Unlocking mitochondrial drug targets: The importance of mitochondrial transport proteins.

A disturbed mitochondrial function contributes to the pathology of many common diseases. These organelles are therefore important therapeutic targets. On the contrary, many adverse effects of drugs can be explained by a mitochondrial off-target effect, in particular, due to an interaction with carrier proteins in the inner membrane. Yet this class of transport proteins remains underappreciated and understudied. The aim of this review is to provide a deeper understanding of the role of mitochondrial carriers in health and disease and their significance as drug targets. We present literature-based evidence that mitochondrial carrier proteins are associated with prevalent diseases and emphasize their potential as drug (off-)target sites by summarizing known mitochondrial drug-transporter interactions. Studying these carriers will enhance our knowledge of mitochondrial drug on- and off-targets and provide opportunities to further improve the efficacy and safety of drugs.

Statins affect human iPSC-derived cardiomyocytes by interfering with mitochondrial function and intracellular acidification.

Statins are effective drugs in reducing cardiovascular morbidity and mortality by inhibiting cholesterol synthesis. These effects are primarily beneficial for the patient's vascular system. A significant number of statin users suffer from muscle complaints probably due to mitochondrial dysfunction, a mechanism that has recently been elucidated. This has raised our interest in exploring the effects of statins on cardiac muscle cells in an era where the elderly and patients with poorer functioning hearts and less metabolic spare capacity start dominating our patient population. Here, we investigated the effects of statins on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-derived CMs). hiPSC-derived CMs were exposed to simvastatin, atorvastatin, rosuvastatin, and cerivastatin at increasing concentrations. Metabolic assays and fluorescent microscopy were employed to evaluate cellular viability, metabolic capacity, respiration, intracellular acidity, and mitochondrial membrane potential and morphology. Over a concentration range of 0.3-100 µM, simvastatin lactone and atorvastatin acid showed a significant reduction in cellular viability by 42-64%. Simvastatin lactone was the most potent inhibitor of basal and maximal respiration by 56% and 73%, respectively, whereas simvastatin acid and cerivastatin acid only reduced maximal respiration by 50% and 42%, respectively. Simvastatin acid and lactone and atorvastatin acid significantly decreased mitochondrial membrane potential by 20%, 6% and 3%, respectively. The more hydrophilic atorvastatin acid did not seem to affect cardiomyocyte metabolism. This calls for further research on the translatability to the clinical setting, in which a more conscientious approach to statin prescribing might be considered, especially regarding the current shift in population toward older patients with poor cardiac function.

Pyruvate dehydrogenase is a potential mitochondrial off-target for gentamicin based on in silico predictions and in vitro inhibition studies.

During the drug development process, organ toxicity leads to an estimated failure of one-third of novel chemical entities. Drug-induced toxicity is increasingly associated with mitochondrial dysfunction, but identifying the underlying molecular mechanisms remains a challenge. Computational modeling techniques have proven to be a good tool in searching for drug off-targets. Here, we aimed to identify mitochondrial off-targets of the nephrotoxic drugs tenofovir and gentamicin using different in silico approaches (KRIPO, ProBis and PDID). Dihydroorotate dehydrogenase (DHODH) and pyruvate dehydrogenase (PDH) were predicted as potential novel off-target sites for tenofovir and gentamicin, respectively. The predicted targets were evaluated in vitro, using (colorimetric) enzymatic activity measurements. Tenofovir did not inhibit DHODH activity, while gentamicin potently reduced PDH activity. In conclusion, the use of in silico methods appeared a valuable approach in predicting PDH as a mitochondrial off-target of gentamicin. Further research is required to investigate the contribution of PDH inhibition to overall renal toxicity of gentamicin.

Statins and Cardiomyocyte Metabolism, Friend or Foe?

Statins inhibit HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis, and are the cornerstone of lipid-lowering treatment. They significantly reduce cardiovascular morbidity and mortality. However, musculoskeletal symptoms are observed in 7 to 29 percent of all users. The mechanism underlying these complaints has become increasingly clear, but less is known about the effect on cardiac muscle function. Here we discuss both adverse and beneficial effects of statins on the heart. Statins exert pleiotropic protective effects in the diseased heart that are independent of their cholesterol-lowering activity, including reduction in hypertrophy, fibrosis and infarct size. Adverse effects of statins seem to be associated with altered cardiomyocyte metabolism. In this review we explore the differences in the mechanism of action and potential side effects of statins in cardiac and skeletal muscle and how they present clinically. These insights may contribute to a more personalized treatment strategy.

