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αKlotho decreases after reduced weight-bearing from both spaceflight and hindlimb unloading

αKlotho decreases after reduced weight-bearing from both spaceflight and hindlimb unloading www.nature.com/npjmgrav BRIEF COMMUNICATION OPEN αKlotho decreases after reduced weight-bearing from both spaceflight and hindlimb unloading 1✉ 2 3 1 4 2 Jeffrey S. Willey , Serena Aunon-Chancellor , Lauren A. Miles , Joseph E. Moore , Xiao W. Mao , Robert W. Wallace and Matthew C. Foy Alpha(α)Klotho, a soluble transmembrane protein, facilitates calcium-phosphorus homeostasis through feedback between bone and kidney and is a potential systemic biomarker for bone-kidney health during spaceflight. We determined if: (1) plasma αKlotho was reduced after both spaceflight aboard the ISS and hindlimb unloading (HU); and (2) deficiency could be reversed with exercise. Both spaceflight and HU lowered circulating plasma αKlotho: plasma αKlotho recovered with exercise after HU. npj Microgravity (2022) 8:18 ; https://doi.org/10.1038/s41526-022-00203-w INTRODUCTION This pilot investigation measured circulating αKlotho levels in mice after exposure to two reduced weight-bearing conditions: Atrophy of skeletal elements has long been identified as a hazard microgravity aboard the ISS, and after hind limb unloading (HU) . for the success of long-duration missions and astronaut quality of Importantly, musculoskeletal degradation has previously been life . US astronauts aboard the International Space Station (ISS) described in hindlimbs from these same mice after 35 days in exhibit loss of bone density when comparing pre- and post-flight 14,15 2,3 microgravity and HU , observing recovery of joint health upon DXA scan measurements . Bone density reduction for a Mars 4 return to full weight-bearing with exercise . Likewise, this study mission has been estimated of up to 36% . Recovery from bone 5,6 examined if deficits in circulating αKlotho after periods HU could be degradation appears to be incomplete . recovered after return to full-weight bearing, with/without perform- The role of the kidneys in skeletal homeostasis is complex. ing exercise. Changes in α-Klotho after acute bouts of exercise are Recent research interest includes the interplay between αKlotho 7 inconsistent when examining resistance vs aerobic exercise (ref. ), and Fibroblast Growth Factor 23 (FGF23) . Alpha Klotho is secreted with long term exercise exhibiting elevated αKlotho in sedentary predominantly in the kidneys, whereas FGF23 is secreted by individuals (ref. ). This hypothesis-generating research aims to osteocytes . FGF23 binds to αKlotho, and increases urine assess if αKlotho can serve as a circulating biomarker for bone phosphorous excretion . Both are required, as seen in mice that status, both in-flight and upon return to weight-bearing. demonstrate phosphate retention if either component is defi- Data are presented mean(SD). Plasma αKlotho in pg/ml was 10 7 cient . FGF23 also increases calcium reabsorption . Through ~50% lower (Fig. 1) after 35 days in orbit aboard the ISS from the these mechanisms, FGF23 eliminates excess phosphorus and FLIGHT mice vs GROUND. No differences were observed between increases plasma calcium, with subsequent decreased calcitriol groups for FGF23 or inorganic phosphorus (Pi) concentration synthesis and parathyroid hormone (PTH) release. In non-weight- (Table 1). Additionally, gastrocnemius muscle mass (g) was lower bearing climates with known bone density loss, αKlotho and (p = 0.0035) in FLIGHT vs GROUND after time in orbit aboard the FGF23 may play a role in skeletal homeostasis. ISS (Table 1). For the hind limb unloading study, plasma αKlotho The current spaceflight countermeasures aimed at preventing after 30 days of tail suspension [HU-30day] was 36% lower vs 6,11 bone loss provide incomplete protection . The Advanced plasma αKlotho from mice that remained full weight bearing Resistive Exercise Device (ARED) is utilized for exercise aboard [GROUND-30day; p < 0.01; Fig. 2]. Likewise, αKlotho was lower in the ISS . Use of the ARED as a single countermeasure largely mice after the initial 30 days of HU vs all mice that were