Cosmic kidney disease: an integrated pan-omic, physiological and morphological study into spaceflight-induced renal dysfunction

Abstract

Missions into Deep Space are planned this decade. Yet the health consequences of exposure to microgravity and galactic cosmic radiation (GCR) over years-long missions on indispensable visceral organs such as the kidney are largely unexplored. We performed biomolecular (epigenomic, transcriptomic, proteomic, epiproteomic, metabolomic, metagenomic), clinical chemistry (electrolytes, endocrinology, biochemistry) and morphometry (histology, 3D imaging, miRNA-ISH, tissue weights) analyses using samples and datasets available from 11 spaceflight-exposed mouse and 5 human, 1 simulated microgravity rat and 4 simulated GCR-exposed mouse missions. We found that spaceflight induces: 1) renal transporter dephosphorylation which may indicate astronauts' increased risk of nephrolithiasis is in part a primary renal phenomenon rather than solely a secondary consequence of bone loss; 2) remodelling of the nephron that results in expansion of distal convoluted tubule size but loss of overall tubule density; 3) renal damage and dysfunction when exposed to a Mars roundtrip dose-equivalent of simulated GCR.

Fig. 1. Study design.

a Infographic representation of the study design. (Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license). b Details of spaceflight missions and simulations included in the project. R return, L launch, PE post-exposure, P plasma, U urine, K kidney; F faecal. a, b N numbers correspond to the number of experimental individuals that were exposed to spaceflight or a simulated spaceflight condition, and to control condition individuals were omitted from the presented counts to ensure paired and unpaired experiments were comparable.

Fig. 2. Human spaceflight urine and plasma physiological measurements.

a Urinary chemistries and b blood plasma endocrine profiles from NASA astronauts (n = 66) exposed to spaceflight up to 180 days measured pre-flight, during (FD flight day) and after returning (R). Dashed lines represent upper and lower normal clinical values, or upper limit where only a single line is present. Data are presented as mean ± SD. Boxed P-values report the repeated measure one-way ANOVA result; all timepoints were compared to pre-flight by pairwise multiple comparison Bonferroni corrected post-hoc tests (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) (For exact P-values see Supplementary Table 1). FE_Ca Fraction Excretion of Calcium. TmP/GFR ratio of tubular maximum reabsorption rate of phosphate to glomerular filtration rate. 1,25-dihydroxyvitamin D3 calcitriol. PTH parathyroid hormone. FGF-23 fibroblast growth factor-23.

Fig. 3. Spaceflight-induced changes in the kidney phosphoproteome.

a Volcano plot of differentially phosphorylated amino acid residues detected in phosphopeptide-enriched kidney protein extract from RR-10 spaceflight-exposed mice (28 days). Regulatory phosphosites in transporters and channels related to the thick ascending limb of the loop of Henle and distal convoluted tubule are highlighted. Green indicates a statistically significant increase in peptide phosphorylation, magenta indicates a decrease, and grey represents no significant change. An unadjusted two-tailed -Log₁₀(Adjusted P-value) of 1.3 was considered statistically significant (indicated by the dashed line intersecting the x-axis). Kir4.1 (KCNJ10); Kir5.1 (KCNJ16); NKCC2 (SLC12A1); NCC (SLC12A3); KCC4 (SLC12A7); pT, phospho-threonine; pS, phospho-serine. b Kinase-Substrate Enrichment Analysis (KSEA) was performed using the Robust Inference of Kinase Activity (RoKAI) App v2.2.1 to predict kinase and phosphatase activity levels using phosphoproteomics from RR-10 spaceflight-exposed mice (28 days) kidney tissue. Bars in blue represent downregulated activity and those in red represent upregulated activity. An adjusted P-value of < 0.05 was considered significant. Data are mean SEM. An FDR of 5% was used as a cutoff. N = 10 biologically independent animals per group (flight vs ground control). 4257 single-site phosphopeptides (Ser/Thr/Tyr) were used as initial input, 495 of which were matched to known kinase/phosphatase targets, that were then used to calculate respective predicted kinase/phosphatase activities. Input and results files for this can be found at OSD 466.c Representative confocal images of RR-10 spaceflight-exposed mice (28 days) kidney sections [n = 7 spaceflight / n = 10 ground control) stained with an anti-NCC pT44, pT48 & pT53 antibody (green; pNCC) and a total NCC antibody (magenta; tNCC) to visualise NCC phosphorylation-dependent activation (green and magenta overlap will appear as white); 50 µm scale bar.

