The tyrosine kinase inhibitor Nintedanib induces lysosomal dysfunctionality: Role of protonation-dependent crystallization processes.

Nintedanib (NIN), a multi-tyrosine kinase inhibitor clinically approved for idiopathic pulmonary fibrosis and lung cancer, is characterized by protonation-dependent lysosomotropic behavior and appearance of lysosome-specific fluorescence emission properties. Here we investigate whether spontaneous formation of a so far unknown NIN matter within the acidic cell compartment is underlying these unexpected emissive properties and investigate the consequences on lysosome functionality. Lysosomes of cells treated with NIN, but not non-protonatable NIN derivatives, exhibited lysosome-associated birefringence signals co-localizing with the NIN-derived fluorescence emission. Sensitivity of both parameters towards vATPase inhibitors confirmed pH-dependent, spontaneous adoption of novel crystalline NIN structures in lysosomes. Accordingly, NIN crystallization from buffer solutions resulted in formation of multiple crystal polymorphs with pH-dependent fluorescence properties. Cell-free crystals grown at lysosomal-like pH conditions resembled NIN-treated cell lysosomes concerning fluorescence pattern, photobleaching dynamics, and Raman spectra. However, differences in birefringence intensity and FAIM-determined anisotropy, as well as predominant association with (intra)lysosomal membrane structures, suggested formation of a semi-solid NIN crystalline matter in acidic lysosomes. Despite comparable target kinase inhibition, NIN, but not its non-protonatable derivatives, impaired lysosomal functionality, mediated massive cell vacuolization, enhanced autophagy, deregulated lipid metabolism, and induced atypical phospholipidosis. Moreover, NIN exerted distinct phototoxicity, strictly dependent on lysosomal microcrystallization events. The spontaneous formation of NIN crystalline structures was also observable in the gut mucosa of orally NIN-treated mice. Summarizing, the here-described kinase inhibition-independent impact of NIN on lysosomal functionality mediates several of its cell biological activities and might contribute to NIN adverse effects.

The role of lysosomal phospholipase A2 in the catabolism of bis(monoacylglycerol)phosphate and association with phospholipidosis.

Bis(monoacylglycerol)phosphate (BMP) is an acidic glycerophospholipid localized to late endosomes and lysosomes. However, the metabolism of BMP is poorly understood. Because many drugs that cause phospholipidosis inhibit lysosomal phospholipase A2 (LPLA2, PLA2G15, LYPLA3) activity, we investigated whether this enzyme has a role in BMPcatabolism. The incubation of recombinant human LPLA2 (hLPLA2) and liposomes containing the naturally occurring BMP (sn-(2-oleoyl-3-hydroxy)-glycerol-1-phospho-sn-1'-(2'-oleoyl-3'-hydroxy)-glycerol (S,S-(2,2',C18:1)-BMP) resulted in the deacylation of this BMP isomer. The deacylation rate was 70 times lower than that of dioleoyl phosphatidylglycerol (DOPG), an isomer and precursor of BMP. The release rates of oleic acid from DOPG and four BMP stereoisomers by LPLA2 differed. The rank order of the rates of hydrolysis were DOPG>S,S-(3,3',C18:1)-BMP>R,S-(3,1',C18:1)-BMP>R,R-(1,1',C18:1)>S,S-(2,2')-BMP. The cationic amphiphilic drug amiodarone (AMD) inhibited the deacylation of DOPG and BMP isomers by hLPLA2 in a concentration-dependent manner. Under these experimental conditions, the IC50s of amiodarone-induced inhibition of the four BMP isomers and DOPG were less than 20 µM and approximately 30 µM, respectively. BMP accumulation was observed in AMD-treated RAW 264.7 cells. The accumulated BMP was significantly reduced by exogenous treatment of cells with active recombinant hLPLA2 but not with diisopropylfluorophosphate-inactivated recombinant hLPLA2. Finally, a series of cationic amphiphilic drugs known to cause phospholipidosis were screened for inhibition of LPLA2 activity as measured by either the transacylation or fatty acid hydrolysis of BMP or phosphatidylcholine as substrates. Fifteen compounds demonstrated significant inhibition with IC50s ranging from 6.8 to 63.3 µM. These results indicate that LPLA2 degrades BMP isomers with different substrate specificities under acidic conditions and may be the key enzyme associated with BMP accumulation in drug-induced phospholipidosis.

