Our team has approached the scientific challenges of studying kidney disease pathophysiology, novel therapeutic agents, and drug/ environmental toxicity by establishing two innovative human models: the proximal tubule kidney chip microphysiological system (PTEC MPS) and the iPSC (induced pluripotent stem cell)-derived kidney organoid. We have developed validation assays for short-duration (2-10 day) exposures in both ground-based laboratories and aboard the International Space Station. The goal of this project is to demonstrate models with extended (6 month) longevity to better understand the role of chronic stressor exposure on kidney disease initiation and progression. Based on previous studies, we hypothesize that human kidney systems will remain viable and functionally stable for at least 6 months and will respond physiologically to extended exposure to stressors.

Phase I – Description and Tasks. Establish extended longevity and functionality (6 months) of kidney microphysiological systems and organoids. The contractor shall: • Determine longevity of the proximal tubule kidney chip (PTEC) MPS at 2.5, 5, and 6 months by employing cell viability (live/dead staining), metabolic function (conversion of 25(OH)VitaminD3 to 1a25(OH)2 VitaminD3), toxic response (resulting in KIM-1 [kidney injury molecule-1] expression), stable expression of immunomodulators (e.g., IL[interleukin]-6, IL-23A, TNF [tumor necrosis factor], IL-32), and global RNA transcript profile. • Determine longevity and stable function of kidney organoids at 2.5, 5, and 6 months by conducting observation of distinct tubules with specific multiple nephron and stromal lineages in segmented structures (immunocytochemical staining), and stable global single cell RNA transcript profile.

Phase II – Description and Tasks. Test structural and functional outcomes after sustained exposure to drugs, environmental toxins, and pathogens and post-exposure cellular recovery. The contractor shall: • Determine functional outcomes of PTEC MPS after exposure to agents known to cause nephrotoxicity after 5 months exposure (PMB [polymyxin B] or other nephrotoxin) followed by 1 month recovery by evaluating structural derangement (de-polarization of transporter proteins), production of injury biomarkers (KIM-1, HO-1 [heme oxygenase-1], cystatin), and altered regulation of immunomodulators (e.g., IL-6, IL-23A, TNF, IL-32). • Evaluate kidney organoid responses to pathogenic stimuli and known nephrotoxins using fluorescent reporters for real-time detection of infection and injury in response to acute (3 days) and extended (5 months) exposure followed by 1 month of recovery.

Organoid updates: Previously, we were able to maintain viable human kidney organoids for six months. We have done additional analysis of kidney organoid structure, function, and gene expression (single cell RNA sequencing) from this experiment. We have also repeated the experiment successfully. The organoid team has now transitioned from Phase 1 to Phase 2 of the project. We are now focused on understanding how nephrotoxicity and pathogens affect kidney organoids in both acute and chronic settings. For pathogens (milestone 6), we have done a trial experiment for 60 days to test 3 different concentrations of IFN-gamma. For nephrotoxicity (milestone 7), we have optimized the doses of gentamicin and cisplatin in the acute setting and validated an antibody for KIM-1 detection that shows cellular damage localizing to the proximal tubular cells.

Our team has approached the scientific challenges of studying kidney disease pathophysiology, novel therapeutic agents, and drug/ environmental toxicity by establishing two innovative human models: the proximal tubule kidney chip microphysiological system (PTEC MPS) and the iPSC (induced pluripotent stem cell)-derived kidney organoid. We have developed validation assays for short-duration (2-10 day) exposures in both ground-based laboratories and aboard the International Space Station. The goal of this project is to demonstrate models with extended (6 month) longevity to better understand the role of chronic stressor exposure on kidney disease initiation and progression. Based on previous studies, we hypothesize that human kidney systems will remain viable and functionally stable for at least 6 months and will respond physiologically to extended exposure to stressors.

Phase I – Description and Tasks. Establish extended longevity and functionality (6 months) of kidney microphysiological systems and organoids. The contractor shall: • Determine longevity of the proximal tubule kidney chip (PTEC) MPS at 2.5, 5, and 6 months by employing cell viability (live/dead staining), metabolic function (conversion of 25(OH)VitaminD3 to 1a25(OH)2 VitaminD3), toxic response (resulting in KIM-1 [kidney injury molecule-1] expression), stable expression of immunomodulators (e.g., IL[interleukin]-6, IL-23A, TNF [tumor necrosis factor], IL-32), and global RNA transcript profile. • Determine longevity and stable function of kidney organoids at 2.5, 5, and 6 months by conducting observation of distinct tubules with specific multiple nephron and stromal lineages in segmented structures (immunocytochemical staining), and stable global single cell RNA transcript profile.