Mitochondrial complex III activity: from invasive muscle biopsies to patient-friendly buccal swab analysis.

Drug-induced mitochondrial dysfunction is a common adverse effect, particularly in case of statins-the most prescribed drugs worldwide. These drugs have been shown to inhibit complex III (CIII) of the mitochondrial oxidative phosphorylation process, which is related to muscle pain. As muscle pain is the most common complaint of statin users, it is crucial to distinguish it from other causes of myalgia to prevent unnecessary cessation of drug therapy. However, diagnosing CIII inhibition currently requires muscle biopsies, which are invasive and not practical for routine testing. Less invasive alternatives for measurement of mitochondrial complex activities are only available yet for complex I and IV. Here, we describe a non-invasive spectrophotometric method to determine CIII catalytic activities using buccal swabs, which we validated in a cohort of statin and non-statin users. Our data indicate that CIII can be reliably measured in buccal swabs, as evidenced by reproducible results above the detection limit. Further validation on a large-scale clinical setting is recommended.

Metabolic impact of genetic and chemical ADP/ATP carrier inhibition in renal proximal tubule epithelial cells.

Mitochondrial dysfunction is pivotal in drug-induced acute kidney injury (AKI), but the underlying mechanisms remain largely unknown. Transport proteins embedded in the mitochondrial inner membrane form a significant class of potential drug off-targets. So far, most transporter-drug interactions have been reported for the mitochondrial ADP/ATP carrier (AAC). Since it remains unknown to what extent AAC contributes to drug-induced mitochondrial dysfunction in AKI, we here aimed to better understand the functional role of AAC in the energy metabolism of human renal proximal tubular cells. To this end, CRISPR/Cas9 technology was applied to generate AAC3-/- human conditionally immortalized renal proximal tubule epithelial cells. This AAC3-/- cell model was characterized with respect to mitochondrial function and morphology. To explore whether this model could provide first insights into (mitochondrial) adverse drug effects with suspicion towards AAC-mediated mechanisms, wild-type and knockout cells were exposed to established AAC inhibitors, after which cellular metabolic activity and mitochondrial respiratory capacity were measured. Two AAC3-/- clones showed a significant reduction in ADP import and ATP export rates and mitochondrial mass, without influencing overall morphology. AAC3-/- clones exhibited reduced ATP production, oxygen consumption rates and metabolic spare capacity was particularly affected, mainly in conditions with galactose as carbon source. Chemical AAC inhibition was stronger compared to genetic inhibition in AAC3-/-, suggesting functional compensation by remaining AAC isoforms in our knockout model. In conclusion, our results indicate that ciPTEC-OAT1 cells have a predominantly oxidative phenotype that was not additionally activated by switching energy source. Genetic inhibition of AAC3 particularly impacted mitochondrial spare capacity, without affecting mitochondrial morphology, suggesting an important role for AAC in maintaining the metabolic spare respiration.

Prolonged Moderate-Intensity Exercise Does Not Increase Muscle Injury Markers in Symptomatic or Asymptomatic Statin Users.

