Interaction of the chemotherapeutic agent oxaliplatin and the tyrosine kinase inhibitor dasatinib with the organic cation transporter 2.

Oxaliplatin (OHP) is effective in colorectal cancer treatment but induces peripheral neurotoxicity (OHP-induced peripheral neurotoxicity, OIPN), diminishing survivor quality of life. Organic cation transporter 2 (OCT2) is a key OHP uptake pathway in dorsal root ganglia. Competing for OCT2-mediated OHP uptake, such as with the tyrosine kinase inhibitor dasatinib, may mitigate OHP side effects. We investigated OHP and dasatinib interaction with OCT2 in human embryonic kidney 293 (HEK293) cells expressing OCT2 within a 10-3 to 10-7 M concentration range. Uptake competition experiments using fluorescent organic cation 4-(4-dimethylaminostyryl)-N-methylpyridinium (ASP+, 1 µM) and mass spectrometry (MS) to determine cellular platinum content indicated that OHP (100 µM) is an OCT2 substrate, mediating OHP cellular toxicity. ASP+ and MS analysis revealed dasatinib as a non-transported inhibitor of hOCT2 (IC50 = 5.9 µM) and as a regulator of OCT2 activity. Dasatinib reduced transporter Vmax, potentially via Y544 phosphorylation suppression. MS analysis showed cellular dasatinib accumulation independent of hOCT2. Although 3 µM dasatinib reduced 100 µM OHP accumulation in hOCT2-HEK293 cells, co-incubation with dasatinib and OHP did not prevent OHP toxicity, possibly due to dasatinib-induced cell viability reduction. In summary, this study demonstrates OHP as an OCT2 substrate and dasatinib as a non-transported inhibitor and regulator of OCT2, offering potential for OIPN mitigation.

Overcoming Biological Barriers: Importance of Membrane Transporters in Homeostasis, Disease, and Disease Treatment 2.0.

This editorial summarizes the seven scientific papers published in the Special Issue "Overcoming Biological Barriers: Importance of Membrane Transporters in Homeostasis, Disease, and Disease Treatment 2 [...].

Modulating the Activity of the Human Organic Cation Transporter 2 Emerges as a Potential Strategy to Mitigate Unwanted Toxicities Associated with Cisplatin Chemotherapy.

Cisplatin (CDDP) stands out as an effective chemotherapeutic agent; however, its application is linked to the development of significant adverse effects, notably nephro- and ototoxicity. The human organic cation transporter 2 (hOCT2), found in abundance in the basolateral membrane domain of renal proximal tubules and the Corti organ, plays a crucial role in the initiation of nephro- and ototoxicity associated with CDDP by facilitating its uptake in kidney and ear cells. Given its limited presence in cancer cells, hOCT2 emerges as a potential druggable target for mitigating unwanted toxicities associated with CDDP. Potential strategies for mitigating CDDP toxicities include competing with the uptake of CDDP by hOCT2 or inhibiting hOCT2 activity through rapid regulation mediated by specific signaling pathways. This study investigated the interaction between the already approved cationic drugs disopyramide, imipramine, and orphenadrine with hOCT2 that is stably expressed in human embryonic kidney cells. Regarding disopyramide, its influence on CDDP cellular transport by hOCT2 was further characterized through inductively coupled plasma isotope dilution mass spectrometry. Additionally, its potential protective effects against cellular toxicity induced by CDDP were assessed using a cytotoxicity test. Given that hOCT2 is typically expressed in the basolateral membrane of polarized cells, with specific regulatory mechanisms, this work studied the regulation of hOCT2 that is stably expressed in Madin-Darby Canine Kidney (MDCK) cells. These cells were cultured in a matrix to induce the formation of cysts, exposing hOCT2 in the basolateral plasma membrane domain, which was freely accessible to experimental solutions. The study specifically tested the regulation of ASP+ uptake by hOCT2 in MDCK cysts through the inhibition of casein kinase II (CKII), calmodulin, or p56lck tyrosine kinase. Furthermore, the impact of this manipulation on the cellular toxicity induced by CDDP was examined using a cytotoxicity test. All three drugs-disopyramide, imipramine, and orphenadrine-demonstrated inhibition of ASP+ uptake, with IC50 values in the micromolar (µM) range. Notably, disopyramide produced a significant reduction in the CDDP cellular toxicity and platinum cellular accumulation when co-incubated with CDDP. The activity of hOCT2 in MDCK cysts experienced a significant down-regulation under inhibition of CKII, calmodulin, or p56lck tyrosine kinase. Interestingly, only the inhibition of p56lck tyrosine kinase demonstrated the capability to protect the cells against CDDP toxicity. In conclusion, certain interventions targeting hOCT2 have demonstrated the ability to reduce CDDP cytotoxicity, at least in vitro. Further investigations in in vivo systems are warranted to ascertain their potential applicability as co-treatments for mitigating undesired toxicities associated with CDDP in patients.