Fig. 4. Multi-mission pan-omic investigation of the contributors to nephrolithiasis risk during spaceflight.

a Multi-omic over-representation analysis of DisGeNET gene-disease associations related to nephrolithiasis. To integrate datasets from different omics modalities, species, missions and tissues, all biomolecules (e.g. phosphopeptides, proteins, transcripts and methylated DNA) were converted to the human orthologs where necessary and linked back to their HGNC gene symbol, aggregated and collapsed to single genes (e.g. multiple phosphosites, isoforms, CpG sites). An unadjusted, two-tailed -Log₁₀(P-value) of 2 was considered significant for ontological term enrichment. b Categorical heatmap of differential abundance directionality in nephrolithiasis-related faecal microbial taxa after spaceflight. An unadjusted, two-tailed -Log₁₀(P-value) of 1.3 was considered significant. c Over-representation analysis of KEGG module metabolic pathways with replication in at least two datasets. An unadjusted, two-tailed -Log₁₀(P-value) of 1.3 was considered significant for ontological term enrichment. a, c Enrichment ratio; the number of differentially regulated hits in a dataset that belong to a given ontological term, normalised to the total number of statistically significant hits in the respective dataset.

Fig. 5. Multi-mission multi-omic consensus on differentially regulated gene products in the kidney.

Categorical heatmap of a, upregulated and b, downregulated gene products in exposure groups (e.g. spaceflight, GCRsim) compared to control groups (e.g. ground control, sham). To integrate datasets from different omics modalities, species, missions and tissues, all biomolecules (e.g. phosphopeptides, proteins, transcripts and methylated DNA) were converted to the human orthologs where necessary and linked back to their HGNC gene symbol, aggregated and collapsed to single genes (e.g. multiple phosphosites, isoforms, CpG sites). These differentially regulated gene products (DRGP) were ranked and represented in descending order using the following rules: (1) only DRGPs with a P-value < 0.05 by Wald test were counted as significant and plotted; (2) Each DRGP was assigned a score of +1 each time it was upregulated or −1 each time it was downregulated; (3) only DRGPs observed in proteome and transcriptome kidney-specific datasets were used for the calculation of the ranking score to avoid confounders, although additional datasets (epiproteome, epigenome and those from plasma/exosomes sources) were plotted for visualisation comparison purposes; (4) The resulting sum of scores for each DRGP was then calculated and multiplied by the number of times the DRGP was observed in the kidney-specific proteome and transcriptome datasets; (5) To increase confidence only DRGPs with a product score of absolute value 9 or higher (but excluding a score of 12) were plotted as these will have a directionality consensus of at least 3 datasets above the number of disagreeing datasets.

Fig. 6. Biomolecular evidence for spaceflight-induced nephron remodelling.

a A curated list of multi-omic over-representation analysis of GO—biological process ontological terms related to remodelling. b A curated list of multi-omic over-representation analysis of KEGG pathway ontological terms related to remodelling. a, b To integrate datasets from different omics modalities, species, missions and tissues, all biomolecules (e.g. phosphopeptides, proteins, transcripts and methylated DNA) were converted to the human orthologs where necessary and linked back to their HGNC gene symbol, aggregated and collapsed to single genes (e.g. multiple phosphosites, isoforms, CpG sites). An unadjusted, two-tailed -Log₁₀(P-value) of 2 was considered significant for ontological term enrichment. Enrichment ratio; the number of differentially regulated hits in a dataset that belong to a given ontological term, normalised to the total number of statistically significant hits in the respective dataset. c STRING protein-protein interaction network of previously absent inflammation-associated and collagen I-associated ECM proteins that appeared in cosmonauts’ urine (n = 10) after 166–199 days of spaceflight exposure.

Fig. 7. Anatomical evidence for spaceflight-induced nephron remodelling.

a Morphometry of kidney mass was assessed by wet weights in 1 G, the weight of both left and right kidneys were averaged, and the kidney weights were normalised against bodyweight (BW) for the same animal and expressed as a percentage. All ground control and sham animals that received no exposure treatment were grouped into control. Animals that only received either full or simplified galactic cosmic radiation (GCR) simulations were grouped into GCR. Animals that only underwent hindlimb unloading microgravity simulation were grouped into microgravity (MG). Animals that underwent a combination of GCR and MG, or were exposed to spaceflight, were grouped into GCR + MG. Data are presented as mean ± SD. One-way ANOVA with pairwise Dunnett’s multiple comparison post-hoc tests (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). N numbers are control = 48, GCR = 30, MG = 28, MG + GCR = 39 biologically independent animals per group. b Representative confocal images showing the annotations of cortex regions (magenta borders) and distal convoluted tubules (DCT, green) in RR-10 spaceflight-exposed mice (28 days) from whole slide images of kidney sections immunolabelled with tNCC/pNCC as DCT markers. 200 µm scale bar. c Morphometric analysis of DCTs assessed the average area per tubule (top Y-axis), the density of no. of tubules per unit area of cortex (middle Y-axis), and the total summed area of all DCTs as percentage of cortex area. Data are mean ± SD. P-value of  0.05 by students t-test was considered significant. N = 10 biologically independent animals per group. Source data are provided as a Source Data file.

Fig. 8. Biomolecular evidence for spaceflight-induced kidney damage.

