Magnesium attenuates cisplatin-induced nephrotoxicity by regulating the expression of renal transporters

Author’s Accepted Manuscript Magnesium attenuates cisplatin-induced nephrotoxicity by regulating the expression of renal transporters Yoshitaka Saito, Keisuke Okamoto, Masaki Kobayashi, Katsuya Narumi, Takehiro Yamada, Ken Iseki www.elsevier.com/locate/ejphar

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S0014-2999(17)30365-5 http://dx.doi.org/10.1016/j.ejphar.2017.05.034 EJP71224

To appear in: European Journal of Pharmacology Received date: 25 January 2017 Revised date: 7 May 2017 Accepted date: 17 May 2017 Cite this article as: Yoshitaka Saito, Keisuke Okamoto, Masaki Kobayashi, Katsuya Narumi, Takehiro Yamada and Ken Iseki, Magnesium attenuates cisplatin-induced nephrotoxicity by regulating the expression of renal t r a n s p o r t e r s , European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2017.05.034 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Research Paper Magnesium attenuates cisplatin-induced nephrotoxicity by regulating the expression of renal transporters

Yoshitaka Saito1, 3, Keisuke Okamoto2, 3, Masaki Kobayashi*, 1, Katsuya Narumi2, Takehiro Yamada1, Ken Iseki*, 1, 2

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Department of Pharmacy, Hokkaido University Hospital: Kita 14-jo, Nishi 5-chome,

Kita-ku, Sapporo 060-8648, Japan 2

Laboratory of Clinical Pharmaceutics & Therapeutics, Faculty of Pharmaceutical

Sciences, Hokkaido University: Kita 12-jo, Nishi 6-chome, Kita-ku, Sapporo 060-0812, Japan 3

These authors contributed equally as co-first authors.

*, To whom correspondence should be addressed. Laboratory of Clinical Pharmaceutics & Therapeutics, Division of Pharmasciences, Faculty of Pharmaceutical Sciences, Hokkaido University Tel/Fax: +81-11-706-3772/3235 and +81-11-706-3770; E-mail: [email protected] and [email protected]

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ABSTRACT Cisplatin (CDDP)-induced nephrotoxicity (CIN) is one of the most serious toxicities caused by this potent antitumor agent. It has been reported that Mg premedication attenuates CIN in clinical trials; however, the mechanism underlying its nephroprotection is not fully understood. Therefore, the aim of this study was to determine whether Mg administration affects CDDP accumulation by regulating the expression level of renal transporters. Rats were divided into control, Mg (40 mg/kg) alone, 2.5 mg/kg CDDP with (20 and 40 mg/kg) and without Mg, 5 mg/kg CDDP groups. These substances were administered on the same day and 7 days later their kidneys were removed. The expression levels of renal transporters and platinum (Pt) accumulation were analyzed. The serum creatinine level was significantly increased by CDDP administration and treatment with Mg significantly ameliorated such elevation. The expressions of the renal organic cation transporter 2 (rOct2) and renal multidrug and toxin extrusion protein 1 (rMate1) were downregulated and upregulated, respectively following co-administration with Mg, which significantly reduced the renal Pt accumulation in the 2.5 mg/kg CDDP-treated group. Moreover, Mg dose-dependently downregulated rOct2, not affecting rMate expression, resulting in the attenuation of CIN. Mg co-administration protected the downregulation of the transient receptor

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potential subfamily Melastatin 6 (rTrpm6), but not the epidermal growth factor (rEgf), as a result, Mg co-injection attenuated CDDP-induced hypomagnesemia. In conclusion, Mg co-administration reduced Pt accumulation by regulating the expression of the renal transporters, rOct2 and rMate1 and, thereby, attenuated CIN.

Keywords: Cisplatin; Nephrotoxicity; Magnesium; rOct2; rMate1; Drug Transport Across Membranes

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1. Introduction Nephrotoxicity is one of the major, most serious, and dose-limiting toxicities caused by cisplatin (cis-dichloro-diammine platinum, CDDP), which is a widely used chemotherapeutic agent for the lung, gastric, head and neck, ovarian, and urological malignancies (Go and Adjei. 1999). CDDP-induced nephrotoxicity (CIN) is recognized to be cumulative, dose-related, and usually reversible, occurring in 30–40% of patients administered with CDDP (Pabla and Dong. 2008; Miller et al. 2010; Yoshida et al. 2014). CIN has been suggested to especially affect the S3 segment of the proximal tubule located in the outer medulla, and the thick ascending limb of the loop of Henle (Dobyan et al. 1980). The mechanism of CIN induction is thought to be DNA damage, oxidative stress, mitochondrial dysfunction, inhibition of protein synthesis, and increased tumor necrosis factor (TNF) family (Tsuruya et al. 2003; Brady et al. 1990; Park et al. 2002). Hypomagnesemia has been reported to occur in approximately 90% of the patients administered with CDDP (Lajer and Daugaard. 1999), and damage to the renal tubular cells and Ca2+/Mg2+ sensing receptor causes CDDP-induced hypomagnesemia and hypocalcemia (Lajer and Daugaard. 1999; Vickers et al. 2004). It has also been reported that the transient receptor potential subfamily Melastatin 6 (rTrpm6), which is present