Contribution and Expression of Organic Cation Transporters and Aquaporin Water Channels in Renal Cancer.

The body homeostasis is maintained mainly by the function of the kidneys, which regulate salt and water balance and excretion of metabolism waste products and xenobiotics. This important renal function is determined by the action of many transport systems, which are specifically expressed in the different parts of the nephron, the functional unit of the kidneys. These transport systems are involved, for example, in the reabsorption of sodium, glucose, and other important solutes and peptides from the primary urine. They are also important in the reabsorption of water and thereby production of a concentrated urine. However, several studies have shown the importance of transport systems for different tumor entities. Transport systems, for example, contributed to the proliferation and migration of cancer cells and thereby on tumor progression. They could also serve as drug transporters that could enable drug resistance by outward transport of, for example, chemotherapeutic agents and other drugs. Although many renal transporters have been characterized in detail with respect to the significance for proper kidney function, their role in renal cancer progression is less known. Here, we describe the types of renal cancer and review the studies that analyzed the role of organic cation transporters of the SLC22-family and of the aquaporin water channel family in kidney tumors.

Overcoming Biological Barriers: Importance of Membrane Transporters in Homeostasis, Disease and Disease Treatment.

This editorial summarizes the 22 scientific papers published in the Special Issue "Overcoming Biological Barriers: Importance of Membrane Transporters in Homeostasis, Disease, and Disease Treatment" of the International Journal of Molecular Sciences [...].

Regulation of Transporters for Organic Cations by High Glucose.

Endogenous positively charged organic substances, including neurotransmitters and cationic uremic toxins, as well as exogenous organic cations such as the anti-diabetic medication metformin, serve as substrates for organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs). These proteins facilitate their transport across cell membranes. Vectorial transport through the OCT/MATE axis mediates the hepatic and renal excretion of organic cations, regulating their systemic and local concentrations. Organic cation transporters are part of the remote sensing and signaling system, whose activity can be regulated to cope with changes in the composition of extra- and intracellular fluids. Glucose, as a source of energy, can also function as a crucial signaling molecule, regulating gene expression in various organs and tissues. Its concentration in the blood may fluctuate in specific physiological and pathophysiological conditions. In this work, the regulation of the activity of organic cation transporters was measured by incubating human embryonic kidney cells stably expressing human OCT1 (hOCT1), hOCT2, or hMATE1 with high glucose concentrations (16.7 mM). Incubation with this high glucose concentration for 48 h significantly stimulated the activity of hOCT1, hOCT2, and hMATE1 by increasing their maximal velocity (Vmax), but without significantly changing their affinity for the substrates. These effects were independent of changes in osmolarity, as the addition of equimolar concentrations of mannitol did not alter transporter activity. The stimulation of transporter activity was associated with a significant increase in transporter mRNA expression. Inhibition of the mechanistic target of rapamycin (mTOR) kinase with Torin-1 suppressed the transporter stimulation induced by incubation with 16.7 mM glucose. Focusing on hOCT2, it was shown that incubation with 16.7 mM glucose increased hOCT2 protein expression in the plasma membrane. Interestingly, an apparent trend towards higher hOCT2 mRNA expression was observed in kidneys from diabetic patients, a pathology characterized by high serum glucose levels. Due to the small number of samples from diabetic patients (three), this observation must be interpreted with caution. In conclusion, incubation for 48 h with a high glucose concentration of 16.7 mM stimulated the activity and expression of organic cation transporters compared to those measured in the presence of 5.6 mM glucose. This stimulation by a diabetic environment could increase cellular uptake of the anti-diabetic drug metformin and increase renal tubular secretion of organic cations in an early stage of diabetes.

