achieved a constant urinary flow rate, three to six
10min collection periods were performed. Blood samples were
withdrawn in the midpoint of the urine collection periods. Inulin and
oxalate concentration in plasma and urine were determined from
their 3H- and 14C-radioactivity, respectively. One hour after end of
preparation a priming dose of l gCi 14C-oxalate (spec. act.
77 mCi/mmol) and 3H-inulin (spec. act. 695 mCi/mmol) was given,
followed by a maintaining infusion of 0.05 pCi/min of either isotopes.
0028- 1298/ 78/ 0304/ 0277/ $01. 00
278 Naunyn-Schmiedeberg's Arch. PharmacoL 304 (1978)
Micropuncture experiments were performed using three protocols.
The preparation of the rats was the same as in the clearance studies,
except that the animals were prepared for micropuncture of the dorsal
surface of the left kidney in a standard fashion. The kidney was placed
in a holder and bathed by paraffin oil heated to 37 ~ C. The urine was
collected from a ureteral catheter. Isotonic saline was infused at a rate
of 2.2ml/h. Proximal transit time was measured by i.v. injection of
lissamine green, pH 7.4 (Steinhausen, 1963). Rats with a proximal
tubular transit time greater than 10 s were discarded.
In free flow micropuncture experiments (5 rats) single nephron
glomerular filtration rate (SNGFR) and single nephron clearance of
oxalate (SNCox) were measured from proximal and distal puncture
sites. Proximal or distal tubules were identified by injection of 0.02 ml
lissamine green (I0%, pH 7.4) according to Steinhausen (1963).
The tubular fluid was collected quantitatively as described by
Wright and Giebisch (/972). SNGFR and SNCoz were calculated
from the collected tubular fluid volume and the tubular fluid to
plasma ratio (TF/P), obtained from the 3H- and 14C-radioactivity,
respectively. A priming dose of 100 ~Ci of 3H-inulin and 100 ~Ci 14C-
oxalate was given, followed by a constant infusion of 5 gCi/min of
either isotopes.
In stopflow microperfusion experiments (3 rats) tubular segments
were identified with perfusion fluid. The perfusate had the following
composition, 143mmol NaC1, 40- 80 cpm/nl 3H-inulin, 8-
32 cpm/nl 14C-oxalate, 1 mg/ml lissamine green, adjusted to pH 7.0.
A second pipette filled with Sudan black stained castor oil was
introduced downstream from the perfusion pipette. Then, the
perfusion pipette was withdrawn, leaving a hole to allow drainage of
glomerular filtrate, and was then inserted downstream from the
second pipette. After setting the pump (Hampel, Frankfurt, Federal
Republic of Germany; Model 1976) to the proper perfusion rate, the
tubule was blocked upstream by injection of castor oil. The tubular
perfusate was collected with a third pipette in the usual manner.
Measurement of fluid delivered by the pump was calculated by the
amount of 3H-inulin being quantitatively collected, assuming 100
recovery. The volume of the collected fluid was measured in a constant
bore capillary. The tubular fluid to perfusion fluid ratio (TF/PF) of
inulin and oxalate were calculated from the 3H- and14C -
radioactivities, respectively.
Infreeflow microperfusion experiments (n = 7 rats) perfusate was
added to the endogenous filtrate at mid to end proximal tubular sites.
The difference between stop flow and free flow perfusion experiments
is the presence, respectively absence of the endogenous filtrate which
by itself could influence the ionized oxalate fraction within the
tubular lumen.
3H- and a 4C-radioactivity in plasma, urine and tubular fluid were
measured in a liquid scintillation counter (isocap/300, Nuclear
Chicago Div.). As scintillator a "Rotiszint 22" cocktail (Roth,
Karlsruhe, Federal Republic of Germany) was used. One litre of the
scintillator contained 5 g PPO, 0.2 g POPOP, 867 g Toluene and 350 g
Triton X 100. Quench correction curves for both nuclides in plasma,
urine, tubular fluid and lissamine green stained perfusion fluid were
obtained from known standards. The counts of the radioactivity were
then converted into dpm on the base of the quench curves, using a
Diehl Alphatronic Computer. 3H-inulin and l~C-oxalate were pur-
chased from Amersham-Buchler Company, Braunschweig, Federal
Republic of Germany. The experimental data were expressed as
means _+S.E.M. Significance was calculated using Student's t-test for
paired and unpaired data.