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on the distal convoluted tubule (DCT) and the epidermal growth factor (rEgf) interact and have important roles in Mg reabsorption in the DCT (Ledeganck et al. 2013). Furthermore, renal mRNA expression of rTrpm6 and rEgf significantly decreased after 2.5 mg/kg CDDP administration, leading to increased Mg excretion (Ledeganck et al. 2013). Mg is the second most common intracellular cation in the human body (Tong and Rude. 2005) and acts as a cofactor for approximately 300 cellular enzymes that modulate cellular energy metabolism involving adenosine triphosphate (ATP), muscle Na+/K+-pump activity, calcium channel activity, and stabilization of membrane structures, as well as RNA/DNA polymerase, which is responsible for mRNA translation, transcription, and replication of DNA (Wacker and Parisi. 1968; Dørup et al. 1988; Reinhart. 1991; Mildvan and Loeb. 1979). Yokoo et al. reported that the organic cation transporter 2 (rOct2), which is predominantly expressed in the basolateral membranes of proximal tubules and is responsible for CDDP accumulation into the S3 segment, is expressed Mg-dependently and hypomagnesemia causes upregulation of rOct2 (Yokoo et al. 2009). They also suggested that the expression level of multidrug and toxin extrusion protein 1 (rMate1), which transports CDDP from the proximal tubule into the urine, tended to decrease in hypomagnesemic rats (Yokoo et al. 2009).

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Clinically, Willox et al. and Bodnar et al. reported that oral and intravenous Mg supplementation ameliorates CDDP-induced serum creatinine or urinary N-acetyl-β-D-glucosaminidase (NAG) elevation (Willox et al. 1986; Bodnar et al. 2008). We have also reported that intravenous Mg premedication alone prevents the incidence of CIN and ameliorates its severity (Saito et al. 2017). However, the mechanism underlying renal protection by Mg co-administration with CDDP is not fully understood. Therefore, this present study was conducted to determine how Mg co-administration attenuates CIN, focusing on the renal transporter expression level.

2. Materials and Methods 2.1. Chemicals CDDP was purchased from Sigma-Aldrich Chemical Corp., (St. Louis, USA). The rOct2- and rMate1-specific primary antibodies were from Santa Cruz Biotechnology (Santa Cruz, USA), rTrpm6 was from GeneTex (California, USA), and rActin was from Merck Millipore (Billerica, USA). Creatinine was measured using a kit (L type Wako creatinine F or CRE･M) purchased from Wako (Osaka, Japan). Serum Mg was determined using a Metallo Assay Mg LS kit from Metallogenics (Chiba, Japan). All other chemicals and reagents were commercially available and of the highest purity

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possible. 2.2. Animals Male Wistar rats (7-weeks-old) weighing 210–230 g were obtained from JLA (Tokyo, Japan). All animals were housed in a standard animal maintenance facility in a temperature (23°C) and moisture (60 ± 10%) controlled room with a 12-h light-dark cycle. All rats were allowed free access to demineralized water and diet pellets. All animal experiments were conducted according to the guidelines for the Care and Use of Laboratory Animals of Hokkaido University. 2.3. Experimental design CDDP 2.5 or 5 mg/kg (1 or 2 mg/ml, respectively) or saline was administered intraperitoneally as a therapeutic agent while Mg sulfate 20 mg/kg (8 mg/ml) or 40 mg/kg (16 mg/ml), or saline was administered intraperitoneally as a prophylactic agent 4 h prior to treatment with the therapeutic agent. The interval between the administration of the therapeutic and prophylactic agents was set at 4 h, which was the shortest time during which the prophylactic agent did not affect the blood levels of the therapeutic agent in our preliminary study. Thirty-six rats were divided into six groups and treated as follows: (1) Control group, saline prophylactically and therapeutically.

