- Email: [email protected]
Effects of extracellular cadmium on renal basolateral organic anion transport
Toxicology Letters 98 (1998) 189 – 194
Effects of extracellular cadmium on renal basolateral organic anion transport Helge Hohage a,*, Thomas Mehrens a, Ulrike Mergelsberg b, Marina Lo¨hr b, Joachim Greven b a
Medical Department D, Uni6ersity of Mu¨nster, Albert Schweitzer Str. 33, D-48129 Mu¨nster, Germany b Department of Pharmacology and Toxicology, RWTH, Aachen, Germany Received 8 April 1998; received in revised form 29 June 1998; accepted 30 June 1998
Abstract Heavy metal ions are well-known nephrotoxic agents. Usually, they induce a damage of various renal transport systems. Cd2 + , however, has been shown to enhance renal basolateral organic cation transport. Therefore, the effects of Cd2 + on the basolateral p-aminohippuric acid (PAH) uptake of microdissected nonperfused rabbit kidney S2 proximal tubule segments were investigated. Incubation with Cd2 + induced a bell-shaped concentration response curve with a 2-fold increase of the PAH transport at a Cd2 + concentration of 10 − 6 M. Since Cd2 + has been described to activate protein kinase C (PKC), the effects of the PKC inhibitor staurosporine were also tested. Staurosporine (10 − 8 M) could, however, not reduce the effects of Cd2 + . Cd2 + may exert its effects by an as yet unknown mechanism that is different from the PKC-activating mechanism of phorbol esters. A stimulation of the tricarboxylic acid cycle in kidney cells by Cd2 + may, however, increase the delivery of a-ketoglutarate. Indeed, incubation of proximal tubules with a-ketoglutarate increased PAH transport across the basolateral membrane significantly. Thus, the observed effects of Cd2 + may be due to an enhanced transport of p-aminohippuric acid by stimulation of exchange of PAH with a-ketoglutarate. The modulation of renal organic anion transport may be another aspect of Cd2 + nephrotoxicity. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Renal proximal tubule; Renal organic anion transport system; Cadmium; Protein kinase C
1. Introduction
* Corresponding author. Tel.: + 49 251 8346984; fax: + 49 251 8347528; e-mail: [email protected]
Heavy metals ions are well-known nephrotoxic compounds. In vitro studies could show that these ions directly impair brush border membranes, thereby reducing the transport of Na + -dependent
0378-4274/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-4274(98)00127-1
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H. Hohage et al. / Toxicology Letters 98 (1998) 189–194
Fig. 1. Schematic drawing of the incubation chamber.
transport systems, such as glucose (Lee et al., 1991) or amino acids (Foulkes, 1991) similar to alterations observed in the Fanconi syndrome. In addition, cell death and apoptosis in proximal tubule segments may also occur (Tanimoto et al., 1993). Furthermore, the heavy metal ion Cd2 + has relatively specific damaging effects on cell – cell junctions (Prozialeck and Niewenhuis, 1991). In contrast to the effects on glucose and amino acid transport, a stimulation of the transport of weak organic cations across the basolateral membrane of proximal tubules by Cd2 + was described (Nikiforov and Ostretsova, 1994). Therefore, in this work the effects of Cd2 + on renal PAH transport were determined in isolated, nonperfused proximal S2 segments of the rabbit. This experimental model allows the examination of the transport of organic anions across the basolateral membrane of the proximal tubule, independent of hemodynamic alterations that may also be induced by these ions.
