Biology subjects

Kelly, E. J.

Publications and source records attributed to Kelly, E. J..

9 recordsLinked to original sources

bioRxiv2026

Voclosporin Preserves Mitochondrial Function Compared With Cyclosporine A in Perfused Human Proximal Tubule Microphysiological Systems

Key PointsO_LIPerfused human kidney MPS revealed CsA-associated sublethal tubular stress that was not detected by conventional 2D viability assays or by KIM-1 release in 3D MPS. C_LIO_LIAt matched exposure, VCS preserved mitochondria and activated ER chaperones and iron detoxification, with no p21 arrest compared to CsA. C_LIO_LIMechanistic separation supports VCSs nephroprotection potential and early mechanism-based biomarkers to guide CNI choice. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/737071v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@c38ab8org.highwire.dtl.DTLVardef@1c8638forg.highwire.dtl.DTLVardef@100485aorg.highwire.dtl.DTLVardef@18e3d70_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG BackgroundCalcineurin inhibitors (CNIs) are indispensable for transplantation immunosuppression, yet cyclosporine A (CsA) produces renal toxicity. Voclosporin (VCS), a CsA analog, is proposed to be less nephrotoxic, but mechanisms remain unclear. MethodsPrimary human proximal tubule epithelial cells (PTECs) were exposed to CsA or VCS in 2D monolayers and perfused 3D kidney microphysiological system (MPS). Viability was assessed in 2D cultures by MTS, mitochondrial membrane potential ({Delta}{Psi}m) by TMRM flow cytometry, and soluble injury and inflammatory biomarkers in MPS effluents by ELISA and MSD multiplex assays. RNA sequencing of 3D-cultured PTECs was used to identify differentially expressed genes and pathways. ResultsIn 2D PTECs, neither drug reduced viability. In 3D MPS effluents, KIM-1 did not distinguish CsA from VCS, whereas the MSD biomarker panel showed larger aggregate deviation with CsA. Confocal tomography showed CsA-associated mitochondrial fragmentation, whereas VCS preserved reticular mitochondrial architecture. TMRM flow cytometry showed a treatment-dependent difference in TMRM-positive cells, with VCS yielding the highest TMRM-positive fraction and exceeding CsA, supporting preservation of {Delta}{Psi}m relative to CsA. RNA-seq identified 1188 CsA-specific and 185 VCS-specific differentially expressed genes, with 304 shared. Pathway analysis indicated CsA enrichment of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress, p21-associated G2/M checkpoint arrest, and transcriptional signatures consistent with ferroptosis priming, while VCS mainly induced ER chaperone and ER-associated degradation gene programs without activating canonical UPR sensors and showed limited cell-cycle suppression. ConclusionsA physiologically relevant 3D kidney MPS revealed sublethal tubular stress from CsA that is masked in 2D culture, including mitochondrial depolarization, proteostatic stress, and ferroptosis priming. At matched exposure, VCS preserved mitochondrial function and proteostasis while eliciting a narrower, adaptive ER quality control response. These data support VCS as a nephron-sparing immunosuppressant and 3D MPS as a mechanism-based platform for evaluating renal safety of drugs and nominating early sub-lethal tubular injury biomarkers.

pharmacology and toxicology↗

bioRxiv2026

Proteomic profiling of drug and nutrient transporter expression by small intestinal region in neonates, pediatrics, and adults

Transporters localized to the small intestinal epithelium govern the absorption of many orally administered drugs and nutrients, and thorough characterization of transporter protein abundance is key to accurate prediction of small molecule absorption. Previous studies have reported more than 100 solute carrier and ATP-binding cassette superfamilies expressed in the intestine, but only a small subset of these transporter proteins have been quantified in native intestinal tissue. In addition, a majority of published transporter abundance data for the intestine was collected with adult tissue. Here, we report the identification and relative quantification of 87 solute carrier and 18 ATP-binding cassette superfamily transporters by intestinal region in neonatal, pediatric, and adult small intestinal mucosa using data-independent acquisition mass spectrometry. This study identified transporters uniquely expressed by intestinal region in all three age groups and region-associated abundance for 23 SLC/ABC transporters in pediatrics and adults. The protein abundance data reported here provide a valuable resource for the prediction of nutrient and drug absorption and efflux across age and will inform future studies of specific transporters and age-associated differences in transporter abundance. Significance statementWe report the relative abundance of key xenobiotic transporters along the length of the small intestine in neonates, pediatrics, and adults based on data-independent acquisition mass spectrometry. Intestinal region-associated transporter expression was observed for drug transporters and under-studied nutrient transporters across age groups.

