Bianchi C, Bocci V, Carone FA, Rabkin R (eds): Kidney, Proteins and Drugs. Contrib Nephrol. Basel, Karger, 1990, vol 83, pp 208-212
Cisplatin Nephrotoxicity and PlatinumMetallothioneins: Uptake and Toxicity in Proximal Tubular Cells from Rat Kidney Pieter 1. Boogaard, Gerard 1. Mulder, 1. Fred Nagelkerke Division of Toxicology, Center for Bio-Pharmaceutical Sciences, Leiden University, Leiden, The Netherlands
Cisplatin (CDDP) is a very potent cytostatic drug. Its use, however, is limited by nephrotoxicity, the mechanism of which is poorly understood. Metallothionein (MT), a low-molecular-weight protein, binds heavy metal ions, including Pt2 + [1,2]. This binding to MT detoxifies metal ions for most organs [3-5], but it can be a toxifying pathway for the kidney. When the metal-MT complex is released into the plasma, it is taken up by the renal proximal tubular cells (PTC). Subsequent intracellular degradation of the complex releases the metal ion, which causes nephrotoxicity [3]. Several observations indicate that MT might play this dual role in the case of CD D P as well: CDDP induces MT expression which correlates inversely with nephrotoxicity [2], and CDDP produces in the PTC, which are the target cells, the same type of lesions as several heavy metals [6, 7]. MT plays a crucial role in the uptake and accumulation of these metals in the PTC [3]. Therefore, we investigated the uptake and toxicity of CDDP and platinum metallothionein (Pt-MT) in freshly isolated PTC from rat kidney and primary cultures of these cells.
Proximal Tubular Cells. PTC were isolated from male Wistar rats (150-200 g) as described previously [8]. PTC in suspension were incubated in Hanks' buffer (pH 7.40), supplemented with 0.25 mM HEPES and 2.5% w/v BSA, at a density of 3 x 106 cells/ml at 37°C on a rotatory shaker. Alternatively, PTC were cultured on plastic plates in a modified DMEMHam F 12 medium [9].
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Materials and Methods
Cisplatin Nephrotoxicity and Platinum-Metallothioneins
209
Preparation ot 95m Pt-CDD P. Labeled CDDP was synthesized by G. Baldew (Interfacul-
tair Reactor Instituut, Delft, The Netherlands) according to Hoeschele et al [lO] from NazPtC14 . Purity was> 98% as checked by TLC. Preparation 195mpt_MT. CdCIz·aq (3.6mglkg) was administered i. p. to male Wistar rats (200 g) 3 times a week for 3 weeks. The livers were homogenized in ice-cold lO mM TRIS HCI buffer pH 7.80, and centrifugated at lOO,OOOg for 2h. The supernatant was fractionated on Sephadex G75. Fractions containing MT (characterized by 254/280 nm absorbance ratio and retention volume of 2 times void volume) were applied on Sephadex DEAE A25 and eluted with a linear gradient from lO to 250 mM TRIS-HCI buffer pH 7.80. The 2 isoforms of MT that eluted were pooled and desalted by Sephadex G25 elution using 25 mM (N~hCOrbuffer pH 7.80 and lyophilization. The MT was dissolved in 0.1 M HCI in order to dissociate the Cd2+ from the apoprotein and eluted on Sephadex G25. The apoprotein was reconstituted with a 2-fold molar excess 195mpt2+ (as NazPtC14 ) and separated from excess 195pt2+ by elution on Sephadex G25 with 25 mM (N~hC03-buffer pH 7.80 and lyophilized. Cytotoxicity and Uptake Determinations. Toxicity was assessed by uptake of (X-methylglucose (X-MG). (X-MG and other uptake experiments were performed as described previously [8, 9, 11].
Results Preparation of 195mpt_MT. Two radioactive peaks eluted from the Sephadex G25 column after reconstitution of 230 nmol MT (pure on SDSPAGE) with 3.2 !-tmol Naz19SmPtC14' The first peak, eluting in the void volume, contained all protein and 47% of the total radioactivity. The MT bound 7 atoms of Pt (fig. 1).
Cytotoxicity of95m Pt-CD DP and 195m Pt-MT. The lowest concentration at which CDDP inhibited (X-MG uptake significantly (by 21 ± 2% in 3 h) in isolatedPTCwas IO!-tM [11]. In cultured PTC, Pt-MT(10 !-tMPt) decreased (X-MG by 43 ± 7% in4h. CDDP and Pt-MT had LCso in isolated PTCof250 and 30 !-tM, respectively.
Downloaded by: Université de Paris 193.51.85.197 - 2/3/2020 9:29:10 PM
Uptake of 195m Pt-CDDP and 195m Pt-MT. The uptake oflabeled CDDP and Pt-MTwas studied in freshly isolated and cultured PTC. CDDP uptake in isolated PTC depended linearly both on the Pt concentration in the medium and time. Cultured PTC incubated with IO!-tM CDDP for 6 h, had taken up only 2-3% of the amountofPt that was taken up in 5h when 15!-tM Pt-MTwas used (fig. 2). Uptake ofCDDP by isolated PTCwas not affected by coincubation with 2.5% w/v BSA or 10mM lysine. However, BSA reduced the uptake of Pt-MT by approximately 40%; addition of lysine to the medium reduced the uptake by another 10% (fig. 3).