2. Materials and methods We used a microdissection technique similar to that described previously (Brandle and Greven, 1991; Hohage et al., 1994a). New Zealand white rabbits (Charley River, Germany) of either sex, weighing 1.5–2.1 kg (mean: 1.790.2 kg), were killed by cervical dislocation. Afterwards, both
kidneys were rapidly removed. Tissue slices were immediately cut out of the other kidney and transferred to a dish with dissection solution (for composition see below). The time between cervical dislocation and transfer of the tissue slices to the dissection solution did not exceed 10 min. In each experiment, 12–20 segments of proximal tubules from each kidney were microdissected according to the method of Burg et al. (1966). The dissection was performed mechanically at 4°C under a stereomicroscope (Wild, Herbrugg, Switzerland) with a 16–40-fold magnification, without using enzymatic agents. Segments of the proximal tubule were identified as S1, S2, S3 segments according to the criteria established by Woodhall et al. (1978). After the dissection, the superficial tubules were transferred in a droplet of dissection solution to an incubation chamber (Fig. 1), which was cooled on crushed ice during the dissection period. The dissection period did not exceed 30 min. The incubation chamber was placed and heated on an inverted microscope (Zeiss, Oberkochen, Germany, model IM 35). The incubation chamber contained 0.8 ml of incubation medium, including Cd2 + (for composition see below). The surface of the bath medium was superfused with 95% O2 and 5% CO2 in order to keep pH and pO2 constant. At the beginning and end of each experiment, the pH of the bath medium was measured. Experiments were disregarded if the difference between these two mea-
H. Hohage et al. / Toxicology Letters 98 (1998) 189–194
surements exceeded 0.15 pH units. A constant bath temperature within the incubation chamber was maintained by adjusting a current flow through a platinum – iridium wire, which surrounded the incubation chamber. Constant monitoring of the bath temperature was accomplished via a temperature probe that was introduced into the incubation medium via a slot in the incubation chamber. A servonulling arrangement regulated the current flow (Yellow Springs Instruments, CA, model 72). Unless stated otherwise, the temperature was kept constant at 37.5°C. In order to substitute water loss by evaporation, 50 ml of deionized water were added to the incubation medium every 10 min. This time interval was chosen to limit changes in osmolality to less than 10%. At the beginning and end of each experiment, the osmolality was measured by cryoscopy (Knauer Osmometer, Wissenschafterlicher Gera¨tebau KG, Dr. Ing Knaur, Berlin, Germany). If the difference in osmolality between the beginning and the end exceeded 10%, the experiment was disregarded. After the dissection period, incubation medium, Cd2 + and the radioactive compounds were given to the bath and the chamber was heated and insufflated under the described condition. The tubules were added and observed at a ×120 magnification using the criteria indicated by Chonko (no visible lumen) (Chonko et al., 1979). Unless stated otherwise, the tubules were incubated for 10 min with the radio-labeled substances (incubation period), because other experiments in our laboratory had shown that the PAH uptake achieved an equilibrium during this time. At the end of the incubation, formalin in a final bath concentration of 2% was added and a further inspection of the tubules was performed at a × 120 magnification to assure that the tubules were totally collapsed. Afterwards, each tubule was photographed separately to permit subsequent determination of its volume (see below). Each tubule was then taken out of the bath separately, using a glass needle, and transferred to a droplet (300 ml) of 0.75 N nitric acid. The droplet of nitric acid was covered with mineral oil to prevent evaporation. After 45 min of extraction, the whole droplet was aspirated into a glass
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pipette and transferred to a plastic vial (Packard Instruments, Groningen, Netherlands) containing 5 ml of scintillation cocktail (Packard Emulsifier Safe, Packard Instruments). The radioactivity was counted for 10 min in a Beckman liquid scintillation counter (LS 6000 SC, Beckman, Fullerton CA). In previous studies, quenching was checked by the external standard channels ratio method (Brandle and Greven, 1991; Hohage et al., 1994a,b). Since the volume of the probe (25 ml) was small compared with the volume of the scintillation cocktail (5 ml), a virtually constant external standard channels ratio was found in all probes varying between 1.40 and 1.41. Because the counting efficiency was equal in all probes, and since we were only interested in the cell-tobath activity ratio, cpm values were not corrected to dpm values. To correct the 14C-spillover in the 3 H window and vice versa, single label 14C-probes and 3H-probes were measured under these conditions. 3H and 14C values of the dual label probe were corrected according to this spillover. Tubular volume was calculated as described previously (Brandle and Greven, 1991). The tissue water volume was calculated by multiplying the tubular volume by a factor of 0.7 (Barfuss and Schafer, 1979).
2.1. Solutions and chemicals To study the cellular uptake of 3H-labeled PAH (p-aminohippuric acid, specific activity 5 Ci/mM), PAH was added to the bath solution at a concentration of 6.5 × 10 − 7 M. In order to measure the extracellular contamination, 14C-labeled inulin (specific activity 10.7 mCi/mM, Amersham International, Buckinghamshire, UK) was also added to the bath medium. In some experiments, the tubules were incubated with 50 mM a-ketoglutarate. Experiments were disregarded if the bath radioisotope concentrations sampled at the beginning and the end of the incubation period differed by more than 10%. The solution used for dissection consisted of (mM): NaCl 115, KCl 3.8, NaHCO3 25, KH2PO4 1.3, MgSO4 · 7H2O 1.25, CaCl2 · 2H2O 2.5, glucose 8.3, L-alanine 2.2, trisodiumcitratedihydrate 1, sodium lactate 1.5, glycine 1.8. The bath solution had the same com-
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position, but Dextran (mol. wt. 40000) at a concentration of 30 g/l was added to the solution. The osmolality of both solutions was adjusted to a final value of 290 mosm/kg H2O. The pH was adjusted to a final value of 7.4 after gassing with 95% O2 and 5% CO2.
