Activation of sclerostin inhibits Isg20-Mediated aerobic glycolysis ameliorating renal Fibrosis: the renoprotective mechanism of hederagenin in CKD

Abstract

Sclerostin (Sost) functions as an inhibitor of the Wnt/β-catenin signaling pathway, which is known to promote kidney cell epithelial-to-mesenchymal transition (EMT), and fibrosis in chronic kidney disease (CKD). However, the renoprotective effects of Sost in kidney diseases and its therapeutic potential as a target remain unknown. To clarify the protective role of Sost in CKD kidneys, we utilized ultrasound microbubble-mediated renal in situ gene transfection to overexpress and knockdown Sost in kidney. Subsequently, we employed the TurboID-based protein interaction technique to screen for Sost-binding proteins and combined it with ECAR/OCR to elucidate the regulation of glycolytic pathways by Sost and its binding proteins. Sost is predominantly expressed in renal tubules and that its expression is significantly diminished in renal tissues of CKD patients, as well as in UUO and folic acid (FA) induced CKD mouse. Overexpression of Sost in vivo and in vitro ameliorated tubular injury and fibrosis. Employing the TurboID technique, we uncovered an interaction between Sost and the potential glycolysis-associated protein Isg20, an interferon-stimulated ribonuclease. This binding interaction serves to inhibit Isg20-mediated aerobic glycolysis and subsequent fibrosis within the kidney of CKD. For Sost agonists screening, we found that HDG exerts salient anti-fibrotic and renal protective effects in CKD, which are likely attributable to its significant upregulation of Sost expression, thereby inhibiting Isg20-mediated glycolysis. In summary, we demonstrate that upregulation of Sost by HDG inhibits glycolysis and renal fibrosis in CKD through binding and suppressing of Isg20, and targeting Sost may develop novel approaches to treat CKD.

Keywords: CKD; Fibrosis; Hederagenin; Isg20; Sost.

Graphical abstract

Fig. 1

Expression of SOST is found reduced in the renal tubules of CKD-afflicted kidneys. a. Immunofluorescence and Hematoxylin-Eosin staining in CKD patients for SOST; b, c. Alterations in SOST expression within renal tissue of CKD patients, as derived from GEO database studies; d, e. Comparative Hematoxylin-Eosin and Masson's Trichrome staining in the kidneys of control versus UUO and FA mice; f. Western Blot analysis demonstrating SOST levels in UUO mouse kidneys; g. Western Blot results for SOST in a folate-induced murine model of chronic kidney disease; h. Quantitative analysis of SOST protein expression via Western Blot; i, j. Temporal variations in SOST and Fibronectin in TCMK1 cells under TGF-β1 stimulation, revealed through Western Blot; k, l. Protein levels of Fibronectin and Collagen Type I Alpha 1 in SOST-overexpressing TCMK1 cells treated with TGF-β1 as shown by Western Blot; m. Examination of renal architecture and the deposition of fibrosis in UUO mouse models subjected to Sost overexpression intervention was conducted using H&E and Masson's trichrome staining techniques; n, o. Western blot analysis reveals the impact of Sost overexpression on fibrosis-associated protein expression in kidneys of UUO mice. Compared to Ctrl/Sham group, ∗∗P < 0.01, ∗∗∗P < 0.001; compared to TGF-β1 group, ^&&P < 0.01, ^&&&P < 0.001. compared to UUO group, ^###P < 0.001.

Fig. 2

Sost exhibits an inhibitory interaction with the expression of Isg20. a. Schematic of TurboID labeling and mass spectrometry detection workflow; b. Proteomic differential abundance mapping via mass spectrometry identification; c. Predictive analysis of the top 21 proteins binding to Sost; d, e. Confirmation of the interaction between SOST and ISG20 through COIP assay; f-h. Western Blot outcomes of Isg20 in the renal tissues of mice induced with UUO and folic acid-induced CKD; i. Immunofluorescence staining of ISG20 in the kidneys of patients with CKD; j. Expression variations of ISG20 in the kidneys of CKD patients as reported in the GEO database; k, l. Western blot analysis of Fn and Col1 in TCMK1 cells treated with TGF-β1,with and without Isg20 knockdown; m, n. Western blot assessment of Isg20 protein levels following overexpression of Sost; o. Immunofluorescence staining of ISG20 and Sost in the kidneys of UUO mice. Compared to control/Sham groups, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; compared to the TGF-β1 group, ^&P < 0.05,^&&P < 0.01, ^&&&P < 0.001.

