ABSTRACT
Aryl hydrocarbon receptor (AHR) activation by tryptophan (Trp) catabolites enhances tumor malignancy and suppresses anti-tumor immunity. The context specificity of AHR target genes has so far impeded systematic investigation of AHR activity and its upstream enzymes across human cancers. A pan-tissue AHR signature, derived by natural language processing, revealed that across 32 tumor entities, interleukin-4-induced-1 (IL4I1) associates more frequently with AHR activity than IDO1 or TDO2, hitherto recognized as the main Trp-catabolic enzymes. IL4I1 activates the AHR through the generation of indole metabolites and kynurenic acid. It associates with reduced survival in glioma patients, promotes cancer cell motility, and suppresses adaptive immunity, thereby enhancing the progression of chronic lymphocytic leukemia (CLL) in mice. Immune checkpoint blockade (ICB) induces IDO1 and IL4I1. As IDO1 inhibitors do not block IL4I1, IL4I1 may explain the failure of clinical studies combining ICB with IDO1 inhibition. Taken together, IL4I1 blockade opens new avenues for cancer therapy.
Subject(s)
L-Amino Acid Oxidase/metabolism , Receptors, Aryl Hydrocarbon/metabolism , Adult , Aged , Animals , Cell Line , Cell Line, Tumor , Disease Progression , Female , Glioma/immunology , Glioma/metabolism , Glioma/therapy , HEK293 Cells , Humans , Immune Checkpoint Inhibitors/pharmacology , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/therapy , Male , Mice , Mice, Inbred C57BL , Middle Aged , RatsABSTRACT
The kidney is a frequent target for organ-specific toxicity as a result of its primary function in controlling body fluids, for example, via resorption of amino acids, peptides, nutrients, ions, xenobiotics and water from the primary urine as well as excretion of metabolic waste products and hydrophilic and amphiphilic xenobiotics. Compounds exhibiting dose-limiting nephrotoxicity include drugs from highly diverse classes and chemical structures, e.g., antibiotics (gentamicin), chemotherapeutics (cisplatin), immunosuppressants (cyclosporine A and tacrolimus) or bisphosphonates (zoledronate). All of these compounds elicit nephrotoxicity primarily by injuring renal proximal tubule epithelial cells (RPTECs). However, prediction of a compound's nephrotoxic potential in humans to support early unmasking of risk-bearing drug candidates remains an unmet challenge, mainly due to the complex kidney anatomy as well as pronounced inter- and intraspecies differences and lack of relevant and validated human in vitro models. Accordingly, we used the recently established human RPTEC/TERT1 cell line to carry out toxicity studies with a focus on impairment of functional characteristics, i.e., transepithelial electrical resistance (TEER), vectorial transport of water, cations, and anions. Results were compared to real-time cytotoxicity assessments using cellular impedance (xCELLigence assay) and the routine cell viability readout (MTT). As expected, most toxins caused exposure time- and concentration-dependent cytotoxicity. However, for some compounds (cyclosporine A and tacrolimus), transport processes were strongly impaired in absence of a concomitant decrease in cell viability. In conclusion, these data demonstrate that functional parameters are important, highly sensitive and meaningful additional readouts for nephrotoxicity assessment in human renal proximal tubule epithelial cells.
Subject(s)
Animal Testing Alternatives/methods , Epithelial Cells/drug effects , Kidney Diseases/chemically induced , Kidney Tubules, Proximal/drug effects , Pharmaceutical Preparations , Xenobiotics/toxicity , Biological Transport , Cell Line , Dose-Response Relationship, Drug , Epithelial Cells/metabolism , Epithelial Cells/pathology , Humans , Kidney Diseases/metabolism , Kidney Tubules, Proximal/metabolism , Kidney Tubules, Proximal/pathology , Pharmaceutical Preparations/metabolism , Sensitivity and Specificity , Water/metabolism , Xenobiotics/pharmacokineticsABSTRACT
The proximal tubule is the primary site for renal solute reabsorption and secretion and thus a main target for drug-induced toxicity. Current nonclinical methods using 2D cell cultures are unable to fully recapitulate clinical drug responses mainly due to limited in vitro functional lifespan. Since extracellular matrices are known to be key regulators of cell development, culturing cells on classic 2D plastic surfaces inevitably results in loss of differentiation. Hence, 3D models of the human proximal tubule that recapitulate the in vivo morphology would allow for improved drug screening and disease modeling. Here, the development and characterization of a 3D proximal tubule model using RPTEC/TERT1 cells is presented. RPTEC/TERT1 cells self-assembled in matrigel to form highly differentiated and stable 3D tubular structures characterized by a branched network of monolayered cells encircling a cell-free lumen thus mimicking the proximal tubule. In vitro tubuli resembled the polarity of a proximal tubule epithelium as indicated by polar expression of Na+/K+- ATPase and ZO-3. Furthermore, 3D cultured RPTEC/TERT1 cells showed overall increased mRNA expression of xenobiotic transporters e.g. OCTs and MATEs and de novo expression of OAT3 when compared to cultures on plastics or membrane inserts. Finally, this model was used to assess delayed cisplatin-induced nephrotoxicity and demonstrated increased sensitivity when compared to 2D culture. Thus, the easy-to-use model described here may prove to be useful for mechanistic investigations, e.g. in discovery of compounds interfering with tubule formation, differentiation and polarization, as well for the detection and understanding of pharmaceutical induced nephrotoxicity.
Subject(s)
Cell Differentiation , Kidney Tubules, Proximal/ultrastructure , RNA, Messenger/metabolism , Animals , Cell Line , Cells, Cultured , HumansABSTRACT
Recent FDA Drug Safety Communications report an increased risk for acute kidney injury in patients treated with the gliflozin class of sodium/glucose co-transport inhibitors indicated for treatment of type 2 diabetes mellitus. To identify a potential rationale for the latter, we used an in vitro human renal proximal tubule epithelial cell model system (RPTEC/TERT1), physiologically representing human renal proximal tubule function. A targeted metabolomics approach, contrasting gliflozins to inhibitors of central carbon metabolism and mitochondrial function, revealed a double mode of action for canagliflozin, but not for its analogs dapagliflozin and empagliflozin. Canagliflozin inhibited the glutamate dehydrogenase (GDH) and mitochondrial electron transport chain (ETC) complex I at clinically relevant concentrations. This dual inhibition specifically prevented replenishment of tricarboxylic acid cycle metabolites by glutamine (anaplerosis) and thus altered amino acid pools by increasing compensatory transamination reactions. Consequently, canagliflozin caused a characteristic intracellular accumulation of glutamine, glutamate and alanine in confluent, quiescent RPTEC/TERT1. Canagliflozin, but none of the classical ETC inhibitors, induced cytotoxicity at particularly low concentrations in proliferating RPTEC/TERT1, serving as model for proximal tubule regeneration in situ. This finding is testimony of the strong dependence of proliferating cells on glutamine anaplerosis via GDH. Our discovery of canagliflozin-mediated simultaneous inhibition of GDH and ETC complex I in renal cells at clinically relevant concentrations, and their particular susceptibility to necrotic cell death during proliferation, provides a mechanistic rationale for the adverse effects observed especially in patients with preexisting chronic kidney disease or previous kidney injury characterized by sustained regenerative tubular epithelial cell proliferation.