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(2) Mg group, 40 mg/kg Mg prophylactically and saline therapeutically. (3) 2.5 mg/kg CDDP group, saline prophylactically and 2.5 mg/kg CDDP therapeutically. (4) 2.5 mg/kg CDDP-Mg group, 40 mg/kg Mg prophylactically and 2.5 mg/kg CDDP therapeutically. (5) 5 mg/kg CDDP group, saline prophylactically and 5 mg/kg CDDP therapeutically. (6) 2.5 mg/kg CDDP-half Mg group, 20 mg/kg Mg prophylactically and 2.5 mg/kg CDDP therapeutically. Blood samples were obtained from the right jugular vein at baseline (before prophylactic agent administration) and 120 and 168 h after the therapeutic agent was injected. The body weight of the animals was measured following the same timing as the blood sampling. After blood collection at 168 h, the rats were anesthetized with ether, euthanized, and the kidneys were collected immediately and the tissue was stored with the serum samples at -80°C until analyzed. 2.4. Western blot analysis The expression levels of rOct2, rMate1 and rTrpm6 were assessed using western blot analysis. The cortex from the kidney was sliced and total protein extracts were prepared by homogenizing in lysis buffer containing 1.0% Triton X-100, 0.1% sodium dodecyl

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sulfate (SDS), and 4.5 M urea. The homogenate was sonicated for 15 min at 4°C, centrifuged at 12100 × g for 15 min at 4°C, and then the samples were denatured at 100°C for 3 min in a loading buffer containing 0.1 M Tris-hydrochloride, 4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue (BPB). Then, the samples were separated using 10% for rOct2 and rMate1, 7.5% for rTrpm6 SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene difluoride membranes (Bio-Rad, Tokyo, Japan) by semidry electroblotting at 15 V for 90 min. The membranes were blocked with phosphate-buffered saline (PBS) containing 0.05% Tween 20 (PBS/T) and 1% non-fat dry milk for 1 h at room temperature. After washing with PBS/T, the membranes were incubated overnight with primary antibodies against rOct2 (1:200), rMate1 (1:100), and rTrpm6 (1:200), washed thrice with PBS/T for 10 min each time, incubated for 1 h with a goat anti-rabbit IgG-horseradish peroxidase (HRP) secondary antibody (Santa Cruz Biotechnology, Santa Cruz, USA) against rOct2, rMate1 and rTrpm6 (1:2000,1:200 and 1:2000, respectively), and then washed thrice with PBS/T for 10 min each time. After washing with PBS/T, the bound antibodies were detected using the Image Quant LAS 4000 (GE Healthcare UK Ltd., Amersham Place, UK) by enhanced chemiluminescence. The protein concentration in the clear supernatant was determined using the method of Lowry et al. (Lowry et al. 1951).

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2.5. Isolation of total RNA and reverse transcription-polymerase chain reaction (RT-PCR) analysis Total RNA was extracted from the homogenized kidney tissue using an ISOGEN II (Nippon Gene, Tokyo, Japan) kit according to the manufacturer’s protocol. RNA concentration was determined by measuring the absorbance at a wavelength of 260 nm. One microgram of total RNA was used to prepare complementary DNA by reverse transcription using ReverTra Ace (Toyobo, Osaka, Japan). The levels of mRNAs for tissue Inhibitor of Metalloproteinase-1 (rTimp-1), rTrpm6 and rEgf were measured using real-time polymerase chain reaction (qPCR). Table 1 shows the primer sequences used for the PCR amplification and the products were normalized to amplified rActin, the internal reference gene. Standard curves were constructed for each target and housekeeping gene, as well as between the threshold cycles (Ct) and the log 10 (copy numbers) using the Applied Biosystems (Agilent Technologies, Santa Clara, USA) sequence detection system software, version 1.9.1. The software calculates the relative amount of the target gene and the housekeeping gene based on the Ct. 2.6. Pt accumulation in kidney The platinum (Pt) accumulated in the kidney was measured using inductively coupled plasma-mass spectrometry (ICP-MS) at the Creative Research Institution of Hokkaido