2.2. Calculations Owing to the fact that all the tubules used were totally collapsed during the incubation period, we regard the uptake of PAH by each tubule as representing the quantity transported over the basolateral membrane. To calculate the amount of the intracellularly stored 3H-activity (cellular 3H-activity), the 3H-activity of the tubule extract was corrected for contamination with the bath solution, using an equation described previously (Brandle and Greven, 1991). Values are presented as mean9 S.E.M. As the samples showed no normal distribution, the values were transformed logarithmically. When comparing two means, the statistical significance of differences was determined by means of the Student’s t-test for paired or unpaired observations. A P value of 0.05 or smaller was considered statistically significant. Whenever more than two means were to be compared, analysis of variance was used. When the F value was significant [P50.05], the group means were analyzed using the Student–Newmanns – Keuls test for significant differences.
ney. Since in recent experiments we could demonstrate that tubular PAH uptake showed a linear accumulation during the first 10 min of the incubation period and reached a maximum after about 15 min, an incubation period of 15 min was used in all subsequent experiments (Hohage et al., 1994a). The PAH concentration in the incubation solution was chosen as 6.5× 10 − 7 M because previous studies had shown that at higher concentrations, significant amounts of PAH were secreted into the lumen of the tubule (Hohage et al., 1994a). Incubation with Cd2 + (Fig. 2) in concentrations ranging from 10 − 8 to 10 − 4 M led to a bell-shaped concentration effect curve of the renal PAH transport. The maximum accumulation occurred at a concentration of 10 − 6 M. Simultaneous incubation of Cd2 + (10 − 6 M) with the protein kinase C inhibitor staurosporine in a concentration of 10 − 8 M, which has been shown to inhibit PKC activity, did not change the basolateral PAH transport significantly (Fig. 3). To investigate the influence of an enhanced delivery, the tubules were preincubated for 10 min with a-ketoglutarate. Under these conditions, PAH uptake increased significantly by 529 7% (Fig. 4).
2.3. Materials p-(glycyl-2-3H)-Aminohippuric acid (specific activity 5 Ci/mmol) was obtained from DuPont, Dreieich, Germany, (hydroxy(14C)methyl)inulin (specific activity 10.7 mCi/mM) from Amersham Buchler, Braunschweig, Germany. Staurosporine, a-ketoglutarate and CdCl2 were purchased from Sigma, Deisenhofen, Germany.
3. Results The basolateral PAH transport studies were performed on isolated S2 segments of rabbit kid-
Fig. 2. Concentration-dependent effects of Cd2 + on the renal PAH transport for an incubation period of 10 min. PAH was added to the incubation medium in a final concentration of 6.5 ×10 − 7 M. The incubation time always was 15 min; 24 – 36 tubules from at least two animals were used. * Denotes P B0.05 vs. control.
H. Hohage et al. / Toxicology Letters 98 (1998) 189–194
Fig. 3. Effects of Cd2 + and simultaneous administrated staurosporine on the renal PAH transport for an incubation period of 10 min. PAH was added to the incubation medium in a final concentration of 6.5 ×10 − 7 M. Con, control; Cd2 + , Cd2 + 10 − 6 M, Cd2 + +Stau, Cd2 + 10 − 6 M +staurosporine 10-8 M. The incubation time always was 15 min; 24–36 tubules from at least two animals were used. * denotes PB 0.05 vs. control.
4. Discussion Heavy metal ions such as Cd2 + are well-known nephrotoxic compounds reducing the activity of a wide variety of Na + -dependent renal transporters. In contrast to expected effects, an increase of the transport of weak organic cations across the basolateral membrane has been reported following Cd2 + application (Nikiforov and Ostretsova, 1994). The purpose of the present study was, therefore, to investigate the influence of Cd2 + on the transport of organic anions in rabbit kidney proximal tubule S2 segments.