R e s u l t s
The mean values of urine volume, inulin clearance (CIN)
and oxalate clearance (Coz) before and after adminis-
tration of 200 rag/ks probenecid i.v. are summarized
Table 1. Mean values of urine volume (V), glomerular filtration rate
(GFR), clearance of oxalate (Cox) and Cox/CIN before and after
intravenous administration of 200 mg/kg probenecid in 6 rats. Values
are expressed per 100 g of body weight
i/ CIN Cox Cox
(gl/min) (ml/min) (ml/min) C1~
Control Mean 20.0 1.07 1.42" 1.36
S.E.M. 6.4 0.1/ 0./2 0.04
Probenecid Mean 15.5 1.13 1.26 1.11"*
S.E.M. 5.7 0.06 0.08 0.03
* P< 0.05 when compared to GFR
** P < 0.005 when compared to control periods
0 10
I 9
8
6
O 5
3
2
1
I I I I | I I I I
1 2 3 4 5 6 7 8 9 10
TF/p i nul i n_ 3 H
Fig. 1. Free flow micropuncture data. Correlation between TF/Pox
and TF/P1N from proximal (9 to distal (e) tubular puncture sites.
The regression line (~ = 0.23 + 0.76 x; r = 0.97) is depicted below the
line of identity
D.
in Table 1. In 4 rats, 20 mg/kg probenecid did not affect
the clearance ratio of Cox/CIN (data not shown).
There was no consistent change of urine volume and
of Cilv following 200ms/ks probenecid i.v. In the
control periods as well as in the probenecid periods, the
oxalate clearance was significantly higher as compared
to GFR. The clearance ratio Cox/CIN decreased signifi-
cantly from 1.36 +_ 0.037 t o 1.11 + 0.026 in the
probenecid periods (P < 0.005).
Since the oxalate clearance exceeded that of inulin
and probenecid could lower the oxalate excretion,
micropuncture experiments were performed in order to
localize the assumed tubular oxalate secretion.
The SNCox obtained from proximal tubular punc-
ture sites averaged 21.7 + 1.94 nl/min and from distal
sites 21.8 4-_ 1.42nl/min, being statistically not diffe-
rent. The corresponding SNGFR values were 25.1 +
1.64nl/min for proximal and 26.9 _+ 1.48nl/min for
distal puncture sites.
R. Ha ut ma nn and H. Osswal d: Renal Handl i ng of Oxal at e in Rat s 279
P O x
P In
1
I I I I I I I I I T F
2 3 4 5 6 7 8 9 " [ 0 ~ - I n
Fi g. 2. The f r act i onal del i very (TF/Pox/TF/P~N), obt ai ned in free fl ow
mi cr opunct ur e exper i ment s, pl ot t ed versus t he TF/ Pm as an est i mat e
o f t he t ubul ar l engt h. The sl ope of t he regressi on line is not
si gni fi cant l y di fferent f r om zero
(.9
v -
I
t~
LI.
D..
I - "
2.0
1. 8
1. 6
1. 4
1. 2
1. 0
0 . 8
, o 9 9
| 9
i , , , i i i , i , , i
0.8 1.0 1.2 1.4 1.6 1.8 2.0
TF/pF I n u l i n - 3 H
Fi g. 4. Tubul ar fluid t o per f usi on fluid rat i os o f i nul i n (TF/PF~N) and
oxal at e (TF/PFox) f r om st op fl ow mi cr oper f usi on experi ment s. The
regressi on line (7 = 0.24 + 0.61 x; r = 0.81) is shown bel ow t he line
of i dent i t y
1.0
9 (18
O 0 6 9 9
0 0 . 4
I I / i q i
0 0.2 0.4 0.6 0.8 1.0
TF/p F Inulin-all
Fig.3. Tubular fluid to perfusion fluid ratios of inulin (TF/PFrN) and
oxalate (TF/PFox) from free flow microperfusion experiments. The
regression line (7 = 0.02 + 0.93 x; r = 0.98) is shown below the line
of identity
In Fig. 1 TF/Pox was pl ot t ed against TF/PtN. The
slope of the cal cul at ed regression line (7 = 0.23 + 0.76
x; r = 0.97) was significantly di fferent f r om one,
i ndi cat i ng back di ffusi on of oxal at e out of the t ubul ar
l umen r at her t han secretion. The fract i onal delivery of
oxal at e was appar ent l y not influenced whet her it was
obt ai ned f r om pr oxi mal (0.86 +_ 0.05, n = 11) or distal
(0.81 ___ 0.03, n = 9) punct ur e sites. In Fi g. 2 the
fract i onal delivery (TF/Pox/TF/PIN) is pl ot t ed against
t he TF/PIN as an est i mat e of t he t ubul ar length. In 3 out
of these 5 rats whol e ki dney clearances of oxal at e and
inulin were measur ed si mul t aneousl y, and the cl earance
rat i o Cox/C1N w a s always f ound to be hi gher (1.2 + 0.1)
t han t hat of t he punct ur ed nephrons. Thus, t he net
addi t i on of oxal at e i nt o the final uri ne seems to occur
beyond the site of mi cr opunct ur e.
