TY - JOUR
AU - Krähenbühl, Stephan
AB - Abstract Background. Patients treated with cisplatin or carboplatin show increased renal excretion of carnitine. It is currently unclear whether this is also the case for oxaliplatin and which are the responsible mechanisms. Methods. We investigated 22 patients treated either with a single dose of cisplatin, carboplatin or oxaliplatin. Carnitine and kidney function parameters were determined in plasma and urine. Inhibition and mRNA expression of OCTN2, the principle carnitine transporter, were assessed in L6 cells overexpressing OCTN2 and in 293-EBNA cells, respectively. Results. Renal excretion of free and short-chain acylcarnitine increased already at the day of administration was maximal the day after and had normalized 1 week after administration of cisplatin, carboplatin or oxaliplatin. The renal excretion fractions for free carnitine and acylcarnitines increased 4–10 times during treatment with platin derivatives. Renal excretions of α1-microglobulin and other proximal tubular markers were also increased, compatible with a proximal tubular defect. Direct inhibition of OCTN2 expressed in L6 cells by cisplatin, oxaliplatin or platinum 2+ could not be demonstrated, and experiments using urine from patients treated with cisplatin inhibited OCTN2 activity no more than expected from the carnitine content in the respective urine sample. Cisplatin was associated with a time- and concentration-dependent decrease of OCTN2 mRNA and protein expression in 293-EBNA cells. Conclusions. All platin derivatives investigated are associated with renal tubular damage in humans without significantly affecting glomerular function. The rapid onset and complete reversibility of this effect favour a functional mechanism such as impaired expression of OCTN2 in proximal tubular cells. carboplatin, carnitine, cisplatin, OCTN2, oxaliplatin Introduction Patients treated with cisplatin frequently develop nephropathy, which can be of either glomerular or proximal tubular origin [ 1,2 ]. In comparison, carboplatin is considered to be less nephrotoxic than cisplatin [ 1 , 3 ]. Carboplatin is not typically associated with a decrease in glomerular filtration, but may cause a transient elevation in urinary enzymes without clinical relevance [ 1 ]. For oxaliplatin, neurotoxicity may become dose limiting whereas nephrotoxicity is rare [ 4 ]. We have shown previously that patients treated with cisplatin rapidly develop a significant increase in the renal clearance of carnitine and acylcarnitines, resulting in a massive increase in renal excretion of carnitine and acylcarnitines [ 5 ]. These findings have recently been confirmed by other researchers, who could demonstrate that carboplatin is also associated with renal loss of carnitine [ 6 ]. After stopping therapy with cisplatin or carboplatin, renal carnitine excretion normalized rapidly and completely [ 5,6 ]. The rapid and complete reversibility of this effect after stopping the administration of platinum derivatives excludes a structural damage of the proximal tubule with a high probability and is compatible with an interaction of cisplatin and carboplatin with the proximal tubular reabsorption of carnitine. Carnitine is essential for the transport of long-chain fatty acids into the mitochondrial matrix, where they can be metabolized by β-oxidation [ 7 ]. Most carnitine is taken up from the diet, with a minor portion being provided by endogenous biosynthesis [ 7–9 ]. Carnitine can be acylated to form acylcarnitines but is otherwise not metabolized [ 7,8 ]. Its elimination occurs as carnitine or acylcarnitine primarily via urinary excretion [ 7,8 ]. After glomerular filtration, it is reabsorbed by 98–99% in the proximal tubule [ 5 , 7 , 10 ]. Proximal tubular reabsorption of carnitine is accomplished by OCTN2, an organic cation transporting protein with a high affinity for carnitine, transporting carnitine in a sodium-dependent way [ 11–13 ]. Some xenobiotics, in particular carnitine analogues and certain cephalosporins, can significantly inhibit carnitine transport via OCTN2 [ 14,15 ]. The current studies were undertaken to compare the effect of carboplatin and oxaliplatin on the urinary excretion of carnitine in humans to cisplatin. Furthermore, the mechanisms involved were investigated by correlating the effect of carnitine excretion with markers of proximal tubular damage and by studying the effect of the platin derivatives on carnitine transport by OCTN2 and on OCTN2 expression in tubular cells and kidney cells. Subjects and methods Materials [ 3 H]acetyl-CoA which was obtained from New England Nuclear (Boston, MA, USA). L-[ 3 H]-carnitine hydrochloride (81 Ci/mmol) was obtained from Amersham-Pharmacia Biotech (Little Chalfont, Buckinghamshire, England). l -carnitine was obtained from Fluka (Buchs, Switzerland). Cisplatin [cis-PtCl 2 (NH 3 ) 2 ], oxaliplatin [ cis -[(1 R ,2 R )-1,2-cyclohexanediamine- N , N ] [oxalato(2-)- O , O ] platinum] and platinum(II) chloride (PtCl 2 ) were purchased from Sigma (Buchs, Switzerland). Other chemicals were from Sigma (Buchs, Switzerland) or Merck (Dietikon, Switzerland). Patients