BACKGROUND: Statin use may exacerbate exercise-induced skeletal muscle injury caused by reduced coenzyme Q10 (CoQ10) levels, which are postulated to produce mitochondrial dysfunction. OBJECTIVES: We determined the effect of prolonged moderate-intensity exercise on markers of muscle injury in statin users with and without statin-associated muscle symptoms. We also examined the association between leukocyte CoQ10 levels and muscle markers, muscle performance, and reported muscle symptoms. METHODS: Symptomatic (n = 35; age 62 ± 7 years) and asymptomatic statin users (n = 34; age 66 ± 7 years) and control subjects (n = 31; age 66 ± 5 years) walked 30, 40, or 50 km/d for 4 consecutive days. Muscle injury markers (lactate dehydrogenase, creatine kinase, myoglobin, cardiac troponin I, and N-terminal pro-brain natriuretic peptide), muscle performance, and reported muscle symptoms were assessed at baseline and after exercise. Leukocyte CoQ10 was measured at baseline. RESULTS: All muscle injury markers were comparable at baseline (P > 0.05) and increased following exercise (P < 0.001), with no differences in the magnitude of exercise-induced elevations among groups (P > 0.05). Muscle pain scores were higher at baseline in symptomatic statin users (P < 0.001) and increased similarly in all groups following exercise (P < 0.001). Muscle relaxation time increased more in symptomatic statin users than in control subjects following exercise (P = 0.035). CoQ10 levels did not differ among symptomatic (2.3 nmol/U; IQR: 1.8-2.9 nmol/U), asymptomatic statin users (2.1 nmol/U; IQR: 1.8-2.5 nmol/U), and control subjects (2.1 nmol/U; IQR: 1.8-2.3 nmol/U; P = 0.20), and did not relate to muscle injury markers, fatigue resistance, or reported muscle symptoms. CONCLUSIONS: Statin use and the presence of statin-associated muscle symptoms does not exacerbate exercise-induced muscle injury after moderate exercise. Muscle injury markers were not related to leukocyte CoQ10 levels. (Exercise-induced Muscle Damage in Statin Users; NCT05011643).

Impact of tyrosine kinase inhibitors on glucose control and insulin regulation in patients with chronic myeloid leukemia.

Treatment with tyrosine kinase inhibitors (TKIs), especially nilotinib, often results in hyperglycemia, which may further increase cardiovascular disease risk in patients with chronic myeloid leukemia (CML). The mechanism underlying the TKI-induced glucose dysregulation is not clear. TKIs are suggested to affect insulin secretion but also insulin sensitivity of peripheral tissue has been proposed to play a role in the pathogenesis of TKI-induced hyperglycemia. Here, we aimed to assess whether skeletal muscle glucose uptake and insulin responses are altered in nondiabetic patients with CML receiving TKI treatment. After a glycogen-depleted exercise bout, an intravenous glucose bolus (0.3 g/kg body weight) was administered to monitor 2-h glucose tolerance and insulin response in 14 patients with CML receiving nilotinib, 14 patients with CML receiving imatinib, and 14 non-CML age- and gender-matched controls. A dynamic [18F]-FDG PET scan during a hyperinsulinemic-euglycemic clamp was performed in a subgroup of 12 male patients with CML to assess m. quadriceps glucose uptake. We showed that patients with CML treated with nilotinib have an increased insulin response to intravenous glucose administration after muscle glycogen-depleted exercise. Despite the increased insulin response to glucose administration in patients with CML receiving nilotinib, glucose disappearance rates were significantly slower in nilotinib-treated patients when compared with controls in the first 15 min after glucose administration. Although [18F]-FDG uptake in m. quadriceps was not different, patients receiving nilotinib showed a trend toward decreased glucose infusion rates during euglycemic clamping when compared with patients receiving imatinib. Together, these findings indicate disturbed skeletal muscle glucose handling in patients with CML receiving nilotinib therapy.NEW & NOTEWORTHY In this study, we have shown that non-diabetic patients with CML receiving nilotinib therapy show early signs of disturbed skeletal muscle glucose handling, which was not observed in imatinib-treated patients. These observations in nilotinib users may reflect decreased muscle insulin sensitivity, which could serve as a potential target to counteract glycemic dysregulation, and is of clinical importance since these patients have an increased cardiovascular disease risk.

Time to Change: A Systems Pharmacology Approach to Disentangle Mechanisms of Drug-Induced Mitochondrial Toxicity.

An increasing number of commonly prescribed drugs are known to interfere with mitochondrial function, which is associated with almost half of all Food and Drug Administration black box warnings, a variety of drug withdrawals, and attrition of drug candidates. This can mainly be attributed to a historic lack of sensitive and specific assays to identify the mechanisms underlying mitochondrial toxicity during drug development. In the last decade, a better understanding of drug-induced mitochondrial dysfunction has been achieved by network-based and structure-based systems pharmacological approaches. Here, we propose the implementation of a tiered systems pharmacology approach to detect adverse mitochondrial drug effects during preclinical drug development, which is based on a toolset developed to study inherited mitochondrial disease. This includes phenotypic characterization, profiling of key metabolic alterations, mechanistic studies, and functional in vitro and in vivo studies. Combined with binding pocket similarity comparisons and bottom-up as well as top-down metabolic network modeling, this tiered approach enables identification of mechanisms underlying drug-induced mitochondrial dysfunction. After validation of these off-target mechanisms, drug candidates can be adjusted to minimize mitochondrial activity. Implementing such a tiered systems pharmacology approach could lead to a more efficient drug development trajectory due to lower drug attrition rates and ultimately contribute to the development of safer drugs. SIGNIFICANCE STATEMENT: Many commonly prescribed drugs adversely affect mitochondrial function, which can be detected using phenotypic assays. However, these methods provide only limited insight into the underlying mechanisms. In recent years, a better understanding of drug-induced mitochondrial dysfunction has been achieved by network-based and structure-based system pharmacological approaches. Their implementation in preclinical drug development could reduce the number of drug failures, contributing to safer drug design.