In or der to exami ne t he out war d di ffusi on of
oxal at e which coul d mask at least in part s a secretion,
free flow and stop flow mi cr oper f usi on experi ment s
were per f or med. The difference of free fl ow and stop
flow mi cr oper f usi on is t he presence, respectively absen-
ce of t he endogenous filtrate which by itself coul d
influence t he i oni zed oxal at e fract i on within the t ubul ar
l umen and, t herefore, t he diffusible amount of oxalate.
The results of free flow mi cr oper f used proxi mal
convol ut ed tubules as well as l oops of Henl e are shown
in Fig. 3. TF/PFox is pl ot t ed al ong the ordi nat e,
TF/ PFIN al ong t he abscissa (regression line: 7 = - 0.02
+ 0.93 x; r = 0.98). The slope was di fferent f r om one
( P < 0.05). The mean recovery of oxal at e was 89.5 _+
1.5%.
Fi gur e4 showsTF/PFox and TF/ PFt N values ob-
t ai ned under stop flow mi cr oper f usi on condi t i ons.
Pr oxi mal convol ut ed tubules and l oops of Henl e were
perfused. Agai n the slope of t he regression line (7 =
0.24 + 0.61 x; r = 0.81) was significantly di fferent
f r om one ( P < 0.05). The mean recovery was 78.8 +
1.8 %, i ndi cat i ng a hi gher oxal at e out war d di ffusi on
t han under free flow mi cr oper f usi on condi t i ons. Agai n
the percent age of the recovered oxal at e was i ndepend-
ent of t he perfused nephr on length.
The oxal at e out war d flux (d?ox; pmol / mi n) cal cul at -
ed f r om st op flow perfusi on experi ment s is correl at ed
with t he t ubul ar l oad of oxal at e as i l l ust rat ed in Fig. 5.
The regression line was ? = 0.01 + 0.21 x; r = 0.95.
280 Naunyn-Schmiedeberg's Arch. Pharmacol. 304 (1978)
E
"5
E
3
~EH
1.5
1.0
0.5
g o
I
2
I I
4 8
t u b u l a r l o a d ( p m o l / m i n )
Fig.5. Stop flow microperfusion experiments. Calculated fluxes of
oxalate (qSo~) out of the tubular lumen are plotted versus the tubular
load. The regression line is described with 7 = 0.01 + 0.21 x; r = 0.95
Di s c u s s i o n
Studies of the renal clearance of oxalate in man and
laboratory animals have led to different conclusions. In
the dog, using isotopic oxalate, Cattell et al. (1962) have
demonstrated three mechanisms in the renal excretion
of oxalate; glomerular filtration, secretion in the proxi-
mal part of the nephron, and reabsorption by passive
back diffusion. Net tubular secretion of oxalate was
demonstrated with an oxalate/inulin clearance ratio
averaging 1.28. Clearance of oxalate was reduced by
caronamide, probenecid, and para-amino-hippurate
(PAH), but was unaffected by urine pH. In contrast to
these findings, Zarembski and Hodgkinson (1963)
reported the renal clearance of oxalate in normal adults
to be 3. 4-5. 0ml per rain and found an oxalate
clearance of 12. 4- 51.3 ml per min in seven hyperoxalu-
ric subjects, with oxatate/creatinine clearance ratios
varying between 0.23 and 0.94.
The basis for studying renal oxalate excretion by
means of its 14C isotope is the free ultrafilterability of
oxalate and the absence of oxalate metabolism (Wil-
liams et al., 1975; Weinhouse and Friedmann, 1951;
Elder and Wyngaarden, 1960).