Double Hit of Hydroxichloroquine and Amiodarone Induced Renal Phospholipidosis in a Patient with Monoclonal Gammopathy and Sclerodermiform Syndrome: A Case Report and Review of the Literature.

Phospholipidosis is a rare disorder which consists of an excessive intracellular accumulation of phospholipids and the appearance of zebra bodies or lamellar bodies when looking at them using electron microscopy. This disease is associated with certain genetic diseases or is secondary to drugs or toxins. Drug-induced phospholipidosis encompasses many types of pharmaceuticals, most notably chloroquine, amiodarone or ciprofloxacin. Clinically and histologically, renal involvement can be highly variable, with the diagnosis not being made until the zebra bodies are seen under an electron microscope. These findings may require genetic testing to discount Fabry disease, as its histological findings are indistinguishable. Most of the chemicals responsible are cationic amphiphilic drugs, and several mechanisms have been hypothesized for the formation of zebra bodies and their pathogenic significance. However, the relationship between drug toxicity and phospholipid accumulation, zebra bodies and organ dysfunction remains enigmatic, as do the renal consequences of drug withdrawal. We present, to our knowledge, the first case report of acute renal injury with a monoclonal gammopathy of renal significance, lesions, and sclerodermiform syndrome, with zebra bodies that were associated with the initiation of a hydroxychloroquine and amiodarone treatment, as an example of drug-induced-phospholipidosis.

Construction of Discrimination Models of Cationic Drugs for Phospholipidosis Induction Potential by Using Interaction Data with Immobilized Artificial Membrane as Well as Physicochemical Properties.

Accurate prediction of the phospholipidosis-induction risk of drugs at early stages is important in drug development. So far, discrimination models for predicting the induction risk of cationic drugs have been proposed, but it is still challenging to accurately predict the risk of cationic drugs with intermediate hydrophobicity (logP). In this study, we introduced a parameter (Δlogk40) reflecting not only hydrophobic interaction but also interactions with the polar headgroup between cationic drugs and phospholipids, obtained with liquid chromatography using an immobilized artificial membrane column. The parameter was used along with other physicochemical properties as features to construct discrimination models. Linear discriminant analysis, the modified Mahalanobis discriminant analysis, support vector machine, and random forest were employed for model construction. The results showed that all discrimination models exhibited good predictive performance, with the modified Mahalanobis discriminant analysis and random forest providing the best results for cationic drugs, suggesting that the usefulness of the parameter reflecting complex interactions between cationic drugs and immobilized artificial membrane for constructing discrimination models to predict the induction risk. Furthermore, by applying the parameter as a feature in constructing discrimination models, we demonstrated an improvement in the predictive performance for drugs with intermediate hydrophobicity.

Hydroxychloroquine-Induced Phospholipidosis - A Forgotten Complication of a Common Drug.

Hydroxychloroquine (HCQ) has immunomodulatory and immunosuppressive properties and is used in many rheumatological conditions like systemic lupus erythematosus, rheumatoid arthritis, and Sjogren's syndrome. It is usually a widely used and well-tolerated DMARD (Disease Modifying Anti Rheumatic Drugs). Its most feared toxicities include retinopathy and, rarely, cardiomyopathy. Among its other reported side effects is drug-induced phospholipidosis. Here, we report two cases of HCQ-induced phospholipidosis based on renal biopsy electron microscopy. HCQ-induced phospholipidosis, although uncommon, must be considered as one of the differentials in a patient with persistent proteinuria.

Does administration of hydroxychloroquine/amiodarone affect the efficacy of enzyme replacement therapy for Fabry mice?

