Chronic kidney disease of unknown etiology (CKDu) is a pervasive condition not prompted by diabetes or hypertension but instead by environmental stimuli and occupational associated risks. Often detected only in advanced stages, CKDu necessitates interventions such as dialysis and kidney transplant, significantly burdening healthcare systems globally and disproportionally affecting rural populations. Ochratoxin-A (OTA), an abundant, ubiquitous, natural contaminant found in food products and is among the postulated risk factors for (CKDu). While animal studies indicate dysregulation of mitochondrial dynamics and production of superoxides via redox cycling as potential mechanisms of OTA-associated nephrotoxicity, its exposure risk in humans and kidney health remain poorly understood. This study investigates the biological pathways that contribute to the toxicity of OTA in the proximal tubule, leading to CKDu. Luminescence-based imaging approach evaluated the occurrence of OTA-induced oxidative stress in human proximal tubular epithelial cells (PTEC) by detecting cytoplasmic reactive oxygen species (ROS). Preliminary mRNA transcriptomic analysis has indicated the down-regulation of glutathione pathways, a major antioxidant pathway removing cellular oxygen. I probed OTA-treated PTECs with a reduced glutathione detection reagent, ThiolTracker Violet, to investigate the cell’s response capability for oxidative stress and detoxify xenobiotics. To verify oxidative stress mediated by OTA in live cells, CellROX Green Reagent probe treated PTECs were brought under confocal microscopy to visualize mitochondrial phenotype. The results of this investigation seek to reveal the metabolic response to OTA cytotoxicity in the human kidney and elucidate its role in CKDu progression to address diagnostic challenges and confront unmet medical needs.

Adipose tissue, also known as body fat, is vital in storing energy. It is composed of adipocytes and present in different depots. In this study we focused on omental (OM) adipose tissue, which is found between organs near the stomach, and subcutaneous (SC) adipose tissue, which is found under the skin. Functional differences have been observed among adipose depots. SC adipose tissue is responsible for insulation while OM adipose tissue has endocrine functions. OM adipocytes have been observed to be smaller and more variable in size compared to SC adipocytes in individuals with obesity. I hypothesized that the size of both OM and SC adipocytes is associated with increasing BMI. I tested this hypothesis using SC and OM adipose tissue biopsies collected during elective surgeries from metabolically healthy participants (20 females, 11 males) with a range of ages (25-65 years) and BMIs (21-56). Afterwards, the tissues were fixed and stained with H&E. I drew 2-5 squares per slide and counted the number of adipocytes within each square. The size difference between OM and SC adipocytes was tested using a Wilcoxon signed-rank test and a significant difference (p=0.004) was observed. A correlation between BMI and the size of OM (p = 0.008) or SC (p = 0.009) adipocytes was detected with weighted linear regressions. Sex was not observed to be a significant covariate. These findings expand on prior data by including lean individuals, and patients with obesity who are otherwise metabolically healthy. The results show there is a clear difference in the size of adipocytes. The adipocyte size in both depots correlated with BMI. This data shows that progressive obesity and adipose tissue enlargement is due to the enlargement of the adipocytes rather than an increase in the number of adipocytes in both OM and SC depots.

My field of research holds significant promise for advancing our understanding of the intricate interplay between immune cells and hormonal regulation, with broader implications for both basic science and clinical applications. In this study, I am advancing in vitro models crucial for toxicology and fertility research, specifically addressing the understudied role of resident macrophages within the testis niche. The research question aims to explore the avenues of generating M2 testicular macrophages in vitro from peripheral blood mononuclear cells (PBMCs). M2 macrophages are anti-inflammatory and are associated with wound healing and repair, unlike pro-inflammatory M1 macrophages. I hypothesize that adding macrophage colony-stimulating factor (M-CSF) combined with exposure to testosterone-rich conditioned media from a primary testis cell culture will induce the polarization of monocytes towards the M2 phenotype. The methodology involves the in vitro culture of primary PBMCs obtained from male Sprague-Dawley rats, with assessments based on surface marker expression (CD68 and CD163) and the evaluation of pro-inflammatory interleukin-6 and anti-inflammatory (interleukin-10) cytokine gene expression. M-CSF growth factor is added to monocytes to induce change to macrophages. The conditioned media for macrophages will be generated from primary neonatal rat testis cells cultured with 500 mIU/mL luteinizing hormone (LH). The media contains a higher LH concentration than in vivo, which helps produce a relevant testosterone concentration in the macrophage in vitro culture. RNA for RT-qPCR (reverse transcription–quantitative PCR) is extracted. Cells are fixed for ICC immunocytochemistry of markers (CD68/163). The implications of this research are substantial, ranging from the ability to study inflammation mechanisms in vitro to achieving a more accurate representation of the testicular hormonal environment. Furthermore, the study sheds light on the intracrine function of immune cells, emphasizing their capacity to generate sex steroids and derivatives that mediate intracrine, autocrine, and paracrine effects.

