Identification of prognostic biomarkers for antibiotic associated nephrotoxicity in cystic fibrosis.

BACKGROUND: Our objective was to discover novel urinary biomarkers of antibiotic-associated nephrotoxicity using an ex-vivo human microphysiological system (MPS) and to translate these findings to a prospectively enrolled cystic fibrosis (CF) population receiving aminoglycosides and/or polymyxin E (colistin) for a pulmonary exacerbation. METHODS: We populated the MPS with primary human kidney proximal tubule epithelial cells (PTECs) from three donors and modeled nephrotoxin injury through exposure to 50 µg/mL polymyxin E for 72 h. We analyzed gene transcriptional responses by RNAseq and tested MPS effluents. We translated candidate biomarkers to a CF cohort via analysis of urine collected prior to, during and two weeks after antibiotics and patients were followed for a median of 3 years after antibiotic use. RESULTS: Polymyxin E treatment resulted in a statistically significant increase in the pro-apoptotic Fas gene relative to control in RNAseq of MPS: fold-change = 1.63, FDR q-value = 7.29 × 10-5. Effluent analysis demonstrated an acute rise of soluble Fas (sFas) concentrations that correlated with cellular injury. In 16 patients with CF, urinary sFas concentrations were significantly elevated during antibiotic treatment, regardless of development of AKI. Over a median of three years of follow up, we identified seven cases of incident chronic kidney disease (CKD). Urinary sFas concentrations during antibiotic treatment were significantly associated with subsequent development of incident CKD (unadjusted relative risk = 2.02 per doubling of urinary sFas, 95 % CI = 1.40, 2.90, p < 0.001). CONCLUSIONS: Using an ex-vivo MPS, we identified a novel biomarker of proximal tubule epithelial cell injury, sFas, and translated these findings to a clinical cohort of patients with CF.

Microphysiological systems in absorption, distribution, metabolism, and elimination sciences.

The use of microphysiological systems (MPS) to support absorption, distribution, metabolism, and elimination (ADME) sciences has grown substantially in the last decade, in part driven by regulatory demands to move away from traditional animal-based safety assessment studies and industry desires to develop methodologies to efficiently screen and characterize drugs in the development pipeline. The past decade of MPS development has yielded great user-driven technological advances with the collective fine-tuning of cell culture techniques, fluid delivery systems, materials engineering, and performance enhancing modifications. The rapid advances in MPS technology have now made it feasible to evaluate critical ADME parameters within a stand-alone organ system or through interconnected organ systems. This review surveys current MPS developed for liver, kidney, and intestinal systems as stand-alone or interconnected organ systems, and evaluates each system for specific performance criteria recommended by regulatory authorities and MPS leaders that would render each system suitable for evaluating drug ADME. Whereas some systems are more suitable for ADME type research than others, not all system designs were intended to meet the recently published desired performance criteria and are reported as a summary of initial proof-of-concept studies.

Microphysiological system modeling of ochratoxin A-associated nephrotoxicity.

Ochratoxin A (OTA) is one of the most abundant mycotoxin contaminants in food stuffs and possesses carcinogenic, nephrotoxic, teratogenic, and immunotoxic properties. Specifically, a major concern is severe nephrotoxicity, which is characterized by degeneration of epithelial cells of the proximal tubules and interstitial fibrosis. However, the mechanism of OTA toxicity, as well as the genetic risk factors contributing to its toxicity in humans has been elusive due to the lack of adequate models that fully recapitulate human kidney function in vitro. The present study attempts to evaluate dose-response relationships, identify the contribution of active transport proteins that govern the renal disposition of OTA, and determine the role of metabolism in the bioactivation and detoxification of OTA using a 3D human kidney proximal tubule microphysiological system (kidney MPS). We demonstrated that LC50 values of OTA in kidney MPS culture (0.375-1.21 µM) were in agreement with clinically relevant toxic concentrations of OTA in urine. Surprisingly, no enhancement of kidney injury biomarkers was evident in the effluent of the kidney MPS after OTA exposure despite significant toxicity observed by LIVE/DEAD staining. Instead, these biomarkers decreased in an OTA concentration-dependent manner. Furthermore, the effect of 1-aminobenzotriazole (ABT) and 6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol (NBDHEX), pan-inhibitors of P450 and glutathione S-transferase (GST) enzymes, respectively, on OTA-induced toxicity in kidney MPS was examined. These studies revealed significant enhancement of OTA-induced toxicity by NBDHEX (3 µM) treatment, whereas ABT (1 mM) treatment decreased OTA-induced toxicity, suggesting roles for GSTs and P450 enzymes in the detoxification and bioactivation of OTA, respectively. Analysis of transcriptional changes using RNA-sequencing of kidney MPS treated with different concentrations of OTA revealed downregulation of several nuclear factor (erythroid derived-2)-like 2 (NRF2)-regulated genes by OTA treatment, including GSTs. The transcriptional repression of GSTs is likely playing a key role in OTA toxicity via attenuation of glutathione conjugation/detoxification. The sequential molecular events may explain the mechanism of toxicity associated with OTA. Additionally, OTA transport studies using kidney MPS in the presence and absence of probenecid (1 mM) suggested a role for organic anionic membrane transporter(s) in the kidney specific disposition of OTA. Our findings provide a clearer understanding of the mechanism of OTA-induced kidney injury, which may support changes in risk assessment, regulatory agency policies on allowable exposure levels, and determination of the role of genetic factors in populations at risk for OTA nephrotoxicity.