and treatments The study had been accepted by the Ethics Committee of the Canton of Basel, and all patients gave written informed consent before entry into the study. Twenty-two patients (6 females and 16 males with a mean age of 60 ± 10 years) undergoing a chemotherapy including single-dose cisplatin (10 males), oxaliplatin (4 females and 4 males) or carboplatin (2 females and 2 males) were recruited at the oncology clinics and at the women's hospital of the university hospital of Basel. None of the patients studied had a creatinine clearance <60 mL/min/1.73 m 2 at entry (determined using the Cockcroft and Gault formula [ 16 ]) and none of them was ingesting any nephrotoxic drugs. Five of the patients treated with cisplatin were suffering from non-small cell carcinoma of the lung, two from non-Hodgkin lymphoma, two from an urothelial carcinoma and one from a carcinoma of the oropharynx. They were treated with an average cisplatin dose of 80 ± 4 mg/m 2 (range 50–100 mg/m 2 ). Other drugs administered included gemcitabine (two patients), etoposide (two patients), rituximab (one patient), cytosar (one patient), taxotere (one patient), navelbine (one patient) and/or prednisone (two patients). All the patients were studied during the first chemotherapy cycle except one of them, who underwent the fourth cycle. Six of the patients treated with oxaliplatin suffered from colon carcinoma and two from rectum carcinoma. The average oxaliplatin dose was 125 ± 15 mg/m 2 (range 88–130 mg/m 2 ). Other drugs administered included capecitabine (seven patients), methotrexate (immunosuppressive dose, one patient) and prednisone (one patient). The patients were in cycle 1, 3, 4, 5 or 6, respectively. From the patients treated with carboplatin, two had a cancer of the ovary and two a non-small cell carcinoma of the lung. The average dose (AUC) of carboplatin was 5.25 ± 0.25 mg mL −1 min (range 5–6 mg mL −1 min). Other drugs administered included taxol (two patients). The patients were in chemotherapy cycle 1, 3, 6 or 8, respectively. In addition, all patients received an antiemetic prophylaxis with a 5HT 3 -receptor antagonist and with dexamethasone. None of the additional drugs administered has been shown to interact with OCTN2 [ 5 , 17 ] or to have a structure with a high probability to interact with OCTN2 [ 17 ]. Study protocol The patients were studied in cycles of 24 h. A cycle began at 08:00 when the patient was asked to collect a sample of the second urine in the morning, which was used for the determination of the urinary protein pattern. After that, urine collection over the next 24 h was started. At the end of the urine-collection period, a venous blood sample was drawn and plasma was obtained by centrifugation, terminating the cycle. Such cycles were performed on the day before treatment (Day 0), the day of treatment (Day 1), the 2 days following treatment (Days 2 and 3) and 1 week after termination of treatment (Day 7). Urine and plasma samples were stored at −20°C until analysis. Carnitine determination The carnitine concentrations in plasma and urine were determined using the radio-enzymatic method originally described by Cederblad [ 18 ] and modified by Brass and Hoppel [ 19 ]. The method is suitable for the determination of free, total acid soluble and long-chain acylcarnitine (chain length of acyl groups ≥10). Short-chain acylcarnitine was obtained by subtracting free from total acid soluble carnitine and total carnitine by the addition of total acid soluble and long-chain acylcarnitine. Analysis of magnesium, creatinine and proteinuria Concentrations of magnesium in urine and creatinine in plasma and urine were determined by routine methods at the Department of Clinical Chemistry at the University Hospital of Basel. To locate a possible kidney damage associated with the drugs administered, we analysed the urinary protein pattern by a previously published method using a Beckman–Coulter array nephelometry system (Beckman–Coulter, Brea, CA, USA) [ 20 ]. Cell lines L6 cells (rat skeletal muscle myoblasts, obtained from American Type Culture Collection, Rockville, MD, USA) were stably transfected with human kidney carnitine transporter (hOCTN2) (L6-hOCTN2 cells) as previously described [ 17 ]. L6-hOCTN2 cells, L6 cells and 293-EBNA cells (human primary embryonal kidney cell line, American Type Culture Collection) were grown in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate and 100 U/mL penicillin and 100 μg/mL streptomycin (all substances from Invitrogen, Basel, Switzerland) at 37°C with 5% CO 2 and 95% humidity. Testing for mycoplasma was negative throughout the study. l -carnitine-uptake experiments The cellular uptake of l -[ 3 H]-carnitine was measured at 30°C in cells grown to confluency on 12-well plates (Becton Dickinson, Basel, Switzerland). The sodium uptake buffer contained 116 mM NaCl, 5.3 mM KCl, 0.8 mM MgSO 4 , 26.2 mM NaHCO 3 , 1 mM NaH 2 PO 4 and 5.5 mM d -glucose, pH = 7.4. The total uptake of l -[ 3 H]-carnitine was determined by incubating the cells with 0.5 ml of sodium uptake buffer containing 50 μM l -carnitine (5 μCi/0.5 ml) for 8 min at 30°C. For the