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α-Klotho released from HK-2 cells inhibits osteogenic differentiation of renal interstitial fibroblasts by inactivating the Wnt–β-catenin pathway

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Abstract

Randall’s plaques (RP) are well established as precursor lesions of idiopathic calcium oxalate (CaOx) stones, and the process of biomineralization driven by osteogenic-like cells has been highlighted in RP formation, but the mechanism is poorly understood. Given the inhibitory role of α-Klotho (KL), an aging suppressor protein with high expression in kidneys, in ectopic calcification and the close association between KL gene polymorphisms and urolithiasis susceptibility, we determined the potential role of KL in RP formation. This study found that both soluble KL (s-KL) and transmembrane KL (m-KL) were downregulated, and that s-KL but not m-KL was inversely correlated with upregulation of osteogenic markers in RP tissues. Additionally, s-KL expression was markedly suppressed in human renal interstitial fibroblasts (hRIFs) and slightly suppressed in HK-2 cells after osteogenic induction, intriguingly, which was echoed to the greater osteogenic capability of hRIFs than HK-2 cells. Further investigations showed the inhibitory effect of s-KL on hRIF osteogenic differentiation in vitro and in vivo. Moreover, coculture with recombinant human KL (r-KL) or HK-2 cells suppressed osteogenic differentiation of hRIFs, and this effect was abolished by coculture with KL-silenced HK-2 cells or the β-catenin agonist SKL2001. Mechanistically, s-KL inactivated the Wnt–β-catenin pathway by directly binding to Wnt2 and upregulating SFRP1. Further investigations identified activation of the Wnt–β-catenin pathway and downregulation of SFRP1 and DKK1 in RP tissues. In summary, this study identified s-KL deficiency as a pathological feature of RP and revealed that s-KL released from HK-2 cells inhibited osteogenic differentiation of hRIFs by inactivating the Wnt–β-catenin pathway, not only providing in-depth insight into the role of s-KL in renal interstitial biomineralization but also shedding new light on the interaction of renal tubular epithelial cells with interstitial cells to clarify RP formation.

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Data availability

The datasets used and/or analyzed during this study are available from the corresponding author on reasonable request.

Abbreviations

ARS:

Alizarin Red staining

ALP:

Alkaline phosphatase

CaOx:

Calcium oxalate

CaP:

Calcium phosphate

CaOx-SF:

CaOx stone formers

HC:

Healthy controls

hRIFs:

Human renal interstitial fibroblasts

KL :

KLOTHO

NRP:

Normal renal papillae

RP:

Randall’s plaques

r-KL:

Recombinant human KL

SFRPs:

Secreted Frizzled-related proteins

s-KL:

Soluble α-Klotho

m-KL:

Transmembrane α-Klotho

UKCR:

Urinary s-KL-to-creatinine ratio

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Acknowledgements

Thank National Natural Science Foundation of China, Natural Science Foundation of Hunan Province for providing funding for this study and Central South University Independent Exploration and Innovation Project for Graduate Students.

Funding

This work was supported by the National Natural Science Foundation of China (81770705 to Hequn Chen; 82000761 to Yu Cui); Natural Science Foundation of Hunan Province (2019JJ40488 to Zhiyong Chen); Central South University Independent Exploration and Innovation Project for Graduate Students (2021zzts0348 to Zewu Zhu).

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Authors and Affiliations

  1. Department of Urology, Xiangya Hospital, Central South University, Changsha, 410008, Hunan, China

    Zewu Zhu, Shuhao Ruan, Yingcheng Jiang, Fang Huang, Weiping Xia, Jinbo Chen, Yu Cui, Cheng He, Feng Zeng, Yang Li, Zhiyong Chen & Hequn Chen

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  1. Zewu Zhu
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Contributions

ZZ, ZC and HC performed study concept and designed the experiment. ZZ, SR, YJ, FH, WX, JC, YC, CH and FZ contributed to the development of methodology and the acquisition of data. ZZ, YL and ZC contributed to analysis and interpretation of data. ZZ, ZC and HC contributed to writing and revision of the manuscript. ZZ, YC and HC contributed to the administrative, technical and material support. ZC and HC contributed to study supervision. All authors read and approved the final paper.

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Correspondence to Zhiyong Chen or Hequn Chen.

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Approval was granted by Xiangya Hospital Ethics Committee (Approval Number: 202103089) and the Institutional Experimental Animal Committee of Central South University (Proof Number: 2021sydw0033).

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Written informed consents for surgical procedures and getting samples were obtained from all included patients prior to surgery. The study was performed in accordance with the Declaration of Helsinki.

