Giuliano Ciarimboli

Cisplatin Nephrotoxicity Is Critically Mediated via the Human Organic Cation Transporter 2

Cis-platin is an effective anti-neoplastic agent, but it is also highly nephrotoxic. Here, we clearly identify the human organic cation transporter 2 (hOCT2) as the critical transporter for cis-platin nephrotoxicity in isolated human proximal tubules and offer a potential mechanism for reducing nephrotoxicity in clinical practice. Interaction of cis-platin with hOCT2 in kidney or hOCT1 in liver was investigated with the fluorescent cation 4-[4-(dimethyl-amino)styril]-methylpyridinium in stably transfected HEK293 cells and for the first time in tissues physiologically expressing these transporters, human proximal tubules, and human hepatocyte couplets. Cis-platin (100 micromol/L) inhibited transport via hOCT2-HEK293 but not hOCT1-HEK293. In human proximal tubules cis-platin competed with basolateral organic cation transport, whereas it had no effect in tubules from a diabetic kidney or in hepatocytes. In hOCT2-HEK293 cells treated for 15 hours, incubation with cis-platin induced apoptosis, which was completely suppressed by contemporaneous incubation with the hOCT2 substrate cimetidine (100 micromol/L). These findings demonstrate that uptake of cis-platin is mediated by hOCT2 in renal proximal tubules, explaining its organ-specific toxicity. A combination of cis-platin with other substrates that compete for hOCT2 offers an effective option to decrease nephrotoxicity in the clinical setting.

Organic cation transporter 2 mediates cisplatin-induced oto- and nephrotoxicity and is a target for protective interventions.

The use of the effective antineoplastic agent cisplatin is limited by its serious side effects, such as oto- and nephrotoxicity. Ototoxicity is a problem of special importance in children, because deafness hampers their language and psychosocial development. Recently, organic cation transporters (OCTs) were identified in vitro as cellular uptake mechanisms for cisplatin. In the present study, we investigated in an in vivo model the role of OCTs in the development of cisplatin oto- and nephrotoxicity. The functional effects of cisplatin treatment on kidney (24 hours excretion of glucose, water, and protein) and hearing (auditory brainstem response) were studied in wild-type and OCT1/2 double-knockout (KO) mice. No sign of ototoxicity and only mild nephrotoxicity were observed after cisplatin treatment of knockout mice. Comedication of wild-type mice with cisplatin and the organic cation cimetidine protected from ototoxicity and partly from nephrotoxicity. For the first time we showed that OCT2 is expressed in hair cells of the cochlea. Furthermore, cisplatin-sensitive cell lines from pediatric tumors showed no expression of mRNA for OCTs, indicating the feasibility of therapeutic approaches aimed to reduce cisplatin toxicities by competing OCT2-mediated cisplatin uptake in renal proximal tubular and cochlear hair cells. These findings are very important to establish chemotherapeutical protocols aimed to maximize the antineoplastic effect of cisplatin while reducing the risk of toxicities.

Regulation of organic cation transport

Transport of organic cations (OC) is important for the recycling of endogenous OC and also a necessary step for detoxification of exogenous OC in the body. Even though the identification and characterisation of numerous OC transporters in recent years has allowed the elucidation of molecular mechanisms underlying OC transport, elucidation of the regulation of this transport is just beginning. This review summarises the general properties of OC transport and then analyses the literature on the regulation of these processes. Studies on short- and long-term regulation of OC transport are considered separately. Important aspects of short-term regulation have been clarified and the regulatory pathways of several OC transporters have been characterised. Short-term regulation appears to be transporter subtype-, tissue- and species-dependent and to involve transporter phosphorylation. Transporter phosphorylation may alter the affinity for substrates or/and expression on the plasma membrane. Even though several studies have shown long-term regulation of OC transport, the pathophysiological meaning of these changes are not well understood. In this case, regulation seems to be subtype-, tissue- and gender-specific. Further research is necessary to clarify this important issue of regulation of OC transport.

Proximal Tubular Secretion of Creatinine by Organic Cation Transporter OCT2 in Cancer Patients

Purpose: Knowledge of transporters responsible for the renal secretion of creatinine is key to a proper interpretation of serum creatinine and/or creatinine clearance as markers of renal function in cancer patients receiving chemotherapeutic agents. Experimental Design: Creatinine transport was studied in transfected HEK293 cells in vitro and in wild-type mice and age-matched organic cation transporter 1 and 2–deficient [Oct1/2(−/−)] mice ex vivo and in vivo. Clinical pharmacogenetic and transport inhibition studies were done in two separate cohorts of cancer patients. Results: Compared with wild-type mice, creatinine clearance was significantly impaired in Oct1/2(−/−) mice. Furthermore, creatinine inhibited organic cation transport in freshly isolated proximal tubules from wild-type mice and humans, but not in those from Oct1/2(−/−) mice. In a genetic association analysis (n = 590), several polymorphisms around the OCT2/SLC22A2 gene locus, including rs2504954 (P = 0.000873), were significantly associated with age-adjusted creatinine levels. Furthermore, in cancer patients (n = 68), the OCT2 substrate cisplatin caused an acute elevation of serum creatinine (P = 0.0083), consistent with inhibition of an elimination pathway. Conclusions: Collectively, this study shows that OCT2 plays a decisive role in the renal secretion of creatinine. This process can be inhibited by OCT2 substrates, which impair the usefulness of creatinine as a marker of renal function. Clin Cancer Res; 18(4); 1101–8. ©2012 AACR.