full- maintains preflight skeletal health over a 6-month mission in weight bearing throughout the entire 80 day study, with or orbit , but bone loss is observed by 1 year post-flight, indicating without exercise [GROUND-No exercise; GROUND-Climbing; and progressive atrophy despite a return to full weight-bearing and GROUND-Running; p < 0.05 for all comparisons)]. In contrast, after from astronauts also receiving bisphosphonate therapy . Addi- returning to full weight-bearing from the 30 day period of HU, tionally, a device as large and complex as ARED is likely not plasma αKlotho levels remained lower in the mice that performed feasible on an exploration class mission due to limitations in mass no exercise [HU-No Exercise] vs plasma from the full weight and habitable space. These findings add concerns for the current bearing mice as measured on Day 30 (p < 0.05). Additionally, these ability to preserve bone health outside low earth orbit. mice that had not performed exercise after the 30 day period of Currently, no reliable real-time analysis of biomarkers or skeletal HU were also significantly lower than all GROUND groups on Day imaging on orbit exists. Identification of novel circulating 80, regardless of exercise (GROUND-No exercise; GROUND- biomarkers for musculoskeletal health, such as αKlotho, could Climbing; GROUND-Running). In contrast, running exercise for prove crucial to protective measures as the space community 49 days after the initial 30 day HU period [HU-Running] resulted in turns its eyes towards Mars. partial recovery of plasma Klotho, with no difference vs the full 1 2 Department of Radiation Oncology, Wake Forest School of Medicine, Section of Radiation Biology, Winston-Salem, NC, USA. Department of Internal Medicine, Louisiana State 3 4 University Medical Center, Baton Rouge, LA, USA. Department of Nephrology, Wake Forest School of Medicine, Winston-Salem, NC, USA. Department of Basic Sciences, Loma Linda University, Loma Linda, CA, USA. Department of Internal Medicine, Louisiana State University Medical Center, Division of Nephrology, Baton Rouge, LA, USA. email: jwilley@wakehealth.edu Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; J.S. Willey et al. p<0.01 *** ** * 60 * Fig. 1 αKlotho concentration is reduced after spaceflight. FLIGHT mice exhibited lower αKlotho vs GROUND. P value from unpaired t test provided above bars. Bars: Mean(SD). Table 1. Descriptive information from both the spaceflight study to Fig. 2 αKlotho concentration is reduced after hind limb unload- the International Space Station, and the ground-based hind limb ing, but can recover with subsequent exercise. αKlotho was lower than GROUND in plasma after 30 days HU, but recovery occurred unloading (HU) study. with exercise *p ≤ 0.05; **p < 0.01; ***p < 0.001 determined via Tukey’s Post Hoc test after performed after ANOVA. Bars: Mean(SD). Spaceflight study Hind limb unloading study GROUND FLIGHT GROUND- HU-30day 30day FGF23 (pg/ml) 26.14(12.45) 27.10(14.79) NA NA Pi (mg/dL) 4.26(0.29) 4.06(0.18) 7.21(3.27) 8.39(3.62) Gastrocnemius 159.0(13.1) 134.6(18.8)# NA NA mass (g) Data are presented mean(SD). # indicates diffference for FLIGHT vs GROUND at p = 0.0035 via unpaired t-test. NA: data not collected (Gastrocnemius mass) or presented in Fig. 3 (FGF 23). weight bearing group at Day 30 (GROUND-30 Day), but still having lower concentration (p < 0.05) vs GROUND-No Exercise. Climbing exercise [HU-Climbing] for 49 days provided full recovery of αKlotho (Fig. 2), being similar to all GROUND groups. No differences were observed between any groups for FGF23 concentration (Fig. 3). Moreover, Pi level between GROUND- Fig. 3 Plasma FGF23 was unaltered after hind limb unloading. 