Fig. 4. Stimulation of PAH accumulation after incubation with 50 mM a-ketoglutarate. Con, control; KG, a-ketoglutarate incubation; 24 – 36 tubules from at least two animals were used. * Denotes P B0.05 vs. control.
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Interestingly, following Cd2 + application, a bell-shaped concentration response relationship was observed. Cd2 + has been described to interact with the protein kinase C system (Beyersmann et al., 1994). Thus, it seemed likely that stimulation of protein kinase C by Cd2 + may also contribute to the enhanced PAH transport since this second messenger enhances the transport of organic anions across the basolateral membrane of proximal tubules (Hohage et al., 1994a). However, the PKC inhibitor staurosporine failed to inhibit the effects of Cd2 + on PAH transport leading to the assumption, that the effects observed following Cd2 + application are not due to direct stimulation of PKC. Nevertheless, Cd2 + may change the conformation of PKC, resulting in an enhanced activity of this molecule and loss of staurosporine sensitivity. This assumption is supported by the fact that Cd2 + interacts with Zn2 + binding sites, which have been described within the PKC molecule (Berg, 1986). Cd2 + induces a characteristic bell-shaped dose effect relationship, which has also been obtained in previous studies when the effect of PMA on renal proximal tubular transporters were measured (Hohage et al., 1994a,b). A down-regulation mechanism of the activated PKC has been suggested to explain this phenomenon. The drop in the dose–effect relationship, however, may also reflect toxic effects of Cd2 + . Similar to our results, a stimulation of weak organic acid uptake in rat renal tubules by Cd2 + was reported (Nikiforov and Ostretsova, 1994), which was related to an activation of the tricarboxylic acid cycle and an export of reducing equivalents from the mitochondria. Indeed, this mechanism also seems to contribute to the enhanced PAH accumulation seen in our experiments. After incubation with a-ketoglutarate, PAH accumulation was increased by 529 7%. Similar effects of a-ketoglutarate have been reported previously by Pritchard (1995). Furthermore, very low Cd2 + concentrations have been shown to stimulate DNA synthesis and cell growth in the renal cell line LLC-PK1 (von Zglinicki et al., 1992), which may also contribute to the effects observed.
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In summary, the data presented provide evidence that the transport of organic anions across the basolateral membrane can be stimulated by Cd2 + , probably due to an enhanced PAH/a-ketoglutarate exchange. An effect of Cd2 + on protein kinase C seems to be excluded.
Acknowledgements The authors are grateful to K. Breidbach and H. Erven for their assistance in the manufacture of the incubation chamber and M. Peters for typing the manuscript. Helpful and critical discussion with E. Schlatter is also gratefully acknowledged.
References Barfuss, D.W., Schafer, J.A., 1979. Active amino acid absorption by proximal convoluted and proximal straight tubules. Am. J. Physiol. 236, F149–F162. Berg, J.M., 1986. More metal-binding fingers. Nature 319, 264 – 265. Beyersmann, D., Block, C., Malviya, A.N., 1994. Effects of cadmium on nuclear protein kinase C. Environ. Health Perspect. 102 (Suppl. 3), 177–180. Brandle, E., Greven, J., 1991. Transport of cimetidine across the basolateral membrane of rabbit kidney proximal tubules: characterization of transport mechanisms. J. Pharmacol. Exp. Ther. 258, 1038–1045. Burg, M., Grantham, J., Abramow, M., Orloff, J., 1966. Preparation and study of fragments of single rabbit nephrons. Am. J. Physiol. 210, 1293–1298. Chonko, A.M., James, M.J., Welling, J.M., Barfuss, S.W., Schaefer, J.A., 1979. Microperfusion of isolated tubules.
.