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Cisplatin Nephrotoxicity and PlatinumMetallothioneins: Uptake and Toxicity in Proximal Tubular Cells from Rat Kidney Pieter 1. Boogaard, Gerard 1. Mulder, 1. Fred Nagelkerke Division of Toxicology, Center for Bio-Pharmaceutical Sciences, Leiden University, Leiden, The Netherlands
Cisplatin (CDDP) is a very potent cytostatic drug. Its use, however, is limited by nephrotoxicity, the mechanism of which is poorly understood. Metallothionein (MT), a low-molecular-weight protein, binds heavy metal ions, including Pt2 + [1,2]. This binding to MT detoxifies metal ions for most organs [3-5], but it can be a toxifying pathway for the kidney. When the metal-MT complex is released into the plasma, it is taken up by the renal proximal tubular cells (PTC). Subsequent intracellular degradation of the complex releases the metal ion, which causes nephrotoxicity [3]. Several observations indicate that MT might play this dual role in the case of CD D P as well: CDDP induces MT expression which correlates inversely with nephrotoxicity [2], and CDDP produces in the PTC, which are the target cells, the same type of lesions as several heavy metals [6, 7]. MT plays a crucial role in the uptake and accumulation of these metals in the PTC [3]. Therefore, we investigated the uptake and toxicity of CDDP and platinum metallothionein (Pt-MT) in freshly isolated PTC from rat kidney and primary cultures of these cells.
Proximal Tubular Cells. PTC were isolated from male Wistar rats (150-200 g) as described previously [8]. PTC in suspension were incubated in Hanks' buffer (pH 7.40), supplemented with 0.25 mM HEPES and 2.5% w/v BSA, at a density of 3 x 106 cells/ml at 37°C on a rotatory shaker. Alternatively, PTC were cultured on plastic plates in a modified DMEMHam F 12 medium [9].
Downloaded by: Université de Paris 193.51.85.197 - 2/3/2020 9:29:10 PM
Materials and Methods
Cisplatin Nephrotoxicity and Platinum-Metallothioneins
209
Preparation ot 95m Pt-CDD P. Labeled CDDP was synthesized by G. Baldew (Interfacul-
tair Reactor Instituut, Delft, The Netherlands) according to Hoeschele et al [lO] from NazPtC14 . Purity was> 98% as checked by TLC. Preparation 195mpt_MT. CdCIz·aq (3.6mglkg) was administered i. p. to male Wistar rats (200 g) 3 times a week for 3 weeks. The livers were homogenized in ice-cold lO mM TRIS HCI buffer pH 7.80, and centrifugated at lOO,OOOg for 2h. The supernatant was fractionated on Sephadex G75. Fractions containing MT (characterized by 254/280 nm absorbance ratio and retention volume of 2 times void volume) were applied on Sephadex DEAE A25 and eluted with a linear gradient from lO to 250 mM TRIS-HCI buffer pH 7.80. The 2 isoforms of MT that eluted were pooled and desalted by Sephadex G25 elution using 25 mM (N~hCOrbuffer pH 7.80 and lyophilization. The MT was dissolved in 0.1 M HCI in order to dissociate the Cd2+ from the apoprotein and eluted on Sephadex G25. The apoprotein was reconstituted with a 2-fold molar excess 195mpt2+ (as NazPtC14 ) and separated from excess 195pt2+ by elution on Sephadex G25 with 25 mM (N~hC03-buffer pH 7.80 and lyophilized. Cytotoxicity and Uptake Determinations. Toxicity was assessed by uptake of (X-methylglucose (X-MG). (X-MG and other uptake experiments were performed as described previously [8, 9, 11].
Results Preparation of 195mpt_MT. Two radioactive peaks eluted from the Sephadex G25 column after reconstitution of 230 nmol MT (pure on SDSPAGE) with 3.2 !-tmol Naz19SmPtC14' The first peak, eluting in the void volume, contained all protein and 47% of the total radioactivity. The MT bound 7 atoms of Pt (fig. 1).
Cytotoxicity of95m Pt-CD DP and 195m Pt-MT. The lowest concentration at which CDDP inhibited (X-MG uptake significantly (by 21 ± 2% in 3 h) in isolatedPTCwas IO!-tM [11]. In cultured PTC, Pt-MT(10 !-tMPt) decreased (X-MG by 43 ± 7% in4h. CDDP and Pt-MT had LCso in isolated PTCof250 and 30 !-tM, respectively.
Downloaded by: Université de Paris 193.51.85.197 - 2/3/2020 9:29:10 PM
Uptake of 195m Pt-CDDP and 195m Pt-MT. The uptake oflabeled CDDP and Pt-MTwas studied in freshly isolated and cultured PTC. CDDP uptake in isolated PTC depended linearly both on the Pt concentration in the medium and time. Cultured PTC incubated with IO!-tM CDDP for 6 h, had taken up only 2-3% of the amountofPt that was taken up in 5h when 15!-tM Pt-MTwas used (fig. 2). Uptake ofCDDP by isolated PTCwas not affected by coincubation with 2.5% w/v BSA or 10mM lysine. However, BSA reduced the uptake of Pt-MT by approximately 40%; addition of lysine to the medium reduced the uptake by another 10% (fig. 3).
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