Dietary phosphorus restriction enables multigenerational breeding of α-Klotho-deficient rats

Sarai, C.; Totsuka, Y.; Horii, T.; Tamura, S.; Gouda, E.; Kojima, S.; Kouki, T.; Ohno, N.; Yoshiki, A.; Ohmori, T.; Kurosu, H.; Endo, H.; Tominaga, K.; Hatada, I.; Yamamoto, T.; Kuro-o, M.; Nagao, Y.

doi:10.64898/2026.10.17.759802

bioRxiv · 10.64898/2026.10.17.759802

Dietary phosphorus restriction enables multigenerational breeding of α-Klotho-deficient rats

Sarai, C.·Totsuka, Y.·Horii, T.·Tamura, S.·Gouda, E.·Kojima, S.·Kouki, T.·Ohno, N.·Yoshiki, A.·Ohmori, T.·Kurosu, H.·Endo, H.·Tominaga, K.·Hatada, I.·Yamamoto, T.·Kuro-o, M.·Nagao, Y.

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Abstract

Severe -Klotho deficiency causes early mortality and reproductive impairment, limiting the maintenance and physiological analysis of rat models. We generated rats carrying a CRISPR/Cas9-mediated -Klotho deletion spanning exons 1-5 and evaluated dietary phosphorus restriction primarily in the Wistar-Imamichi background. Maternal low-phosphorus (LP) feeding from mating through lactation was associated with higher overall survival to weaning (92.6% versus 85.8% under normal-phosphorus conditions) and a higher proportion of KO animals among weaned survivors (34/113 versus 23/145). Maternal LP feeding also reduced plasma phosphorus in KO pups. Continued LP feeding after weaning supported growth, gonadal development, and natural KO x KO breeding. Across 15 litters, 74 of 85 offspring survived to weaning, and KO colonies were maintained to at least the fifth generation. We additionally characterized 24-h metabolic rhythms in LP-maintained KO animals using indirect calorimetry. These profiles remained heterogeneous under a light-dark cycle. In conclusion, dietary phosphorus restriction provides a reliable strategy for the multigenerational maintenance of severe -Klotho-deficient rats, establishing a maintainable model to investigate residual metabolic and physiological phenotypes.

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Sarai, C., Totsuka, Y., Horii, T., Tamura, S., Gouda, E., Kojima, S., Kouki, T., Ohno, N., Yoshiki, A., Ohmori, T., Kurosu, H., Endo, H., Tominaga, K., Hatada, I., Yamamoto, T., Kuro-o, M., Nagao, Y.. 2026-10-18. Dietary phosphorus restriction enables multigenerational breeding of α-Klotho-deficient rats. https://doi.org/10.64898/2026.10.17.759802

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