In: Maldonado M. (ed.), ‘Methods in Pharmacology’ (vol. 4B), New York: Plemann Press. Foulkes, E.C., 1991. Nature of Cd and Hg effects on epithelial amino acid transport in vivo and role of chelators. Toxicology 69, 177 – 185. Hohage, H., Lohr, M., Querl, U., Greven, J., 1994a. The renal basolateral transport system for organic anions: properties of the regulation mechanism. J. Pharmacol. Exp. Ther. 269, 659 – 664. Hohage, H., Mo¨rth, D.M., Querl, I.U., Greven, J., 1994b. Regulation by protein kinase C of the contraluminal transport system for organic cations in rabbit kidney S2 proximal tubules. J. Pharmacol. Exp. Ther. 268, 897 – 901. Lee, H.Y., Kim, K.R., Park, Y.S., 1991. Transport kinetics of glucose and alanine in renal brush border membrane vesicles of cadmium intoxicated rabbits. Pharmacol. Toxicol. 69, 390 – 395. Nikiforov, A.A., Ostretsova, I.B., 1994. Stimulation of weak organic acid uptake in rat renal tubules by cadmium and nystatin. Biochem. Pharmacol. 47, 815 – 820. Pritchard, J.B., 1995. Intracellular a-ketoglutarate controls the efficacy of renal organic anion transport. J. Pharmacol. Exp. Ther. 274, 1278 – 1284. Prozialeck, W.C., Niewenhuis, R.J., 1991. Cadmium (Cd2 + ) disrupts (Ca2 + )-dependent cell – cell junctions and alters the pattern of E-cadherin immunofluorescence in LLCPK1 cells. Biochem. Biophys. Res. Commun. 181, 1118 – 1124. Tanimoto, A., Hamada, T., Koide, O., 1993. Cell death and regeneration of renal proximal tubular cells in rats with subchronic cadmium intoxication. Toxicol. Pathol. 21, 341 – 352. von Zglinicki, T., Edwall, C., Ostlund, E., Lind, B., Nordberg, M., Ringertz, M., Wroblenski, J., 1992. Very low cadmium concentrations stimulate DNA synthesis and cell growth. J. Cell. Sci. 103, 1073 – 1081. Woodhall, P.B., Tsiher, C.C., Simonton, C.A., Robinson, R.R., 1978. Relationship between p-aminohippurate secretion and cellular morphology in rabbit proximal tubules. J. Clin. Invest. 51, 1320 – 1329.
.
Effects of extracellular cadmium on renal basolateral organic anion transport Helge Hohage a,*, Thomas Mehrens a, Ulrike Mergelsberg b, Marina Lo¨hr b, Joachim Greven b a
Medical Department D, Uni6ersity of Mu¨nster, Albert Schweitzer Str. 33, D-48129 Mu¨nster, Germany b Department of Pharmacology and Toxicology, RWTH, Aachen, Germany Received 8 April 1998; received in revised form 29 June 1998; accepted 30 June 1998
Abstract Heavy metal ions are well-known nephrotoxic agents. Usually, they induce a damage of various renal transport systems. Cd2 + , however, has been shown to enhance renal basolateral organic cation transport. Therefore, the effects of Cd2 + on the basolateral p-aminohippuric acid (PAH) uptake of microdissected nonperfused rabbit kidney S2 proximal tubule segments were investigated. Incubation with Cd2 + induced a bell-shaped concentration response curve with a 2-fold increase of the PAH transport at a Cd2 + concentration of 10 − 6 M. Since Cd2 + has been described to activate protein kinase C (PKC), the effects of the PKC inhibitor staurosporine were also tested. Staurosporine (10 − 8 M) could, however, not reduce the effects of Cd2 + . Cd2 + may exert its effects by an as yet unknown mechanism that is different from the PKC-activating mechanism of phorbol esters. A stimulation of the tricarboxylic acid cycle in kidney cells by Cd2 + may, however, increase the delivery of a-ketoglutarate. Indeed, incubation of proximal tubules with a-ketoglutarate increased PAH transport across the basolateral membrane significantly. Thus, the observed effects of Cd2 + may be due to an enhanced transport of p-aminohippuric acid by stimulation of exchange of PAH with a-ketoglutarate. The modulation of renal organic anion transport may be another aspect of Cd2 + nephrotoxicity. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Renal proximal tubule; Renal organic anion transport system; Cadmium; Protein kinase C
1. Introduction
* Corresponding author. Tel.: + 49 251 8346984; fax: + 49 251 8347528; e-mail: [email protected]
Heavy metals ions are well-known nephrotoxic compounds. In vitro studies could show that these ions directly impair brush border membranes, thereby reducing the transport of Na + -dependent
0378-4274/98/$ - see front matter © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0378-4274(98)00127-1
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H. Hohage et al. / Toxicology Letters 98 (1998) 189–194
Fig. 1. Schematic drawing of the incubation chamber.