sodium-independent l -carnitine uptake, the sodium uptake buffer was replaced by the choline uptake buffer (similar to sodium uptake buffer, but sodium completely replaced by choline). The sodium-dependent l -carnitine uptake was determined as the difference between the uptake in the presence of the sodium and the choline buffer [ 17 ]. For the inhibition experiments with platin derivatives, preincubation with the respective platin preparation was performed for the time indicated in the Result section at 30°C in the presence of sodium or choline uptake buffer, followed by the determination of carnitine uptake as described above. The platin derivatives were dissolved in NaCl 0.9% or H 2 O and the pH adjusted to 7.4 with 1N KOH as needed. The solutions were used either freshly or 24 h after the preparation as described in the Results section. The final concentrations in the uptake experiments ranged from 1 to 100 μM. Incubations for inhibition experiments with urine were composed of 500 μl incubation solution and 250 μl urine. The final buffer concentration was identical to the incubations as described above and the final carnitine concentration was 50 μmol/L (plus carnitine from the urine, see the Results section). OCTN2 mRNA expression in 293-EBNA cells treated with cisplatin 293-EBNA cells were grown to confluency and then exposed to 50 or 100 μmol/L cisplatin for 8–48 h as described in the Results section. Total RNA was isolated using RNeasy® as described by the manufacturer (Qiagen, Hombrechtikon, Switzerland). The quality of the total RNA samples was analysed using Agilent's Pico-Chips (Agilent Technologies Schweiz, Basel, Switzerland). Superscript TM II together with Oligo (dT) and Random Hexamer primers (Invitrogen, Basel, Switzerland) were used for reverse transcription of 2 μg total RNA. The real-time PCR and quantification of OCTN2 mRNA were performed as previously described using GAPDH mRNA as an internal standard [ 17 ]. OCTN2 protein expression in 293-EBNA cells treated with cisplatin The cells were cultured and exposed to cisplatin as described above for OCTN2 mRNA quantification. The cells (10 6 ) were lysed using 1% Nonidet P-40 (NP-40) and a protease inhibitor cocktail (Roche AG, Basel, Switzerland) plus 1 mM phenylmethylsulfonyl fluoride (PMSF). After centrifugation, supernatants were combined with the same volume of loading buffer (2% SDS, 10% glycerol, 100 mM TrisHCl pH 6.8, bromophenol blue, 5 mM DTT) and loaded (50 μg protein) on a 10% polyacrylamide sodium dodecyl sulfate (SDS) gel. The procedure including separation by electrophoresis, transfer to nitrocellulose filters and detection and quantification of OCTN2 protein bands was performed as previously described [ 17 ]. Polyclonal antibodies against OCTN2 had been prepared as previously described [ 17 ]. Cytotoxicity Cytotoxicity of cisplatin was estimated with the ToxiLight® Bioassay (Cambrex Bio Science Verviers, Belgium). The assay is based on the conversion of ADP to ATP by adenylate kinase, an enzyme released by cellular destruction. ATP is detected by the luminescent firefly luciferase assay. The protocol was as described by the manufacturer. In short, 20 μl of cell supernatant was transferred to a luminescence compatible 96-well plate and 100 μl of the reconstituted detection reagent were added. After 5 min, the emitted light was measured in a HTS 7000 Plus Bio Assay Reader (Perkin Elmer, Schwerzenbach, Switzerland). Calculations and statistics Excretion fractions were calculated by dividing the urine to plasma ratio of the analyte investigated (free carnitine, short-chain acylcarnitines, total carnitine) by the urine to plasma ratio of creatinine obtained in the same urine. Values are given as mean (SEM). Statistical analyses were performed using SigmaStat version 3.5 (Scientific Solutions, Pully-Lausanne, Switzerland). Time series (Figures 1 and 4 and Tables 1–3) or unpaired groups (Figures 2 and 3) were analysed using one-way analysis of variance for repeated measures or one-way analysis of variance for unpaired data, respectively, followed by the Holm–Sidak method to localize significant differences to baseline or control values. A P < 0.05 was considered to be statistically significant. Fig. 1 View largeDownload slide Excretion fractions of free carnitine (free Cn), short-chain acylcarnitine (SCA Cn) and total carnitine (total Cn) in patients treated with cisplatin ( n = 10), carboplatin ( n = 4) or oxaliplatin ( n = 8). Excretion fractions were calculated as described in the Method section and are given as a percentage. *P < 0.05 versus Day 0 (the day before the start of chemotherapy). Fig. 1 View largeDownload slide Excretion fractions of free carnitine (free Cn), short-chain acylcarnitine (SCA Cn) and total carnitine (total Cn) in patients treated with cisplatin ( n = 10), carboplatin ( n = 4) or oxaliplatin ( n = 8). Excretion fractions were calculated as described in the Method section and are given as a percentage. *P < 0.05 versus Day 0 (the day before the start of chemotherapy). Fig. 2 View largeDownload slide Inhibition of the transport of 3 H- l -carnitine into L6 cells expressing hOCTN2. Measurements