30day and HU-30day was similar (Table 1). Sample limitation Plasma FGF23 was similar across groups and time points. Bars: Mean prevented the assessment of other groups. (SD). A reduction in plasma αKlotho levels was observed in both FLIGHT and HU mice vs controls. While a mechanistic/causal link adverse effects of microgravity on bone, as well as muscle, these between weight-bearing conditions and αKlotho are not deter- findings suggest an important link between this hormone level mined, both reduced weight-bearing conditions did result in and skeletal health. Histologic changes of bone with altered lowered αKlotho. For HU mice, levels of αKlotho increased kidney physiology in relation to these hormonal changes are following exercise, with partial recovery in the running group needed to better understand this relationship. The similar changes and full recovery in the climbing group, suggesting an important relationship between exercise/potentially elevated weight-bearing in αKlotho found in both FLIGHT mice and HU mice suggest that and αKlotho level improvement. These data are aligned with HU mice may provide an appropriate terrestrial analog for recovery of joint health upon a return to full weight-bearing with examining the relationship between αKlotho and health along exercise . Moreover, for FLIGHT mice, where muscle masses were the musculoskeletal tissue-kidney axis. collected, the decrease in αKlotho post-flight occurred with reduced gastrocnemius mass, which itself is not unexpected but METHODS αKlotho may also be affected by skeletal muscle activity . When considering the importance αKlotho plays in maintaining The animal and environmental details of the ISS study (Rodent musculoskeletal skeletal health, viewed within the context of the Research-9 mission), the accompanying HU study, and all npj Microgravity (2022) 18 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA G O R UND FLIGHT Ground-30day HU-30day GROUND-No Exercise G O i R UND-Runn ng GROUND-Climbing HU-No Exercise HU-Running HU-Climbing Ground-30day HU-30day GROUND-No Exercise GROUND-Running GROUND-Climbing HU-No Exercise HU-Running HU-Climbing 1234567890():,; Klotho Concentration (pg/ml) FGF23 Concentraiton (pg/ml) Klotho Concentraiton (pg/ml) J.S. Willey et al. approved IACUC protocols from Wake Forest School of Medicine, 7. Kuro, O. M. The Klotho proteins in health and disease. Nat. Rev. Nephrol. 15,27–44 (2019). NASA Ames, and the Kenney Space Center have been published . 8. Kuro, O. M. A potential link between phosphate and aging-lessons from Klotho- All mice were male, C57BL/6 (Jackson Labs) that were 10 weeks at deficient mice. Mech. Ageing Dev. 131, 270–275 (2010). the start of each study. 9. Hu, M. C., Shiizaki, K., Kuro-o, M. & Moe, O. W. Fibroblast growth factor 23 and Klotho: Physiology and pathophysiology of an endocrine network of mineral ISS mission metabolism. Annu. Rev. Physiol. 75, 503–533 (2013). 10. Kuro, O. M. Phosphate and Klotho. Kidney Int. https://doi.org/10.1038/ki.2011.26 Groups included FLIGHT and GROUND control . This investigation (2011). had access to n = 4–5 plasma samples/group collected after 11. Sibonga, J. et al. Resistive exercise in astronauts on prolonged spaceflights pro- 35 days on orbit; all available samples were analyzed for this study. vides partial protection against spaceflight-induced bone loss. Bone 128, 112037 (2019). HU study 12. Smith, S. M. et al. Benefits for bone from resistance exercise and nutrition in long- duration spaceflight: Evidence from biochemistry and densitometry. J. Bone Min. Groups included weight-bearing GROUND mice, or HU via tail Res. 27, 1896–1906 (2012). suspension, as described .OnDay 30,plasmawas isolated from a 13. Steczina, S. et al. Dietary countermeasure mitigates simulated spaceflight- cohort of GROUND (GROUND-30day) and HU (HU-30day) mice. induced osteopenia in mice. Sci. Rep. 10, 6484 (2020). Remaining HU mice were then removed from tail suspension and 14. da Silveira, W. A. et al. Comprehensive multi-omics analysis reveals mitochondrial were thus weight-bearing the remainder of the study. All mice that stress as a central biological hub for spaceflight impact. Cell 183, 1185–1201 were previously GROUND or HU were enrolled into one of 3 exercise e1120 (2020). groups, as described ,fromDays31–80, performing: 1] no exercise; 15. Kwok, A. T. et al. Spaceflight and hind limb unloading induces an arthritic pheno- type in knee articular cartilage and menisci of rodents. Sci. Rep. 11, 10469 (2021). 2] climbing exercise 3X weekly, or; 3] running exercise 3X weekly in 16. Iturriaga, T. et al. Acute impacts of different types of exercise on circulating alpha- order to determine if recovery was possible with aerobic (running) or Klotho protein levels. Front. Physiol. 