Download Reabsorption of the Nephrotoxin Ochratoxin A Along the Rat

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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 280:220 –224, 1997
Vol. 280, No. 1
Printed in U.S.A.
Reabsorption of the Nephrotoxin Ochratoxin A Along the Rat
Nephron In Vivo1
MATTHIAS ZINGERLE, STEFAN SILBERNAGL and MICHAEL GEKLE
Physiologisches Institut, Universität Würzburg, D-97070 Würzburg, Germany
Accepted for publication September 16, 1996
The kidney plays an important role in OTA toxicity. On the
one hand it is the main target of this mycotoxin and on the
other hand it plays an important role in OTA excretion
(Kuiper-Goodman and Scott, 1989; Delacruz and Bach,
1990). OTA impairs renal hemodynamics (Gekle and Silbernagl, 1993a), urine concentrating mechanisms (Gekle et al.,
1993; Krogh et al., 1974), secretion of organic anions (Gekle
and Silbernagl, 1994) and increases the incidence of renal
adenoma and carcinoma (National Toxicology Program,
1989; Kuiper-Goodman and Scott, 1989). Due to the ubiquitous ocurrence of OTA in improperly stored food and animal
food there is a high risk of exposure for man and animals.
Once ingested OTA is reabsorbed very effectively from the
gastrointestinal tract and reaches the circulation (Kumagai
and Aibara, 1982). In blood, OTA is bound to more than 99%
to serum proteins (mainly albumin), contributing to its long
half-life in the body (Chu, 1971; Chu, 1974). Renal elimination of OTA contributes to at least 50% of total clearance
(Kuiper-Goodman and Scott, 1989). Because effective filtration is hindered by the binding to albumin, the main route of
infusion and from 20 to 10% during distal infusion when pH
increased from 6.0 to 7.4. The dipeptides carnosine and glyclysarcosine reduced OTA reabsorption significantly. L-Phenylalanine showed no significant inhibitory action. From our results
we conclude: 1) there is substantial reabsorption of OTA along
the nephron; 2) one-third of reabsorption takes place in the
distal tubule and/or the collecting duct, two-thirds in the proximal tubule; 3) “distal” reabsorption can be explained at least in
part by nonionic diffusion, because it was not saturable but pH
dependent; 4) “proximal” reabsorption was in part mediated by
the H1-dipeptide cotransporter; 5) reabsorption of filtered and
secreted OTA delays its excretion and may lead to accumulation of the toxin in renal tissue and 6) inhibition of OTA reabsorption (e.g., by urine alkalinization) should help to accelerate
OTA excretion and thus reduce its toxicity.
OTA into the tubular lumen is secretion in the proximal
tubule (Gekle and Silbernagl, 1994; Stein et al., 1984; Sokol
et al., 1988). The concentration of free OTA in final urine
exceeds the concentration of free OTA in plasma (Gekle and
Silbernagl, 1994).
The fate of OTA which has reached the tubular lumen is
not clear so far. There is only little information available
from cell culture studies (Gekle et al., 1993b) with respect to
possible reabsorption of OTA. For this reason we investigated
possible reabsorption of OTA along the nephron by in vivo
microinjection and determined the underlying mechanisms
of transport. Our results show that the mycotoxin is reabsorbed in proximal and distal sections of the nephron by the
H1-dipeptide cotransporter (Silbernagl et al., 1987) and probably in part by nonionic diffusion. Reabsorption of OTA is of
toxicological importance because it contributes to the long
half-life of the mycotoxin in the body and could furthermore
lead to recycling and accumulation in certain parts of the
kidney (Bauer et al., 1995).
Materials and Methods
Received for publication June 19, 1996.
1
This study was supported by the Deutsche Forschungsgemeinschaft DFG
Si 170/7-2.
Preparation of animals for microinfusion experiments in
vivo. Male Wistar rats (Charles River, Sulzfeld, Germany) weighing
ABBREVIATIONS: OTA, ochratoxin A; FE, fractional excretion; FR, fractional reabsorption; TES, N-tris(hydroxymethyl)methyl-2-aminomethane
sulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; MDCK, Madin Darby canine kidney; Jmax, maximum transport rate.