Phase II – Description and Tasks. Test structural and functional outcomes after sustained exposure to drugs, environmental toxins, and pathogens and post-exposure cellular recovery. The contractor shall: • Determine functional outcomes of PTEC MPS after exposure to agents known to cause nephrotoxicity after 5 months exposure (PMB [polymyxin B] or other nephrotoxin) followed by 1 month recovery by evaluating structural derangement (de-polarization of transporter proteins), production of injury biomarkers (KIM-1, HO-1 [heme oxygenase-1], cystatin), and altered regulation of immunomodulators (e.g., IL-6, IL-23A, TNF, IL-32). • Evaluate kidney organoid responses to pathogenic stimuli and known nephrotoxins using fluorescent reporters for real-time detection of infection and injury in response to acute (3 days) and extended (5 months) exposure followed by 1 month of recovery.

We have also been able to maintain viable kidney organoids for at least six months. Kidney organoid structure and organization was consistent throughout the experiment. To analyze function, we demonstrated proximal tubule function in the kidney organoids via dextran absorption over the course of the experiment, supporting the idea that these cells were functional after 180 days. We have collected and analyzed RNA from organoids on day 25 and day 180 for single cell RNA sequencing.

Dr. Edward Kelly, a Co-Investigator on this grant, also contributed to a National Academies of Sciences Decadal Survey publication entitled, "Thriving in space: Ensuring the future of biological and physical sciences research: A decadal survey for 2023-2032." [Ed. Note: See Bibliography.]

Our team has approached the scientific challenges of studying kidney disease pathophysiology, novel therapeutic agents, and drug/ environmental toxicity by establishing two innovative human models: the proximal tubule kidney chip microphysiological system (PTEC MPS) and the iPSC (induced pluripotent stem cell)-derived kidney organoid. We have developed validation assays for short-duration (2-10 day) exposures in both ground-based laboratories and aboard the International Space Station. The goal of this project is to demonstrate models with extended (6 month) longevity to better understand the role of chronic stressor exposure on kidney disease initiation and progression. Based on previous studies, we hypothesize that human kidney systems will remain viable and functionally stable for at least 6 months and will respond physiologically to extended exposure to stressors.

Phase I – Description and Tasks. Establish extended longevity and functionality (6 months) of kidney microphysiological systems and organoids. The contractor shall: • Determine longevity of the proximal tubule kidney chip (PTEC) MPS at 2.5, 5, and 6 months by employing cell viability (live/dead staining), metabolic function (conversion of 25(OH)VitaminD3 to 1a25(OH)2 VitaminD3), toxic response (resulting in KIM-1 [kidney injury molecule-1] expression), stable expression of immunomodulators (e.g., IL[interleukin]-6, IL-23A, TNF [tumor necrosis factor], IL-32), and global RNA transcript profile. • Determine longevity and stable function of kidney organoids at 2.5, 5, and 6 months by conducting observation of distinct tubules with specific multiple nephron and stromal lineages in segmented structures (immunocytochemical staining), and stable global single cell RNA transcript profile.

Phase II – Description and Tasks. Test structural and functional outcomes after sustained exposure to drugs, environmental toxins, and pathogens and post-exposure cellular recovery. The contractor shall: • Determine functional outcomes of PTEC MPS after exposure to agents known to cause nephrotoxicity after 5 months exposure (PMB [polymyxin B] or other nephrotoxin) followed by 1 month recovery by evaluating structural derangement (de-polarization of transporter proteins), production of injury biomarkers (KIM-1, HO-1 [heme oxygenase-1], cystatin), and altered regulation of immunomodulators (e.g., IL-6, IL-23A, TNF, IL-32). • Evaluate kidney organoid responses to pathogenic stimuli and known nephrotoxins using fluorescent reporters for real-time detection of infection and injury in response to acute (3 days) and extended (5 months) exposure followed by 1 month of recovery.