RESUMEN
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). Given the profound bone loss observed in microgravity and PTECs produce the active form of vitamin D, we 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 months) exposure to microgravity.
RESUMEN
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.
RESUMEN
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.
RESUMEN
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.
RESUMEN
MglA is a transcriptional regulator of genes that contribute to the virulence of Francisella tularensis, a highly infectious pathogen and the causative agent of tularemia. This study used a label-free shotgun proteomics method to determine the F. tularensis subsp. novicida (F. novicida) proteins that are regulated by MglA. The differences in relative protein amounts between wild-type F. novicida and the mglA mutant were derived directly from the average peptide precursor ion intensity values measured with the mass spectrometer by using a suite of mathematical algorithms. Among the proteins whose relative amounts changed in an F. novicida mglA mutant were homologs of oxidative and general stress response proteins. The F. novicida mglA mutant exhibited decreased survival during stationary-phase growth and increased susceptibility to killing by superoxide generated by the redox-cycling agent paraquat. The F. novicida mglA mutant also showed increased survival upon exposure to hydrogen peroxide, likely due to increased amounts of the catalase KatG. Our results suggested that MglA coordinates the stress response of F. tularensis and is likely essential for bacterial survival in harsh environments.