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ABSTRACT
Ochratoxin A (OTA) is a widespread nephrotoxin excreted to a
substantial degree via the kidney. We investigated whether
[3H]OTA is reabsorbed from the tubular lumen along the
nephron and thus recycles within the kidney. Superficial early
proximal and early distal tubules of male Wistar rats were
micropunctured in situ. Microinfusion of OTA into superficial
nephrons showed that it was reabsorbed in proximal as well as
distal parts of the nephron. Reabsorption during early distal
microinfusion was not saturable in the range of 0 to 5z1024
mol/liter and accounted for 20% of OTA infused. Reabsorption
during early proximal microinfusion was partially saturable and
reached values up to 70% of OTA infused. The apparent Km for
OTA reabsorption was 236z1026 mol/liter and maximum transport rate 970 fmol/min/nephron. OTA reabsorption was pH
dependent and decreased from 70 to 40% during proximal
1997
Renal Reabsorption of Ochratoxin A
Results
26
Up to 70% of OTA (10 mol/l) infused into the rat nephron
is reabsorbed (fig. 1), depending on the site of infusion and on
the pH of the microinfusate. In the “distal” parts of the
nephron (distal tubule and collecting duct) FR was ;20%.
Thus, up to 50% of OTA was reabsorbed in the proximal
tubule and possibly in the short loops of Henle (“proximal”
part of the nephron) (fig. 1A). “Distal” reabsorption was not
saturated when infusing OTA concentrations up to 5z1024
mol/liter, because FR did not decrease significantly compared
to FR when 1026 mol/liter OTA was infused (fig. 1c). The
constancy of “distal” FR made it possible to determine “proximal” FR as the difference of total FR minus 20% (5 “distal”
FR).
In contrast to “distal” FR, “proximal” FR reabsorption was
Fig. 1. Fractional reabsorption (FR) of OTA during early proximal and
early distal microinfusion. (A) FR of OTA during earl proximal microinfusion was significantly higher as compared to FR during early distal
microinfusion. Increasing the concentration of OTA led to a decrease of
FR during proximal microinfusion (B) but not during distal microinfusion
(C). n 5 6 for all values.
a saturable process as shown in figures 1B and 2. FR decreased with increasing concentrations of OTA in the infusion solution. Figure 2A shows the dependence of “proximal”
reabsorption (determined after subtraction of “distal” reabsorption as described above) on OTA concentration. Analysis
of the data either by nonlinear curve fitting or after linearization (fig. 2b, Hill plot) yields an apparent Km of
236z1026659z1026 mol/liter. The maximum transport rate
was estimated as 970 6 75 fmol/min (fig. 2A).
To determine a possible pH dependence of OTA transport,
FR at two different pH values of the microinfusate was determined (1026 mol/liter OTA; solutions buffered as described in “Materials and Methods”). As shown in figure 3
there was a clear dependence on pH for “proximal” and “distal” reabsorption. Total FR was ;70% at pH 6.0 but only
;35% at pH 7.4. “Distal” FR was ;20% at pH 6.0 and ;10%
at pH 7.4. Thus, “proximal” FR decreased from 50 to 25%
when pH increased from 6.0 to 7.4.
Glycylsarcosine and carnosine (1022 mol/liter), both substrates for the H1-dipeptide cotransporter in the nephron
(Silbernagl et al., 1987), inhibited FR of OTA significantly as
shown in figure 4. By contrast, “distal” FR was not affected by
the addition of dipeptides: FR during infusion with 1026
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210 to 290 g were fed on an Altromin standard diet and had free
access to water. The rats were anesthetized with 120 mg/kg body
weight Inactin (Byk-Gulden, Constance, Germany). After inserting a
polyethylene tube into the trachea and two catheters into the jugular
vein, the animals were infused i.v. with Ringer solution at a rate of
50 ml/min. The kidney was prepared for micropuncture as previously
described (Gekle and Silbernagl, 1994).