A curated list of enriched gene-disease associations are presented for DisGeNET ontological terms relevant to kidney health. These were ranked and represented in descending order using the following rules: 1) No. of mission datasets it replicated in; 2) most significant P-value; 3) greatest enrichment. To integrate datasets from different omics modalities, species, missions and tissues, all biomolecules (e.g. phosphopeptides, proteins, transcripts and methylated DNA) were converted to the human orthologs where necessary and linked back to their HGNC gene symbol, aggregated and collapsed to single genes (e.g. multiple phosphosites, isoforms, CpG sites). An unadjusted, two-tailed Log₁₀(P-value) of 2 was considered significant for ontological term enrichment. Enrichment ratio; the number of differentially regulated hits in a dataset that belong to a given ontological term, normalised to the total number of statistically significant hits in the respective dataset.

Fig. 9. Acute impact of galactic cosmic radiation (GCR) on kidney health.

a Schematic representation of an axial cross section of mouse kidney hemisected at through the hilum as shown. Annotations of gross anatomical regions of the kidney used for spatial RNA expression analyses. b Representative images of miR-125b staining (small red dots) in the outer medulla of haematoxylin-stained kidney sections from BNL-3 simGCRsim-exposed mice (~ 1.5-year-dose equivalent) harvested ~ 24 h post-exposure. White arrows indicate concentrations of miR-125b staining around capillaries in the interstitium; 50 µm scale bar. Data are mean ± SD. A P-value of < 0.05 by students t-test was considered significant. Average; the simple arithmetic mean of the four anatomical regions, n = 6 biologically independent animals per group c Gross anatomical and microanatomy features annotated on exemplar slide-seq pucks of BNL-1 simGCRsim-exposed mice (~ 1.5-year-dose equivalent) harvested ~ 24 h post-exposure. Samples for pucks were taken to maximise the anatomical coverage from the approximate regions shown in the green (autofluorescence) axial section of mouse kidney. Beads are assigned identities according to their highest probable cell type classification. For scale, each dot represents a 10 µm bead. d The table shows the differentially regulated mRNA transcripts from c, captured by different cell typed beads after exposure to GCR. An adjusted P-value of < 0.05 by students t-test was considered significant. Abbreviations: OSOM outer stripe of outer medulla. ISOM inner stripe of outer medulla. IM Inner medulla. DCT distal convoluted tubule. Early PT or PTS1 early proximal tubule S1. Endo; endothelial cell. IC; intercalated cell. CTAL/MTAL or LOH; (cortical or medullary) thick ascending limb of the loop of Henle. Macro; macrophage. Neutro neutrophil. NP nephron progenitor cell. PC principal cell. PCT or PTS1/2 proximal convoluted tubule S1 + S2; Podo podocyte. PST or PTS3 proximal straight tubule S3. G glomerulus. DTL descending thin limb of the loop of Henle. ATL ascending thin limb of the loop of Henle. MD macula densa. CNT connecting tubule. CCD cortical collecting duct. OMCD outer medullary collecting duct. IMCD inner medullary collecting duct. Endo endothelial cell. IC intercalated cell. Macro macrophage. Neutro neutrophil. NP nephron progenitor cel.; Podo podocyte. The schematic map of a whole kidney’s cross section and the visualisation by actin filament fluorescent were published in The FEBS Journal 287 (2020) 1176–1194 [doi:10.1111/febs.15088] © 2019 Kumaran et al. and is licensed under CC BY 4.0. Source data are provided as a Source Data file.

Fig. 10. Chronic impact of galactic cosmic radiation (GCR) on kidney health.

a Representative images of haematoxylin and eosin (H&E), Masson’s trichrome, Martius scarlet blue (MSB), Periodic acid–Schiff (PAS), Picrosirius red, and Von Kossa stained kidney sections from NSRL22A GCRsim-exposed mice (~ 2.5-year dose-equivalent) harvested ~ 6 months post-exposure. Arrows indicate microthrombi in the glomerular capillary tufts visible in H&E, Masson, MSB and PAS stains. Scale bar: 50 µm (left panel) and 100 µm (right). b Urine (Protein:Cr, Glucose:Cr, Magnesium:Cr, FE_Na, FE_Ca, FE_Mg(Corr), FE_PO4) and plasma (sodium, chloride) physiological measurements from NSRL22A GCRsim-exposed mice ( ~ 2.5-year dose-equivalent) harvested ~ 6 months post-exposure that were normalised to sham controls for illustrative purposes. Boxed P-values report the two-way ANOVA treatment group factor result. Data are mean ± SD. A P-value of < 0.05 was considered significant. Cr Creatinine, FE Fraction Excretion, Corr corrected for albumin, Ca calcium, Na sodium, Mg magnesium, P_O4 phosphate, N = 12 biologically independent animals per sex per group. ‘Source data are provided as a Source Data file.