12, 716473 (2021). a resistance (climbing) regimen, yielding the following six groups: 17. Amaro-Gahete, F. J. et al. Exercise training increases the S-Klotho plasma levels in GROUND-No exercise; GROUND-Climbing; GROUND-Running; HU-No sedentary middle-aged adults: A randomised controlled trial. The FIT-AGEING exercise; HU-Climbing; and HU-Running. Climbing exercises were study. J. Sports Sci. 37, 2175–2183 (2019). performed 3X/week,. This investigation had access to n=4plasma 18. Avin, K. G. et al. Skeletal muscle as a regulator of the longevity protein, Klotho. samples/group, all of which were analyzed. Front. Physiol. 5, 189 (2014). Blood was collected in [K2]ethylenediaminetetraacetic acid (EDTA)- containing syringes by cardiac puncture and centrifuged for 10 min at ACKNOWLEDGEMENTS 3000 rpm at 4 °C; Plasma was isolated and ELISA was used to detect NASA Space Biology grant NNX15AB50G(JSW). αKlotho (R&D Systems™ #DY5334) and FGF23 (Abcam #ab213863); a commercial Pi kit was also used (Pointe Scientific #P7516-500). Comparisons between FLIGHT and GROUND mice were AUTHOR CONTRIBUTIONS performed using a two-way unpaired t-test; ANOVA was J.S.W.: conceptualization, study design, performance of study, data analysis/ performed for the HU study, with Tukeys post hoc tests. α ≤ interpretation, manuscript preparation. S.A.C. and M.C.F.: conceptualization, data 0.05; the assumption of equal variance was used to test equality of interpretation, manuscript preparation. L.A.M. and R.W.W.: conceptualization, manu- variance. Analyses were performed using GraphPad Prizm 8.4.0. script preparation. J.E.M.: performance of study, data analysis, manuscript prepara- The data that support the findings of this study are available on tion. X.W.M.: study design, tissue collection, manuscript preparation. request from the corresponding author. COMPETING INTERESTS Reporting summary The authors declare no competing interests. Further information on research design is available in the Nature Research Reporting Summary linked to this article. ADDITIONAL INFORMATION Supplementary information The online version contains supplementary material DATA AVAILABILITY available at https://doi.org/10.1038/s41526-022-00203-w. The authors declare that data supporting the findings of this study are available within the figures of the article, and/or are available on request from the Correspondence and requests for materials should be addressed to Jeffrey S. Willey. corresponding author (J.S.W.) Reprints and permission information is available at http://www.nature.com/ reprints Received: 15 October 2021; Accepted: 13 May 2022; Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. REFERENCES 1. Vico, L. & Hargens, A. Skeletal changes during and after spaceflight. Nat. Rev. Open Access This article is licensed under a Creative Commons Rheumatol. 14, 229 (2018). Attribution 4.0 International License, which permits use, sharing, 2. Keyak, J. H., Koyama, A. K., LeBlanc, A., Lu, Y. & Lang, T. F. Reduction in proximal adaptation, distribution and reproduction in any medium or format, as long as you give femoral strength due to long-duration spaceflight. Bone 44, 449–453 (2009). appropriate credit to the original author(s) and the source, provide a link to the Creative 3. Vico, L. & Hargens, A. Skeletal changes during and after spaceflight. Nat. Rev. Commons license, and indicate if changes were made. The images or other third party Rheumatol. 14, 229–245 (2018). material in this article are included in the article’s Creative Commons license, unless 4. Axpe, E. et al. A human mission to Mars: Predicting the bone mineral density loss indicated otherwise in a credit line to the material. If material is not included in the of astronauts. PLoS One 15, e0226434 (2020). article’s Creative Commons license and your intended use is not permitted by statutory 5. Sibonga, J. D. et al. Recovery of spaceflight-induced bone loss: bone mineral regulation or exceeds the permitted use, you will need to obtain permission directly density after long-duration missions as fitted with an exponential function. Bone from the copyright holder. To view a copy of this license, visit http://creativecommons. 41, 973–978 (2007). org/licenses/by/4.0/. 6. Orwoll, E. S. et al. Skeletal health in long-duration astronauts: Nature, assessment, and management recommendations from the NASA Bone Summit. J. Bone Min. Res. 28, 1243–1255 (2013). © The Author(s) 2022 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 18 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png npj Microgravity Springer Journals