As a standard therapy for Fabry disease, enzyme replacement therapy (ERT) with recombinant human α-galactosidase A (α-Gal) has been successfully used, and the instructions for this drug state that "it should not be co-administrated with cationic amphiphilic drugs such as hydroxychloroquine (HCQ) and amiodarone (AMI), since these drugs have the potential to inhibit intracellular α-Gal activity". However, there would be cases in which HCQ or AMI is required for patients with Fabry disease, considering their medical efficacy and application. Thus, we examined the impact of HCQ/AMI on recombinant human α-Gal by in vitro, cellular, and animal experiments. The results revealed that HCQ/AMI affected the enzyme activity of α-Gal incorporated into cultured fibroblasts from a Fabry mouse when the cells were cultured in medium containing these drugs and the enzyme, although their direct inhibitory effect on the enzyme is not strong. These lysosomotropic drugs may be trapped and concentrated in lysosomes, followed by inhibition of α-Gal. On the other hand, no reduction of α-Gal activity incorporated into the organs and tissues, or acceleration of glycoshingolipid accumulation was observed in Fabry mice co-administered with HCQ/AMI and the enzyme, compared with in the case of usual ERT. As HCQ/AMI administered are catabolized in the liver, these drugs possibly do not affect ERT for Fabry mice, different from in the case of cultured cells in an environment isolated from the surroundings.

Uncovering the toxicity mechanisms of a series of carboxylic acids in liver cells through computational and experimental approaches.

Hepatotoxicity poses a significant concern in drug design due to the potential liver damage that can be caused by new drugs. Among common manifestations of hepatotoxic damage is lipid accumulation in hepatic tissue, resulting in liver steatosis or phospholipidosis. Carboxylic derivatives are prone to interfere with fatty acid metabolism and cause lipid accumulation in hepatocytes. This study investigates the toxic behaviour of 24 structurally related carboxylic acids in hepatocytes, specifically their ability to cause accumulation of fatty acids and phospholipids. Using high-content screening (HCS) assays, we identified two distinct lipid accumulation patterns. Subsequently, we developed structure-activity relationship (SAR) and quantitative structure-activity relationship (QSAR) models to determine relevant molecular substructures and descriptors contributing to these adverse effects. Additionally, we calculated physicochemical properties associated with lipid accumulation in hepatocytes and examined their correlation with our chemical structure characteristics. To assess the applicability of our findings to a wide range of chemical compounds, we employed two external datasets to evaluate the distribution of our QSAR descriptors. Our study highlights the significance of subtle molecular structural variations in triggering hepatotoxicity, such as the presence of nitrogen or the specific arrangement of substitutions within the carbon chain. By employing our comprehensive approach, we pinpointed specific molecules and elucidated their mechanisms of toxicity, thus offering valuable insights to guide future toxicology investigations.

Livogrit prevents Amiodarone-induced toxicity in experimental model of human liver (HepG2) cells and Caenorhabditis elegans by regulating redox homeostasis.

Treatment with cationic amphiphilic drugs like Amiodarone leads to development of phospholipidosis, a type of lysosomal storage disorder characterized by excessive deposition of phospholipids. Such disorder in liver enhances accumulation of drugs and its metabolites, and dysregulates lipid profiles, which subsequently leads to hepatotoxicity. In the present study, we assessed pharmacological effects of herbal medicine, Livogrit, against hepatic phospholipidosis-induced toxicity. Human liver (HepG2) cells and in vivo model of Caenorhabditis elegans (N2 and CF1553 strains) were used to study effect of Livogrit on Amiodarone-induced phospholipidosis. In HepG2 cells, Livogrit treatment displayed enhanced uptake of acidic pH-based stains and reduced phospholipid accumulation, oxidative stress, AST, ALT, cholesterol levels, and gene expression of SCD-1 and LSS. Protein levels of LPLA2 were also normalized. Livogrit treatment restored Pgp functionality which led to decreased cellular accumulation of Amiodarone as observed by UHPLC analysis. In C. elegans, Livogrit prevented ROS generation, fat-6/7 gene overexpression, and lysosomal trapping of Amiodarone in N2 strain. SOD-3::GFP expression in CF1553 strain normalized by Livogrit treatment. Livogrit regulates phospholipidosis by regulation of redox homeostasis, phospholipid anabolism, and Pgp functionality hindered by lysosomal trapping of Amiodarone. Livogrit could be a potential therapeutic intervention for amelioration of drug-induced phospholipidosis and prevent hepatotoxicity.