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University (Adhim et al. 2012). Briefly, each sample (0.1 ml) was reduced to ash by repeated treatment with nitric acid (for poisonous metal determination, Wako, Osaka, Japan), hydrogen peroxide (for atomic absorption spectrochemical analysis, Wako, Osaka, Japan), and perchloric acid (for poisonous metal determination, Wako, Osaka, Japan) under heating at 200 °C. The sample ash was dissolved in 5 ml of 60% nitric acid and then analyzed by ICP-MS using a Shimadzu ICPE-9000E (Shimadzu, Kyoto, Japan). Pt levels (m/z 195) in the three representative samples were measured. Contamination from tubes and other potential sources of Pt was carefully avoided. The concentration of Pt in each sample was calculated using linear regression of a Pt standard curve prepared using a Pt standard solution. The Pt standard curve exhibited linear regression in the range of 1–10000 µg/L (r = 0.9999). 2.7. Cell culture The human lung adenocarcinoma A549 cell line (American Type Culture Collection, ATCC, Manassas, USA) was cultured in low glucose Dulbecco’s modified Eagle’s medium (DMEM, Wako, Osaka, Japan) supplemented with 10% fetal calf serum (Biocera, Seongnam, Korea) in a humidified atmosphere of 5% CO2 at 37°C. 2.8. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay The cells were separately seeded in 96-well plates at a density of 8,000 cells/well in

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the culture medium. After their adherence to the culture dish, the cells were incubated with specific mixtures of 2 or 8 μg/ml of CDDP and control (saline) or 1.25 or 2.5 mM Mg sulfate for 48 h. Then, the cells were cultured for an additional 48 h and cell survival was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay according to the manufacturer’s instructions. Absorbance was measured for the resulting reaction solution at 535 nm for samples and at 650 nm for the reference wavelength by using a spectrophotometer. 2.9. Statistical analysis The data was analyzed statistically using an unpaired Student’s t-test or ANOVA followed by Tukey’s or Dunnett’s post-hoc test. Differences were considered statistically significant at P < 0.05.

3. Results 3.1. Body weight change The body weight changes of the various groups are shown in Table 2. All the CDDP-treated rats showed a body weight increase that was significantly lower than that of the control rats was. The difference was more significant in 5 mg/kg CDDP group than it was in 2.5 mg/kg CDDP group on day 6, and the difference persisted until

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day 8. Furthermore, the Mg administration did not affect the body weight increase regardless of the CDDP dose. 3.2. Change in serum creatinine and Mg level, renal rTimp-1 mRNA level The variation in the serum creatinine level is shown in Table 3, which reveals that levels of the 2.5- and 5 mg/kg CDDP groups were significantly higher than that of the control group was on day 6, and were maintained until day 8. Renal mRNA level of rTimp-1on day 8 in 2.5- and 5 mg/kg CDDP groups was significantly higher than that in the control group (Table 3). Furthermore, the serum creatinine increase was CDDP-dose dependently enhanced. On the other hand, the elevation of the serum creatinine level on day 8 and renal rTimp-1 mRNA expression was significantly attenuated in the 2.5 mg/kg CDDP-Mg group compared with the 2.5 mg/kg CDDP group. The change in the serum Mg level is also shown in Table 3, and the level was more significantly decreased in the 2.5 and 5 mg/kg CDDP groups than it was in the control group, while Mg co-administration with 2.5 mg/kg CDDP significantly improved the CDDP-induced serum Mg depletion on days 6 and 8. 3.3. Comparison of renal transporter expression level The expression level of rOct2 and rMate1 on day 8 is shown in Fig. 1. The rats in the

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2.5 mg/kg CDDP group showed a significant increase in the expression level of renal rOct2 compared with the control group. On the other hand, compared to the control group, the rOct2 level was significantly downregulated by up to 50% in the Mg and 2.5 mg/kg CDDP-Mg groups. The rOct2 expression level was significantly lower in the 5 mg/kg CDDP group than it was in the 2.5 mg/kg CDDP group. Furthermore, the rMate1 expression level of the 2.5 mg/kg CDDP-Mg-treated rats was significantly higher than that of rats in the control and 2.5 mg/kg CDDP groups. 3.4. Comparison of renal Pt accumulation The Pt accumulation in the kidney was compared between the group administered 2.5 mg/kg CDDP alone and those treated with 2.5 mg/kg CDDP combined with Mg. The renal accumulation of Pt in the 2.5 mg/kg CDDP-Mg group was significantly lower than that in the 2.5 mg/kg CDDP group was (Fig. 2). 3.5.Involvement of Mg dose against CIN We also examined the nephroprotective effect of 20 mg/kg Mg co-administration with 2.5 mg/kg CDDP. Serum creatinine elevation was not significantly reduced by 20 mg/kg co-administration (Fig. 3A). The treatment did significantly downregulate the elevated renal rOct2 expression level by 2.5 mg/kg CDDP and upregulated the renal rMate1 expression level compared to that in the 2.5 mg/kg CDDP group (Fig. 3B, 3C).

