Development of a kidney microphysiological system hardware platform for microgravity studies.

Determining the physiological effects of microgravity on the human kidney is limited to relatively insensitive tests of biofluids (blood and urine) that do not return abnormal results until more than 50% of kidney function is lost. We have developed an "organ on chip" microphysiological model of the human kidney proximal tubule (PT-MPS) that can recapitulate many kidney functions and disease states and could play a critical role in determining mechanisms of early kidney dysfunction in microgravity. However, the ground-based PT-MPS system is incompatible with spaceflight as it requires a large pneumatic system coupled to a cell incubator for perfusion and intensive hand-on manipulation. Herein, we report the hardware engineering and performance of the Kidney Chip Perfusion Platform (KCPP), a small, advanced, semi-autonomous hardware platform to support kidney microphysiological model experiments in microgravity. The KCPP is composed of five components, the kidney MPS, the MPS housing and valve block, media cassettes, fixative cassettes, and the programable precision syringe pump. The system has been deployed twice to the ISSNL (aboard CRS-17 and CRS-22). From each set of ISSNL experiments and ground-based controls, we were able to recover PT-MPS effluent for biomarker analysis and RNA suitable for transcriptomics analysis demonstrating the usability and functionality of the KCPP.

Investigating the association between CYP2J2 inhibitors and QT prolongation: a literature review.

Drug withdrawal post-marketing due to cardiotoxicity is a major concern for drug developers, regulatory agencies, and patients. One common mechanism of cardiotoxicity is through inhibition of cardiac ion channels, leading to prolongation of the QT interval and sometimes fatal arrythmias. Recently, oxylipin signaling compounds have been shown to bind to and alter ion channel function, and disruption in their cardiac levels may contribute to QT prolongation. Cytochrome P450 2J2 (CYP2J2) is the predominant CYP isoform expressed in cardiomyocytes, where it oxidizes arachidonic acid to cardioprotective epoxyeicosatrienoic acids (EETs). In addition to roles in vasodilation and angiogenesis, EETs bind to and activate various ion channels. CYP2J2 inhibition can lower EET levels and decrease their ability to preserve cardiac rhythm. In this review, we investigated the ability of known CYP inhibitors to cause QT prolongation using Certara's Drug Interaction Database. We discovered that among the multiple CYP isozymes, CYP2J2 inhibitors were more likely to also be QT-prolonging drugs (by approximately 2-fold). We explored potential binding interactions between these inhibitors and CYP2J2 using molecular docking and identified four amino acid residues (Phe61, Ala223, Asn231, and Leu402) predicted to interact with QT-prolonging drugs. The four residues are located near the opening of egress channel 2, highlighting the potential importance of this channel in CYP2J2 binding and inhibition. These findings suggest that if a drug inhibits CYP2J2 and interacts with one of these four residues, then it may have a higher risk of QT prolongation and more preclinical studies are warranted to assess cardiovascular safety.

Development of a Kidney Microphysiological System Hardware Platform for Microgravity Studies.

Study of the physiological effects of microgravity on humans is limited to non-invasive testing of astronauts. Microphysiological models of human organs recapitulate many functions and disease states. Here we describe the development of an advanced, semi-autonomous hardware platform to support kidney microphysiological model experiments in microgravity.

Impact of microgravity on a three-dimensional microphysiologic culture of the human kidney proximal tubule epithelium: cell response to serum and vitamin D.

The microgravity environment aboard the International Space Station (ISS) provides a unique stressor that can help understand underlying cellular and molecular drivers of pathological changes observed in astronauts with the ultimate goals of developing strategies to enable long-term spaceflight and better treatment of diseases on Earth. We used this unique environment to evaluate the effects of microgravity on kidney proximal tubule epithelial cell (PTEC) response to serum exposure and vitamin D biotransformation capacity. To test if microgravity alters the pathologic response of the proximal tubule to serum exposure, we treated PTECs cultured in a microphysiological system (PT-MPS) with human serum and measured biomarkers of toxicity and inflammation (KIM-1 and IL-6) and conducted global transcriptomics via RNAseq on cells undergoing flight (microgravity) and respective controls (ground). We also treated 3D cultured PTECs with 25(OH)D3 (vitamin D) and monitored vitamin D metabolite formation, conducted global transcriptomics via RNAseq, and evaluated transcript expression of CYP27B1, CYP24A1, or CYP3A5 in PTECs undergoing flight (microgravity) and respective ground controls. We demonstrated that microgravity neither altered PTEC metabolism of vitamin D nor did it induce a unique response of PTECs to human serum, suggesting that these fundamental biochemical pathways in the kidney proximal tubule are not significantly altered by short-term exposure to microgravity. Given the prospect of extended spaceflight, more study is needed to determine if these responses are consistent with extended (> 6 month) exposure to microgravity.