Restoring cellular NAD(P)H levels by PPARα and LXRα stimulation to improve mitochondrial complex I deficiency.

Mitochondrial complex I (CI), the first multiprotein enzyme complex of the oxidative phosphorylation system, plays a crucial role in cellular energy production. CI deficiency is associated with a variety of clinical phenotypes, including Leigh syndrome. At the cellular level, an increased NAD(P)H concentration is one of the hallmarks in CI-deficiency. AIMS: Here, we aimed to attenuate increased NAD(P)H levels by stimulation of ATP-dependent cassette (ABC)A1 and ABCG1-mediated cellular cholesterol efflux with various PPARα and LXRα agonists. MAIN METHODS: Mitochondrial CI-deficient fibroblasts and chemically-induced CI-deficient HeLa cells were used to study the dose-dependent effects of various PPARα and LXRα agonists on cellular NAD(P)H levels and cholesterol efflux. KEY FINDINGS: In patient-derived mitochondrial CI-deficient fibroblasts, GW590735, astaxanthin, oleoylethanolamide, and GW3965 significantly reduced the enhanced NAD(P)H levels in CI-deficient fibroblasts. Similar effects were observed in chemically-induced CI-impaired HeLa cells, in which BMS-687453, Wy14643, GW7647, T0901317, DMHCA also demonstrated a beneficial effect. Surprisingly, no effect on ABCA1- and ABCG1-mediated cholesterol efflux in HeLa cells and fibroblasts was found after treatment with these compounds. The reduction in NAD(P)H levels by GW590735 could be partially reversed by inhibition of fatty acid synthase and ß-oxidation, which suggests that its beneficial effects are possibly mediated via stimulation of fatty acid metabolism rather than cholesterol efflux. SIGNIFICANCE: Collectively, PPARα and LXRα stimulation resulted in attenuated cellular NAD(P)H levels in CI-impaired HeLa cells and patient-derived fibroblasts and could eventually have a therapeutic potential in CI deficiency.

Dissecting Drug-Induced Cytotoxicity and Metabolic Dysfunction in Conditionally Immortalized Human Proximal Tubule Cells.

Fourteen to 26 percent of all hospitalized cases of acute kidney injury are explained by drug-induced toxicity, emphasizing the importance of proper strategies to pre-clinically assess renal toxicity. The MTT assay is widely used as a measure of cell viability, but largely depends on cellular metabolic activity. Consequently, MTT as a single assay may not be the best way to assess cytotoxicity of compounds that reduce mitochondrial function and cellular metabolic activity without directly affecting cell viability. Accordingly, we aim to highlight the limitations of MTT alone in assessing renal toxicity of compounds that interfere with metabolic activity. Therefore, we compared toxic effects observed by MTT with a fluorescent assay that determines compromised plasma membrane permeability. Exposure of proximal tubule epithelial cells to nephrotoxic compounds reduced cellular metabolic activity concentration- and time-dependently. We show that compared to our fluorescence-based approach, assessment of cellular metabolic activity by means of MTT provides a composite readout of cell death and metabolic impairment. An approach independent of cellular metabolism is thus preferable when assessing cytotoxicity of compounds that induce metabolic dysfunction. Moreover, combining both assays during drug development enables a first discrimination between compounds having a direct or indirect mitochondrial toxic potential.

Key features of inhibitor binding to the human mitochondrial pyruvate carrier hetero-dimer.