Role of Mouse Organic Cation Transporter 2 for Nephro- and Peripheral Neurotoxicity Induced by Chemotherapeutic Treatment with Cisplatin.

Cisplatin (CDDP) is an efficient chemotherapeutic agent broadly used to treat solid cancers. Chemotherapy with CDDP can cause significant unwanted side effects such as renal toxicity and peripheral neurotoxicity. CDDP is a substrate of organic cation transporters (OCT), transporters that are highly expressed in renal tissue. Therefore, CDDP uptake by OCT may play a role in causing unwanted toxicities of CDDP anticancer treatment. In this study, the contribution of the mouse OCT2 (mOCT2) to CDDP nephro- and peripheral neurotoxicity was investigated by comparing the effects of cyclic treatment with low doses of CDDP on renal and neurological functions in wild-type (WT) mice and mice with genetic deletion of OCT2 (OCT2-/- mice). This CDDP treatment protocol caused significant impairment of kidneys and peripherical neurological functions in WT mice. These effects were significantly reduced in OCT2-/- mice, however, less profoundly than what was previously measured in mice with genetic deletion of both OCT1 and 2 (OCT1-2-/- mice). Comparing the apparent affinities (IC50) of mOCT1 and mOCT2 for CDDP, the mOCT1 displayed a higher affinity for CDDP than the mOCT2 (IC50: 9 and 558 µM, respectively). Also, cellular toxicity induced by incubation with 100 µM CDDP was more pronounced in cells stably expressing mOCT1 than in cells expressing mOCT2. Therefore, in mice, CDDP uptake by both OCT1 and 2 contributes to the development of CDDP undesired side effects. OCT seem to be suitable targets for establishing treatment protocols aimed at decreasing unwanted CDDP toxicity and improving anticancer treatment with CDDP.

TrkC Is Essential for Nephron Function and Trans-Activates Igf1R Signaling.

BACKGROUND: Injury to kidney podocytes often results in chronic glomerular disease and consecutive nephron malfunction. For most glomerular diseases, targeted therapies are lacking. Thus, it is important to identify novel signaling pathways contributing to glomerular disease. Neurotrophic tyrosine kinase receptor 3 (TrkC) is expressed in podocytes and the protein transmits signals to the podocyte actin cytoskeleton. METHODS: Nephron-specific TrkC knockout (TrkC-KO) and nephron-specific TrkC-overexpressing (TrkC-OE) mice were generated to dissect the role of TrkC in nephron development and maintenance. RESULTS: Both TrkC-KO and TrkC-OE mice exhibited enlarged glomeruli, mesangial proliferation, basement membrane thickening, albuminuria, podocyte loss, and aspects of FSGS during aging. Igf1 receptor (Igf1R)-associated gene expression was dysregulated in TrkC-KO mouse glomeruli. Phosphoproteins associated with insulin, erb-b2 receptor tyrosine kinase (Erbb), and Toll-like receptor signaling were enriched in lysates of podocytes treated with the TrkC ligand neurotrophin-3 (Nt-3). Activation of TrkC by Nt-3 resulted in phosphorylation of the Igf1R on activating tyrosine residues in podocytes. Igf1R phosphorylation was increased in TrkC-OE mouse kidneys while it was decreased in TrkC-KO kidneys. Furthermore, TrkC expression was elevated in glomerular tissue of patients with diabetic kidney disease compared with control glomerular tissue. CONCLUSIONS: Our results show that TrkC is essential for maintaining glomerular integrity. Furthermore, TrkC modulates Igf-related signaling in podocytes.