The clearance studies (Table 1) demonstrate that
also in the rat the oxalate clearance exceeds that of
inulin. The ratio Cox/Cis = 1.36 is in good agreement
with the data in other species.
The decrease of Co~/CIN after administration of
probenecid reported here corresponds with the data of
Cattell et al. (1962), but is in contrast to those of
Zarembski and Hodgkinson (1963). Therefore, effects
of probenecid other than inhibition of anionic secretion
may be involved. Garcia and Yendt (1970) observed an
increased calcium excretion after probenecid in man.
On the other hand, Hodgkinson and Zarembski (1968)
showed a direct relationship between urinary excretion
of oxalic acid and calcium.
The attempt to localize the suggestive secretion of
oxalate with micropuncture of superficial nephrons
failed to show any TF/Pox exceeding that of TF/PIN in
proximal as well as in distal tubules. Although net
secretion of oxalate into the tubular fluid at any
puncture site was absent, the final urine showed a
Cox/Cru ratio of 1.24. Therefore, it seems unlikely that
oxalate is secreted in the proximal tubule in any
appreciable amount. In mid proximal tubular loops the
fractional delivery of oxalate was already reduced by
about 15 ~, and this value was not found to be different
at more distal tubular puncture sites. Therefore, out-
ward diffusion of oxalate in early proximal loops
appears likely (Fig.2). In a most recently published
micropuncture study Weinman et al. (1978) followed
the fractional delivery of oxalate and inulin along the
proximal tubule of rats and reported a 1.2-fold higher
oxalate delivery compared to inulin, regardless if early
or late proximal tubules were punctured. Probenecid
could not reduce this ratio. The discrepancy between
this study and our findings remains unexplained so far.
Deetjen et al., (1977) concluded from microperfu-
sion and -injection studies in rats that oxalate is secreted
into the proximal tubule, causing excess of oxalate
clearance compared to that of inulin. However, this
conclusion is based on oxalate excretion following its
injection into peril~ubular capillaries. Since oxalate is
freely diffusible across biological membranes (Hassel-
bach and Makinose, 1961 ; Fanburg and Gergely, 1965;
Binder, 1974; Hautmann et al., 1978), the observed-
effect could be explained by simple diffusion of oxalate
from the peritubular capillaries into the tubular lumen.
The stop flow microperfusion experiments (Fig.4)
revealed a mean recovery of intratubular infused 14C-
oxalate of 79 ~o, indicating outward diffusion of oxala-
te. The tubular load of oxalate correlated closely with
its outward flux consistent with a diffusion mechanism
(Fig.5).
Free flow microperfusion experiments (Fig. 3) were
performed to observe the oxalate recovery in the
presence of endogenous tubular fluid, bearing the idea
in mind that calcium in the tubular fluid might trap the
oxalate ion. Calcium oxalate by itself cannot diffuse
across biological membranes (Binder, 1974; Madorsky
and Finlayson, 1977). The obtained recovery in this
type of experiments was 89.5~, being statistically
significant higher than that of stop flow microperfusion
experiments. This result suggests the importance of the
calcium ion in controlling oxalate fluxes across the
tubular epithelium.
R. Haut mann and H. Osswald: Renal Handl i ng of Oxalate in Rats 281
Two alternatives could explain the observed discrep-
ancy between clearance and micropuncture data.
First, the oxalate inward transport in excess to the
filtrate is located in the terminal nephron. Or, second,
the deep nephrons deliver more oxalate into the
collecting ducts than the punctured superficial neph-
rons.
In our experiments reported here and in the experi-
ments in dogs (Schmitz et al., 1977), when oxalate was
injected into the renal artery, probenecid could only
reduce two thirds of the amount of oxalate excreted in
excess to that of inulin. To what extent the probenecid
sensitive and insensitive fraction of oxalate excretion is
dependent on calcium gradients across tubular epithe-
lium remains to be elucidated.
Acknowledgements. We wish t o t hank Dr. Greiner, Dept. of Pharma-
cology, RWTH, Aachen, Federal Republic of Germany, for prepa-
ring the comput er pr ogr am for the quench correction curve. Probene-
cid was kindly supplied by Merck, Sharp & Dohme, Munich, Federal
Republic of Germany.
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Received April 24/Accepted July 13, 1978