Oxysterols in Cell Viability, Phospholipidosis and Extracellular Vesicles Production in a Lung Cancer Model.

The study carried out systematic research on the influence of selected oxysterols on cells viability, phospholipidosis and the level of secreted extracellular vesicles. Three oxidized cholesterol derivatives, namely 7α-hydroxycholesterol (7α-OH), 7- ketocholesterol (7-K) and 24(S)-hydroxycholesterol (24(S)-OH) were tested in three different concentrations: 50 µM, 100 µM and 200 µM for 24 h incubation with A549 lung cancer cell line. All the studied oxysterols were found to alter cells viability. The lowest survival rate of the cells was observed after 24 h of 7-K treatment, slightly better for 7α-OH while cells incubated with 24(S)-OH had the best survival rate among the oxysterols used. 7-K increased phospholipids accumulation in cells, however, most noticeable effect was noticed for 24(S)-OH. Changes in the level of extracellular vesicles secreted in cells culture after the treatment with oxysterols were also observed. It was found that all oxysterols used increased the level of secreted vesicles, both exosomes and ectosomes. The strongest effect was noticed for 24(S)-OH. Taken together, these results suggest that 7-K is the most potent inducer of cancer cell death, while 7α-OH is slightly less potent in this respect. The lower cytotoxic effect of 24(S)-OH correlates with greater phospholipids accumulation, extracellular vesicles production and better cells survival.

Hydroxychloroquine-Induced Renal Phospholipidosis: Case Report and Review of Differential Diagnoses.

Introduction: Renal phospholipidosis describes the accumulation of phospholipids in the lysosomes of kidney cells, in particular podocytes. Originally, this was described primarily in the context of the lysosomal storage disorder Fabry disease. It is now known that a variety of drugs can lead to the accumulation of lysosomal phospholipids. Case Presentation: We present the case of a 69-year-old female patient suffering chronic kidney disease and systemic lupus erythematosus who underwent a kidney biopsy because of a further increase in serum creatinine levels. There was no evidence of lupus nephritis, but electron microscopy showed zebra bodies as a morphological sign of phospholipidosis. This was most likely drug-induced after 25 years of continuous medication with hydroxychloroquine. A renal biopsy 2 years and 6 months earlier, when the renal function of the patient was distinctively better, showed no signs of renal phospholipidosis. Afterward, medication with hydroxychloroquine was discontinued, and renal function parameters remained stable in the 1-year course. Conclusion: This case raises the question of how severely impaired renal function affects the risk of hydroxychloroquine-induced renal phospholipidosis and underlines that hydroxychloroquine should be administered with caution in patients with kidney insufficiency. Moreover, we provide a review of the causes of renal phospholipidosis, which have been described in the literature and give an overview of possible differential diagnoses in cases with histologically proven phospholipidosis in renal biopsies.

AMALPHI: A Machine Learning Platform for Predicting Drug-Induced PhospholIpidosis.