Cellular retinoic acid binding proteins (CRABP) are soluble proteins that play an important role in binding to retinoids, primarily all-trans-retinoic acid (atRA). RA isomers and their metabolites play a vital role in embryonic development and the central nervous system. CRABP binding regulates retinoid signaling. Retinoid binding affinity to CRABP is described by the equilibrium dissociation constant K_d. CRABP1 interacts with atRA metabolizing enzymes, where relative concentrations of atRA and CRABP1 determine the rate of atRA metabolism. Therefore, measurements of the accurate K_d for endogenous retinoids to CRABP1 are an important aspect of understanding retinoid signaling during development. My goal was to determine the binding affinities of various endogenous retinoids to CRABP1, using a modern fluorescence spectrometric assay and lower CRABP1 concentrations compared to historical methods. The Norris equation was fit to the data to determine the K_d from the decrease of peak tryptophan fluorescence as retinoid bound to CRABP1. The calculated K_d was <1 nM for atRA, 3-10 nM for 4-oxo-atRA, 50-100 nM for 9-cis RA, and near 1 uM for 4-oxo-13-cis and 13-cis RA. When the K_d is much lower than CRABP1 concentration, binding of the retinoid to CRABP1 results in depletion of the retinoid in solution and hence an overestimation of the K_d value. As this work uses lower CRABP1 concentrations than previous studies, the predicted K_d estimates for retinoids that tightly bind to CRABP1 would be lower and more accurate. This is supported by the data. While the K_d of atRA is similar to its endogenous concentration in human plasma, the K_d of the other retinoids tested are much higher than their concentrations, suggesting that small fractions of these retinoids are bound to CRABP1 in vivo. Knowing these K_d values may give insight into how altered CRABP1 concentrations impact development through their modulation of RA concentration.

Organs-on-a-chip (OOAC) are biomimetic structures that replicate the physiological environments of human organs. OOACs are growing in popularity as they provide control of parameters including shear stress, concentration gradient, and biological interactions between cells and biofluids; they can be used in pharmacokinetic, physiological, and toxicological studies. The Kelly-Yeung lab works with kidney OOACs to study toxicology and pharmacokinetics. An important component of the chips is the hydrogel that provides a tubular scaffold and biological substrate for the kidney cells. The hydrogel mixture typically consists of decellularized kidney matrix, Collagen I (Col-I), and two types of cell culture media (PTEC and 199x). The matrix mimics the kidney microenvironment, the Col-I is a stabilizer and tissue regeneration agent, and the two cell culture media are used to mimic the extracellular fluids. More matrix in the hydrogel is ideal since it will better mimic a kidney. However, the matrix by itself is not structurally stable, hence the need for a stabilizing agent. The goal of this project is to maximize the ratio of matrix to Col-I while maintaining a stable hydrogel. In order to determine the optimal ratio, stiffness testing of the hydrogels will be performed via AFM (atomic force microscopy) and a Parallel Plate Rheometer to find out how much matrix can be used before the stiffness of the hydrogel composition is compromised. At this stage, we have started testing the collagen hydrogel with the rheometer to gain base measurements before adding the kidney matrix. We aim to incorporate the kidney matrix, achieving measurements closely mirroring those obtained with the collagen hydrogel alone, while supporting healthy cell growth. Creating a more accurate OOAC based on optimized kidney extracellular matrices will help improve the models and recapitulate the effects of drugs, toxins, and diseases on human kidneys more precisely.

