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We used this unique environment to evaluate the effects of microgravity on kidney proximal tubule epithelial cell (PTEC) response to serum exposure and vitaminD 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)D 3 (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. 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The treatment conditions were control (PTEC maintenance media), vitamin D, or 2% human serum. After delivery and installation on board the ISS, cells were acclimated to microgravity for 6 days. Maintenance media cassettes were switched to treatment media cassettes containing either maintenance media, 2% human serum, or 3\u2009\u00b5M vitamin D binding protein (DBP)\u2009+\u20091.5\u2009\u00b5M vitamin D (25(OH)D3) and treated for 48\u2009h prior to fixation with either RNA later for RNAseq or formalin. Ground controls were conducted using minute-to-minute time matching with on-station timing, with a 36\u2009h delay. To prepare 2% human serum treatment media, normal human serum (Valley Biomedical, HS1021) was diluted in PTEC maintenance media to a final concentration of 2%. Vitamin D treatment media consisted of PTEC maintenance media supplemented with 1.5\u2009\u00b5M 25(OH)D3 (Toronto Research Chemicals, C125700) and 3\u2009\u00b5M DBP (Athens Research, 16-16-070307). 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Aligned reads were assessed for strandedness using RSeQC infer_experiment.py (version 5.0.4), gene body coverage using RSeQC geneBody_coverage.py (version 5.0.4), inner distance was assessed using RSeQC inner_distance.py (version 5.0.4), and distribution of RNA features using RSeQC read_distribution.py (version 5.0.4). Aligned reads from all samples were quantified using RSEM (version 1.3.3), with strandedness set to none and RSEM count log files were compiled with MultiQC (version 1.26). To create a set of rRNA-removed quantification data, Ensembl IDs mapping to ribosomal RNA (rRNA) were removed from the RSEM gene count data. Both the original set of RSEM quantification data and the rRNA-removed data were subject to differential gene expression analysis as follows. A runsheet was generated with dp_tools (version 1.3.8) and the runsheet and quantification data were imported to R (version 4.4.2) with tximport (version 1.34.0) and normalized with DESeq2 (version 1.46.0) median of ratios method. Normalized gene counts were subject to differential expression analysis. Differential expression analysis was performed in R (version 4.4.2) using DESeq2 (version 1.46.0); all groups were compared pairwise using the Wald test and the likelihood ratio test was used to generate the F statistic p-value. 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