Microinfusion experiments. After identification of the nephron
section by i.v. injection of lissamine green SF (Chroma-Gesellschaft,
Köngen, Germany) at a bolus dose of 0.02 ml of a 100 g/liter solution
titrated with NaOH to pH 7.4, the tubule was micropunctured using
glass capillaries. They had ground tips (outer tip diameter 10–12
mm) and were mounted on a microperfusion pump (Sonnenberg and
Deetjen, 1964). Puncture sites were 1) the earliest superficial loop of
the proximal tubule (“early proximal”), which represents a distance
from the glomerulus of ;0.3-1.2 mm and 2) the first superficial loop
of the distal tubule (“early distal”). The microinfused solution was a
Ringer solution (see below) containing [14C]inulin and [3H]-OTA.
Microinfusion at a rate of 20 nl/min lasted for 10 min. Starting
shortly before microinfusion, the ipsilateral urine was collected from
a uretheral catheter in 15-min fractions for 60 min, and radioactivity
of each fraction was counted in a liquid scintillation spectrometer
(Packard Instruments, Frankfurt, Germany). Urinary 3H-OTA recovery (fractional excretion) was calculated from ([3H]urine z
[14C]perfusion)/([14C]urine z [3H]perfusion). FR is 1 2 fractional excretion. As a control, the urine of the contralateral kidney was
collected from a bladder catheter. Radioactivity did not exceed background level.
Materials. The Ringer solution consisted of (in mmol/l): 156.4
Na1, 5.4 K1, 1.7 Ca21, 162.8 Cl2 and 2.4 HCO32, pH 7.4. In the
experimental series in which the pH dependence of OTA reabsorption was tested the Ringer solution was buffered with 10 mmol/liter
N-tris(hydroxymethyl)methyl-2-aminomethane sulfonic acid (pKa 5
7.5) and 10 mmol/liter 2-(N-morpholino)ethanesulfonic acid (pKa 5
6.15). This solution was titrated to pH 7.4 or 6.0. 14C-inulin (0.4
GBq/g) was obtained from Du Pont de Nemours (Dreieich, Germany),
3
H-OTA (1.37z1014 Bq/mol) from Moravek Biochemicals (Brea, CA),
N-tris(hydroxymethyl)methyl-2-aminomethane sulfonic acid and
2-(N-morpholino)ethanesulfonic acid from Serva (Heidelberg, Germany). All other chemicals were purchased from Merck (Darmstadt,
Germany). The purity of 3H-OTA was checked by high performance
liquid chromatography as described previously (Gekle and Silbernagl, 1994).
Statistics. Two to three tubules were studied per rat and the data
are presented as mean values 6 S.E.M. n gives the number of
tubules studied. Significance was tested by unpaired t test. Differences were considered significant if P , .05. Curve fitting was
performed by the least square method (Sigma Plot; Jandel, Corte
Madera, CA).
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Zingerle et al.
Vol. 280
Fig. 2. A, Concentration-dependence of proximal FR, calculated as
described in “Results.” B, Hill-plot of proximal FR. The slope was not
significantly different from unity (0.93). Km 5 236 z 1026 6 59 z 1026
mol/liter. Jmax 5 970 6 75 fmol/min. n 5 6 for all values.
mol/liter 3H-OTA was 21 6 1% (n 5 6) in the absence and
27 6 4% (n 5 3) in the presence of 20z1022 mol/liter glycylsarcosine. L-Phenylalanine (2z1022 mol/liter), a substrate of
the high capacity carrier for neutral amino acids (Silbernagl,
1992), led to a small reduction of FR that did not reach
significance. Under control conditions (1026 mol/liter 3HOTA) FR was 61.7 6 5.4% (n 5 6) and in the presence of
20z1022 mol/liter L-phenylalanine 53.5 6 1.4% (n 5 4).
Discussion
The results of this study show clearly that OTA, once it has
reached the tubular lumen either by secretion or filtration,
undergoes significant reabsorption along the nephron. About
two-thirds of total reabsorption (which amounts to 60 –70% of
the tubular load) occurs before OTA reached the distal tubule, i.e., in the proximal convoluted tubule and/or in the
short loops of Henle. The remaining reabsorption takes place
in the distal tubule and/or the collecting duct.