were performed in the presence of a sodium gradient and in the presence of a choline gradient as described in the Method section. The difference between these two measurements is the sodium-dependent portion of the transport, which is shown in the Figure. Cisplatin, oxaliplatin and platinum(II) chloride did not inhibit transport of 3 H- l -carnitine. Butyrobetaine, the direct biosynthetic carnitine precursor known to inhibit OCTN2 [ 13 ], was used as a positive control. The data are given as a percentage of the control values ( 3 H- l -carnitine uptake in L6 cells without the presence of an inhibitor = 344 ± 28 pmol/min/mg protein). *P < 0.05 versus control values. Fig. 2 View largeDownload slide Inhibition of the transport of 3 H- l -carnitine into L6 cells expressing hOCTN2. Measurements were performed in the presence of a sodium gradient and in the presence of a choline gradient as described in the Method section. The difference between these two measurements is the sodium-dependent portion of the transport, which is shown in the Figure. Cisplatin, oxaliplatin and platinum(II) chloride did not inhibit transport of 3 H- l -carnitine. Butyrobetaine, the direct biosynthetic carnitine precursor known to inhibit OCTN2 [ 13 ], was used as a positive control. The data are given as a percentage of the control values ( 3 H- l -carnitine uptake in L6 cells without the presence of an inhibitor = 344 ± 28 pmol/min/mg protein). *P < 0.05 versus control values. Fig. 3 View largeDownload slide Inhibition of the transport of 3 H- l -carnitine into L6 cells expressing hOCTN2 by urine samples. Urine samples (Day 0, Day 1, Day 2) were tested for inhibition of carnitine uptake by L6 cells as described in the Method section. The total carnitine concentration in the assays (assay plus urine) is given in parentheses. There is a significant inhibition of 3 H- l -carnitine transport by urine, corresponding roughly to the carnitine concentration in the urines. The data are given as a percentage of the control values ( 3 H- l -carnitine uptake in L6 cells without the presence of an inhibitor = 313 ± 28 pmol/min/mg protein). Fig. 3 View largeDownload slide Inhibition of the transport of 3 H- l -carnitine into L6 cells expressing hOCTN2 by urine samples. Urine samples (Day 0, Day 1, Day 2) were tested for inhibition of carnitine uptake by L6 cells as described in the Method section. The total carnitine concentration in the assays (assay plus urine) is given in parentheses. There is a significant inhibition of 3 H- l -carnitine transport by urine, corresponding roughly to the carnitine concentration in the urines. The data are given as a percentage of the control values ( 3 H- l -carnitine uptake in L6 cells without the presence of an inhibitor = 313 ± 28 pmol/min/mg protein). Fig. 4 View largeDownload slide Influence of cisplatin on the expression of hOCTN2 mRNA and protein in cultured 293-EBNA cells. mRNA extracted from cultured 293-EBNA cells was transformed to the corresponding cDNA as described in the Method section. This cDNA was used to determine the concentration of hOCTN2 and GAPDH mRNA by real-time PCR. Expression of hOCTN2 mRNA is given normalized to the expression of GAPDH. Protein expression of OCTN2 was performed by western blotting as described in the Method section. Values are given normalized to actin expression. Cisplatin is associated with a time and concentration-dependent decrease in hOCTN2 mRNA and protein expression. *P < 0.05 versus control values. Fig. 4 View largeDownload slide Influence of cisplatin on the expression of hOCTN2 mRNA and protein in cultured 293-EBNA cells. mRNA extracted from cultured 293-EBNA cells was transformed to the corresponding cDNA as described in the Method section. This cDNA was used to determine the concentration of hOCTN2 and GAPDH mRNA by real-time PCR. Expression of hOCTN2 mRNA is given normalized to the expression of GAPDH. Protein expression of OCTN2 was performed by western blotting as described in the Method section. Values are given normalized to actin expression. Cisplatin is associated with a time and concentration-dependent decrease in hOCTN2 mRNA and protein expression. *P < 0.05 versus control values. Table 1 Carnitine plasma concentrations in patients treated with cisplatin, carboplatin or oxaliplatin on Day 1   Before treatment  During (Day 1) and after treatment (Days 2–7)    Day 0  Day 1  Day 2  Day 3  Day 7  Cisplatin ( n = 10)              Free carnitine  44.8 (5.0)  65.8 (7.0) *  64.9 (3.9) *  55.6 (4.6)  34.3 (2.9)   Short-chain acylcarnitine  2.3 (0.5)  5.2 (1.2)  4.5 (0.6)  3.7 (0.8)  3.4 (1.3)   Total carnitine  54.3 (5.9)  81.1 (8.7) *  79.2 (4.6) *  68.2 (4.8)  42.6 (3.9)  Oxaliplatin ( n = 8)              Free carnitine  31.3 (4.7)  60.8 (5.5) *  41.1 (5.9)  31.3 (4.2)  29.2 (3.1)   Short-chain acylcarnitine  3.4 (0.6)  3.8 (0.9)  4.5 (0.7)  2.7 (0.9)  3.9 (0.9)   Total carnitine  39.0 (5.6)  72.1 (6.1) *  50.7 (6.7)  38.8 (5.2)  37.2 (3.8)  Carboplatin ( n = 4)              Free carnitine  30.8 (4.2)  47.7 (6.2)  40.0 (2.1)  36.3 (1.0)  36.3 (6.6)   Short-chain acylcarnitine  5.6 (1.1)  3.2 (1.2)  4.6 (0.2)  3.0 (0.4)  3.8 (1.0)   Total carnitine  41.3 (6.0)  58.1 (6.5)  49.8 (1.7)  44.5 (1.2)  45.3 (6.9)    Before treatment  During (Day 1) and after treatment (Days 2–7)    Day 0  Day 1  Day 2  Day 3  Day 7  Cisplatin ( n = 10)              Free carnitine  44.8 (5.0)  65.8 (7.0) *  64.9 (3.9) *  55.6 (4.6)  34.3 (2.9)   Short-chain acylcarnitine  2.3 (0.5)  5.2 (1.2)  4.5 (0.6)  3.7 (0.8)  3.4 (1.3)   Total carnitine  54.3 (5.9)  81.1 (8.7) *  79.2 (4.6) *  68.2 (4.8)  42.6 (3.9)  Oxaliplatin ( n = 8)              Free carnitine  31.3 (4.7)  60.8 (5.5) *  41.1 (5.9)  31.3 (4.2)  29.2 (3.1)   Short-chain acylcarnitine  3.4 (0.6)  3.8 (0.9)  4.5 (0.7)  2.7 (0.9)  3.9 (0.9)   Total carnitine  39.0 (5.6)  72.1 (6.1) *  50.7 (6.7)  38.8 (5.2)  37.2 (3.8)  Carboplatin ( n = 4)              Free carnitine  30.8 (4.2)  47.7 (6.2)  40.0 (2.1)  36.3 (1.0)  36.3 (6.6)   Short-chain acylcarnitine  5.6 (1.1)  3.2 (1.2)  4.6 (0.2)  3.0 (0.4)  3.8 (1.0)   Total carnitine  41.3 (6.0)  58.1 (6.5)  49.8 (1.7)  44.5 (1.2)  45.3 (6.9)  Carnitine concentrations were determined radioenzymatically as described in the Method section. Units are μmol/24 h. Values are given as mean (SEM). *P < 0.05 versus Day 0. View Large Table 2 Urinary excretion of carnitine in patients treated with cisplatin, carboplatin or oxaliplatin   Before treatment  During (Day 1) and after treatment (Days 2–7)    Day 0  Day 1  Day 2  Day 3  Day 7  Cisplatin ( n = 10)              Free carnitine  134 (54)  439 (152)  1580 (540) *  861 (312)  183 (114)   Short-chain acylcarnitine  91.4 (16.6)  91.3 (21.8)  357 (117) *  138 (40)  80.5 (10.6)   Total carnitine  225 (69)  531 (161)  1940 (640) *  999 (351)  264 (124)  Oxaliplatin ( n = 8)              Free carnitine  72.1 (41.6)  555 (178)  553 (152) *  121 (74)  33.3 (25.1)   Short-chain acylcarnitine  64.4 (15.4)  163 (76)  184 (38)  56.1 (11.2)  73.0 (14.6)   Total carnitine  137 (52)  718 (247)  738 (189) *  177 (69)  106 (37)  Carboplatin ( n = 4)              Free carnitine  41.0 (19.9)  359 (164)  403 (215)  73.4 (25.3)  36.3 (16.3)   Short-chain acylcarnitine  81.1 (21.2)  177 (49)  148 (42)  95.8 (9.7)  115 (34)   Total carnitine  122 (41)  536 (212)  551 (257)  169 (32)  151 (50)    Before treatment  During (Day 1) and after treatment (Days 2–7)    Day 0  Day 1  Day 2  Day 3  Day 7  Cisplatin ( n = 10)              Free carnitine  134 (54)  439 (152)  1580 (540) *  861 (312)  183 (114)   Short-chain acylcarnitine  91.4 (16.6)  91.3 (21.8)  357 (117) *  138 (40)  80.5 (10.6)   Total carnitine  225 (69)  531 (161)  1940 (640) *  999 (351)  264 (124)  Oxaliplatin ( n = 8)              Free carnitine  72.1 (41.6)  555 (178)  553 (152) *  121 (74)  33.3 (25.1)   Short-chain acylcarnitine  64.4 (15.4)  163 (76)  184 (38)  56.1 (11.2)  73.0 (14.6)   Total carnitine  137 (52)  718 (247)  738 (189) *  177 (69)  106 (37)  Carboplatin ( n = 4)              Free carnitine  41.0 (19.9)  359 (164)  403 (215)  73.4 (25.3)  36.3 (16.3)   Short-chain acylcarnitine  81.1 (21.2)  177 (49)  148 (42)  95.8 (9.7)  115 (34)   Total carnitine  122 (41)  536 (212)  551 (257)  169 (32)  151 (50)  Carnitine concentrations were determined radio-enzymatically as described in the Method section. Units are μmol/24 h. Values are given as mean (SEM). *P < 0.05 versus Day 0. View Large Table 3 Urinary excretion of magnesium and proteins by patients treated with cisplatin, carboplatin or oxaliplatin   Before treatment  During (Day 1) and after treatment (Days 2–7)    Day 0  Day 1  Day 2  Day 3  Day 7  Cisplatin ( n = 10)              Magnesium  2.54 (0.47)  2.80 (0.48)  3.69 (0.67)  5.33 (0.65) *  3.55 (0.77)   IgG  0.72 (0.58)  0.88 (0.58)  0.91 (0.44)  1.52 (1.06)  2.90 (1.30)   Transferrin  0.12 (0.08)  0.12 (0.07)  0.15 (0.11)  0.43 (0.24)  1.35 (0.52)   Albumin  2.25 (0.90)  2.54 (0.91)  3.21 (1.36)  5.42 (3.43)  12.8 (5.1)   α1-micro-globulin  1.62 (0.27)  1.97 (0.45)  4.09 (1.12) *  4.88 (0.96) *  3.26 (0.51) *  Oxaliplatin ( n = 8)              Magnesium  2.45 (0.55)  2.58 (0.66)  3.42 (0.62)  3.16 (0.38)  3.05 (0.48)   IgG  0.22 (0.11)  0.38 (0.13)  0.87 (0.15)  0.51 (0.09)  0.34 (0.13)   Transferrin  0.03 (0.02)  0.13 (0.07)  0.33 (0.14)  0.08 (0.05)  0.09 (0.05)   Albumin  0.44 (0.11)  0.66 (0.16)  2.60 (1.08)  1.02 (0.21)  1.19 (0.38)   α1-micro-globulin  1.67 (0.08)  1.95 (0.22)  3.85 (0.77) *  2.60 (0.48) *  1.85 (0.23)  Carboplatin ( n = 4)              Magnesium  4.64 (0.41)  2.93 (0.48)  3.82 (0.79)  3.53 (0.19)  2.84 (0.45)   IgG  1.71 (1.15)  1.23 (0.32)  1.08 (0.55)  0.77 (0.42)  1.47 (0.67)   Transferrin  0.47 (0.06)  0.39 (0.08)  0.50 (0.29)  0.32 (0.16)  0.41 (0.19)   Albumin  8.33 (2.04)  6.38 (1.53)  4.80 (2.45)  4.21 (1.32)  5.62 (2.07)   α1-micro-globulin  2.11 (0.34)  2.01 (0.26)  4.27 (0.91)  4.11 (1.10)  2.76 (0.51)    Before treatment  During (Day 1) and after treatment (Days 2–7)    Day 0  Day 1  Day 2  Day 3  Day 7  Cisplatin ( n = 10)              Magnesium  2.54 (0.47)  2.80 (0.48)  3.69 (0.67)  5.33 (0.65) *  3.55 (0.77)   IgG  0.72 (0.58)  0.88 (0.58)  0.91 (0.44)  1.52 (1.06)  2.90 (1.30)   Transferrin  0.12 (0.08)  0.12 (0.07)  0.15 (0.11)  0.43 (0.24)  1.35 (0.52)   Albumin  2.25 (0.90)  