Multiple Sclerosis (MS) is a chronic central nervous system (CNS) disease in which the immune system attacks the myelin surrounding nerve cells, resulting in decreased communication between the brain and the rest of the body, and severe physical and neurological defects. Gut metabolites produced by gut microbiota play a significant role in maintaining host immune homeostasis, and research has shown that short-chain fatty acids (SCFAs) have therapeutic potential. When tested in the mouse model of MS, experimental autoimmune encephalomyelitis (EAE), delivering butyrate-conjugated block copolymer micelles to the distal gut ameliorated EAE progression by increasing the amount of regulatory T cells and cytokines, and strengthening intestinal barrier functions. For prolonged SCFA delivery to the distal gut, a SCFA-conjugated hybrid lipid-polymer nanopolymer carrier is required. A nanoparticle is required because butyrate has a strong odor/taste and requires many pills per day to have significant suspension. To synthesize this nanoparticle, we optimized the nanogenerator to determine its effects on size and charge. This included modulating the total flow rate, flow rate ratio, input concentration ratio (lipid:polymer). We hypothesize that larger size, smaller surface area-to-volume ratio, and anionic surface charge will prolong retention and time in the distal GI. Once the nanoparticle is optimized, the potential of SCFA-conjugated nanocarriers to ameliorate EAE progression will be measured by comparing EAE mice and healthy controls, using the same outcome measures. This will allow us to select which polymer-conjugated SCFAs to incorporate into the optimized nanocarriers. We believe the SCFAs will inhibit proinflammatory macrophage activation, induce anti-inflammatory Treg cells, and promote demyelination and axonal degeneration to reduce the effects of MS in mice. In humans, delivering these nanoparticles as a drug will serve as a much more efficient means of treating MS when compared to the current treatment procedures.

Cannabis is the most commonly used drug of abuse used by approximately 2.5% of the world's population. Tetrahydrocannabinol (THC) is the major psychoactive component of cannabis. Cannabidiol (CBD) on the other hand is a pharmacologically active component of cannabis that is not psychoactive. THC is metabolized via cytochrome P450 enzyme-mediated oxidation to 11-hydroxy-THC (11-OH-THC) and subsequently to 11-carboxy-THC (11-COOH-THC). 11-COOH-THC then undergoes glucuronidation to form an acyl glucuronide (AG), 11-nor-9-carboxy-Δ9-tetrahydrocannabinol glucuronide (11-COOH-THC-Glucuronide). CBD is also metabolized via an analogous pathway to THC, where CBD is oxidized to 7-hydroxy cannabidiol (7-OH-CBD) and then subsequently to 7-COOH-CBD and ultimately glucuronidated to form the AG 7-COOH-CBD-Glucuronide. The THC and CBD AGs are important because they circulate at high concentrations in biological samples. Thus, identifying the enzymes responsible for the formation of cannabinoid AGs is important for understanding inter-individual variability and disease effects of cannabinoid exposures and pharmacological effects. Glucuronidation is governed by UDP-glucuronosyltransferases (UGTs). The goal of this study is to identify and characterize the UGTs that form THC and CBD AGs, to define in what organs these glucuronides are formed, and to determine whether disease states may alter 11-COOH-THC and 7-COOH-CBD glucuronidation. To accomplish this I incubated human liver microsomes (HLMs) and recombinant UGTs with 11-COOH-THC (5 µM) or 7-COOH-CBD (5 µM). The incubations were done at pH 7.4, 37℃ for 5 (THC-COOH) or 45 (7-COOH-CBD) minutes. Formation of the THC and CBD AGs was detected in HLMs and with UGT1A1, UGT1A3, UGT1A7, UGT1A8, UGT1A9, and UGT2B7. UGT2B4 and UGT2B17 were specific to THC AG formation and UGT1A10 was specific to CBD AG formation. Kinetic assessments such as intrinsic clearance measurements and inhibition assays will further the understanding of the importance of the specific UGT isoforms responsible for cannabinoid acyl glucuronide formation in vivo.