Thus, the long half-life of OTA in the body (Delacruz and
Fig. 4. The dipeptides glycylsarcosine and carnosine (1022 mol/liter)
inhibited FR during early proximal infusion significantly. The microinfusion solution was buffered to pH 6.0. The concentration of OTA was
1026 mol/liter. Number of experiments in parentheses.
Bach, 1990) is probably not only the result of its binding to
plasma proteins but also due to tubular reabsorption. The
low specific activity of 3H-OTA made it impossible to reduce
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Fig. 3. pH-dependence of FR during early proximal and early distal
microinfusion. The microinfusate was buffered to the respective pH
values as described in “Materials and Methods.” The concentration of
OTA was 1026 mol/liter. n 5 6 for all values.
1997
223
the H1-dipeptide cotransporter of the proximal tubule. These
results are in good agreement with the data obtained for
proximal tubule-derived OK cells (Gekle et al., 1993b). Uptake across the apical membrane of OK cells was also pHdependent and inhibitable by dipeptides. The reason for the
incomplete inhibition of OTA reabsorption by the dipeptides
might be that due to the constant flow of native tubular fluid
the concentration of the dipeptides at the transporter site
was diluted to suboptimal concentrations. In this case the
carrier would not have been saturated completely. Furthermore, the low-affinity H1-dipeptide cotransporter, with a Km
value of 21z1023 mol/liter in brush-border vesicles (Silbernagl
et al., 1987) may also be involved. Obviously, this transporter
was not saturated. As already discussed above, nonionic diffusion could also be involved to some extent in OTA reabsorption because the pH of tubular fluid drops from 7.4 to
;6.7 along the proximal tubule.
According to previous studies (Friis et al., 1988; Sokol et
al., 1988; Gekle and Silbernagl, 1994) the renal organic anion
transport system is responsible for the blood-to-lumen translocation of OTA in the proximal tubule. Thus, this transport
system also should lead to an enrichment of OTA in proximal
tubular cells. Reabsorption of the toxin by the H1-dipeptide
cotransporter and/or by nonionic diffusion reduces transepithelial net-secretion and thereby its elimination. Furthermore, reabsorption enhances the accumulation of OTA
within proximal tubular cells but is probably the predominant mechanism for accumulation in postproximal tubular
cells, that have no organic anion transport system. Of course,
proximal secretion in the prerequisite for postproximal reabsorption and accumulation.
L-phenylalanine did reduce OTA reabsorption in tendency
but not significantly. Thus, although OTA is a substrate of
phenylalanine hydroxylase (Creppy et al., 1990) and interacts with phenylalanine t-RNA synthetase (Konrad and Roschenthaler, 1977) it seems not to be a substrate of the amino
acid carrier(s) responsible for L-phenylalanine reabsorption.
We cannot exclude completely that the effective concentration of L-phenylalanine at the carrier site was also diluted
and a possible inhibitory effect masked. Due to the lower
affinity of L-phenylalanine reabsorption (Km ;6 z 1023 mol/
liter) (Silbernagl, 1992) as compared to the high affinity
dipeptide carrier a reduction of the effective concentration
would lead more readily to a lack of effect as compared to
dipeptides.
Our data indicate that the H1-dipeptide cotransporter is
the predominant carrier system for OTA reabsorption in
nephron sections before the distal tubule. Although, the proximal tubule seems to be the most probable candidate for the
site of reabsorption, the thick ascending limb of the short
loops of Henle can not be excluded as an additional site of
reabsorption because there is evidence for dipeptide reabsorption in this part of the nephron (Silbernagl et al., 1987).