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Supplementary Information

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18_2021_3972_MOESM1_ESM.tif

Supplementary file1. Supplementary Figure 1. 2% agarose gel electrophoresis was performed to verify the PCR products amplified by each pair of primers (TIF 8167 KB)

18_2021_3972_MOESM2_ESM.tif

Supplementary file2. Supplementary Figure 2. (A) The DAB staining density of soluble α-Klotho (s-KL) and osteogenic markers (Runx2, MSX2, OCN) was quantified by average gray value using the IHC Toolbox plugin in ImageJ (Randall’s Plaques (RP), n=6; normal renal papillae (NRP), n=6). (B–D) Correlation analysis between the relative protein expression levels of s-KL and transmembrane KL (m-KL), Runx2, and OCN in RP tissues; n=18. (E–G) Correlation analysis between the relative protein expression levels of m-KL and osteogenic markers (Runx2, MSX2, and OCN) in RP tissues; n=18. (H–K) Quantification of s-KL and osteogenic marker expression determined by WB in human renal interstitial fibroblasts (hRIFs) 1, 4, 7, and 14 days after culture in either osteogenic medium (OM) or normal medium (NM); n=3. (L–O) Quantification of s-KL and osteogenic marker expression determined by WB in HK-2 cells 1, 4, 7, and 14 days after culture in either OM or NM; n=3 (TIF 515 KB)

18_2021_3972_MOESM3_ESM.tif

Supplementary file3. Supplementary Figure 3. (A–B) qRT-PCR was used to determine the mRNA expression level of α-Klotho (KL) in human interstitial fibroblasts (hRIFs) transfected with Len-ctrl, Len-KL, Len-sh-ctrl, Len-sh1-KL, Len-sh2-KL, and Len-sh3-KL; n=3. Len-sh-KL-3, with the highest efficiency, was used to silence KL in hRIFs in the following experiments. (C) qRT-PCR was used to determine the relative mRNA expression level of KL in hRIFs and HK-2 cells; n=3. (D–E) Western blots and quantitative analysis of soluble α-Klotho (s-KL) and transmembrane KL (m-KL) in hRIFs and HK-2 cells; n=3. (F–G) qRT-PCR was used to determine the mRNA expression level of KL in HK-2 cells transfected with Len-ctrl, Len-KL, Len-sh-ctrl, Len-sh1-KL, Len-sh2-KL, and Len-sh3-KL; n=3. Len-sh-KL-1, with the highest efficiency, was used to silence KL in HK-2 cells in the following experiments. (H) Representative western blots of Wnt9a in whole-cell lysates of hRIFs and coimmunoprecipitation with the anti-s-KL antibody, IgG or unconjugated beads as a negative control (NC); n=3. Immunoblotting=IB; immunoprecipitation=IP. (I) Representative western blots of Wnt5b in whole-cell lysates of hRIFs and coimmunoprecipitation with the anti-s-KL antibody, IgG or unconjugated beads; n=3. (J) Quantitative analysis of the western blot results for SFRP1 and SFRP4 in hRIFs retransfected with siRNAs targeting SFRP1 and induced with OM for 7 days; n=3. (K) Quantitative analysis of the western blot results for SFRP1 and SFRP4 in hRIFs retransfected with siRNAs targeting SFRP4 and induced with OM for 7 days; n=3 (TIF 10454 KB)

18_2021_3972_MOESM4_ESM.tif

Supplementary file4. Supplementary Figure 4. (A–B) Correlation analysis between the relative protein expression levels of SFRP1 and MSX2 and OCN in RP (Randall’s plaque) tissues; n=18. (C–D) Correlation analysis between the relative protein expression levels of DKK1 and Runx2 and MSX2 in RP tissues; n=18. (E–I) Correlation analysis between the ratio of p-β-catenin to β-catenin and the relative protein expression levels of SFRP1, DKK1, Runx2, MSX2, and OCN in RP tissues; n=18. (J–L) Correlation analysis between the relative protein expression level of transmembrane α-Klotho (m-KL) and the ratio of p-β-catenin to β-catenin, SFRP1 expression, and DKK1 expression in RP tissues; n=18 (TIF 8065 KB)

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Zhu, Z., Ruan, S., Jiang, Y. et al. α-Klotho released from HK-2 cells inhibits osteogenic differentiation of renal interstitial fibroblasts by inactivating the Wnt–β-catenin pathway. Cell. Mol. Life Sci. 78, 7831–7849 (2021). https://doi.org/10.1007/s00018-021-03972-x

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