Physiology, Biochemistry and Pharmacology of Transporters for Organic Cations 2.0.

This editorial summarizes the 12 scientific papers published in the Special Issue "Physiology, Biochemistry, and Pharmacology of Transporters for Organic Cations 2 [...].

Exacerbation of Cisplatin Cellular Toxicity by Regulation of the Human Organic Cation Transporter 2 through Angiotensin II.

Cisplatin (CDDP) is an efficient chemotherapeutic drug, whose use is associated with the development of serious undesired toxicities, such as nephrotoxicity. The human organic cation transporter 2 (hOCT2), which is highly expressed in the basolateral membrane domain of renal proximal tubules seems to play an important role in the development of CDDP nephrotoxicity. The role of angiotensin II (AII) signaling by binding to the AII receptor type 1 (AT1R) in the development and/or progression of CDDP nephrotoxicity is debated. Therefore, in this work, the regulation of hOCT2 activity by AII and its role in the development of CDDP cellular toxicity was investigated. To do this, hOCT2 was overexpressed by viral transduction in Madin-Darby Canine Kidney (MDCK) cells which were cultivated on a filter. This approach allows the separation of an apical and a basolateral membrane domain, which are easily accessible for experimentation. In this system, hOCT2 was mainly localized on the basolateral plasma membrane domain of the cells. The transporter was functional since a specific uptake of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+) with an affinity (Km) of 35 µM was only detectable by the addition of ASP+ to the basolateral compartment of hOCT2 expressing MDCK (hOCT2-MDCK) cells. Similarly, CDDP toxicity was evident mainly by CDDP addition to the basolateral compartment of hOCT2-MDCK cells cultivated on a filter. The addition of 1 nM AII stimulated hOCT2 function via PKC activation and worsened CDDP cytotoxicity via binding to AT1R. Therefore, the AII signaling pathway may be implicated in the development and/or progression of CDDP nephrotoxicity. This signaling pathway may be a target for protective interventions for example by blocking AT1R in the kidneys. However, it should be further investigated whether these findings obtained in a cell culture system may have translational relevance for the clinical situation. For toxicity experiments, a 100 µM CDDP concentration was used, which is high but allows us to identify clearly toxic effects due to hOCT2. In summary, down-regulation of hOCT2 activity by the inhibition of the AII signaling pathway may protect against CDDP nephrotoxicity.

Role of Organic Cation Transporter 2 in Autophagy Induced by Platinum Derivatives.

The human organic cation transporter 2 (hOCT2) mediates renal and neuronal cellular cisplatin and oxaliplatin uptake, and therefore plays a significant role in the development of side effects associated with these chemotherapeutic drugs. Autophagy is induced by cisplatin and oxaliplatin treatment and is believed to promote cell survival under stressful conditions. We examined in vitro the role of hOCT2 on autophagy induced by cisplatin and oxaliplatin. We also explored the effect of autophagy on toxicities of these platinum derivatives. Our results indicate that autophagy, measured as LC3 II accumulation and reduction in p62 expression level, is induced in response to cisplatin and oxaliplatin in HEK293-hOCT2 but not in wild-type HEK293 cells. Furthermore, inhibition of autophagy is associated with higher toxicity of platinum derivatives, and starvation was found to offer protection against cisplatin-associated toxicity. In conclusion, activation of autophagy could be a potential strategy to protect against unwanted toxicities induced by treatment with platinum derivatives.

Interaction of Masitinib with Organic Cation Transporters.