Serum Protein Exposure Activates a Core Regulatory Program Driving Human Proximal Tubule Injury.

BACKGROUND: The kidneys efficiently filter waste products while retaining serum proteins in the circulation. However, numerous diseases compromise this barrier function, resulting in spillage of serum proteins into the urine (proteinuria). Some studies of glomerular filtration suggest that tubules may be physiologically exposed to nephrotic-range protein levels. Therefore, whether serum components can directly injure the downstream tubular portions of the kidney, which in turn can lead to inflammation and fibrosis, remains controversial. METHODS: We tested the effects of serum protein exposure in human kidney tubule microphysiologic systems and with orthogonal epigenomic approaches since animal models cannot directly assess the effect of serum components on tubules. RESULTS: Serum, but not its major protein component albumin, induced tubular injury and secretion of proinflammatory cytokines. Epigenomic comparison of serum-injured tubules and intact kidney tissue revealed canonical stress-inducible regulation of injury-induced genes. Concordant transcriptional changes in microdissected tubulointerstitium were also observed in an independent cohort of patients with proteinuric kidney disease. CONCLUSIONS: Our results demonstrate a causal role for serum proteins in tubular injury and identify regulatory mechanisms and novel pathways for intervention.

Gutsy science: In vitro systems of the human intestine to model oral drug disposition.

The intestine has important gate-keeping functions that can profoundly affect the systemic blood exposure of orally administered drugs. Thus, characterizing a new molecular entity's (NME) disposition within the intestine is of utmost importance in drug development. While currently used in vitro systems, such as Ussing chamber, precision-cut intestinal slices, immortalized cell lines, and primary enterocytes provide substantial knowledge about drug absorption and the intestinal first-pass effect, they remain sub-optimal for quantitatively predicting this process and the oral bioavailability of many drugs. Use of novel in vitro systems such as intestinal organoids and intestinal microphysiological systems have provided substantial advances over the past decade, expanding our understanding of intestinal physiology, pathology, and development. However, application of these emerging in vitro systems in the pharmaceutical science is in its infancy. Preliminary work has demonstrated that these systems more accurately recapitulate the physiology and biochemistry of the intact intestine, as it relates to oral drug disposition, and thus they hold considerable promise as preclinical testing platforms of the future. Here we review currently used and emerging in vitro models of the human intestine employed in pharmaceutical science research. We also highlight aspects of these emerging tools that require further study.

Kidney Organoid and Microphysiological Kidney Chip Models to Accelerate Drug Development and Reduce Animal Testing.

Kidneys are critical for the elimination of many drugs and metabolites via the urine, filtering waste and maintaining proper fluid and electrolyte balance. Emerging technologies incorporating engineered three-dimensional (3D) in vitro cell culture models, such as organoids and microphysiological systems (MPS) culture platforms, have been developed to replicate nephron function, leading to enhanced efficacy, safety, and toxicity evaluation of new drugs and environmental exposures. Organoids are tiny, self-organized three-dimensional tissue cultures derived from stem cells that can include dozens of cell types to replicate the complexity of an organ. In contrast, MPS are highly controlled fluidic culture systems consisting of isolated cell type(s) that can be used to deconvolute mechanism and pathophysiology. Both systems, having their own unique benefits and disadvantages, have exciting applications in the field of kidney disease modeling and therapeutic discovery and toxicology. In this review, we discuss current uses of both hPSC-derived organoids and MPS as pre-clinical models for studying kidney diseases and drug induced nephrotoxicity. Examples such as the use of organoids to model autosomal dominant polycystic kidney disease, and the use of MPS to predict renal clearance and nephrotoxic concentrations of novel drugs are briefly discussed. Taken together, these novel platforms allow investigators to elaborate critical scientific questions. While much work needs to be done, utility of these 3D cell culture technologies has an optimistic outlook and the potential to accelerate drug development while reducing the use of animal testing.

