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Uremic toxin p‑cresyl sulfate enhances the calcification of aortic valvular interstitial cells via klotho/sirtuin‑1 signaling

  • Authors:
    • Shao-Jung Li
    • Tzu-Yu Cheng
    • Yu-Hsun Kao
    • Wei-Yu Chen
    • Cheng-Chih Chung
    • Nguyen Ngoc Trang
    • Pao-Huan Chen
    • Yi-Jen Chen
  • View Affiliations / Copyright

    Affiliations: Division of Cardiovascular Surgery, Department of Surgery, School of Medicine, College of Medicine, Taipei Medical University, Taipei 110301, Taiwan, R.O.C., Division of Cardiovascular Surgery, Department of Surgery, Wan Fang Hospital, Taipei Medical University, Taipei 116079, Taiwan, R.O.C., Cardiovascular Research Center, Wan Fang Hospital, Taipei Medical University, Taipei 116079, Taiwan, R.O.C., Department of Pathology, Wan Fang Hospital, Taipei Medical University, Taipei 116079, Taiwan, R.O.C., Radiology Center, Bach Mai Hospital, Hanoi 100000, Vietnam, Department of Psychiatry, Taipei Medical University Hospital, Taipei 110301, Taiwan, R.O.C.
    Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 162
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    Published online on: April 1, 2026
       https://doi.org/10.3892/mmr.2026.13872
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Abstract

Calcific aortic valve disease (CAVD), a valvular heart disease with severe complications, is common in patients with chronic kidney disease (CKD). P‑cresyl sulfate (PCS) is a protein‑binding uremic toxin that induces chronic inflammation. Klotho and sirtuin‑1 (SIRT1) represent potential therapeutic agents for mitigating CKD‑induced vascular calcifications. We hypothesized that PCS could enhance valvular interstitial cell (VIC) calcification, which could be modulated by klotho/SIRT1 signaling. Alizarin Red S staining, western blotting and immunohistochemical analysis were performed in order to examine calcification and klotho/SIRT1 signaling in isolated porcine VICs following various 7‑day treatments. VIC treatments included incubation with PCS (10 and 100 µM), klotho (100 pM), the hypoxia‑inducible factor‑1α (HIF‑1α) inhibitor PX‑478 (0.5 µM) and the SIRT1 activator SRT1720 (1 mM). Furthermore, the present study established a PCS‑induced rat model of CKD and analyzed the effects of klotho on runt‑related transcription factor 2 (RUNX2) expression in rat aortic valves in vivo. Treatment with PCS increased VIC calcification, NF‑κB acetylation and the expression of RUNX2 and HIF‑1α expression but was shown to reduce klotho expression. Klotho supplementation attenuated the PCS‑induced enhancement of VIC calcification and mitigated PCS‑mediated increases in NF‑κB acetylation and RUNX2 expression. Additionally, the SIRT1 activator SRT1720 attenuated the PCS‑mediated enhancement of VIC calcification and was shown to upregulate klotho and downregulate RUNX2 in PCS‑treated VICs. Furthermore, the present study demonstrated that klotho supplementation mitigated CKD‑mediated RUNX2 upregulation in the aortic valves of PCS‑treated CKD model rats. The present study demonstrated that PCS induced VIC calcification by activating HIF‑1α signaling and downregulating klotho. Treatment with klotho or SRT1720 was shown to attenuate PCS‑mediated activation of the NF‑κB/RUNX2 signaling pathway, suggesting that these agents demonstrate notable therapeutic potential for targeting PCS‑induced CAVD.
View Figures

Figure 1

Effect of PCS on VIC calcification.
(A) The upper panel contains representative images of Alizarin Red
S-stained VICs that were cultured with various concentrations of
PCS. VIC calcification was quantified by determining the total area
of positive staining (red) per field (3 fields were observed per
treatment group), as shown in the lower panel (n=6). Scale bar, 50
µm. (B) Western blotting and semi-quantification of RUNX2 protein
expression in control and PCS-treated VICs (n=7). β-actin was used
as an internal control. **P<0.01 and ***P<0.005. PCS,
p-cresol; RUNX2, runt-related transcription factor 2; VIC, valvular
interstitial cell.

Figure 2

Effect of PCS on klotho and HIF-1α
expression. Western blotting and semi-quantification of (A) klotho
and (B) HIF-1α protein expression in control and PCS-treated
valvular interstitial cells. PCS-treated cells were incubated with
PCS (100 µM) for 7 days (n=5). β-actin was used as an internal
control. *P<0.05. PCS, p-cresol; HIF-1α, hypoxia-inducible
factor-1α.

Figure 3

Effect of HIF-1α inhibition on
PCS-induced VIC calcification. The upper panel depicts
representative images of Alizarin Red S-stained VICs that have been
incubated with or without the HIF-1α inhibitor PX-478 (0.5 µM) and
with or without PCS (100 µM). VIC calcification was quantified by
determining the total area of positive staining (red) per field (3
fields were observed per treatment group), as shown in the lower
panel (n=3). Scale bar, 25 µm. *P<0.05. PCS, p-cresol; VIC,
valvular interstitial cell; HIF-1α, hypoxia-inducible
factor-1α.

Figure 4

Effect of recombinant klotho on
PCS-induced VIC calcification. The upper panel depicts
representative images of Alizarin Red S-stained VICs incubated with
or without klotho (100 pM) and with or without PCS (100 µM). VIC
calcification was quantified by determining the total area of
positive staining (red) per field (3 fields were observed per
treatment group), as shown in the lower panel (n=4). Scale bar, 50
µm. **P<0.01 and ***P<0.005. PCS, p-cresol; VIC, valvular
interstitial cell.