Tyrosine kinase inhibitors (TKI) such as Masitinib were reported to be useful as therapeutic options in malignant disorders and nonmalignant diseases, like coronavirus disease 2019 (COVID-19). Most kinases must be translocated into targeted cells by the action of specific transport proteins, as they are hydrophilic and not able to cross cell membranes freely. Accordingly, the efficacy of TKI in target cells is closely dependent on the expression of their transporters. Specifically, Masitinib is an organic cation and is expected to interact with organic cation transporters (OCT and Multidrug and Toxin Extrusion proteins-MATE-). The aim of this work was to characterize the interaction of Masitinib with different OCTs. Human embryonic kidney 293 cells stably transfected with murine or human OCT were used for the experiments. The interaction of Masitinib with OCTs was investigated using quenching experiments. The intracellular accumulation of this drug was quantified using high performance liquid chromatography. Our results identified interactions of Masitinib with almost all investigated mouse (m) and human (h) OCTs and hMATE1 and indicated OCT1 and hOCT2 to be especially potent Masitinib translocators across cell membranes. Interestingly, some important differences were observed for the interaction with murine and human OCTs. In the future, investigations concerning further in vitro and in vivo properties of Masitinib and its efficacy related to transporter-related uptake mechanisms under pathophysiological conditions should be performed. Clinical trials in humans and other animals with Masitinib have already shown promising results. However, further research is necessary to understand the disease specific transport mechanisms of Masitinib to contribute to a successful and responsible therapy employment.

Influence of Cation Transporters (OCTs and MATEs) on the Renal and Hepatobiliary Disposition of [11C]Metoclopramide in Mice.

PURPOSE: To investigate the role of cation transporters (OCTs, MATEs) in the renal and hepatic disposition of the radiolabeled antiemetic drug [11C]metoclopramide in mice with PET. METHODS: PET was performed in wild-type mice after administration of an intravenous microdose (<1 µg) of [11C]metoclopramide without and with co-administration of either unlabeled metoclopramide (5 or 10 mg/kg) or the prototypical cation transporter inhibitors cimetidine (150 mg/kg) or sulpiride (25 mg/kg). [11C]Metoclopramide PET was also performed in wild-type and Slc22a1/2(-/-) mice. Radiolabeled metabolites were measured at 15 min after radiotracer injection and PET data were corrected for radiolabeled metabolites. RESULTS: [11C]Metoclopramide was highly metabolized and [11C]metoclopramide-derived radioactivity was excreted into the urine. The different investigated treatments decreased (~2.5-fold) the uptake of [11C]metoclopramide from plasma into the kidney and liver, inhibited metabolism and decreased (up to 3.8-fold) urinary excretion, which resulted in increased plasma concentrations of [11C]metoclopramide. Kidney and liver uptake were moderately (~1.3-fold) reduced in Slc22a1/2(-/-) mice. CONCLUSIONS: Our results suggest a contribution of OCT1/2 to the kidney and liver uptake and of MATEs to the urinary excretion of [11C]metoclopramide in mice. Cation transporters may contribute, next to variability in the activity of metabolizing enzymes, to variability in metoclopramide pharmacokinetics and side effects.

Physiology, Biochemistry, and Pharmacology of Transporters for Organic Cations.

This editorial summarizes the 13 scientific papers published in the Special Issue "Physiology, Biochemistry, and Pharmacology of Transporters for Organic Cations" of the International Journal of Molecular Sciences [...].

Properties of Transport Mediated by the Human Organic Cation Transporter 2 Studied in a Polarized Three-Dimensional Epithelial Cell Culture Model.