OBJECTIVE: The mitochondrial pyruvate carrier (MPC) has emerged as a promising drug target for metabolic disorders, including non-alcoholic steatohepatitis and diabetes, metabolically dependent cancers and neurodegenerative diseases. A range of structurally diverse small molecule inhibitors have been proposed, but the nature of their interaction with MPC is not understood, and the composition of the functional human MPC is still debated. The goal of this study was to characterise the human MPC protein in vitro, to understand the chemical features that determine binding of structurally diverse inhibitors and to develop novel higher affinity ones. METHODS: We recombinantly expressed and purified human MPC hetero-complexes and studied their composition, transport and inhibitor binding properties by establishing in vitro transport assays, high throughput thermostability shift assays and pharmacophore modeling. RESULTS: We determined that the functional unit of human MPC is a hetero-dimer. We compared all different classes of MPC inhibitors to find that three closely arranged hydrogen bond acceptors followed by an aromatic ring are shared characteristics of all inhibitors and represent the minimal requirement for high potency. We also demonstrated that high affinity binding is not attributed to covalent bond formation with MPC cysteines, as previously proposed. Following the basic pharmacophore properties, we identified 14 new inhibitors of MPC, one outperforming compound UK5099 by tenfold. Two are the commonly prescribed drugs entacapone and nitrofurantoin, suggesting an off-target mechanism associated with their adverse effects. CONCLUSIONS: This work defines the composition of human MPC and the essential MPC inhibitor characteristics. In combination with the functional assays we describe, this new understanding will accelerate the development of clinically relevant MPC modulators.

Ndufs4 knockout mouse models of Leigh syndrome: pathophysiology and intervention.

Mitochondria are small cellular constituents that generate cellular energy (ATP) by oxidative phosphorylation (OXPHOS). Dysfunction of these organelles is linked to a heterogeneous group of multisystemic disorders, including diabetes, cancer, ageing-related pathologies and rare mitochondrial diseases. With respect to the latter, mutations in subunit-encoding genes and assembly factors of the first OXPHOS complex (complex I) induce isolated complex I deficiency and Leigh syndrome. This syndrome is an early-onset, often fatal, encephalopathy with a variable clinical presentation and poor prognosis due to the lack of effective intervention strategies. Mutations in the nuclear DNA-encoded NDUFS4 gene, encoding the NADH:ubiquinone oxidoreductase subunit S4 (NDUFS4) of complex I, induce 'mitochondrial complex I deficiency, nuclear type 1' (MC1DN1) and Leigh syndrome in paediatric patients. A variety of (tissue-specific) Ndufs4 knockout mouse models were developed to study the Leigh syndrome pathomechanism and intervention testing. Here, we review and discuss the role of complex I and NDUFS4 mutations in human mitochondrial disease, and review how the analysis of Ndufs4 knockout mouse models has generated new insights into the MC1ND1/Leigh syndrome pathomechanism and its therapeutic targeting.

Characterization of drug-induced human mitochondrial ADP/ATP carrier inhibition.

An increasing number of commonly prescribed drugs are known to interfere with mitochondrial function, causing cellular toxicity, but the underlying mechanisms are largely unknown. Although often not considered, mitochondrial transport proteins form a significant class of potential mitochondrial off-targets. So far, most drug interactions have been reported for the mitochondrial ADP/ATP carrier (AAC), which exchanges cytosolic ADP for mitochondrial ATP. Here, we show inhibition of cellular respiratory capacity by only a subset of the 18 published AAC inhibitors, which questions whether all compound do indeed inhibit such a central metabolic process. This could be explained by the lack of a simple, direct model system to evaluate and compare drug-induced AAC inhibition. Methods: For its development, we have expressed and purified human AAC1 (hAAC1) and applied two approaches. In the first, thermostability shift assays were carried out to investigate the binding of these compounds to human AAC1. In the second, the effect of these compounds on transport was assessed in proteoliposomes with reconstituted human AAC1, enabling characterization of their inhibition kinetics. Results: Of the proposed inhibitors, chebulinic acid, CD-437 and suramin are the most potent with IC50-values in the low micromolar range, whereas another six are effective at a concentration of 100 µM. Remarkably, half of all previously published AAC inhibitors do not show significant inhibition in our assays, indicating that they are false positives. Finally, we show that inhibitor strength correlates with a negatively charged surface area of the inhibitor, matching the positively charged surface of the substrate binding site. Conclusion: Consequently, we have provided a straightforward model system to investigate AAC inhibition and have gained new insights into the chemical compound features important for inhibition. Better evaluation methods of drug-induced inhibition of mitochondrial transport proteins will contribute to the development of drugs with an enhanced safety profile.