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Moreover, 40 mg/kg Mg significantly downregulated rOct2 expression level compared to the effect observed with 20 mg/kg Mg whereas the dosage of Mg did not affect the rMate1 expression. Pt accumulation was significantly reduced by only 40 mg/kg Mg co-administration (Fig. 3D). 3.6. Renal mRNA expression of rTrpm6 and rEgf, protein expression of rTrpm6 The renal mRNA expression level of rTrpm6 and rEgf is shown in Fig. 4. Compared to the control group, the renal mRNA expression of rTrpm6 was significantly decreased in all the groups except the 2.5 mg/kg CDDP-Mg group (Fig. 4A). The rEgf expression level was significantly decreased in all groups compared to the control group (Fig. 4B). Mg administration alone reduced the renal rEgf mRNA level; however, CDDP administration consistently decreased the mRNA level of rEgf with or without Mg co-administration. On the other hand, the expression level of rTrpm6 was significantly downregulated by CDDP and Mg administration (Fig. 4C).

4. Discussion Nephrotoxicity is one of the most limiting adverse effects of CDDP, and it is necessary to manage CIN to enhance the safety and efficacy of CDDP-containing chemotherapy regimens. In addition, it is important to develop new prophylactic

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strategies for preventing and treating CIN, and to determine their mechanisms of action. Therefore, in this study, we evaluated the nephroprotective effect of Mg co-administration with CDDP and its mechanism against CIN, focusing on possible effects on the expression of renal transporters. It has been reported that Mg co-administration with CDDP significantly ameliorates the incidence and severity of CIN (Yoshida et al. 2014; Willox et al. 1986; Bodnar et al. 2008; Saito et al. 2017). In this study, the serum creatinine increase was CDDP-dose related, and Mg co-injection significantly attenuated the elevation of serum creatinine level and renal rTimp-1 mRNA expression induced by administration of 2.5 mg/kg CDDP. As previously mentioned, Pt renal pharmacokinetics is mainly modulated by rOct2 and rMate1 (Ludwig et al. 2004; Nakamura et al. 2010; Yonezawa and Inui. 2011). It has been suggested that Mg depletion enhances Pt accumulation, while Mg replacement attenuates the augmented Pt accumulation induced by Mg deficiency (Solanki et al. 2014). However, the study did not address additional Mg co-administration against CIN in a baseline situation. It has also been shown that CDDP administration regulates the expression level of rOct2 and rMate1, which are responsible for CDDP accumulation in the kidney (Yokoo et al. 2009; Solanki et al. 2014; Erman et al. 2014; Morisaki et al. 2008). There are two possible pathways for

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the variation in renal rOct2 and rMate1 expression levels. CDDP may have directly changed the expression of these renal transporters. Alternatively, CDDP may have decreased Mg re-absorption by downregulating the rEgf-rTrpm6 pathway, resulting in the reduction of serum Mg level, and subsequently, the lack of serum Mg induced the variation in the expression of these renal transporters. Our results are the first to show that the expression levels of rOct2 and rMate1 are down- and upregulated, respectively by additional Mg co-administration with 2.5 mg/kg CDDP, which is close to the clinical dose with decreased renal Pt accumulation. There was a significant decrease in the expression level of rOct2 to about 40% and an increase in rMate1 by 1.7-fold in the 2.5 mg/kg CDDP-Mg group compared to 2.5 mg/kg CDDP group. Although the Pt accumulation showed a difference of only 15% between both groups, this difference may have been sufficient to generate significant renal damage. We also speculated that the transport activity of these transporters might be changed by Mg co-administration with CDDP and, therefore, the Pt accumulation is possibly compensatory. Alternatively, another transport mechanism such as the copper transporter 1 (rCtr1), which has been reported to transport CDDP and to be a channel-like transporter (Yonezawa and Inui, 2011), might be involved, resulting in only a 15% difference in Pt accumulation following the 2.5 mg/kg CDDP injection. On the other hand, some reports suggest the

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non-nephroprotective effect of Mg against CIN (Ashrafi et al. 2012; Soltani et al. 2013). However, these results are inconsistent with the clinical results, therefore, it was speculated that administration methods of CDDP and Mg have affected the contradictory findings. We also examined the dose-dependent nephroprotective effect of Mg using 2.5 mg/kg CDDP. There was no reports of Mg intravenous co-administration in the CDDP-administered rats. In our clinical trials, 20 mEq MgSO4 premedication has attenuated CIN, and the mean body weight of the patients was 58 kg (Saito et al. 2017), we calculated the dose of MgSO4 for 40 mg/kg. Serum creatinine elevation was significantly reduced by only 40 mg/kg Mg co-administration. The treatment in both dose did significantly downregulate the elevated renal rOct2 expression level by 2.5 mg/kg CDDP and upregulated the renal rMate1 expression level compared to that in the 2.5 mg/kg CDDP group. Moreover, Mg dose-dependently significantly downregulated rOct2 expression level and reduced the renal Pt accumulation. From these results, it appears that the renal protective effect of Mg could be dose-related and that the nephroprotective mechanism might be the changes in renal rOct2 expression level. Solanki et al. also reported that mice fed a Mg-deficient diet developed renal oxidative stress, which was enhanced by CDDP administration but completely