The renal secretory clearance for organic cations (neurotransmitters, metabolism products and drugs) is mediated by transporters specifically expressed in the basolateral and apical plasma membrane domains of proximal tubule cells. Here, human organic cation transporter 2 (hOCT2) is the main transporter for organic cations in the basolateral membrane domain. In this study, we stably expressed hOCT2 in Madin-Darby Canine Kidney (MDCK) cells and cultivated these cells in the presence of an extracellular matrix to obtain three-dimensional (3D) structures (cysts). The transport properties of hOCT2 expressed in MDCK cysts were compared with those measured using human embryonic kidney cells (HEK293) stably transfected with hOCT2 (hOCT2-HEK cells). In the MDCK cysts, hOCT2 was expressed in the basolateral membrane domain and showed a significant uptake of the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+) with an affinity (Km) of 3.6 ± 1.2 µM, similar to what was measured in the hOCT2-HEK cells (Km = 3.1 ± 0.2 µM). ASP+ uptake was inhibited by tetraethylammonium (TEA+), tetrapentylammonium (TPA+), metformin and baricitinib both in the hOCT2-HEK cells and the hOCT2- MDCK cysts, even though the apparent affinities of TEA+ and baricitinib were dependent on the expression system. Then, hOCT2 was subjected to the same rapid regulation by inhibition of p56lck tyrosine kinase or calmodulin in the hOCT2-HEK cells and hOCT2- MDCK cysts. However, inhibition of casein kinase II regulated only activity of hOCT2 expressed in MDCK cysts and not in HEK cells. Taken together, these results suggest that the 3D cell culture model is a suitable tool for the functional analysis of hOCT2 transport properties, depending on cell polarization.

Rapid Regulation of Human Multidrug and Extrusion Transporters hMATE1 and hMATE2K.

Vectorial transport of organic cations (OCs) in renal proximal tubules is mediated by sequential action of human OC transporter 2 (hOCT2) and human multidrug and toxic extrusion protein 1 and 2K (hMATE1 and hMATE2K), expressed in the basolateral (hOCT2) and luminal (hMATE1 and hMATE2K) plasma membranes, respectively. It is well known that hOCT2 activity is subjected to rapid regulation by several signaling pathways, suggesting that renal OC secretion may be acutely adapted to physiological requirements. Therefore, in this work, the acute regulation of hMATEs stably expressed in human embryonic kidney cells was characterized using the fluorescent substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+) as a marker. A specific regulation of ASP+ transport by hMATE1 and hMATE2K measured in uptake and efflux configurations was observed. In the example of hMATE1 efflux reduction by inhibition of casein kinase II, it was also shown that this regulation is able to modify transcellular transport of ASP+ in Madin-Darby canine kidney II cells expressing hOCT2 and hMATE1 on the basolateral and apical membrane domains, respectively. The activity of hMATEs can be rapidly regulated by some intracellular pathways, which sometimes are common to those found for hOCTs. Interference with these pathways may be important to regulate renal secretion of OCs.

Tofacitinib and Baricitinib Are Taken up by Different Uptake Mechanisms Determining the Efficacy of Both Drugs in RA.

BACKGROUND: Rheumatoid arthritis (RA) is a systemic autoimmune disease in which synovial fibroblasts (SF) play a key role. Baricitinib and Tofacitinib both act intracellularly, blocking the ATP-binding side of JAK proteins and thereby the downstream signalling pathway via STAT-3. Therefore, we investigated the role of organic cation transporters (OCTs) in Baricitinib and Tofacitinib cellular transport. METHODS: OCT expression was analysed in SF isolated from RA and osteoarthritis (OA) patients, as well as peripheral blood mononuclear cells. The interaction of Baricitinib and Tofacitinib with OCTs was investigated using quenching experiments. The intracellular accumulation of both drugs was quantified using LC/MS. Target inhibition for both drugs was tested using Western blot for phosphorylated JAK1 and STAT3 upon stimulation with IL-6. RESULTS: MATE-1 expression increased in OASF compared to RASF. The other OCTs were not differentially expressed. The transport of Baricitinib was not OCT dependent. Tofacitinib; however, was exported from RASF in a MATE-1 dependent way. Tofacitinib and Baricitinib showed comparable inhibition of downstream signalling pathways. CONCLUSION: We observed different cellular uptake strategies for Baricitinib and Tofacitinib. Tofacitinib was exported out of healthy cells due to the increased expression of MATE1. This might make Tofacitinib the favourable drug.

The proteome microenvironment determines the protective effect of preconditioning in cisplatin-induced acute kidney injury.