2.54 (0.91)  3.21 (1.36)  5.42 (3.43)  12.8 (5.1)   α1-micro-globulin  1.62 (0.27)  1.97 (0.45)  4.09 (1.12) *  4.88 (0.96) *  3.26 (0.51) *  Oxaliplatin ( n = 8)              Magnesium  2.45 (0.55)  2.58 (0.66)  3.42 (0.62)  3.16 (0.38)  3.05 (0.48)   IgG  0.22 (0.11)  0.38 (0.13)  0.87 (0.15)  0.51 (0.09)  0.34 (0.13)   Transferrin  0.03 (0.02)  0.13 (0.07)  0.33 (0.14)  0.08 (0.05)  0.09 (0.05)   Albumin  0.44 (0.11)  0.66 (0.16)  2.60 (1.08)  1.02 (0.21)  1.19 (0.38)   α1-micro-globulin  1.67 (0.08)  1.95 (0.22)  3.85 (0.77) *  2.60 (0.48) *  1.85 (0.23)  Carboplatin ( n = 4)              Magnesium  4.64 (0.41)  2.93 (0.48)  3.82 (0.79)  3.53 (0.19)  2.84 (0.45)   IgG  1.71 (1.15)  1.23 (0.32)  1.08 (0.55)  0.77 (0.42)  1.47 (0.67)   Transferrin  0.47 (0.06)  0.39 (0.08)  0.50 (0.29)  0.32 (0.16)  0.41 (0.19)   Albumin  8.33 (2.04)  6.38 (1.53)  4.80 (2.45)  4.21 (1.32)  5.62 (2.07)   α1-micro-globulin  2.11 (0.34)  2.01 (0.26)  4.27 (0.91)  4.11 (1.10)  2.76 (0.51)  Magnesium was determined in the 24 h urines and proteins in the second urine sample in the morning using established methods as described in the Method section. Units are mmol/24 h for magnesium and mg × mmol creatinine −1 for proteins. Values are given as mean (SEM). *P < 0.05 versus Day 0. View Large Results We studied 22 patients (6 females and 16 males) with a median age of 60 years (range 38–77) undergoing chemotherapy containing either cisplatin (10 males), oxaliplatin (4 females, 4 males) or carboplatin (2 females, 2 males). The effect of the platin derivatives on the plasma carnitine concentration is shown in Table 1 . The normal values for free carnitine, short-chain acylcarnitine and total carnitine in plasma in our laboratory are ( n = 178 healthy blood donors, mean ± 1.96 × standard deviation) 40.8 ± 27.8, 8.7 ± 16.8 and 54.7 ± 33.4, respectively. Importantly, none of the patients studied had any abnormal carnitine concentration (total, free, short-chain acylcarnitine) at study entry, irrespective of the chemotherapy cycle. During treatment with cisplatin, oxaliplatin or carboplatin, the plasma total carnitine concentration increased by 49%, 85% or 41%, respectively. On the day of chemotherapy (day 1), 6 out of 10 patients treated with cisplatin, 4 out of 8 treated with oxaliplatin and none out of 4 treated with carboplatin had exceeded the upper limit of normal regarding plasma total carnitine. Similar increases were also seen for free carnitine and for the short-chain acylcarnitines. On Day 7, the plasma concentrations of total, free and short-chain acylcarnitine of all patients were again within the limits of normal. The daily excretion of total, free and short-chain acylcarnitines in urine is displayed in Table 2 . The carnitine excretion pattern was qualitatively similar for all platin derivatives. The excretion of the different carnitine fractions started to increase already on Day 1, was highest on Day 2 and had normalized again on Day 7. Considering total carnitine excretion, the maximal increase on Day 2 was 762% for cisplatin, 439% for oxaliplatin and 352% for carboplatin. As shown in Figure 1 , most of this increase can be explained by impaired renal reabsorption of free and short-chain acylcarnitines. Regarding free carnitine, the excretion fraction increased from 2.95% to 18.8% on Day 2 for cisplatin, from 0.98% to 5.1% on Day 2 for carboplatin and from 0.99% to 11.3% on Day 2 for oxaliplatin. On Day 7, the excretion fractions had reached pretreatment values again. For the excretion of short-chain acylcarnitines, a similar pattern as for free carnitine could be observed (see Figure 1 ). In contrast to the renal handling of carnitine, the creatinine clearance was not affected by the treatment with any of the platin derivatives. Overall ( n = 22 patients), the mean (SEM) creatinine clearance was 90.2 (8.8) mL/min before chemotherapy and 103 (12) mL/min on Day 1, 96.2 (10.6) mL/min on Day 2, 93.8 (9.4) mL/min on Day 3 and 92.0 (9.1) mL/min on Day 7. In order to find out possible reasons for the impaired renal handling of carnitine during treatment with platin derivatives, several in vivo and in vitro investigations were performed. In vivo , we determined markers of glomerular (IgG, transferrin, albumin) and of tubular damage (α1-microglobulin, β2-microglobulin, retinol-binding protein and magnesium) in urine. As shown in Table 3 , the glomerular markers showed no significant change during treatment with platin derivatives. In contrast, the urinary excretion of α1-microglobulin increased on Days 2 or 3 after chemotherapy for all platin derivatives (reaching statistical significance for cisplatin and oxaliplatin) and had clearly decreased or normalized on Day 7. Similarly, urinary excretion of β2-microglobulin and retinol-binding protein were increased in most patients at Days 2 and 3 after chemotherapy and showed a clear decrease on Day 7 (data not shown). The excretion of magnesium showed a similar pattern, reaching statistical significance on Day 3 for cisplatin. These data suggested an effect of platin derivatives on tubular function, including the proximal tubule as shown in previous studies [ 5,6 ]. In order to find out the reasons for the toxicity of the platin derivatives on the proximal tubule, we next studied carnitine transport into L6 cells overexpessing OCTN2. As shown in Figure 2 , neither cisplatin nor oxaliplatin or platinum(II) chloride inhibited sodium-dependent transport of 3 H- l -carnitine into L6 cells, suggesting that these platin derivatives or ions do not inhibit hOCTN2. The cisplatin concentrations used (1–100 mM) are in the range that is achieved after infusion of 80 mg cisplatin per m 2 [ 21 ]. Cisplatin ( cis -PtCl 2 (NH 3 ) 2 ) can be hydrolyzed in aqueous solutions to cis -PtCl(NH 3 ) 2 (OH 2 ) + and cis -Pt(NH 3 ) 2 (OH 2 ) 22+ [ 22 ]. These hydrolization products of cisplatin are considered to be active metabolites of the drug [ 23 ] and may be able to inhibit OCTN2. To test this hypothesis, cisplatin stock solutions were prepared up to 48 h in advance in water and the inhibition experiments repeated. The result was identical to that shown in Figure 2 (not shown), excluding this possibility. In order to test the hypothesis that cisplatin has to enter the cells before inhibiting OCTN2, we exposed the cells up to 24 h with cisplatin and the other platin derivatives and repeated the experiment as described in Figure 2 . Again, we found no impairment of sodium-dependent transport of 3 H- l -carnitine under these conditions (results not shown). Since most cisplatin is metabolized in primates and metabolites are excreted in the urine [ 24 ], it could be possible that such metabolites inhibit OCTN2. To test this hypothesis, we assessed inhibition of OCTN2 directly by urine of patients treated with cisplatin. As shown in Figure 3 , urine inhibited OCTN2 activity significantly, but both before (Day 0) and after treatment with cisplatin (Days 1 and 2). A comparison with the inhibition of the assay by carnitine solutions revealed that the inhibition by urine could be explained sufficiently by the carnitine in the urine. The hypothesis that cisplatin metabolites in the urine inhibit OCTN2 could therefore also be excluded. Finally, cisplatin could decrease the expression of OCTN2. In order to test this hypothesis, we used 293-EBNA cells, a human embryonic kidney cell line expressing OCTN2. Cytotoxicity experiments showed no toxicity for cisplatin up to 100 μM for 48 h. As shown in Figure 3 , exposure of 293-EBNA cells to different subtoxic concentrations of cisplatin revealed a concentration-dependent down-regulation of hOCTN2 mRNA after an incubation period of 8, 24 and 48 h. Similarly, OCTN2 protein expression was also decreased after 12 and 24 h of incubation with cisplatin. Discussion Our study demonstrates that all platin derivatives tested, namely cisplatin, carboplatin and oxaliplatin, are associated with increased renal excretion of free and short-chain acylcarnitines due to a rise in their renal excretion fraction. Similar results have been previously reported for cisplatin [ 5 ] and carboplatin [ 6 ], but so far not for oxaliplatin. Increased filtration of total carnitine could not sufficiently explain the observed increase in urinary excretion, since the plasma values increased only by a factor of ∼1.5, but urinary excretion by a factor of 4–10. The effect on renal carnitine excretion was qualitatively similar for all three platin derivatives investigated, but most accentuated for cisplatin. The renal excretion of carnitine started to increase already on the day of drug administration, reached a maximum on the next day and then started to decrease with a complete normalization 1 week after the administration of the platin derivative. Although the effect on renal carnitine reabsorption was quite dramatic, none of the patients developed carnitine plasma concentrations below our normal values. It is possible, however, that specific patients may develop plasma carnitine concentrations below normal during repetitive cycles of platin-based chemotherapies, in particular those with a low oral intake of carnitine [ 25 ]. Carnitine plasma levels may therefore be determined in patients having been treated with platin-based chemotherapies who develop signs of carnitine deficiency such as fatigue, myopathy and/or encephalopathy. In symptomatic patients with decreased plasma carnitine concentrations, treatment with carnitine could possibly be beneficial. The observed increase in the plasma carnitine concentration, which occurs despite an increased renal excretion fraction of carnitine, is difficult to explain. Since the maximal transport capacity of the renal reabsorption of free carnitine is reached at ∼60 μmol/L [ 26 ], it should not have been reached by most of our patients. Alternatively, cisplatin may interact with tissue distribution of carnitine and acylcarnitines, but the transport processes of carnitine into tissues and out of tissues are, with the exception of the kidney, currently not well described. Renal carnitine reabsorption is accomplished by OCTN2, a sodium-dependent carnitine carrier expressed in the proximal tubules [ 11 , 13 ]. In our patients, a proximal tubular damage was not only shown by the increased excretion of carnitine, but also