Individual PKC-Phosphorylation Sites in Organic Cation Transporter 1 Determine Substrate Selectivity and Transport Regulation

To elucidate the molecular mechanisms underlying stimulation of rat organic cation transporter type 1 (rOCT1) by protein kinase C (PKC) activation, functional properties and regulation of rOCT1 stably expressed in HEK293 cells after site-directed mutagenesis of putative PKC phosphorylation-sites were compared with wild-type (WT) rOCT1 using microfluorometric measurements with the fluorescence organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP(+)). Either substitutions of single (S286A, S292A, T296A, S328A, and T550A) or of all five PKC-sites (5x-PKC) with alanine suppressed PKC-induced stimulation of ASP(+) uptake, whereas regulation by p56(lck) tyrosine kinase was conserved in all mutants. Remarkably, the apparent affinities for TEA(+), TPA(+), and quinine were changed differently in each mutant (EC(50) in WT, S286A, S292A, T296A, S328A, T550A, and 5x-PKC in mumol: TEA(+): 105, 153, 56, 1135, 484, 498, 518; TPA(+): 0.1, 2.1, 0.3, 1.0, 43, 0.3, 2.2; quinine: 1.5, 3.0, 2.5, 4.8, 81, 7.6, 8.9, respectively). After mutations, no effects of PKC activation on apparent affinity of rOCT1 for these substrates could be detected, in contrast to what was observed in WT. PKC activation had no significant effect on rOCT1 trafficking from intracellular pools to the cell membrane. Substitution of all PKC sites suppressed PKC-induced phosphorylation of rOCT1. In conclusion, it was found that the presence of all five potential PKC phosphorylation sites is necessary for the PKC-induced stimulation of rOCT1. The different effects on the EC(50) values by the different mutations suggest that the large intracellular loop participates in building the substrate binding pocket of rOCT1 or specifically modulates its structure.

Regulation of the human organic cation transporter hOCT1

The human organic cation transporter type 1 (hOCT1) is an important transport system for small organic cations in the liver. Organic cation transporters are regulated by different signaling pathways, but the regulation of hOCT1 has not yet been studied. In this work, we have for the first time investigated the regulation of hOCT1. hOCT1 was expressed in Chinese hamster ovary cells (CHO‐hOCT1) and in human embryonic kidney cells (HEK293‐hOCT1). Its activity was monitored using microfluorimetry with the fluorescent organic cation 4‐(4‐(dimethylamino)styryl)‐N‐methylpyridinium (ASP+) as substrate. hOCT1 expressed in CHO‐cells was inhibited by protein kinase A (PKA) activation (1 µM forskolin, −58 ± 6%, n = 12), calmodulin inhibition (0.1 µM calmidazolium, −68 ± 3%, n = 6; 10 µM ophiobolin A, −48 ± 10%, n = 7), calmodulin‐dependent kinase II inhibition (1 µM KN62, −78 ± 4%, n = 12), and inhibition of p56lck tyrosine kinase (10 µM aminogenistein, −35 ± 7%, n = 12). The apparent affinities for TEA+ were lower in CHO‐hOCT1 than in HEK293‐hOCT1, while those for TPA+ and quinine were almost identical; the rank order of EC50 values (TPA+ > quinine > TEA+) was independent of the expression system. EC50 values for TEA+ in CHO‐hOCT1 or HEK293‐hOCT1 were increased under calmidazolium incubation (6.3 and 1.4 mM, respectively). hOCT1 was inhibited by PKA and endogenously activated by calmodulin, calmodulin‐dependent kinase II, and p56lck tyrosine kinase. Regulation pathways were the same in the two expression systems. Since apparent substrate affinities depend on activity of regulatory pathways, the expression system plays a role in determining the substrate affinities.

Oxaliplatin-induced neurotoxicity is dependent on the organic cation transporter OCT2

Oxaliplatin is an integral component of colorectal cancer therapy, but its clinical use is associated with a dose-limiting peripheral neurotoxicity. We found that the organic cation transporter 2 (OCT2) is expressed on dorsal root ganglia cells within the nervous system where oxaliplatin is known to accumulate. Cellular uptake of oxaliplatin was increased by 16- to 35-fold in cells overexpressing mouse Oct2 or human OCT2, and this process was associated with increased DNA platination and oxaliplatin-induced cytotoxicity. Furthermore, genetic or pharmacologic knockout of Oct2 protected mice from hypersensitivity to cold or mechanical-induced allodynia, which are established tests to assess acute oxaliplatin-induced neurotoxicity. These findings provide a rationale for the development of targeted approaches to mitigate this debilitating toxicity.