Acute kidney injury (AKI) leads to significant morbidity and mortality; unfortunately, strategies to prevent or treat AKI are lacking. In recent years, several preconditioning protocols have been shown to be effective in inducing organ protection in rodent models. Here, we characterized two of these interventions-caloric restriction and hypoxic preconditioning-in a mouse model of cisplatin-induced AKI and investigated the underlying mechanisms by acquisition of multi-layered omic data (transcriptome, proteome, N-degradome) and functional parameters in the same animals. Both preconditioning protocols markedly ameliorated cisplatin-induced loss of kidney function, and caloric restriction also induced lipid synthesis. Bioinformatic analysis revealed mRNA-independent proteome alterations affecting the extracellular space, mitochondria, and transporters. Interestingly, our analyses revealed a strong dissociation of protein and RNA expression after cisplatin treatment that showed a strong correlation with the degree of damage. N-degradomic analysis revealed that most posttranscriptional changes were determined by arginine-specific proteolytic processing. This included a characteristic cisplatin-activated complement signature that was prevented by preconditioning. Amyloid and acute-phase proteins within the cortical parenchyma showed a similar response. Extensive analysis of disease-associated molecular patterns suggested that transcription-independent deposition of amyloid P-component serum protein may be a key component in the microenvironmental contribution to kidney damage. This proof-of-principle study provides new insights into the pathogenesis of cisplatin-induced AKI and the molecular mechanisms underlying organ protection by correlating phenotypic and multi-layered omics data.

An integrative approach to cisplatin chronic toxicities in mice reveals importance of organic cation-transporter-dependent protein networks for renoprotection.

Cisplatin (CDDP) is one of the most important chemotherapeutic drugs in modern oncology. However, its use is limited by severe toxicities, which impair life quality after cancer. Here, we investigated the role of organic cation transporters (OCT) in mediating toxicities associated with chronic (twice the week for 4 weeks) low-dose (4 mg/kg body weight) CDDP treatment (resembling therapeutic protocols in patients) of wild-type (WT) mice and mice with OCT genetic deletion (OCT1/2-/-). Functional and molecular analysis showed that OCT1/2-/- mice are partially protected from CDDP-induced nephrotoxicity and peripheral neurotoxicity, whereas ototoxicity was not detectable. Surprisingly, proteomic analysis of the kidneys demonstrated that genetic deletion of OCT1/2 itself was associated with significant changes in expression of proinflammatory and profibrotic proteins which are part of an OCT-associated protein network. This signature directly regulated by OCT consisted of three classes of proteins, viz., profibrotic proteins, proinflammatory proteins, and nutrient sensing molecules. Consistent with functional protection, CDDP-induced proteome changes were more severe in WT mice than in OCT1/2-/- mice. Laser ablation-inductively coupled plasma-mass spectrometry analysis demonstrated that the presence of OCT was not associated with higher renal platinum concentrations. Taken together, these results redefine the role of OCT from passive membrane transporters to active modulators of cell signaling in the kidney.

Protein Abundance of Clinically Relevant Drug Transporters in The Human Kidneys.

Renal drug transporters such as the organic cation transporters (OCTs), organic anion transporters (OATs) and multidrug resistance proteins (MRPs) play an important role in the tubular secretion of many drugs influencing their efficacy and safety. However, only little is known about the distinct protein abundance of these transporters in human kidneys, and about the impact of age and gender as potential factors of inter-subject variability in their expression and function. The aim of this study was to determine the protein abundance of MDR1, MRP1-4, BCRP, OAT1-3, OCT2-3, MATE1, PEPT1/2, and ORCTL2 by liquid chromatography-tandem mass spectrometry-based targeted proteomics in a set of 36 human cortex kidney samples (20 males, 16 females; median age 53 and 55 years, respectively). OAT1 and 3, OCT2 and ORCTL2 were found to be most abundant renal SLC transporters while MDR1, MRP1 and MRP4 were the dominating ABC transporters. Only the expression levels of MDR1 and ORCTL2 were significantly higher abundant in older donors. Moreover, we found several significant correlations between different transporters, which may indicate their functional interplay in renal vectorial transport processes. Our data may contribute to a better understanding of the molecular processes determining renal excretion of drugs.