Drug-induced phospholipidosis (PLD) involves the accumulation of phospholipids in cells of multiple tissues, particularly within lysosomes, and it is associated with prolonged exposure to druglike compounds, predominantly cationic amphiphilic drugs (CADs). PLD affects a significant portion of drugs currently in development and has recently been proven to be responsible for confounding antiviral data during drug repurposing for SARS-CoV-2. In these scenarios, it has become crucial to identify potential safe drug candidates in advance and distinguish them from those that may lead to false in vitro antiviral activity. In this work, we developed a series of machine learning classifiers with the aim of predicting the PLD-inducing potential of drug candidates. The models were built on a high-quality chemical collection comprising 545 curated small molecules extracted from ChEMBL v30. The most effective model, obtained using the balanced random forest algorithm, achieved high performance, including an AUC value computed in validation as high as 0.90. The model was made freely available through a user-friendly web platform named AMALPHI (https://www.ba.ic.cnr.it/softwareic/amalphiportal/), which can represent a valuable tool for medicinal chemists interested in conducting an early evaluation of PLD inducer potential.

A machine learning and live-cell imaging tool kit uncovers small molecules induced phospholipidosis.

Drug-induced phospholipidosis (DIPL), characterized by excessive accumulation of phospholipids in lysosomes, can lead to clinical adverse effects. It may also alter phenotypic responses in functional studies using chemical probes. Therefore, robust methods are needed to predict and quantify phospholipidosis (PL) early in drug discovery and in chemical probe characterization. Here, we present a versatile high-content live-cell imaging approach, which was used to evaluate a chemogenomic and a lysosomal modulation library. We trained and evaluated several machine learning models using the most comprehensive set of publicly available compounds and interpreted the best model using SHapley Additive exPlanations (SHAP). Analysis of high-quality chemical probes extracted from the Chemical Probes Portal using our algorithm revealed that closely related molecules, such as chemical probes and their matched negative controls can differ in their ability to induce PL, highlighting the importance of identifying PL for robust target validation in chemical biology.

Case report of progressive renal dysfunction as a consequence of amiodarone-induced phospholipidosis.

Background: Amiodarone is associated with a range of unwanted effects on pulmonary, thyroid, and liver function. However, the nephrotoxic side effect caused by renal phospholipidosis has hardly received any attention up to now. Case summary: This is a case of an 86-year-old Caucasian male with an acute on chronic kidney disease 4 months after the initiation of amiodarone. A renal biopsy demonstrated the intracellular accumulation of phospholipids that have previously been demonstrated in association with organ dysfunction because of amiodarone use. Serum creatinine levels subsequently improved from 388 to 314 µmol/L after stopping amiodarone over the course of 2 months. Discussion: In this case, a diagnosis of partially reversible acute on chronic kidney disease caused by lysosomal phospholipidosis due to amiodarone use was deemed highly likely. Lysosomal dysfunction leads to the accumulation of intra-lysosomal phospholipids (phospholipidosis). This accumulation is accompanied by progressive organ damage and dysfunction, including renal dysfunction, in rare instances. Guidelines advise regular surveillance for liver, lung, and thyroid toxicity during amiodarone treatment but do not mention the potential for renal toxicity. This case suggests that it might be prudent to include screening for renal toxicity in this surveillance.

Drug-induced phospholipidosis is not correlated with the inhibition of SARS-CoV-2 - inhibition of SARS-CoV-2 is cell line-specific.

Recently, Tummino et al. reported that 34 compounds, including Chloroquine and Fluoxetine, inhibit SARS-CoV-2 replication by inducing phospholipidosis, although Chloroquine failed to suppress viral replication in Calu-3 cells and patients. In contrast, Fluoxetine represses viral replication in human precision-cut lung slices (PCLS) and Calu-3 cells. Thus, it is unlikely that these compounds have similar mechanisms of action. Here, we analysed a subset of these compounds in the viral replication and phospholipidosis assays using the Calu-3 cells and PCLS as the patient-near system. Trimipramine and Chloroquine induced phospholipidosis but failed to inhibit SARS-CoV-2 replication in Calu-3 cells, which contradicts the reported findings and the proposed mechanism. Fluoxetine, only slightly induced phospholipidosis in Calu-3 cells but reduced viral replication by 2.7 orders of magnitude. Tilorone suppressed viral replication by 1.9 orders of magnitude in Calu-3 cells without causing phospholipidosis. Thus, induction of phospholipidosis is not correlated with the inhibition of SARS-CoV-2, and the compounds act via other mechanisms. However, we show that compounds, such as Amiodarone, Tamoxifen and Tilorone, with antiviral activity on Calu-3 cells, also inhibited viral replication in human PCLS. Our results indicate that antiviral assays against SARS-CoV-2 are cell-line specific. Data from Vero E6 can lead to non-transferable results, underlining the importance of an appropriate cell system for analysing antiviral compounds against SARS-CoV-2. We observed a correlation between the active compounds in Calu-3 cells and PCLS.