pharmacology and toxicology↗

bioRxiv2025

Differential retinoic acid responses across testicular development in vitro

Spermatogonial differentiation is controlled by distinct spatial and temporal activities of metabolizing enzymes and signaling molecule formation. In vitro models are reductionist ways to examine the interactions between Sertoli cells and spermatogonial stem cells, which may be therapeutic targets for infertility that cause non-obstructive azoospermia, due to either Sertoli-cell only syndrome, hypospermatogenesis or maturation arrest. Here, we find that nanomolar doses of isotretinoin are sufficient to drive Stra8 expression in vitro, an interaction that is both dose-dependent and inhibitable. We compare complex in vitro models (CIVMs) seeded from cells isolated post-natal day 5 (PND 5) and post-natal day 10 (PND 10) Sprague Dawley rat testis. The CIVMs maintain metabolic capacity to produce bioactive retinoids form retinol. We also investigate the impact of common media supplementations on spermatogonial phenotype and find that they can impact the expression of Stra8 and Plzf as markers of early differentiating and undifferentiated spermatogonia, respectively. These results highlight the power of in vitro models to investigate the dynamics of the spermatogonial niche.

pharmacology and toxicology↗

bioRxiv2025

Regulation of Renal Transporters by Pro-inflammatory Cytokines in Human Proximal Tubular Epithelial Cells: Identification of the Perpetrator and Mechanisms

IntroductionInfection and inflammation elevate circulating pro-inflammatory cytokines that can affect renal drug clearance. Accordingly, we sought to (i) quantify the extent of modulation of renal drug-metabolizing enzymes and transporters (DMETs) by cytokines and (ii) identify the mechanism(s) underlying these effects. MethodsFresh primary human proximal tubular epithelial cells (PTECs) were cultured on extracellular matrix-coated Transwells. PTECs were exposed every 24 h, for 48 h, to IL-6, IL-1{beta}, TNF-, IFN-{gamma}, IL-4, or IL-10 (0.1 or 1 ng/mL), individually or as a cocktail. mRNA expression of 25 renal DMETs was quantified by RT-qPCR. Individual activity of OAT1-4, OCT2, and OCTN1 was measured. To determine mechanisms of these effects, selective MAPK/NF-{kappa}B inhibitors (ERK [PD98059], p38MAPK [SB203580], JNK [SP600125], and NF-{kappa}B [PDTC]), individually or as a cocktail, were used. IL-6, soluble IL-6 receptor (sIL-6R), and IL-6 + sIL-6R were used to probe endogenous/exogenous IL-6 classic versus trans-signaling. ResultsIL-1{beta} was the predominant modulator, downregulating mRNA expression of OAT1-3, OCT2, OAT4, MATE2-K, MRP2, and OATP4C1, and upregulating mRNA expression of OCTN1 and MRP3. TNF- downregulated OAT1-3 mRNA expression to an extent similar to IL-1{beta}, but did not affect other transporters. Activity changes for the major uptake transporters mirrored mRNA directionality. MAPK/NF-{kappa}B blockade by the inhibitor cocktail reduced IL-6 secretion while completely reversing the IL-1{beta}-driven downregulation of OAT1-3 mRNA. JNK inhibition alone restored OAT1/3 mRNA. Inhibition of p38MAPK blunted OAT2 mRNA downregulation. OCTN1 mRNA induction required NF-{kappa}B. Downregulation of OAT4/OCT2 mRNA was largely MAPK/NF-{kappa}B-independent. IL-6 alone, sIL-6R alone, or IL-6 + sIL-6R did not reproduce IL-1{beta}-driven changes in transporter mRNA. ConclusionsIL-1{beta} is the principal driver of cytokine-mediated regulation of human renal transporters in PTECs via JNK/p38MAPK/NF-{kappa}B nodes. These mechanistic, exposure-verified data provide inputs for physiologically based pharmacokinetic predictions of renal secretory clearance and pathway-mediated drug interactions during inflammation. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/690608v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@177e372org.highwire.dtl.DTLVardef@1f56bc4org.highwire.dtl.DTLVardef@17612e3org.highwire.dtl.DTLVardef@d2291f_HPS_FORMAT_FIGEXP M_FIG C_FIG Translational StatementSystemic inflammation increases cytokine concentrations and alters drug pharmacokinetics. Yet, cytokine regulation of renal drug transporters remains poorly defined, even though the kidney clears many anti-infective drugs via active secretion. Using an optimized primary human proximal tubular epithelial cell model that preserves expression and function of major renal transporters, we found that IL-1{beta} is the predominant cytokine that downregulates the mRNA and activity of OAT1-3, OCT2, and OAT4, while upregulating the mRNA and activity of OCTN1. We further showed that IL-1{beta}-driven downregulation of OAT1/3 occurs through JNK signaling, OAT2 through p38MAPK, and OCTN1 through NF-{kappa}B. These data provide quantitative inputs for physiologically based pharmacokinetic models to predict how inflammation alters renal transporter-mediated drug clearance, informing dose adjustment and risk assessment for disease-drug and drug-drug interactions in patients with inflammatory kidney disease or systemic infections. They also highlight signaling nodes where anti-inflammatory therapies might inadvertently modify renal drug transport.