The Availability of Culturally Preferred Fruits, Vegetables and Whole Grains in Corner Stores and Non-Traditional Food Stores.

Chronic health inequities for communities of color is partially attributed to a lack of healthy preferred food access. This manuscript explores whether corner stores and non-traditional food stores stock fruits, vegetables and whole grain foods that the area cultural communities may prefer as part of complying with a local ordinance. This exploratory analysis identified corner and non-traditional food stores located in immigrant populations of color and African American neighborhoods as part of a larger study. Culturally preferred foods were identified from a list of food items in the parent (STORE) study and used to assess changes in availability. Stores did not have a great variety of culturally relevant foods pre- or post-ordinance, and overall findings show no significant changes over time and/or between ordinance and control community. Further interventions are needed to address cultural food availability in stores near communities of color.

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.

Variable clinical features and genotype-phenotype correlations in 18 patients with late-onset Pompe disease.

BACKGROUND: Pompe disease is a lysosomal storage disorder caused by the deficiency of enzyme acid alpha-glucosidase (GAA) which results in accumulation of glycogen, particularly in the skeletal, cardiac, and smooth muscles. The late-onset form with symptoms presenting in childhood through adulthood, is characterized by proximal muscle weakness, respiratory insufficiency, and unlike the infantile-onset form often with no cardiac involvement. METHODS: We report our experience with 18 adult patients (14 males/4 females) with Pompe disease, several of whom had unique findings and novel pathogenic variants. Patients ranged in ages from 22-74 years (mean 53.7 years) and were diagnosed at an age range of 11-65 years (mean 43.6 years), often after a history of progressive muscle disease of several years' duration. All 18 patients were treated with alglucosidase alfa (Lumizyme) and their response to treatment was monitored by measurements of their pulmonary function and muscle weakness, six-minute walk test (6MWT), and other functional studies. RESULTS: Genetic sequencing revealed that 16 out of 18 individuals had the common c.-32-13T>G splicing variant, and six patients, including two sibships had four novel pathogenic variants: c.1594G>A, c.2655_2656delCG, c.1951-1952delGGinsT, and c.1134C>G. A male with the c.1594G>A variant developed an intracerebral aneurysm at the age of 43 years treated with surgery. Two siblings with the c.2655_2656delCG developed very high antibody titers, one of whom developed a severe infusion reaction. Other clinical features included BiPAP requirement in twelve, tinnitus in seven, scoliosis in five, cardiomyopathy in three, one individual was diagnosed with a cerebral aneurysm who underwent successful Penumbra coil placement, and another individual was diagnosed with both Graves' disease and testicular cancer. CONCLUSIONS: Our study illustrates significant variability in the range of clinical features, and the variable clinical response to enzyme replacement therapy. It also alerts us to the importance of careful monitoring and early management of complications. Possible genotype-phenotype associations with the novel mutations identified may emerge with larger studies.

Isoelectric focusing technology quantifies protein signaling in 25 cells.

A previously undescribed isoelectric focusing technology allows cell signaling to be quantitatively assessed in <25 cells. High-resolution capillary isoelectric focusing allows isoforms and individual phosphorylation forms to be resolved, often to baseline, in a 400-nl capillary. Key to the method is photochemical capture of the resolved protein forms. Once immobilized, the proteins can be probed with specific antibodies flowed through the capillary. Antibodies bound to their targets are detected by chemiluminescence. Because chemiluminescent substrates are flowed through the capillary during detection, localized substrate depletion is overcome, giving excellent linearity of response across several orders of magnitude. By analyzing pan-specific antibody signals from individual resolved forms of a protein, each of these can be quantified, without the problems associated with using multiple antibodies with different binding avidities to detect individual protein forms.