Lysosomal trapping of 4-dimethylamino-1-{3-(1-methyl-1H-imidazole-2-yl)propanoyl}piperidine, a hydrophilic and weakly basic amine, in human aortic vascular smooth muscle cells.

Some weakly basic compounds lead to cell death accompanied by cellular vacuolation. The novel analgesic agent, 4-dimethylamino-1-{3-(1-methyl-1H-imidazole-2-yl)propanoyl}piperidine (DMIP), is a hydrophilic and weakly basic compound that induces vacuolation in the vascular smooth muscle cells in dogs. Here, we investigated the vacuolation mechanism and the potential cytotoxicity of DMIP using human aortic vascular smooth muscle cells. When cells were treated with DMIP (0.1, 0.3, and 1 mM) for 6, 24, and 48 h, clear cytoplasmic vacuolation was observed at 1 mM after 24 and 48 h, along with an increase in the intracellular DMIP concentration. The vacuolation and intracellular DMIP were markedly reduced by bafilomycin A1, a vacuolar H+-ATPase inhibitor. The late endosome marker Rab7 and lysosome marker LAMP-2 were highly expressed but the early endosome marker Rab5 and autophagosome marker LC3 were not expressed specifically on the vacuolar membranes. These results suggested that the most vacuoles were enlarged late endosomes/lysosomes, resulting from the accumulation of DMIP by ion trapping. Moreover, DMIP did not affect lysosomal membrane integrity and was less cytotoxic than chloroquine, an inducer of phospholipidosis. The current study provides further insight into the mechanisms of vacuolation and lysosomal trapping induced by the hydrophilic and weakly basic amine DMIP.

Kidney Podocyte Zebra Bodies after Lung Transplantation for Lymphangioleiomyomatosis.

A 55-year-old woman showed progressive renal dysfunction after unilateral deceased-donor lung transplantation for lymphangioleiomyomatosis. A kidney biopsy showed a striped pattern of interstitial fibrosis, suggesting calcineurin inhibitor toxicity, and zebra body accumulation predominantly in the podocytes, characteristics of Fabry disease. Nevertheless, she had no extra-renal symptoms of the disease, and gene testing identified no known pathogenic variant or exon deletion. Our case report and literature review suggest that this atypical lysosomal inclusion may be phospholipidosis induced by sertraline. Potential underlying etiologies linking zebra body deposits may be not only hereditary but also drug-induced phospholipidosis.

Lamellar Bodies in Podocytes Associated With Compound Heterozygous Mutations for Niemann Pick Type C1 Mimicking Fabry Disease, a Case Report.