Figure 5

Effect of klotho supplementation on
PCS-mediated upregulation of RUNX2 and NF-kB acetylation in
valvular interstitial cells. Western blotting and
semi-quantification of (A) RUNX2 expression and (B) NF-kB
acetylation in control cells, cells treated with PCS and cells
treated with PCS + klotho (n=5). β-actin was used as an internal
control. *P<0.05, **P<0.01 and ***P<0.005. PCS, p-cresol;
RUNX2, runt-related transcription factor 2; acetyl-NF-κB,
acetylated-NF-κB.

Figure 6

Effect of SRT1720 on PCS-induced VIC
calcification. The upper panel depicts representative images of
Alizarin Red S-stained VICs incubated with or without SRT1720 (1
mM) and with or without PCS (100 µM). VIC calcification was
quantified by determining the total area of positive staining (red)
per field, as shown in the lower panel (n=4); 3 fields were
observed per treatment group. Scale bar, 50 µm. *P<0.05 and
***P<0.005. PCS, p-cresol; VIC, valvular interstitial cell.

Figure 7

Effect of sirtuin-1 activation on
klotho and RUNX2 expression. (A) Representative western blots
demonstrating the effect of SRT1720 (1 mM) treatment on RUNX2
protein expression in PCS-treated VICs, compared with untreated
controls and VICs treated with PCS alone. RUNX2 expression was
semi-quantified in each group for statistical comparisons between
groups (n=6). (B) Representative western blots demonstrating the
effect of SRT1720 on klotho protein expression in PCS-treated VICs,
compared with untreated controls and VICs treated with PCS alone.
Klotho expression was semi-quantified in each group for statistical
comparisons between groups (n=6). (C) Representative western blots
demonstrating the effect of SRT1720 on acetyl-NF-kB and total NF-kB
protein expression in PCS-treated VICs, compared with untreated
controls and VICs treated with PCS alone. NF-kB and acetyl-NF-kB
expression levels were semi-quantified in each group for
statistical comparisons between groups (n=8). *P<0.05 and
**P<0.01. PCS, p-cresol; RUNX2, runt-related transcription
factor 2; acetyl-NF-κB, acetylated-NF-κB; VIC, valvular
interstitial cell.

Figure 8

Effect of klotho on RUNX2 expression
in the aortic valves of PCS-induced CKD model rats. Representative
immunohistochemistry images displaying RUNX2 expression in rat
aortic valves in control (n=4), PCS-induced CKD (n=5) and
PCS-induced CKD + klotho rats (n=4). Scale bar, 50 µm.
Immunohistochemical results were quantified to statistically
compare RUNX2 expression between treatment groups by % of positive
staining. The arrows within the IHC images in this figure indicate
the RUNX2 expression. ***P<0.005. PCS, p-cresol; CKD, chronic
kidney disease; RUNX2, runt-related transcription factor 2.
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Copy and paste a formatted citation
Spandidos Publications style
Li S, Cheng T, Kao Y, Chen W, Chung C, Trang NN, Chen P and Chen Y: Uremic toxin p‑cresyl sulfate enhances the calcification of aortic valvular interstitial cells via klotho/sirtuin‑1 signaling. Mol Med Rep 33: 162, 2026.
APA
Li, S., Cheng, T., Kao, Y., Chen, W., Chung, C., Trang, N.N. ... Chen, Y. (2026). Uremic toxin p‑cresyl sulfate enhances the calcification of aortic valvular interstitial cells via klotho/sirtuin‑1 signaling. Molecular Medicine Reports, 33, 162. https://doi.org/10.3892/mmr.2026.13872
MLA
Li, S., Cheng, T., Kao, Y., Chen, W., Chung, C., Trang, N. N., Chen, P., Chen, Y."Uremic toxin p‑cresyl sulfate enhances the calcification of aortic valvular interstitial cells via klotho/sirtuin‑1 signaling". Molecular Medicine Reports 33.6 (2026): 162.
Chicago
Li, S., Cheng, T., Kao, Y., Chen, W., Chung, C., Trang, N. N., Chen, P., Chen, Y."Uremic toxin p‑cresyl sulfate enhances the calcification of aortic valvular interstitial cells via klotho/sirtuin‑1 signaling". Molecular Medicine Reports 33, no. 6 (2026): 162. https://doi.org/10.3892/mmr.2026.13872
Copy and paste a formatted citation
x
Spandidos Publications style
Li S, Cheng T, Kao Y, Chen W, Chung C, Trang NN, Chen P and Chen Y: Uremic toxin p‑cresyl sulfate enhances the calcification of aortic valvular interstitial cells via klotho/sirtuin‑1 signaling. Mol Med Rep 33: 162, 2026.
APA
Li, S., Cheng, T., Kao, Y., Chen, W., Chung, C., Trang, N.N. ... Chen, Y. (2026). Uremic toxin p‑cresyl sulfate enhances the calcification of aortic valvular interstitial cells via klotho/sirtuin‑1 signaling. Molecular Medicine Reports, 33, 162. https://doi.org/10.3892/mmr.2026.13872
MLA
Li, S., Cheng, T., Kao, Y., Chen, W., Chung, C., Trang, N. N., Chen, P., Chen, Y."Uremic toxin p‑cresyl sulfate enhances the calcification of aortic valvular interstitial cells via klotho/sirtuin‑1 signaling". Molecular Medicine Reports 33.6 (2026): 162.
Chicago
Li, S., Cheng, T., Kao, Y., Chen, W., Chung, C., Trang, N. N., Chen, P., Chen, Y."Uremic toxin p‑cresyl sulfate enhances the calcification of aortic valvular interstitial cells via klotho/sirtuin‑1 signaling". Molecular Medicine Reports 33, no. 6 (2026): 162. https://doi.org/10.3892/mmr.2026.13872
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