αKlotho decreases after reduced weight-bearing from both spaceflight and hindlimb unloading

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www.nature.com/npjmgrav BRIEF COMMUNICATION OPEN αKlotho decreases after reduced weight-bearing from both spaceflight and hindlimb unloading 1✉ 2 3 1 4 2 Jeffrey S. Willey , Serena Aunon-Chancellor , Lauren A. Miles , Joseph E. Moore , Xiao W. Mao , Robert W. Wallace and Matthew C. Foy Alpha(α)Klotho, a soluble transmembrane protein, facilitates calcium-phosphorus homeostasis through feedback between bone and kidney and is a potential systemic biomarker for bone-kidney health during spaceflight. We determined if: (1) plasma αKlotho was reduced after both spaceflight aboard the ISS and hindlimb unloading (HU); and (2) deficiency could be reversed with exercise. Both spaceflight and HU lowered circulating plasma αKlotho: plasma αKlotho recovered with exercise after HU. npj Microgravity (2022) 8:18 ; https://doi.org/10.1038/s41526-022-00203-w INTRODUCTION This pilot investigation measured circulating αKlotho levels in mice after exposure to two reduced weight-bearing conditions: Atrophy of skeletal elements has long been identified as a hazard microgravity aboard the ISS, and after hind limb unloading (HU) . for the success of long-duration missions and astronaut quality of Importantly, musculoskeletal degradation has previously been life . US astronauts aboard the International Space Station (ISS) described in hindlimbs from these same mice after 35 days in exhibit loss of bone density when comparing pre- and post-flight 14,15 2,3 microgravity and HU , observing recovery of joint health upon DXA scan measurements . Bone density reduction for a Mars 4 return to full weight-bearing with exercise . Likewise, this study mission has been estimated of up to 36% . Recovery from bone 5,6 examined if deficits in circulating αKlotho after periods HU could be degradation appears to be incomplete . recovered after return to full-weight bearing, with/without perform- The role of the kidneys in skeletal homeostasis is complex. ing exercise. Changes in α-Klotho after acute bouts of exercise are Recent research interest includes the interplay between αKlotho 7 inconsistent when examining resistance vs aerobic exercise (ref. ), and Fibroblast Growth Factor 23 (FGF23) . Alpha Klotho is secreted with long term exercise exhibiting elevated αKlotho in sedentary predominantly in the kidneys, whereas FGF23 is secreted by individuals (ref. ). This hypothesis-generating research aims to osteocytes . FGF23 binds to αKlotho, and increases urine assess if αKlotho can serve as a circulating biomarker for bone phosphorous excretion . Both are required, as seen in mice that status, both in-flight and upon return to weight-bearing. demonstrate phosphate retention if either component is defi- Data are presented mean(SD). Plasma αKlotho in pg/ml was 10 7 cient . FGF23 also increases calcium reabsorption . Through ~50% lower (Fig. 1) after 35 days in orbit aboard the ISS from the these mechanisms, FGF23 eliminates excess phosphorus and FLIGHT mice vs GROUND. No differences were observed between increases plasma calcium, with subsequent decreased calcitriol groups for FGF23 or inorganic phosphorus (Pi) concentration synthesis and parathyroid hormone (PTH) release. In non-weight- (Table 1). Additionally, gastrocnemius muscle mass (g) was lower bearing climates with known bone density loss, αKlotho and (p = 0.0035) in FLIGHT vs GROUND after time in orbit aboard the FGF23 may play a role in skeletal homeostasis. ISS (Table 1). For the hind limb unloading study, plasma αKlotho The current spaceflight countermeasures aimed at preventing after 30 days of tail suspension [HU-30day] was 36% lower vs 6,11 