transport systems, such as glucose (Lee et al., 1991) or amino acids (Foulkes, 1991) similar to alterations observed in the Fanconi syndrome. In addition, cell death and apoptosis in proximal tubule segments may also occur (Tanimoto et al., 1993). Furthermore, the heavy metal ion Cd2 + has relatively specific damaging effects on cell – cell junctions (Prozialeck and Niewenhuis, 1991). In contrast to the effects on glucose and amino acid transport, a stimulation of the transport of weak organic cations across the basolateral membrane of proximal tubules by Cd2 + was described (Nikiforov and Ostretsova, 1994). Therefore, in this work the effects of Cd2 + on renal PAH transport were determined in isolated, nonperfused proximal S2 segments of the rabbit. This experimental model allows the examination of the transport of organic anions across the basolateral membrane of the proximal tubule, independent of hemodynamic alterations that may also be induced by these ions.
2. Materials and methods We used a microdissection technique similar to that described previously (Brandle and Greven, 1991; Hohage et al., 1994a). New Zealand white rabbits (Charley River, Germany) of either sex, weighing 1.5–2.1 kg (mean: 1.790.2 kg), were killed by cervical dislocation. Afterwards, both
kidneys were rapidly removed. Tissue slices were immediately cut out of the other kidney and transferred to a dish with dissection solution (for composition see below). The time between cervical dislocation and transfer of the tissue slices to the dissection solution did not exceed 10 min. In each experiment, 12–20 segments of proximal tubules from each kidney were microdissected according to the method of Burg et al. (1966). The dissection was performed mechanically at 4°C under a stereomicroscope (Wild, Herbrugg, Switzerland) with a 16–40-fold magnification, without using enzymatic agents. Segments of the proximal tubule were identified as S1, S2, S3 segments according to the criteria established by Woodhall et al. (1978). After the dissection, the superficial tubules were transferred in a droplet of dissection solution to an incubation chamber (Fig. 1), which was cooled on crushed ice during the dissection period. The dissection period did not exceed 30 min. The incubation chamber was placed and heated on an inverted microscope (Zeiss, Oberkochen, Germany, model IM 35). The incubation chamber contained 0.8 ml of incubation medium, including Cd2 + (for composition see below). The surface of the bath medium was superfused with 95% O2 and 5% CO2 in order to keep pH and pO2 constant. At the beginning and end of each experiment, the pH of the bath medium was measured. Experiments were disregarded if the difference between these two mea-
H. Hohage et al. / Toxicology Letters 98 (1998) 189–194
surements exceeded 0.15 pH units. A constant bath temperature within the incubation chamber was maintained by adjusting a current flow through a platinum – iridium wire, which surrounded the incubation chamber. Constant monitoring of the bath temperature was accomplished via a temperature probe that was introduced into the incubation medium via a slot in the incubation chamber. A servonulling arrangement regulated the current flow (Yellow Springs Instruments, CA, model 72). Unless stated otherwise, the temperature was kept constant at 37.5°C. In order to substitute water loss by evaporation, 50 ml of deionized water were added to the incubation medium every 10 min. This time interval was chosen to limit changes in osmolality to less than 10%. At the beginning and end of each experiment, the osmolality was measured by cryoscopy (Knauer Osmometer, Wissenschafterlicher Gera¨tebau KG, Dr. Ing Knaur, Berlin, Germany). If the difference in osmolality between the beginning and the end exceeded 10%, the experiment was disregarded. After the dissection period, incubation medium, Cd2 + and the radioactive compounds were given to the bath and the chamber was heated and insufflated under the described condition. The tubules were added and observed at a ×120 magnification using the criteria indicated by Chonko (no visible lumen) (Chonko et al., 1979). Unless stated otherwise, the tubules were incubated for 10 min with the radio-labeled substances (incubation period), because other experiments in our laboratory had shown that the PAH uptake achieved an equilibrium during this time. At the end of the incubation, formalin in a final bath concentration of 2% was added and a further inspection of the tubules was performed at a × 120 magnification to assure that the tubules were totally collapsed. Afterwards, each tubule was photographed separately to permit subsequent determination of its volume (see below). Each tubule was then taken out of the bath separately, using a glass needle, and transferred to a droplet (300 ml) of 0.75 N nitric acid. The droplet of nitric acid was covered with mineral oil to prevent evaporation. After 45 min of extraction, the whole droplet was aspirated into a glass
191
pipette and transferred to a plastic vial (Packard Instruments, Groningen, Netherlands) containing 5 ml of scintillation cocktail (Packard Emulsifier Safe, Packard Instruments). The radioactivity was counted for 10 min in a Beckman liquid scintillation counter (LS 6000 SC, Beckman, Fullerton CA). In previous studies, quenching was checked by the external standard channels ratio method (Brandle and Greven, 1991; Hohage et al., 1994a,b). Since the volume of the probe (25 ml) was small compared with the volume of the scintillation cocktail (5 ml), a virtually constant external standard channels ratio was found in all probes varying between 1.40 and 1.41. Because the counting efficiency was equal in all probes, and since we were only interested in the cell-tobath activity ratio, cpm values were not corrected to dpm values. To correct the 14C-spillover in the 3 H window and vice versa, single label 14C-probes and 3H-probes were measured under these conditions. 3H and 14C values of the dual label probe were corrected according to this spillover. Tubular volume was calculated as described previously (Brandle and Greven, 1991). The tissue water volume was calculated by multiplying the tubular volume by a factor of 0.7 (Barfuss and Schafer, 1979).