Microphysiological Systems to Assess Nonclinical Toxicity.

The liver and the kidney are key toxicity target organs during drug development campaigns, as they typically carry the burden of drug transport and metabolism. Primary hepatocytes and proximal tubule epithelial cells grown in traditional in vitro 2-D culture systems do not maintain transporter and metabolic functions, thus limiting their utility for nonclinical toxicology investigations. We have developed a renal and hepatic microphysiological system (MPS) platform that uses a commercially available MPS device as the core cell culture platform for our methodologies. We describe protocols for isolating and propagating human proximal epithelial cells and how to seed and culture a renal MPS to recapitulate the human proximal tubule. We present two methods to culture hepatocytes within an MPS and the steps required to connect a renal MPS to a liver MPS. © 2017 by John Wiley & Sons, Inc.

Characterization of rat or human hepatocytes cultured in microphysiological systems (MPS) to identify hepatotoxicity.

The liver is the main site for drug and xenobiotics metabolism, including inactivation or bioactivation. In order to improve the predictability of drug safety and efficacy in clinical development, and to facilitate the evaluation of the potential human health effects from exposure to environmental contaminants, there is a critical need to accurately model human organ systems such as the liver in vitro. We are developing a microphysiological system (MPS) based on a new commercial microfluidic platform (Nortis, Inc.) that can utilize primary liver cells from multiple species (e.g., rat and human). Compared to conventional monolayer cell culture, which typically survives for 5-7days or less, primary rat or human hepatocytes in an MPS exhibited higher viability and improved hepatic functions, such as albumin production, expression of hepatocyte marker HNF4α and canaliculi structure, for up to 14days. Additionally, induction of Cytochrome P450 (CYP) 1A and 3A4 in cryopreserved human hepatocytes was observed in the MPS. The acute cytotoxicity of the potent hepatotoxic and hepatocarcinogen, aflatoxin B1, was evaluated in human hepatocytes cultured in an MPS, demonstrating the utility of this model for acute hepatotoxicity assessment. These results indicate that MPS-cultured hepatocytes provide a promising approach for evaluating chemical toxicity in vitro.

Differential epigenetic effects of chlorpyrifos and arsenic in proliferating and differentiating human neural progenitor cells.

Understanding the underlying temporal and mechanistic responses to neurotoxicant exposures during sensitive periods of neuronal development are critical for assessing the impact of these exposures on developmental processes. To investigate the importance of timing of neurotoxicant exposure for perturbation of epigenetic regulation, we exposed human neuronal progenitor cells (hNPCs) to chlorpyrifos (CP) and sodium arsenite (As; positive control) during proliferation and differentiation. CP or As treatment effects on hNPCs morphology, cell viability, and changes in protein expression levels of neural differentiation and cell stress markers, and histone H3 modifications were examined. Cell viability, proliferation/differentiation status, and epigenetic results suggest that hNPCs cultures respond to CP and As treatment with different degrees of sensitivity. Histone modifications, as measured by changes in histone H3 phosphorylation, acetylation and methylation, varied for each toxicant and growth condition, suggesting that differentiation status can influence the epigenetic effects of CP and As exposures.

The presence of macrophages and inflammatory responses in an in vitro testicular co-culture model of male reproductive development enhance relevance to in vivo conditions.

Our 3-dimensional testis co-culture system (3D-TCS) represents a promising model of male reproductive toxicity which captures sensitive processes of male reproductive development and contains the main testes cell types (germ, Leydig and Sertoli cells). Macrophages are another cell type important for testicular function and help to modulate immuno-endocrine processes during testes development. Chemicals such as phthalate esters (PE's) affect macrophage function and testosterone production in the testes in vivo. The aim of this study was to determine whether macrophages were present in the 3D-TCS and investigate responses in our model that may be related to immuno-endocrine functions. We observed consistent expression of the resident macrophage marker ED2 as well as increases in inflammatory cytokines produced by macrophages and testes cells (IL-6, TNF-α and KC/GRO) after exposure to toxic PE's. Pathway analysis of gene expression changes after exposure to PE's showed that IL-6 and TNF-α signaling pathways were enriched after treatment with reproductively toxic, but not non-reproductively toxic phthalates. These results indicate that macrophages and inflammatory processes are captured in the 3D-TCS and that these processes are impacted by exposure to reproductive toxicants. These processes represent a major mode of action for in vivo testis toxicity for a variety of compounds and our novel in vitro model is able to capture toxicant perturbation of immune function.