bone loss provide incomplete protection . The Advanced plasma αKlotho from mice that remained full weight bearing Resistive Exercise Device (ARED) is utilized for exercise aboard [GROUND-30day; p < 0.01; Fig. 2]. Likewise, αKlotho was lower in the ISS . Use of the ARED as a single countermeasure largely mice after the initial 30 days of HU vs all mice that were full- maintains preflight skeletal health over a 6-month mission in weight bearing throughout the entire 80 day study, with or orbit , but bone loss is observed by 1 year post-flight, indicating without exercise [GROUND-No exercise; GROUND-Climbing; and progressive atrophy despite a return to full weight-bearing and GROUND-Running; p < 0.05 for all comparisons)]. In contrast, after from astronauts also receiving bisphosphonate therapy . Addi- returning to full weight-bearing from the 30 day period of HU, tionally, a device as large and complex as ARED is likely not plasma αKlotho levels remained lower in the mice that performed feasible on an exploration class mission due to limitations in mass no exercise [HU-No Exercise] vs plasma from the full weight and habitable space. These findings add concerns for the current bearing mice as measured on Day 30 (p < 0.05). Additionally, these ability to preserve bone health outside low earth orbit. mice that had not performed exercise after the 30 day period of Currently, no reliable real-time analysis of biomarkers or skeletal HU were also significantly lower than all GROUND groups on Day imaging on orbit exists. Identification of novel circulating 80, regardless of exercise (GROUND-No exercise; GROUND- biomarkers for musculoskeletal health, such as αKlotho, could Climbing; GROUND-Running). In contrast, running exercise for prove crucial to protective measures as the space community 49 days after the initial 30 day HU period [HU-Running] resulted in turns its eyes towards Mars. partial recovery of plasma Klotho, with no difference vs the full 1 2 Department of Radiation Oncology, Wake Forest School of Medicine, Section of Radiation Biology, Winston-Salem, NC, USA. Department of Internal Medicine, Louisiana State 3 4 University Medical Center, Baton Rouge, LA, USA. Department of Nephrology, Wake Forest School of Medicine, Winston-Salem, NC, USA. Department of Basic Sciences, Loma Linda University, Loma Linda, CA, USA. Department of Internal Medicine, Louisiana State University Medical Center, Division of Nephrology, Baton Rouge, LA, USA. email: jwilley@wakehealth.edu Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; J.S. Willey et al. p<0.01 *** ** * 60 * Fig. 1 αKlotho concentration is reduced after spaceflight. FLIGHT mice exhibited lower αKlotho vs GROUND. P value from unpaired t test provided above bars. Bars: Mean(SD). Table 1. Descriptive information from both the spaceflight study to Fig. 2 αKlotho concentration is reduced after hind limb unload- the International Space Station, and the ground-based hind limb ing, but can recover with subsequent exercise. αKlotho was lower than GROUND in plasma after 30 days HU, but recovery occurred unloading (HU) study. with exercise *p ≤ 0.05; **p < 0.01; ***p < 0.001 determined via Tukey’s Post Hoc test after performed after ANOVA. Bars: Mean(SD). Spaceflight study Hind limb unloading study GROUND FLIGHT GROUND- HU-30day 30day FGF23 (pg/ml) 26.14(12.45) 27.10(14.79) NA NA Pi (mg/dL) 4.26(0.29) 4.06(0.18) 7.21(3.27) 8.39(3.62) Gastrocnemius 159.0(13.1) 134.6(18.8)# NA NA mass (g) Data are presented mean(SD). # indicates diffference for FLIGHT vs GROUND at p = 0.0035 via unpaired t-test. NA: data not collected (Gastrocnemius mass) or presented in Fig. 3 (FGF 23). weight bearing group at Day 30 (GROUND-30 Day), but still having lower concentration (p < 0.05) vs GROUND-No Exercise. Climbing exercise [HU-Climbing] for 49 days provided full recovery of αKlotho (Fig. 2), being similar to all GROUND groups. No differences were observed between any groups for FGF23 concentration (Fig. 3). Moreover, Pi level between GROUND- Fig. 3 Plasma FGF23 was unaltered after hind limb unloading. 