Rationale: Niemann-Pick type C (NPC) is an autosomal recessive lysosomal storage disease (LSD) caused by mutations in NPC1 or NPC2 genes. Mutations result in abnormal cholesterol trafficking, which is manifested by abnormal cholesterol and glycosphingolipid accumulation in lysosomes of various cells. Presenting Concerns of the Patient: The patient had a history of hyperlipidemia, hypertension, depression, and elevated alkaline phosphatase and initially presented for a workup regarding chronic kidney disease stage G3b/A3 with proteinuria of 1.9 g/day. Diagnosis: Kidney biopsy revealed numerous lamellar bodies (LB) in podocytes with differential diagnoses of Fabry disease (FD), nail-patella syndrome (which is associated with LMX1B gene mutations), and drug-induced phospholipidosis per pathology report. Her workup was negative for a galactosidase-alpha (GLA) mutation with normal serum and leukocyte alpha-galactosidase A activity. She was serendipitously discovered to have compound heterozygous mutations in NPC1 genes (one pathogenic and the other a variant of uncertain significance) from the comprehensive lysosomal storage gene panel as part of her genetic workup for FD. Further studies were done to determine the significance of the NPC1 mutation and revealed elevated oxysterols. (The profile was consistent with NPC, with elevated cholestane-3beta,5alpha,6beta-triol and 7-ketocholesterol and normal lyso-sphingomyelin.) Sonogram revealed hepatosplenomegaly (liver measuring 20 cm and spleen 15.8 cm). These findings in conjunction with lysosomal lipid accumulation on kidney biopsy were consistent with NPC. Interventions: She was on 2 cationic amphiphilic agents (CAAs), fluoxetine and atorvastatin, both of which were stopped. There was no significant difference in proteinuria 2 months off CAAs. The treatment of NPC remained supportive care and avoiding medications that can induce seizures or excessive salivary secretion. Novel Findings: The presence of LB is classically described as a feature of FD which is an LSD. Niemann-Pick type C is another example of an LSD and is typically manifested by neurovisceral symptoms and varies by the age of onset. Renal diseases are typically not described as one of the manifestations of NPC. To our knowledge, there is only one report each for Niemann-Pick disease type A/B and NPC with LB on kidney biopsy. The finding reaffirms that the presence of LB indicates lysosomal lipid accumulation from a variety of etiologies and is not a pathognomonic finding of FD. Niemann-Pick type C should be included as one of the diseases capable of causing renal LB.

Justification: La maladie de Niemann-Pick de type C (NPC) est une maladie lysosomale autosomique récessive (MLAR) causée par des mutations sur les gènes NPC1 ou NPC2. Ces mutations se traduisent par un transport anormal du cholestérol, lequel se manifeste par une accumulation anormale de cholestérol et de glycosphingolipides dans les lysosomes de diverses cellules. Présentation du cas: Une patiente avec des antécédents d'hyperlipidémie, d'hypertension, de dépression et de phosphatase alcaline (PA) élevée s'étant initialement présentée pour un bilan relativement à une insuffisance rénale chronique (IRC) de stade G3b/A3 avec protéinurie à 1,9 gramme/jour. Diagnostic: La biopsie rénale a révélé la présence de nombreux corps lamellaires (CL) dans les podocytes avec, selon le rapport pathologique, des diagnostics différentiels pour la maladie de Fabry (MF), l'ostéo-onychodysostose (associée à des mutations du gène LMX1B) et la phospholipidose (PL) induite par les médicaments. Le bilan s'est avéré négatif pour une mutation de la galactosidase-alpha (GLA) puisque l'activité enzymatique sérique et leucocytaire de celle-ci était normale. Dans le bilan génétique de la MF, qui comprenait l'ensemble des gènes de stockage lysosomal, on a découvert par hasard que la patiente présentait des mutations hétérozygotes composées dans les gènes NPC1 (une pathogène et une variante de signification incertaine [VSI]). D'autres examens réalisés pour déterminer l'importance de la mutation NPC1 ont révélé un taux élevé d'oxystérols. (Le profil était conforme à la NPC, avec des taux élevés de cholestane-3beta, 5alpha, 6beta-triol et de 7-cétocholestérol, et un taux normal de lysosphingomyéline.) L'échographie a montré une hépatosplénomégalie (foie de 20 cm et rate de 15,8 cm). Ces résultats, conjointement à l'accumulation de lipides dans les lysosomes révélée par la biopsie rénale, étaient conformes à une NPC. Intervention: La fluoxétine et l'atorvastatine, les deux agents amphiphiles cationiques (AAC) que prenait la patiente, ont été cessés. La protéinurie est demeurée pratiquement inchangée deux mois après l'arrêt des AAC. Le traitement de la NPC s'est limité à prodiguer des soins de soutien et à éviter les médicaments pouvant induire des convulsions ou une sécrétion salivaire excessive. Nouveaux résultats: La présence de CL est généralement décrite comme une caractéristique de la MF, un type de MLAR. La NPC est un autre exemple de MLAR; elle varie selon l'âge du patient à l'apparition et se manifeste généralement par des symptômes neuroviscéraux. Les néphropathies ne sont généralement pas décrites parmi les manifestations de la NPC. À notre connaissance, il n'existe qu'un seul rapport pour la maladie de Niemann-Pick (NPD) de type A/B et pour la NPC avec CL révélés par biopsie rénale. Cette découverte confirme que la présence de CL est indicatrice d'une accumulation de lipides dans les lysosomes à partir d'une variété d'étiologies et qu'il ne s'agit pas d'une preuve pathognomonique de MF. La NPC doit être incluse comme maladie pouvant causer des CL dans les reins.