In conclusion, our data show that there is substantial reabsorption of OTA along the nephron. One-third of the reabsorption takes place in the distal tubule and/or the collecting
duct, two-third in the proximal tubule and/or the loop of
Henle. “Proximal” reabsorption was, at least in part, mediated by the H1-dipeptide cotransporter. “Distal” reabsorption possibly involves nonionic diffusion, because it was not
saturable but pH dependent. Reabsorption of filtered and
secreted OTA delays its excretion and may lead to accumu-
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its concentration in the perfusion solution below 1026 mol/
liter to investigate reabsorption in the nanomolar range. Yet,
as Km was 236 z 1026 mol/liter reabsorption in the low micromolar and nanomolar range can be assumed as being
proportional to the concentration of OTA. Thus, the FR determined during infusion with 1026 mol/liter is the same as
for lower concentrations that occur during natural exposure
(Kuiper-Goodman and Scott, 1989). Furthermore, manipulation of OTA reabsorption (as discussed below) should be a tool
to increase its rate of elimination and consequently reduce its
toxicity. Probably, the beneficial effect of bicarbonate gavage
in murine ochratoxicosis (Yong et al., 1987) can be explained
in this way. Luminal uptake of OTA by renal epithelial cells
may also increase the cellular content of the toxin and
thereby enhance its cytotoxic actions.
Reabsorption in the “distal” parts of the nephron was not
saturable in the concentration range investigated. Thus,
transport was either due to simple diffusion or mediated by a
low-affinity transporter. A nonsaturable transport for OTA
has been also described for the gastrointestinal tract (Kumagai and Aibara, 1982) and it was concluded that absorption is
due to diffusion. Furthermore, “distal” reabsorption decreased with increasing pH in the infusion solution. Because
OTA is a weak organic acid with a pKa value of 7.1 (KuiperGoodman and Scott, 1989) an increase in pH from 6.0 to 7.4
reduces the fraction of uncharged (nondissociated) OTA from
93 to 33%. In the nondissociated form, OTA is well dissolvable in nonpolar solvents (Kuiper-Goodman and Scott, 1989)
and probably also well membrane permeable. Thus, the pHdependence of “distal” OTA reabsorption can be explained by
nonionic diffusion. In this case OTA would be trapped within
tubular cells, where pH is in the range of 7.1, and the intracellular concentration should be 2-fold the extracellular one.
Accumulation of OTA in tubular cells of renal medulla and
papilla (Bauer et al., 1995) can be explained, at least in part,
by intracellular ionic trapping. Our data do not indicate the
involvement of a H1-driven dipeptide cotransport in “distal”
transport. Yet, a small but significant reabsorption of glycylsarcosine during early distal microinfusion has been shown
(Silbernagl et al., 1987). Thus, there is the possibility that a
low affinity H1-dipeptide cotransporter that was not saturated under our experimental conditions contributed to OTA
reabsorption. In collecting duct-derived Madin Darby canine
kidney cell monolayers a pH-dependent and dipeptide-inhibitable reabsorptive transport of OTA has been shown
(Schwerdt et al., 1996). These data point to the possibility
of dipeptide carrier-mediated reabsorption of OTA in renal
collecting duct.
In contrast to “distal” reabsorption, “proximal” reabsorption was saturable pointing to a carrier-mediated transport.
Furthermore, OTA transport was also pH dependent with
decreasing FR as pH increased. This transport pattern can be
well explained by the involvement of the proximal tubular
H1-dipeptide cotransporter (Silbernagl et al., 1987). The apparent affinity of “proximal” reabsorption for OTA (236z1026
mol/liter) is almost the same as as for the high affinity dipeptide carrier (240z1026 mol/liter) (Silbernagl et al., 1987). Furthermore, FR of OTA was significantly reduced in the presence of the dipeptides glycylsarcosine or carnosine that are
both substrates of the H1-dipeptide cotransporter in the
proximal convoluted tubule (Silbernagl et al., 1987). Thus,
our results show that OTA is reabsorbed, at least in part, via
Renal Reabsorption of Ochratoxin A
224
Zingerle et al.
lation of the toxin in renal tissue, thus enhancing its toxicity.
Inhibition of OTA reabsorption (e.g., by urine alkalinization)
should help to accelerate OTA excretion and thus reduce its
toxicity.
Acknowledgment
The authors thank Katharina Völker for introducing M.Z. into the
technique of micropuncture.
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