Quantitative bioimaging of platinum in polymer embedded mouse organs using laser ablation ICP-MS

A novel quantification approach for tissue imaging using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) based on tissue embedding in cold-curing resins (Technovit 7100) is presented. With respect to massive side effects on cisplatin, the platinum distribution at different time intervals after cisplatin treatment of mice was determined quantitatively in different tissues including cochlea, testis and kidney. For this purpose, cold-curing resin blocks spiked with different amounts of platinum acetyl acetonate prior to curing were ablated after sectioning at 5 μm thickness and were analysed using ICP-MS after microwave digestion. High spatial resolution and limits of detection in the low ppb range (8 μg kg(-1)) were achieved using a simple and efficient sample preparation. External calibration using the Technovit 7100 standards proved to yield precise and reproducible quantification results. The distribution and retention behaviour of cisplatin in the organs was investigated using the new calibration method.

The organic cation transporters rOCT1 and hOCT2 are inhibited by cGMP.

The electrogenic cation transporters OCT1 and OCT2 in the basolateral membrane of renal proximal tubules mediate the first step during secretion of organic cations. Previously we demonstrated stimulation and change of selectivity for rat OCT1 (rOCT1) by protein kinase C. Here we investigated the effect of cGMP on cation transport by rOCT1 or human OCT2 (hOCT2) after expression in human embryonic kidney cells (HEK293) or oocytes of Xenopus laevis. In HEK293 cells, uptake was measured by microfluorimetry using the fluorescent cation 4-(4-(dimethyl-amino)styryl)-N-methylpyridinium iodide (ASP + ) as substrate, whereas uptake into Xenopus laevis oocytes was measured with radioactively labelled cations. In addition, ASP +-induced depolarizations of membrane voltages (Vm) were measured in HEK293 cells using the slow whole-cell patch-clamp method. Incubation of rOCT1-expressing HEK293 cells for 10 min with 100 mM 8-Br-cGMP reduced initial ASP + uptake by maximally 78% with an IC50 value of 24 ± 16 mM. This effect was not abolished by the specific PKG inhibitor KT5823, indicating that a cGMP-dependent kinase is not involved. An inhibition of ASP + uptake by rOCT1 in HEK293 cells was also obtained when the cells were incubated for 10 min with 100 mM cGMP, whereas no effect was obtained when cGMP was given together with ASP + . ASP + (100 mM)-induced depolarizations of Vm were reduced in the presence of 8-Br-cGMP (100 mM) by 44 ± 11% (n = 6). Since it could be demonstrated that [3H]cGMP is taken up by an endogeneous cyanine863-inhibitable transporter, the effect of cGMP is probably mediated from inside the cell. Uptake measurements with [14C]tetraethylammonium and [3H]2-methyl-4-phenylpyridinium in Xenopus laevis oocytes expressing rOCT1 performed in the absence and presence of 8-Br-cGMP showed that cGMP does not interact directly with the transporter. The data suggest that the inhibition mediated by cGMP observed in HEK293 cells occurs most likely via a mammalian cGMP-binding protein that interacts with OCT1-2 transporters.

Organic cation transporters OCT1, 2, and 3 mediate high-affinity transport of the mutagenic vital dye ethidium in the kidney proximal tubule

The positively charged fluorescent dyes ethidium (Et(+)) and propidium (Pr(2+)) are widely used as DNA and necrosis markers. Et(+) is cytotoxic and mutagenic. The polyspecific organic cation transporters OCT1 (SLC22A1), OCT2 (SLC22A2), and OCT3 (SLC22A3) mediate electrogenic facilitated diffusion of small (< or =500 Da) organic cations with broad specificities. In humans, OCT2 mediates basolateral uptake by kidney proximal tubules (PT), whereas in rodents OCT1/2 are involved. In mouse kidney, perfused Et(+) accumulated predominantly in the S2/S3 segments of the PT, but not Pr(2+). In cells stably overexpressing human OCTs (hOCTs), Et(+) uptake was observed with K(m) values of 0.8 +/- 0.2 microM (hOCT1), 1.7 +/- 0.5 microM (hOCT2), and 2.0 +/- 0.5 microM (hOCT3), whereas Pr(2+) was not transported. Accumulation of Et(+) was inhibited by OCT substrates quinine, 3-methyl-4-phenylpyridinium (MPP(+)), cimetidine, and tetraethylammonium (TEA(+)). For hOCT1 and hOCT2, the IC(50) values for MPP(+), TEA(+), and cimetidine were higher than for inhibition of previously tested transported substrates. For hOCT2, the inhibition of Et(+) uptake by MPP(+) and cimetidine was shown to be competitive. Et(+) also inhibited transport of 0.1 microM [(3)H]MPP(+) by all hOCT isoforms with IC(50) values between 0.4 and 1.3 microM, and the inhibition of hOCT1-mediated uptake of MPP(+) by Et(+) was competitive. In Oct1/2(-/-) mice, Et(+) uptake in the PT was almost abolished. The data demonstrate that Et(+) is taken up avidly by the PT, which is mediated by OCT1 and/or OCT2. Considering the high affinity of OCTs for Et(+) and their strong expression in various organs, strict safety guidelines for Et(+) handling should be reinforced.