30day and HU-30day was similar (Table 1). Sample limitation Plasma FGF23 was similar across groups and time points. Bars: Mean prevented the assessment of other groups. (SD). A reduction in plasma αKlotho levels was observed in both FLIGHT and HU mice vs controls. While a mechanistic/causal link adverse effects of microgravity on bone, as well as muscle, these between weight-bearing conditions and αKlotho are not deter- findings suggest an important link between this hormone level mined, both reduced weight-bearing conditions did result in and skeletal health. Histologic changes of bone with altered lowered αKlotho. For HU mice, levels of αKlotho increased kidney physiology in relation to these hormonal changes are following exercise, with partial recovery in the running group needed to better understand this relationship. The similar changes and full recovery in the climbing group, suggesting an important relationship between exercise/potentially elevated weight-bearing in αKlotho found in both FLIGHT mice and HU mice suggest that and αKlotho level improvement. These data are aligned with HU mice may provide an appropriate terrestrial analog for recovery of joint health upon a return to full weight-bearing with examining the relationship between αKlotho and health along exercise . Moreover, for FLIGHT mice, where muscle masses were the musculoskeletal tissue-kidney axis. collected, the decrease in αKlotho post-flight occurred with reduced gastrocnemius mass, which itself is not unexpected but METHODS αKlotho may also be affected by skeletal muscle activity . When considering the importance αKlotho plays in maintaining The animal and environmental details of the ISS study (Rodent musculoskeletal skeletal health, viewed within the context of the Research-9 mission), the accompanying HU study, and all npj Microgravity (2022) 18 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA G O R UND FLIGHT Ground-30day HU-30day GROUND-No Exercise G O i R UND-Runn ng GROUND-Climbing HU-No Exercise HU-Running HU-Climbing Ground-30day HU-30day GROUND-No Exercise GROUND-Running GROUND-Climbing HU-No Exercise HU-Running HU-Climbing 1234567890():,; Klotho Concentration (pg/ml) FGF23 Concentraiton (pg/ml) Klotho Concentraiton (pg/ml) J.S. Willey et al. approved IACUC protocols from Wake Forest School of Medicine, 7. Kuro, O. M. The Klotho proteins in health and disease. Nat. Rev. Nephrol. 15,27–44 (2019). NASA Ames, and the Kenney Space Center have been published . 8. Kuro, O. M. A potential link between phosphate and aging-lessons from Klotho- All mice were male, C57BL/6 (Jackson Labs) that were 10 weeks at deficient mice. Mech. Ageing Dev. 131, 270–275 (2010). the start of each study. 9. Hu, M. C., Shiizaki, K., Kuro-o, M. & Moe, O. W. Fibroblast growth factor 23 and Klotho: Physiology and pathophysiology of an endocrine network of mineral ISS mission metabolism. Annu. Rev. Physiol. 75, 503–533 (2013). 10. Kuro, O. M. Phosphate and Klotho. Kidney Int. https://doi.org/10.1038/ki.2011.26 Groups included FLIGHT and GROUND control . This investigation (2011). had access to n = 4–5 plasma samples/group collected after 11. Sibonga, J. et al. Resistive exercise in astronauts on prolonged spaceflights pro- 35 days on orbit; all available samples were analyzed for this study. vides partial protection against spaceflight-induced bone loss. Bone 128, 112037 (2019). HU study 12. Smith, S. M. et al. Benefits for bone from resistance exercise and nutrition in long- duration spaceflight: Evidence from biochemistry and densitometry. J. Bone Min. Groups included weight-bearing GROUND mice, or HU via tail Res. 27, 1896–1906 (2012). suspension, as described .OnDay 30,plasmawas isolated from a 13. Steczina, S. et al. Dietary countermeasure mitigates simulated spaceflight- cohort of GROUND (GROUND-30day) and HU (HU-30day) mice. induced osteopenia in mice. Sci. Rep. 10, 6484 (2020). Remaining HU mice were then removed from tail suspension and 14. da Silveira, W. A. et al. Comprehensive multi-omics analysis reveals mitochondrial were thus weight-bearing the remainder of the study. All mice that stress as a central biological hub for spaceflight impact. Cell 183, 1185–1201 were previously GROUND or HU were enrolled into one of 3 exercise e1120 (2020). groups, as described ,fromDays31–80, performing: 1] no exercise; 15. Kwok, A. T. et al. Spaceflight and hind limb unloading induces an arthritic pheno- type in knee articular cartilage and menisci of rodents. Sci. Rep. 11, 10469 (2021). 