Stimulation of cholesterol biosynthesis in mitochondrial complex I-deficiency lowers reductive stress and improves motor function and survival in mice.

The majority of cellular energy is produced by the mitochondrial oxidative phosphorylation (OXPHOS) system. Failure of the first OXPHOS enzyme complex, NADH:ubiquinone oxidoreductase or complex I (CI), is associated with multiple signs and symptoms presenting at variable ages of onset. There is no approved drug treatment yet to slow or reverse the progression of CI-deficient disorders. Here, we present a comprehensive human metabolic network model of genetically characterized CI-deficient patient-derived fibroblasts. Model calculations predicted that increased cholesterol production, export, and utilization can counterbalance the surplus of reducing equivalents in patient-derived fibroblasts, as these pathways consume considerable amounts of NAD(P)H. We show that fibrates attenuated increased NAD(P)H levels and improved CI-deficient fibroblast growth by stimulating the production of cholesterol via enhancement of its cellular efflux. In CI-deficient (Ndufs4-/-) mice, fibrate treatment resulted in prolonged survival and improved motor function, which was accompanied by an increased cholesterol efflux from peritoneal macrophages. Our results shine a new light on the use of compensatory biological pathways in mitochondrial dysfunction, which may lead to novel therapeutic interventions for mitochondrial diseases for which currently no cure exists.

Effects of clofibrate and KH176 on life span and motor function in mitochondrial complex I-deficient mice.

Mitochondrial complex I (CI), the first multiprotein enzyme complex of the OXPHOS system, executes a major role in cellular ATP generation. Consequently, dysfunction of this complex has been linked to inherited metabolic disorders, including Leigh disease (LD), an often fatal disease in early life. Development of clinical effective treatments for LD remains challenging due to the complex pathophysiological nature. Treatment with the peroxisome proliferation-activated receptor (PPAR) agonist bezafibrate improved disease phenotype in several mitochondrial disease mouse models mediated via enhanced mitochondrial biogenesis and fatty acid ß-oxidation. However, the therapeutic potential of this mixed PPAR (α, δ/ß, γ) agonist is severely hampered by hepatotoxicity, which is possibly caused by activation of PPARγ. Here, we aimed to investigate the effects of the PPARα-specific fibrate clofibrate in mitochondrial CI-deficient (Ndufs4-/-) mice. Clofibrate increased lifespan and motor function of Ndufs4-/- mice, while only marginal hepatotoxic effects were observed. Due to the complex clinical and cellular phenotype of CI-deficiency, we also aimed to investigate the therapeutic potential of clofibrate combined with the redox modulator KH176. As described previously, single treatment with KH176 was beneficial, however, combining clofibrate with KH176 did not result in an additive effect on disease phenotype in Ndufs4-/- mice. Overall, both drugs have promising, but independent and nonadditive, properties for the pharmacological treatment of CI-deficiency-related mitochondrial diseases.

Safety of drug use in patients with a primary mitochondrial disease: An international Delphi-based consensus.

Clinical guidance is often sought when prescribing drugs for patients with primary mitochondrial disease. Theoretical considerations concerning drug safety in patients with mitochondrial disease may lead to unnecessary withholding of a drug in a situation of clinical need. The aim of this study was to develop consensus on safe medication use in patients with a primary mitochondrial disease. A panel of 16 experts in mitochondrial medicine, pharmacology, and basic science from six different countries was established. A modified Delphi technique was used to allow the panellists to consider draft recommendations anonymously in two Delphi rounds with predetermined levels of agreement. This process was supported by a review of the available literature and a consensus conference that included the panellists and representatives of patient advocacy groups. A high level of consensus was reached regarding the safety of all 46 reviewed drugs, with the knowledge that the risk of adverse events is influenced both by individual patient risk factors and choice of drug or drug class. This paper details the consensus guidelines of an expert panel and provides an important update of previously established guidelines in safe medication use in patients with primary mitochondrial disease. Specific drugs, drug groups, and clinical or genetic conditions are described separately as they require special attention. It is important to emphasise that consensus-based information is useful to provide guidance, but that decisions related to drug prescribing should always be tailored to the specific needs and risks of each individual patient. We aim to present what is current knowledge and plan to update this regularly both to include new drugs and to review those currently included.