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inhibited by Mg supplementation (Solanki et al. 2014). It has been reported that Mg possesses antioxidant properties and scavenges oxygen radicals, possibly by affecting the rate of spontaneous dismutation of the superoxide ion (Afanasʹ ev et al. 1995). It known that one of the mechanism of CIN could be oxidative stress, is necessary to determine whether Mg directly relieves the oxidative stress caused by CDDP administration. Furthermore, CDDP is usually administered repeatedly clinically, and it is known that CIN occurs during the first course of CDDP-containing chemotherapy and increases in severity over the course of treatment (Yoshida et al. 2014; Saito et al. 2017). Therefore, it is necessary to investigate how Mg protects against CIN following CDDP multiple administration, as well as its effects on the expression and transport activity of renal transporters, especially rOct2 and rMate1. Ledeganck et al. and Magil et al. have reported that a weekly intraperitoneal injection of 2.5 mg/kg CDDP for three weeks is well tolerated and causes CIN (Ledeganck et al. 2013; Magil et al. 1986). In future studies, we will examine the influence of Mg co-administration on CIN in the same protocol. Further studies are needed to elucidate the mechanisms underlying the effect of Mg on the expression of rOct2 and rMate1, as well as the effects of Mg on their transport activity or oxidative stress under similar clinical conditions.

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Interestingly, this study also revealed that 2.5 mg/kg CDDP administration increased the expression level of rOct2 while 5 mg/kg did not change its expression. In this study, we have adopted the CDDP dose for 2.5 mg/kg (Pinches et al. 2012; Wadey et al. 2014), since this dose of CDDP has been reported to be well tolerated in the multiple administration (Ledeganck et al. 2013; Magil et al. 1986). As expected, 5 mg/kg CDDP administration induced more serious nephrotoxicity than the 2.5 mg/kg CDDP dose did. In previous reports, the rOct2 expression level was significantly downregulated by CDDP administration (Erman et al. 2014; Morisaki et al. 2008; Ulu et al. 2012). The doses of CDDP in these reports were higher than those used in this study were, especially the 2.5 mg/kg. Therefore, it is possible to speculate that the expression level of rOct2, which is critically responsible for CDDP uptake into the proximal tubule, might have been downregulated, which led to the reduction in renal Pt accumulation since higher CDDP doses were administered in their studies. On the other hand, it has been reported that the expression level of rMate1 is downregulated by CDDP administration (Yokoo et al. 2009; Morisaki et al. 2008). In our study, the rMate1 expression level tended to decrease after CDDP injection at both doses; this finding was similar to that in previous reports. The rOct2 expression level shown in this study

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suggests that the dose of CDDP could be a critical factor in the regulation of transporter expression level. It has been shown that rEgf stimulates Mg reabsorption in the DCT via its receptor present on the basolateral membrane and activation of rTrpm6 in the apical membrane, by influencing its mRNA synthesis (Ledeganck et al. 2011, 2013). In this study, it was revealed that Mg administration alone downregulated the expression levels of rTrpm6, and mRNA levels of rTrpm6 and rEgf. This effect could be negative feedback caused by the temporary elevation of serum Mg levels to maintain homeostasis. It was also considered that additional Mg loading compensated for the Mg loss caused by downregulation of rTrpm6 expression, resulting in conservation of the serum Mg level in the Mg group. CDDP administration significantly downregulated rTrpm6 expression, this result might have induced the CDDP-induced serum Mg loss. It was suggested that Mg co-administration protects the downregulation of rTrpm6 mRNA level but not rEgf level following administration of 2.5 mg/kg CDDP. However, Mg administration significantly downregulated the rTrpm6 expression level regardless of the CDDP injection. Whereas, it was also shown that the serum Mg level of the group administered the clinically relevant 2.5 mg/kg CDDP dose, was significantly decreased, and this reduction was improved by Mg co-administration. We proposed the

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following hypotheses for this result: 1) replacement action by Mg administration, 2) CIN was attenuated by Mg co-administration, which ameliorated the downregulation of Mg reabsorption pathways such as claudin-16 (Hou et al. 2010) involved in the prevention of CDDP-induced Mg loss. Moreover, the effect of combination treatment with Mg on CDDP antitumor efficacy was assessed. We used A549 cells to evaluate the antitumor effect on lung adenocarcinoma, for which CDDP is one of the most important treatments. The MTT cell viability assay showed that CDDP dose-dependently depleted the viable cells, and Mg co-incubation did not affect the survival of A549 cell (Supplemental Fig. 1). This result was consistent with those observed in previous clinical trials (Bodnar et al. 2008; Saito et al. 2017).