2.1. Solutions and chemicals To study the cellular uptake of 3H-labeled PAH (p-aminohippuric acid, specific activity 5 Ci/mM), PAH was added to the bath solution at a concentration of 6.5 × 10 − 7 M. In order to measure the extracellular contamination, 14C-labeled inulin (specific activity 10.7 mCi/mM, Amersham International, Buckinghamshire, UK) was also added to the bath medium. In some experiments, the tubules were incubated with 50 mM a-ketoglutarate. Experiments were disregarded if the bath radioisotope concentrations sampled at the beginning and the end of the incubation period differed by more than 10%. The solution used for dissection consisted of (mM): NaCl 115, KCl 3.8, NaHCO3 25, KH2PO4 1.3, MgSO4 · 7H2O 1.25, CaCl2 · 2H2O 2.5, glucose 8.3, L-alanine 2.2, trisodiumcitratedihydrate 1, sodium lactate 1.5, glycine 1.8. The bath solution had the same com-
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position, but Dextran (mol. wt. 40000) at a concentration of 30 g/l was added to the solution. The osmolality of both solutions was adjusted to a final value of 290 mosm/kg H2O. The pH was adjusted to a final value of 7.4 after gassing with 95% O2 and 5% CO2.
2.2. Calculations Owing to the fact that all the tubules used were totally collapsed during the incubation period, we regard the uptake of PAH by each tubule as representing the quantity transported over the basolateral membrane. To calculate the amount of the intracellularly stored 3H-activity (cellular 3H-activity), the 3H-activity of the tubule extract was corrected for contamination with the bath solution, using an equation described previously (Brandle and Greven, 1991). Values are presented as mean9 S.E.M. As the samples showed no normal distribution, the values were transformed logarithmically. When comparing two means, the statistical significance of differences was determined by means of the Student’s t-test for paired or unpaired observations. A P value of 0.05 or smaller was considered statistically significant. Whenever more than two means were to be compared, analysis of variance was used. When the F value was significant [P50.05], the group means were analyzed using the Student–Newmanns – Keuls test for significant differences.
ney. Since in recent experiments we could demonstrate that tubular PAH uptake showed a linear accumulation during the first 10 min of the incubation period and reached a maximum after about 15 min, an incubation period of 15 min was used in all subsequent experiments (Hohage et al., 1994a). The PAH concentration in the incubation solution was chosen as 6.5× 10 − 7 M because previous studies had shown that at higher concentrations, significant amounts of PAH were secreted into the lumen of the tubule (Hohage et al., 1994a). Incubation with Cd2 + (Fig. 2) in concentrations ranging from 10 − 8 to 10 − 4 M led to a bell-shaped concentration effect curve of the renal PAH transport. The maximum accumulation occurred at a concentration of 10 − 6 M. Simultaneous incubation of Cd2 + (10 − 6 M) with the protein kinase C inhibitor staurosporine in a concentration of 10 − 8 M, which has been shown to inhibit PKC activity, did not change the basolateral PAH transport significantly (Fig. 3). To investigate the influence of an enhanced delivery, the tubules were preincubated for 10 min with a-ketoglutarate. Under these conditions, PAH uptake increased significantly by 529 7% (Fig. 4).