Flow stimulates drug transport in a human kidney proximal tubule-on-a-chip independent of primary cilia.

BACKGROUND: Kidney disease modeling and assessment of drug-induced kidney injury can be advanced using three-dimensional (3D) microfluidic models that recapitulate in vivo characteristics. Fluid shear stress (FSS) has been depicted as main modulator improving in vitro physiology in proximal tubule epithelial cells (PTECs). We aimed to elucidate the role of FSS and primary cilia on transport activity and morphology in PTECs. METHODS: Human conditionally immortalized PTEC (ciPTEC-parent) was cultured in a microfluidic 3D device, the OrganoPlate, under a physiological peak FSS of 2.0 dyne/cm2 or low peak FSS of 0.5 dyne/cm2. Upon a 9-day exposure to FSS, albumin-FITC uptake, activity of P-glycoprotein (P-gp) and multidrug resistance-associated proteins 2/4 (MRP2/4), cytotoxicity and cell morphology were determined. RESULTS: A primary cilium knock-out cell model, ciPTEC-KIF3α-/-, was successfully established via CRISPR-Cas9 genome editing. Under physiological peak FSS, albumin-FITC uptake (p = .04) and P-gp efflux (p = .002) were increased as compared to low FSS. Remarkably, a higher albumin-FITC uptake (p = .03) and similar trends in activity of P-gp and MRP2/4 were observed in ciPTEC-KIF3α-/-. FSS induced cell elongation corresponding with the direction of flow in both cell models, but had no effect on cyclosporine A-induced cytotoxicity. CONCLUSIONS: FSS increased albumin uptake, P-gp efflux and cell elongation, but this was not attributed to a mechanosensitive mechanism related to primary cilia in PTECs, but likely to microvilli present at the apical membrane. GENERAL SIGNIFICANCE: FSS-induced improvements in biological characteristics and activity in PTECs was not mediated through a primary cilium-related mechanism.

Brothers in Arms: ABCA1- and ABCG1-Mediated Cholesterol Efflux as Promising Targets in Cardiovascular Disease Treatment.

Atherosclerosis is a leading cause of cardiovascular disease worldwide, and hypercholesterolemia is a major risk factor. Preventive treatments mainly focus on the effective reduction of low-density lipoprotein cholesterol, but their therapeutic value is limited by the inability to completely normalize atherosclerotic risk, probably due to the disease complexity and multifactorial pathogenesis. Consequently, high-density lipoprotein cholesterol gained much interest, as it appeared to be cardioprotective due to its major role in reverse cholesterol transport (RCT). RCT facilitates removal of cholesterol from peripheral tissues, including atherosclerotic plaques, and its subsequent hepatic clearance into bile. Therefore, RCT is expected to limit plaque formation and progression. Cellular cholesterol efflux is initiated and propagated by the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1. Their expression and function are expected to be rate-limiting for cholesterol efflux, which makes them interesting targets to stimulate RCT and lower atherosclerotic risk. This systematic review discusses the molecular mechanisms relevant for RCT and ABCA1 and ABCG1 function, followed by a critical overview of potential pharmacological strategies with small molecules to enhance cellular cholesterol efflux and RCT. These strategies include regulation of ABCA1 and ABCG1 expression, degradation, and mRNA stability. Various small molecules have been demonstrated to increase RCT, but the underlying mechanisms are often not completely understood and are rather unspecific, potentially causing adverse effects. Better understanding of these mechanisms could enable the development of safer drugs to increase RCT and provide more insight into its relation with atherosclerotic risk. SIGNIFICANCE STATEMENT: Hypercholesterolemia is an important risk factor of atherosclerosis, which is a leading pathological mechanism underlying cardiovascular disease. Cholesterol is removed from atherosclerotic plaques and subsequently cleared by the liver into bile. This transport is mediated by high-density lipoprotein particles, to which cholesterol is transferred via ATP-binding cassette transporters ABCA1 and ABCG1. Small-molecule pharmacological strategies stimulating these transporters may provide promising options for cardiovascular disease treatment.