5. Conclusion We demonstrated the renal protective effect of Mg co-administration against CIN. Its renoprotective effect was mediated by regulation of the expression of the renal transporters, rOct2 and rMate1, resulting in the reduction of renal Pt accumulation. These findings could lead further strategies such as the combination of Mg and other substances which have other nephroprotective mechanisms. This present study also

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suggested that Mg nephroprotective effect could be Mg-dose related, suggesting the necessity of the clinical trial to elucidate the most appropriate Mg dose for prevention of CIN. Further studies are needed to investigate the preventive mechanism of Mg co-administration with multiple doses of CDDP which is close to clinical situation.

Conflict of interest statement None declared. Authorship Contributions Participated in research design: Y.S., K.O., M.K., K.N. and K.I. Conducted experiments: Y.S., K.O. and M.K. Contributed new reagents or analytic tools: Y.S., K.O., M.K., K.N., T.Y. and K.I. Performed data analysis: Y.S., K.O., M.K. and K.I. Wrote or contributed to the writing of the manuscript: Y.S., K.O., M.K., K.N., T.Y. and K.I.

Acknowledgments This work was supported by JSPS KAKENHI Grant Number 26926007.

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Table titles

Table 1. Primer sequences of rat tissue inhibitor of metalloproteinase-1 (rTimp-1), rat transient receptor potential subfamily Melastatin 6 (rTrpm6) and rat epidermal growth factor (rEgf)

Genes

Primer sequence

Timp-1

Forward: 5ʹ-TTGCTTGTGGACAGATCAGA-3ʹ Reverse: 5ʹ-GTATTGCCAGGTGCACAAAT-3ʹ

Trpm6

Forward: 5ʹ-TGCGAGATGCCTTAGTGATG-3ʹ

Reverse: 5ʹ-TTACCAGCCCAATGTCGATG-3ʹ Egf

Forward: 5ʹ-TTCCCGTGTTCTTCTGAGTTCC-3ʹ

Reverse: 5ʹ-GCCTCCAGCAGTGTTTTTACATC-3

Table 2. Body weight changes of rats Body weight at baseline, day 6, and day 8 was measured. Variation of body weight from baseline was compared between groups. Values are mean with S.D., n = 6 rats; aP < 0.01

compared with the control group, bP

< 0.05 compared with 2.5 mg/kg CDDP group. Day

1

6

8 33

(Baseline) day 6 – day 1 Control

221.3

±

4.6 Mg

215.8

mg/kg

221.3

CDDP

9.1

2.5

218.3

mg/kg

CDDP

+

28.2 ± 7.5

253.9 ± 7.1

32.6 ± 7.0

30.0 ± 4.6

255.0 ± 7.4

39.1 ± 4.8

14.2 ± 4.2a

247.9 ± 11.0

26.6 ± 3.4

15.2 ± 2.9a

242.6 ± 6.4

24.3 ± 2.4

230.5 ± 5.2

17.2 ± 5.9a, b

± 7.9 ±

3.4 2.5

249.5

day 8 – day 1

245.8 ± 7.6

±

235.5 ± 11.0

±

4.9

233.5 ± 5.6

Mg 5

mg/kg

CDDP

213.3 3.6

±

219.0

5.7 ± 3.8a, b

± 5.2

CDDP, cisplatin Mg, magnesium

Table 3. Variation of serum creatinine and magnesium (Mg) levels, and mRNA level of rTimp-1 Serum creatinine and Mg levels were measured at baseline and on days 6 and 8. Kidneys were removed 7 days after each injection and relative amounts of rTimp-1 mRNA was normalized to rActin. Data is shown as mean with S.D., n = 6 rats; a

P < 0.05 and bP < 0.01 compared with the control group; cP < 0.05 and dP < 0.01

compared with 2.5 mg/kg CDDP group. Day

1 (Baseline)

6

8

Control

0.69 ± 0.25

0.51 ± 0.09

0.56 ± 0.17

Mg

0.71 ± 0.22

0.62 ± 0.22

0.65 ± 0.14

Serum creatinine level (mg/dl)