pharmacology and toxicology↗

bioRxiv2025

Evaluation of toxicological mechanisms of ochratoxin-A in human primary proximal tubule epithelial cells

Ochratoxin-A (OTA) is a ubiquitous mycotoxin contaminant in food products and a known nephrotoxin. OTA is hypothesized to be a potential environmental agent causing chronic kidney disease of unknown etiology (CKDu), however the mechanism of OTA toxicity in the human kidney remains elusive. This study aims to elucidate OTA-induced molecular toxicological pathways using primary human proximal tubule epithelial cells (PTECs). We demonstrated that exposure to OTA (10 M) induces over 7000 differentially expressed genes, including key regulators of mitochondrial fission and fusion. This was confirmed at the cellular level by confocal microscopy, where a breakdown of the mitochondrial network was observed at 100 nM OTA. Crucially, OTA was found to significantly induce reactive oxygen species (mROS) and inhibit basal mitochondrial oxidative phosphorylation as well as glycolysis through measurements of oxygen consumption rate and extracellular acidification, indicating reduced cellular energetics and mitochondrial toxicity. The previously reported downregulation of NRF2 target antioxidant response elements was not able to be recovered with co-administration of NRF2 agonists, sulforaphane or tert-butylhydroquinone, suggesting a possible mechanism of inhibition of NRF2 nuclear translocation or DNA binding. In conclusion, we demonstrate that OTA induces oxidative stress, mitochondrial dysfunction, and reduced ATP production, leading to a senescent-like state in PTECs characteristic of renal disease progression. These findings provide insight into early toxicological endpoints induced by OTA which have been established as pathophysiological changes involved in chronic kidney disease. HighlightsO_LIOTA exposure suppresses oxidative phosphorylation and glycolysis C_LIO_LIOTA exposure disrupts mitochondrial fission and fusion C_LIO_LIOTA induced mitochondrial ROS C_LIO_LIEarly mitochondrial stress may underlie OTA-associated CKDu risk C_LI

pharmacology and toxicology↗

bioRxiv2021

Pango lineage designation and assignment using SARS-CoV-2 spike gene nucleotide sequences

More than 2 million SARS-CoV-2 genome sequences have been generated and shared since the start of the COVID-19 pandemic and constitute a vital information source that informs outbreak control, disease surveillance, and public health policy. The Pango dynamic nomenclature is a popular system for classifying and naming genetically-distinct lineages of SARS-CoV-2, including variants of concern, and is based on the analysis of complete or near-complete virus genomes. However, for several reasons, nucleotide sequences may be generated that cover only the spike gene of SARS-CoV-2. It is therefore important to understand how much information about Pango lineage status is contained in spike-only nucleotide sequences. Here we explore how Pango lineages might be reliably designated and assigned to spike-only nucleotide sequences. We survey the genetic diversity of such sequences, and investigate the information they contain about Pango lineage status. Although many lineages, including the main variants of concern, can be identified clearly using spike-only sequences, some spike-only sequences are shared among tens or hundreds of Pango lineages. To facilitate the classification of SARS-CoV-2 lineages using subgenomic sequences we introduce the notion of designating such sequences to a "lineage set", which represents the range of Pango lineages that are consistent with the observed mutations in a given spike sequence. These data provide a foundation for the development of software tools that can assign newly-generated spike nucleotide sequences to Pango lineage sets.

