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. 2023 Mar 14;13(1):4211.
doi: 10.1038/s41598-023-31117-6.

Differential toxicity profile of secreted and processed α-Klotho expression over mineral metabolism and bone microstructure

Joan Roig-Soriano  1 , Cristina Sánchez-de-Diego  2 , Jon Esandi-Jauregui  1 , Sergi Verdés  1 , Carmela R Abraham  3 , Assumpció Bosch  1   4   5 , Francesc Ventura  2 , Miguel Chillón  6   7   8   9
Affiliations

Differential toxicity profile of secreted and processed α-Klotho expression over mineral metabolism and bone microstructure

Joan Roig-Soriano et al. Sci Rep. .

Abstract

The aging-protective gene α-Klotho (KL) produces two main transcripts. The full-length mRNA generates a transmembrane protein that after proteolytic ectodomain shedding can be detected in serum as processed Klotho (p-KL), and a shorter transcript which codes for a putatively secreted protein (s-KL). Both isoforms exhibit potent pleiotropic beneficial properties, although previous reports showed negative side effects on mineral homeostasis after increasing p-KL concentration exogenously. Here, we expressed independently both isoforms using gene transfer vectors, to assess s-KL effects on mineral metabolism. While mice treated with p-KL presented altered expression of several kidney ion channels, as well as altered levels of Pi and Ca2+ in blood, s-KL treated mice had levels comparable to Null-treated control mice. Besides, bone gene expression of Fgf23 showed a fourfold increase after p-KL treatment, effects not observed with the s-KL isoform. Similarly, bone microstructure parameters of p-KL-treated mice were significantly worse than in control animals, while this was not observed for s-KL, which showed an unexpected increase in trabecular thickness and cortical mineral density. As a conclusion, s-KL (but not p-KL) is a safe therapeutic strategy to exploit KL anti-aging protective effects, presenting no apparent negative effects over mineral metabolism and bone microstructure.

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Conflict of interest statement

Portions of this work are the subject of a patent application held by the Universitat Autonòma de Barcelona (UAB, Spain); the Universitat de Barcelona (UB, Spain); the Institucio Catalana de Recerca i Estudis Avançats (ICREA, Spain); and the Vall d'Hebron Institute of Research (VHIR, Spain). J.R., A.B., and M.C. are included in this patent application. C.R.A. is the co-founder of Klogenix, a company that is seeking to develop Klotho-boosting therapeutics. No other authors possess a conflict of interest.

Figures

Figure 1
Figure 1
(a) Scheme with the different KL isoforms. (b) Experimental design followed. (c) Body weight follow up displayed as increase in grams during the 8 weeks-long treatment. No differences in body weight were observed between groups before the start of the experiment (t = 0) or after 8 weeks. (d) Gene expression of secreted and processed KL in liver 2 months after the treatment. Stripped bars represent gene expression in males and solid bars in females. Data presented as fold change expression compared to Null treated animals. (e) KL protein levels in serum 2 months after the treatment. Mean ± Standard error of the mean (SEM) for (c,e), and relative quantity (RQ) ± standard error of RQ for (d); n = 4–6; #p < 0.05 for differences between males and females, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001 for differences between different treatment groups within the same gender.
Figure 2
Figure 2
(a) Ca2+ and Pi serum levels after 8 weeks of treatment. (b) Expression of ion channels in kidney. (c) Expression of genes involved in vitamin 1,25D activation and degradation. Data are presented as fold change expression compared to Null-treated animals. Mean ± Standard error of the mean (SEM) for (a), and relative quantity (RQ) ± standard error of RQ for (b,c); n = 4–6; *p < 0.05; **p < 0.01; ***p < 0.001 for differences between the treatment groups.
Figure 3
Figure 3
(a) Gene expression of secreted and processed KL in bone tissue 2 months after the treatment. Stripped bar represents gene expression in males and solid bar in females. Statistical significance presented as (*) for representing differences between treatment in male groups, and (#) for comparing treatments in females. (b) Effect of treatment on Fgf23 gene expression. (c) Expression analysis of Klotho down-stream gene c-Fos. (d) Effect of treatment on cell type specific gene expression. (e) Effect of treatment on bone protein matrix gene expression. Data are presented as fold change expression compared to Null treated animals. Relative quantity (RQ) ± standard error of RQ; n = 4–6; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001 for differences between the treatment groups.
Figure 4
Figure 4
(a) MicroCT analysis of different structural variables. Tibial length, BV (bone volume), B.Pm (bone perimeter), CsTh (cross-sectional thickness), Mineral Density (MD), BV/TV (bone volume/tissue volume), Tb.N (trabecular number), Tb.Th (trabecular thickness), Tb.Sp (trabecular space). (b) 3D reconstruction of the analyzed ROI. First and third columns corresponding to cortical bone and second and fourth to trabecular bone. Scale bar = 0.2 mm. (c) Results of three-point bending test. Data represented as maximum load resisted by the bones, force at fracture, stiffness, and total energy absorbed by the bones during the experiment. Data are presented as fold change expression versus Null treated animals. Mean ± Standard error of the mean (SEM), n = 4–6; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001 for differences between the treatment groups.
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