2.5 mg/kg CDDP

0.68 ± 0.08 34

b

0.89 ± 0.12

0.90 ± 0.12b

2.5 mg/kg CDDP + Mg

0.68 ± 0.32

0.71 ± 0.33

0.67 ± 0.08c

5 mg/kg CDDP

0.71 ± 0.05

1.37 ± 0.09b, d

1.20 ± 0.08b, d

Control

2.25 ± 0.30

2.01 ± 0.23

2.15 ± 0.28

Mg

2.36 ± 0.38

2.01 ± 0.30

2.10 ± 0.31

2.5 mg/kg CDDP

2.17 ± 0.32

1.30 ± 0.20b

1.63 ± 0.07b

2.5 mg/kg CDDP + Mg

2.26 ± 0.35

1.79 ± 0.35c

2.12 ± 0.13c

5 mg/kg CDDP

2.16 ± 0.23

1.29 ± 0.17b

1.65 ± 0.18a

Serum magnesium level (mg/dl)

Relative renal rTimp-1 mRNA level Control

1.00 ± 0.28

Mg

0.97 ± 0.35

2.5 mg/kg CDDP

1.48 ± 0.17a

2.5 mg/kg CDDP + Mg

1.19 ± 0.23c

5 mg/kg CDDP

2.29 ± 0.30b, d

CDDP, cisplatin Mg, magnesium

Figure Captions

35

Fig. 1. Change in the expression of rat renal organic cation transporter 2 (rOct2) and rat renal multidrug and toxin extrusion protein 1 (rMate1) Western blot analysis of renal rOct2 and rMate1 of five treatment groups on day 8 after each injection. Antisera specific for rOct2 and rMate1 (55 and 64 kDa, respectively) and 36

rActin were used as primary antibodies. Densitometric ratio of (A) rOct2 and (B) rMate1 to rActin and control rat values were arbitrarily defined as 1.0. Each column is mean with S.D., n = 6 rats; **P < 0.01 compared with the control group; ††P < 0.01 compared with 2.5 mg/kg CDDP group.

Fig. 2. Renal platinum (Pt) accumulation Kidney was removed 7 days after CDDP injection. Pt accumulation in the 2.5 mg/kg CDDP with and without Mg co-administration was analyzed using inductively coupled plasma-mass spectrometry (ICP-MS). Each column is mean with S.D., n = 6 rats.

37

38

Fig. 3. Comparison of 20 mg/kg and 40 mg/kg Mg on serum creatinine and expression levels of renal rOct2 and rMate1, and renal Pt accumulation (A) The serum creatinine level was measured at baseline and on days 6 and 8. Western blot analysis of renal (B) rOct2 and (C) rMate1 in the four treatment groups was

39

assessed on day 8 after each injection. (D) Pt accumulation in the 2.5 mg/kg CDDP without or with 40 mg/kg Mg and 20 mg/kg Mg co-administration on day 8 was analyzed using ICP-MS. Data in each column is shown as mean with S.D., n = 6 rats; P < 0.05 and **P < 0.01 compared with the control group; †P < 0.05 and ††P < 0.01

*

compared with 2.5 mg/kg CDDP group; and ‡P < 0.05 compared with 2.5 mg/kg CDDP-Mg group. The data regarding control, 2.5 mg/kg CDDP, and 2.5 mg/kg CDDP-Mg group has already been shown in Table 3 and Fig. 1, 2.

40

Fig. 4. mRNA and protein expression level of rat renal transient receptor potential subfamily Melastatin 6 (rTrpm6) and rat renal epidermal growth factor (rEgf) mRNA expression in the kidney Kidneys were removed 7 days after each injection and relative amounts of (A) rTrpm6 and (B) rEgf mRNA were normalized to rActin.

41

(C) Western blot analysis of renal rTrpm6 on day 8 after each injection. Antisera specific for rTrpm6 and rActin were used as primary antibodies. Expression level of control was arbitrarily set at 1.0. Each column is mean with S.D., n = 6 rats; *P < 0.05 and **P < 0.01 compared with the control group; †P < 0.05 and ††P < 0.01 compared with 2.5 mg/kg CDDP group.

Supplemental Fig. 1. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay of cisplatin (CDDP) with Mg CDDP at 2 or 8 μg/ml was incubated with A549 cells, and 1.25 or 2.5 mM of Mg or saline was co-incubated with CDDP for 48 h. Each column is mean with S.D. of 3-5 measurements.

42

Graphical abstract

43