Regulation of renal organic cation transporters.

Transporters for organic cations (OCs) facilitate exchange of positively charged molecules through the plasma membrane. Substrates for these transporters encompass neurotransmitters, metabolic byproducts, drugs, and xenobiotics. Consequently, these transporters actively contribute to the regulation of neurotransmission, cellular penetration and elimination process for metabolic products, drugs, and xenobiotics. Therefore, these transporters have significant physiological, pharmacological, and toxicological implications. In cells of renal proximal tubules, the vectorial secretion pathways for OCs involve expression of organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs) on basolateral and apical membrane domains, respectively. This review provides an overview of documented regulatory mechanisms governing OCTs and MATEs. Additionally, regulation of these transporters under various pathological conditions is summarized. The expression and functionality of OCTs and MATEs are subject to diverse pre- and post-translational modifications, providing insights into their regulation in various pathological conditions. Typically, in diseases, downregulation of transporter expression is observed, probably as a protective mechanism to prevent additional damage to kidney tissue. This regulation may be attributed to the intricate network of modifications these transporters undergo, shedding light on their dynamic responses in pathological contexts.

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 [...].

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.

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.

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.

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 [...].

Importance of ACE2 for SARS-CoV-2 Infection of Kidney Cells.

In late 2019, the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the causative agent of coronavirus disease 2019 (COVID-19) emerged in China and spread rapidly around the world, causing an ongoing pandemic of global concern. COVID-19 proceeds with moderate symptoms in most patients, whereas others experience serious respiratory illness that requires intensive care treatment and may end in death. The severity of COVID-19 is linked to several risk factors including male sex, comorbidities, and advanced age. Apart from respiratory complications, further impairments by COVID-19 affecting other tissues of the human body are observed. In this respect, the human kidney is one of the most frequently affected extrapulmonary organs and acute kidney injury (AKI) is known as a direct or indirect complication of SARS-CoV-2 infection. The aim of this work was to investigate the importance of the protein angiotensin-converting enzyme 2 (ACE2) for a possible cell entry of SARS-CoV-2 into human kidney cells. First, the expression of the cellular receptor ACE2 was demonstrated to be decisive for viral SARS-CoV-2 cell entry in human AB8 podocytes, whereas the presence of the transmembrane protease serine 2 (TMPRSS2) was dispensable. Moreover, the ACE2 protein amount was well detectable by mass spectrometry analysis in human kidneys, while TMPRSS2 could be detected only in a few samples. Additionally, a negative correlation of the ACE2 protein abundance to male sex and elderly aged females in human kidney tissues was demonstrated in this work. Last, the possibility of a direct infection of kidney tubular renal structures by SARS-CoV-2 was demonstrated.

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.

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.

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 [...].

IFITM3 Interacts with the HBV/HDV Receptor NTCP and Modulates Virus Entry and Infection.

The Na+/taurocholate co-transporting polypeptide (NTCP, gene symbol SLC10A1) is both a physiological bile acid transporter and the high-affinity hepatic receptor for the hepatitis B and D viruses (HBV/HDV). Virus entry via endocytosis of the virus/NTCP complex involves co-factors, but this process is not fully understood. As part of the innate immunity, interferon-induced transmembrane proteins (IFITM) 1-3 have been characterized as virus entry-restricting factors for many viruses. The present study identified IFITM3 as a novel protein-protein interaction (PPI) partner of NTCP based on membrane yeast-two hybrid and co-immunoprecipitation experiments. Surprisingly, IFITM3 knockdown significantly reduced in vitro HBV infection rates of NTCP-expressing HuH7 cells and primary human hepatocytes (PHHs). In addition, HuH7-NTCP cells showed significantly lower HDV infection rates, whereas infection with influenza A virus was increased. HBV-derived myr-preS1 peptide binding to HuH7-NTCP cells was intact even under IFITM3 knockdown, suggesting that IFITM3-mediated HBV/HDV infection enhancement occurs in a step subsequent to the viral attachment to NTCP. In conclusion, IFITM3 was identified as a novel NTCP co-factor that significantly affects in vitro infection with HBV and HDV in NTCP-expressing hepatoma cells and PHHs. While there is clear evidence for a direct PPI between IFITM3 and NTCP, the specific mechanism by which this PPI facilitates the infection process remains to be identified in future studies.

Impact of Pals1 on Expression and Localization of Transporters Belonging to the Solute Carrier Family.

Pals1 is part of the evolutionary conserved Crumbs polarity complex and plays a key role in two processes, the formation of apicobasal polarity and the establishment of cell-cell contacts. In the human kidney, up to 1.5 million nephrons control blood filtration, as well as resorption and recycling of inorganic and organic ions, sugars, amino acids, peptides, vitamins, water and further metabolites of endogenous and exogenous origin. All nephron segments consist of polarized cells and express high levels of Pals1. Mice that are functionally haploid for Pals1 develop a lethal phenotype, accompanied by heavy proteinuria and the formation of renal cysts. However, on a cellular level, it is still unclear if reduced cell polarization, incomplete cell-cell contact formation, or an altered Pals1-dependent gene expression accounts for the renal phenotype. To address this, we analyzed the transcriptomes of Pals1-haploinsufficient kidneys and the littermate controls by gene set enrichment analysis. Our data elucidated a direct correlation between TGFß pathway activation and the downregulation of more than 100 members of the solute carrier (SLC) gene family. Surprisingly, Pals1-depleted nephrons keep the SLC's segment-specific expression and subcellular distribution, demonstrating that the phenotype is not mainly due to dysfunctional apicobasal cell polarization of renal epithelia. Our data may provide first hints that SLCs may act as modulating factors for renal cyst formation.

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.

Anticancer Platinum Drugs Update.

The discovery of the anticancer properties of platinum derivatives by Rosenberg represents a milestone in the development of chemotherapeutic protocols for tumor treatment [...].

The role of cholesterol recognition (CARC/CRAC) mirror codes in the allosterism of the human organic cation transporter 2 (OCT2, SLC22A2).

The human organic cation transporter 2 (OCT2) is a multispecific transporter with cholesterol-dependent allosteric features. The present work elucidates the role of evolutionarily conserved cholesterol recognition/interaction amino acid consensus sequences (CRAC and CARC) in the allosteric binding to 1-methyl-4-phenylpyridinium (MPP+) in human embryonic kidney 293 cells stably or transiently expressing OCT2. Molecular blind simulations docked two mirroring cholesterol molecules in the 5th putative transmembrane domain, where a CARC and a CRAC sequence lie. The impact of the conserved amino acids that may constitute the CARC/CRAC mirror code was studied by alanine-scanning mutagenesis. At a saturating extracellular concentration of substrate, at which the impact of cholesterol depletion is maximal, five mutants transported MPP+ at a significantly lower rate than the wild-type OCT2 (WT), resembling the behavior of the WT upon cholesterol depletion. MPP+ influx rate as a function of the extracellular concentration of substrate was measured for the mutants R234A, R235A, L252A and R263A. R234A kinetic behavior was similar to that of the WT, whereas R235A, L252A and R263A activity shifted from allosteric to one-binding site kinetics, very much like the WT upon cholesterol depletion. The impact of cholesterol on protein thermal stability was assessed for WT, R234A and R263A. While the thermal stability of WT and R234A was improved by the supplementation with cholesterol, R263A was not sensitive to the presence of cholesterol. To conclude, the disruption of the CARC/CRAC mirror code in the 5th putative transmembrane domain is sufficient to abolish the allosteric interaction between OCT2 and MPP+.

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.