2.3. Materials p-(glycyl-2-3H)-Aminohippuric acid (specific activity 5 Ci/mmol) was obtained from DuPont, Dreieich, Germany, (hydroxy(14C)methyl)inulin (specific activity 10.7 mCi/mM) from Amersham Buchler, Braunschweig, Germany. Staurosporine, a-ketoglutarate and CdCl2 were purchased from Sigma, Deisenhofen, Germany.
3. Results The basolateral PAH transport studies were performed on isolated S2 segments of rabbit kid-
Fig. 2. Concentration-dependent effects of Cd2 + on the renal PAH transport for an incubation period of 10 min. PAH was added to the incubation medium in a final concentration of 6.5 ×10 − 7 M. The incubation time always was 15 min; 24 – 36 tubules from at least two animals were used. * Denotes P B0.05 vs. control.
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Fig. 3. Effects of Cd2 + and simultaneous administrated staurosporine on the renal PAH transport for an incubation period of 10 min. PAH was added to the incubation medium in a final concentration of 6.5 ×10 − 7 M. Con, control; Cd2 + , Cd2 + 10 − 6 M, Cd2 + +Stau, Cd2 + 10 − 6 M +staurosporine 10-8 M. The incubation time always was 15 min; 24–36 tubules from at least two animals were used. * denotes PB 0.05 vs. control.
4. Discussion Heavy metal ions such as Cd2 + are well-known nephrotoxic compounds reducing the activity of a wide variety of Na + -dependent renal transporters. In contrast to expected effects, an increase of the transport of weak organic cations across the basolateral membrane has been reported following Cd2 + application (Nikiforov and Ostretsova, 1994). The purpose of the present study was, therefore, to investigate the influence of Cd2 + on the transport of organic anions in rabbit kidney proximal tubule S2 segments.
Fig. 4. Stimulation of PAH accumulation after incubation with 50 mM a-ketoglutarate. Con, control; KG, a-ketoglutarate incubation; 24 – 36 tubules from at least two animals were used. * Denotes P B0.05 vs. control.
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Interestingly, following Cd2 + application, a bell-shaped concentration response relationship was observed. Cd2 + has been described to interact with the protein kinase C system (Beyersmann et al., 1994). Thus, it seemed likely that stimulation of protein kinase C by Cd2 + may also contribute to the enhanced PAH transport since this second messenger enhances the transport of organic anions across the basolateral membrane of proximal tubules (Hohage et al., 1994a). However, the PKC inhibitor staurosporine failed to inhibit the effects of Cd2 + on PAH transport leading to the assumption, that the effects observed following Cd2 + application are not due to direct stimulation of PKC. Nevertheless, Cd2 + may change the conformation of PKC, resulting in an enhanced activity of this molecule and loss of staurosporine sensitivity. This assumption is supported by the fact that Cd2 + interacts with Zn2 + binding sites, which have been described within the PKC molecule (Berg, 1986). Cd2 + induces a characteristic bell-shaped dose effect relationship, which has also been obtained in previous studies when the effect of PMA on renal proximal tubular transporters were measured (Hohage et al., 1994a,b). A down-regulation mechanism of the activated PKC has been suggested to explain this phenomenon. The drop in the dose–effect relationship, however, may also reflect toxic effects of Cd2 + . Similar to our results, a stimulation of weak organic acid uptake in rat renal tubules by Cd2 + was reported (Nikiforov and Ostretsova, 1994), which was related to an activation of the tricarboxylic acid cycle and an export of reducing equivalents from the mitochondria. Indeed, this mechanism also seems to contribute to the enhanced PAH accumulation seen in our experiments. After incubation with a-ketoglutarate, PAH accumulation was increased by 529 7%. Similar effects of a-ketoglutarate have been reported previously by Pritchard (1995). Furthermore, very low Cd2 + concentrations have been shown to stimulate DNA synthesis and cell growth in the renal cell line LLC-PK1 (von Zglinicki et al., 1992), which may also contribute to the effects observed.
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In summary, the data presented provide evidence that the transport of organic anions across the basolateral membrane can be stimulated by Cd2 + , probably due to an enhanced PAH/a-ketoglutarate exchange. An effect of Cd2 + on protein kinase C seems to be excluded.
Acknowledgements The authors are grateful to K. Breidbach and H. Erven for their assistance in the manufacture of the incubation chamber and M. Peters for typing the manuscript. Helpful and critical discussion with E. Schlatter is also gratefully acknowledged.
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