Repurposing drugs as COVID-19 therapies: A toxicity evaluation.

Drug repurposing is an appealing method to address the Coronavirus 2019 (COVID-19) pandemic because of the low cost and efficiency. We analyzed our in-house database of approved drug screens and compared their activity profiles with results from a severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) cytopathic effect (CPE) assay. The activity profiles of the human ether-à-go-go-related gene (hERG), phospholipidosis (PLD), and many cytotoxicity screens were found significantly correlated with anti-SARS-CoV-2 activity. hERG inhibition is a nonspecific off-target effect that has contributed to promiscuous drug interactions, whereas drug-induced PLD is an undesirable effect linked to hERG blockers. Thus, this study identifies preferred drug candidates as well as chemical structures that should be avoided because of their potential to induce toxicity. Lastly, we highlight the hERG liability of anti-SARS-CoV-2 drugs currently enrolled in clinical trials.

Highly Sensitive Two-Photon Lipid Droplet Tracker for In Vivo Screening of Drug Induced Liver Injury.

Lipid droplets (LDs) are lipid-abundant organelles found in most cell lines and primarily consist of neutral lipids. They serve as a repository of various lipids and are associated with many cellular metabolic processes, including energy storage, membrane synthesis, and protein homeostasis. LDs are prominent in a variety of diseases related to lipid regulation, including obesity, fatty liver disease, diabetes, and atherosclerosis. To monitor LD dynamics in live samples, we developed a highly selective two-photon fluorescent tracker for LDs (LD1). It exhibited outstanding sensitivity with a remarkable two-photon-action cross section (Φδmax > 600 GM), photostability, and low cytotoxicity. In human hepatocytes and in vivo mouse liver tissue imaging, LD1 showed very bright fluorescence with high LD selectivity and minimized background signal to evaluate the stages of nonalcoholic fatty liver disease. Interestingly, we demonstrated that the liver sinusoid morphology became narrower with increasing LD size and visualized the dynamics including fusion of the LDs in vivo. Moreover, real-time and dual-color TPM imaging with LD1 and a two-photon lysosome tracker could be a useful predictive screening tool in the drug development process to monitor impending drug-induced liver injury inducing drug candidates.

Cell-Based Imaging Assay for Detection of Phospholipidosis.

Accumulation of lysosomal phospholipids in cells exposed to cationic amphiphilic drugs is characteristic of drug-induced phospholipidosis. The morphological hallmark of phospholipidosis is the appearance of unicentric or multicentric-lamellar bodies when viewed under an electron microscope (EM). The EM method, the gold standard of detecting cellular phospholipidosis, has downsides, namely, low-throughput, high-costs, and unsuitability for screening a large chemical library. This chapter describes a cell-based high-content phospholipidosis assay using the LipidTOX reagent in a high-throughput screening (HTS) platform. This assay has been optimized and validated in HepG2 and HepRG cells, and miniaturized into a 1536-well plate, thus can be used for high-throughput screening (HTS) to identify chemical compounds that induce phospholipidosis.