2] climbing exercise 3X weekly, or; 3] running exercise 3X weekly in 16. Iturriaga, T. et al. Acute impacts of different types of exercise on circulating alpha- order to determine if recovery was possible with aerobic (running) or Klotho protein levels. Front. Physiol. 12, 716473 (2021). a resistance (climbing) regimen, yielding the following six groups: 17. Amaro-Gahete, F. J. et al. Exercise training increases the S-Klotho plasma levels in GROUND-No exercise; GROUND-Climbing; GROUND-Running; HU-No sedentary middle-aged adults: A randomised controlled trial. The FIT-AGEING exercise; HU-Climbing; and HU-Running. Climbing exercises were study. J. Sports Sci. 37, 2175–2183 (2019). performed 3X/week,. This investigation had access to n=4plasma 18. Avin, K. G. et al. Skeletal muscle as a regulator of the longevity protein, Klotho. samples/group, all of which were analyzed. Front. Physiol. 5, 189 (2014). Blood was collected in [K2]ethylenediaminetetraacetic acid (EDTA)- containing syringes by cardiac puncture and centrifuged for 10 min at ACKNOWLEDGEMENTS 3000 rpm at 4 °C; Plasma was isolated and ELISA was used to detect NASA Space Biology grant NNX15AB50G(JSW). αKlotho (R&D Systems™ #DY5334) and FGF23 (Abcam #ab213863); a commercial Pi kit was also used (Pointe Scientific #P7516-500). Comparisons between FLIGHT and GROUND mice were AUTHOR CONTRIBUTIONS performed using a two-way unpaired t-test; ANOVA was J.S.W.: conceptualization, study design, performance of study, data analysis/ performed for the HU study, with Tukeys post hoc tests. α ≤ interpretation, manuscript preparation. S.A.C. and M.C.F.: conceptualization, data 0.05; the assumption of equal variance was used to test equality of interpretation, manuscript preparation. L.A.M. and R.W.W.: conceptualization, manu- variance. Analyses were performed using GraphPad Prizm 8.4.0. script preparation. J.E.M.: performance of study, data analysis, manuscript prepara- The data that support the findings of this study are available on tion. X.W.M.: study design, tissue collection, manuscript preparation. request from the corresponding author. COMPETING INTERESTS Reporting summary The authors declare no competing interests. Further information on research design is available in the Nature Research Reporting Summary linked to this article. ADDITIONAL INFORMATION Supplementary information The online version contains supplementary material DATA AVAILABILITY available at https://doi.org/10.1038/s41526-022-00203-w. The authors declare that data supporting the findings of this study are available within the figures of the article, and/or are available on request from the Correspondence and requests for materials should be addressed to Jeffrey S. Willey. corresponding author (J.S.W.) Reprints and permission information is available at http://www.nature.com/ reprints Received: 15 October 2021; Accepted: 13 May 2022; Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. REFERENCES 1. Vico, L. & Hargens, A. Skeletal changes during and after spaceflight. Nat. Rev. Open Access This article is licensed under a Creative Commons Rheumatol. 14, 229 (2018). Attribution 4.0 International License, which permits use, sharing, 2. Keyak, J. H., Koyama, A. K., LeBlanc, A., Lu, Y. & Lang, T. F. Reduction in proximal adaptation, distribution and reproduction in any medium or format, as long as you give femoral strength due to long-duration spaceflight. Bone 44, 449–453 (2009). appropriate credit to the original author(s) and the source, provide a link to the Creative 3. Vico, L. & Hargens, A. Skeletal changes during and after spaceflight. Nat. Rev. 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Skeletal health in long-duration astronauts: Nature, assessment, and management recommendations from the NASA Bone Summit. J. Bone Min. Res. 28, 1243–1255 (2013). © The Author(s) 2022 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 18

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Published: Jun 2, 2022

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