genomics↗

bioRxiv2021

Multi-omic Characterization of Human Tubular Epithelial Cell Response to Serum

Proteinuria, the spillage of serum proteins into the urine, is a feature of glomerulonephritides, podocyte disorders and diabetic nephropathy. However, the response of tubular epithelial cells to serum protein exposure has not been systematically characterized. Using transcriptomic profiling we studied serum-induced changes in primary human tubular epithelial cells cultured in 3D microphysiological devices. Serum proteins induced cellular proliferation, cytokine secretion and activated a coordinated stress response. We orthogonally confirmed our findings by comparing the transcriptomic and epigenomic landscapes of intact human kidney cortex and isolated tubular epithelial cells cultured in fetal bovine serum. Importantly, key transcriptomic programs in response to either type of serum exposure remained consistent, including comparisons to an established mouse model of kidney injury. This serum-induced transcriptional response was dominated by switching off of nuclear receptor-driven programs and activation of AP-1 and NF-{kappa}B signatures in the tubular epigenomic landscape. These features of active regulation were seen at canonical kidney injury genes (HAVCR1) and genes associated with COVID-19 (ACE2, IL6). Our data provide a reference map for dissecting the regulatory and transcriptional response of kidney tubular epithelial cells injury induced by serum.

cell biology↗

bioRxiv2020

Bridging the gap between in silico and in vivo: modeling opioid disposition in a kidney proximal tubule microphysiological system

BackgroundOpioid overdose, dependence, and addiction are a major public health crisis. Patients with chronic kidney disease (CKD) are at high risk of opioid overdose, therefore novel methods that provide accurate prediction of kidney clearance (CLr) and systemic disposition of opioids in CKD patients can facilitate the optimization of therapeutic regimens. MethodsWe conducted prediction of kidney clearance and systemic disposition of morphine and its active metabolite morphine-6-glucuronide (M6G) in CKD patients using a vascularized human proximal tubule microphysiological system (VPT-MPS) coupled with a parent-metabolite full body physiologically-based pharmacokinetic (PBPK) model. ResultsThe VPT-MPS, populated with a human umbilical vein endothelial cell (HUVEC) channel and an adjacent human primary proximal tubular epithelial cells (PTEC) channel, successfully demonstrated secretory transport of morphine and M6G from the HUVEC channel into the PTEC channel in a time-dependent manner; transporter inhibitors decreased translocation by 74.3% and 63.6%, respectively. The in vitro data generated by VPT-MPS were incorporated into a mechanistic kidney model and parent-metabolite full body PBPK model to predict CLr and systemic disposition of morphine and M6G. The model successfully predicted CLr within 1.5-fold, and the plasma concentration-time profiles of morphine and M6G in both healthy subjects and CKD patients, with absolute average fold error values <1.5. ConclusionsA microphysiological system together with mathematical modeling successfully predicted kidney clearance and systemic disposition of opioids in CKD patients and healthy subjects.

pharmacology and toxicology↗

bioRxiv2019

Transcriptomic profiling of PBDE-exposed HepaRG cells unveils critical lncRNA- PCG pairs involved in intermediary metabolism

Polybrominated diphenyl ethers (PBDEs) were formally used as flame-retardants and are chemically stable, lipophlic persistent organic pollutants which are known to bioaccumulate in humans. Although its toxicities are well characterized, little is known about the changes in transcriptional regulation caused by PBDE exposure. Long non-coding RNAs (lncRNAs) are increasingly recognized as key regulators of transcriptional and translational processes. It is hypothesized that lncRNAs can regulate nearby protein-coding genes (PCGs) and changes in the transcription of lncRNAs may act in cis to perturb gene expression of its neighboring PCGs. The goals of this study were to 1) characterize PCGs and lncRNAs that are differentially regulated from exposure to PBDEs; 2) identify PCG-lncRNA pairs through genome annotation and predictive binding tools; and 3) determine enriched canonical pathways caused by differentially expressed lncRNA-PCGs pairs. HepaRG cells, which are human-derived hepatic cells that accurately represent gene expression profiles of human liver tissue, were exposed to BDE-47 and BDE-99 at a dose of 25 M for 24 hours. Differentially expressed lncRNA-PCG pairs were identified through DESeq2 and HOMER; significant canonical pathways were determined through Ingenuity Pathway Analysis (IPA). LncTar was used to predict the binding of 19 lncRNA-PCG pairs with known roles in drug-processing pathways. Genome annotation revealed that the majority of the differentially expressed lncRNAs map to PCG introns. PBDEs regulated overlapping pathways with PXR and CAR such as protein ubiqutination pathway and PPAR-RXR activation but also regulate distinctive pathways involved in intermediary metabolism. BDE-47 uniquely regulated signaling by Rho Family GTPases and PBDE-99 uniquely regulates JAK/Stat signaling, bile acid biosynthesis, sirtuin signaling pathway, and autophagy. In conclusion, lncRNAs play essential roles in modifying important pathways involved in intermediary metabolism such as carbohydrate and lipid metabolism.

pharmacology and toxicology↗