by the increase in the excretion of α1-microglobulin, β2-microglobulin and retinol-binding protein [ 27,28 ]. As shown by the renal protein excretion pattern and the creatinine clearance, the tubular damage developed without a significant glomerular damage for all three substances tested. Any mechanism leading to the observed renal damage has to be compatible with the observed time sequence (rapid appearance and recovery) and the localization of the damage in the proximal tubule. The rapid appearance of the damage and complete normalization of the proximal tubular function within 1 week favour a functional as opposed to a structural defect. In contrast, repetitive and high doses of cisplatin have been shown to be associated with proximal tubular necrosis and possibly fibrosis both in animals and in humans [ 29,30 ]. In such patients, recovery was slow and mostly not complete [ 2 ], findings not compatible with the situation observed in our patients. In order to find out possible mechanisms, we performed several studies in L6 cells overexpressing OCTN2 as well as in 293-EBNA cells, a human embryonic kidney cell line. Our studies show that cisplatin and oxaliplatin do not directly inhibit OCTN2, findings which are in agreement with reports in the literature [ 31 ]. In the report of Yonezawa et al. [ 31 ], neither cisplatin nor carboplatin or oxaliplatin inhibited OCTN2. Since cisplatin can be hydrolyzed under conditions of low chloride concentrations [ 22,23 ], we also excluded the possibility that the hydrolyzed forms inhibit OCTN2. Longer periods of preincubation were also not associated with impaired function of OCTN2, suggesting that cisplatin inhibits OCTN2 neither from outside, nor from inside of the cells. Cisplatin is heavily metabolized, <50% of the drug is excreted non-metabolized in the urine [ 3 ], suggesting that a metabolite could be responsible for its toxicity. As already mentioned above, cisplatin can be hydrolized in aqueous solutions with a low chloride concentration [ 22,23 ]. Furthermore, cisplatin can react with nucleophiles such as mercapto groups [ 2 , 23 ]. For instance, cisplatin can form a complex with metallothionein [ 23 ], which can be excreted in the urine by glomerular filtration. Alpha1-microglobulin, whose urinary excretion increased during chemotherapy with platin derivatives, is also cleared by glomerular filtration but can be reabsorbed by binding to megalin in proximal tubular cells [ 32 ]. Megalin is a 600 kDa transmembrane protein belonging to the low-density lipoprotein receptor family with a high expression in proximal tubular cells. It can be regarded as a multiligand endocytic receptor, which is important for the proximal tubular reabsorption of proteins such as α1-microglobulin [ 32 ]. Since metallothionein can also bind to megalin and thereby compete with other ligands [ 33 ], this interaction is a likely explanation for the increased renal excretion of α1-microglobulin in patients treated with cisplatin and other platin derivatives. Such a mechanism is not associated with cell damage and therefore fits our clinical observations. In order to investigate the possibility that metabolites could inhibit OCTN2, we performed incubations with urine from patients treated with cisplatin. Considering the interpretation of these incubations, it is important to realize that the carnitine contained in the urine is leading to tracer dilution, suggesting inhibition of carnitine transport. Taking into account this apparent inhibitory effect, the results obtained in these experiments did not show inhibition of OCTN2. Regarding the mechanism discussed for α1-microglobulin excretion, it can be argued that active metabolites may have been retained in the proximal tubules. This possibility cannot be excluded by our investigations. Cisplatin has been shown to decrease mRNA expression of PPARα [ 34 ], possibly mediated by TNF-α [ 35,36 ]. Since activation of PPARα has been shown to induce mRNA expression of OCTN2 [ 37 ], we speculated that cisplatin could decrease OCTN2 expression. As shown in Figure 4 , this is indeed the case in 293-EBNA cells exposed to cisplatin. Similar to mRNA expression, OCTN2 protein expression was also decreased, further supporting our hypothesis. In conclusion, cisplatin, carboplatin and oxaliplatin are associated with renal tubular damage in humans without significantly affecting glomerular function. The rapid onset and complete reversibility of this effect favour a functional mechanism such as impaired expression of OCTN2 in proximal tubular cells. The study was supported by a grant from the Swiss National Science Foundation to SK (310000-112483). Conflict of interest statement. None declared. 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TI - Urinary excretion of carnitine as a marker of proximal tubular damage associated with platin-based antineoplastic drugs
JF - Nephrology Dialysis Transplantation
DO - 10.1093/ndt/gfp456
DA - 2009-09-07
UR - https://www.deepdyve.com/lp/oxford-university-press/urinary-excretion-of-carnitine-as-a-marker-of-proximal-tubular-damage-MyYWz0GJUj
SP - 426
EP - 433
VL - 25
IS - 2
DP - DeepDyve
ER -