Fig. 3

Isg20 has been found to enhance aerobic glycolysis in renal tubular epithelial cells. a. Immunofluorescence staining of HK2 in renal tissues of CKD patients; b, c. Western blot analysis reveals the levels of fibrosis and glycolysis-associated proteins in TCMK1 cells treated with 2-DG; d, e. Assessment of the impact of ISG20 knockdown on the extracellular acidification rate; f, g. Assessment of the impact of ISG20 knockdown on the oxygen consumption rate; h. Examination of the effect of ISG20 knockdown on lactate production in the cell supernatant; i, j. Western blot analysis evaluates the impact of ISG20 knockdown on glycolysis; k, l. Western blot analysis evaluates the impact of ISG20 overexpression on fibrosis and glycolysis. Compared to Ctrl group, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; compared to the TGF-β1 group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001.

Fig. 4

Knock-down of Sost in kidney exacerbates the fibrosis and glycolysis in UUO mice. a, HE staining of the kidneys in each group; b, Masson staining of the kidneys in each group; c-e, Western blot results of fibrosis and glycolysis indicators in each group. Compared to Ctrl group, ∗∗P < 0.01, ∗∗∗P < 0.001; Compared to the UUO group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001.

Fig. 5

Overexpression of Isg20 attenuates the inhibitory effect of Sost on TGF-β1-induced renal tubular cell fibrosis and glycolysis. a, b. Results of the ECAR assay on Sost overexpression; c, d. Results of the OCR assay in each group; e. Variations in lactate concentration within the supernatant of Sost overexpressing cells; f, g. The western blotting analysis delineates the alterations in the levels of glycolysis-associated proteins within TCMK1 overexpressing Sost subsequent to TGF-β1 treatment. h, i. The western blotting analysis performed to evaluate the fibrosis and glycolysis-associated proteins levels after co-overexpression of Sost and Isg20. Compared to Ctrl group, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; compared to the TGF-β1 group, ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001; compared to the TGF-β1+Sost-OE group, ^@@P < 0.01, ^@@@P < 0.001.

Fig. 6

HDG has demonstrated efficacy in mitigating the interstitial fibrosis of the kidneys associated with CKD and in addressing the Warburg effect. a,b. Histological and Masson's trichrome staining results of the kidneys in UUO and FA mice subjected to HDG intervention; c, d. Immunofluorescent staining of HK2 in the kidneys of UUO and FA mice treated with HDG (50 mg/kg); e,f. Determination of serum creatinine and blood urea nitrogen concentrations in FA mice using assay kits; g, h. Western blot analysis of α-SMA, Fn, and Col1 protein levels in the kidneys of UUO mice following HDG treatment; i. Lactate measurements in the serum of UUO and FA mice upon HDG intervention; j-l. Western blot analysis of glycolysis-associated protein expressions in the kidneys of UUO mice treated with HDG. Compared to Ctrl group, ∗∗∗P < 0.001; Compared to the model group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001.

Fig. 7

HDG can bind to activate Sost and inhibit the expression of Isg20.a-e. WB and Real-time PCR assays were employed to investigate the effects of HDG on the expression levels of sclerostin protein and its mRNA counterpart in the kidneys of mice with UUO and FA; f. Immunofluorescent staining was employed to ascertain the alterations in the expression of Sost within the renal tissue of UUO mouse models following HDG intervention; g. Immunofluorescence staining assays reveal the modulation of Isg20 expression in the renal tissues of mice models with HDG intervention in UUO and FA conditions; h, i.WB detects alterations in Isg20 protein expression in the kidneys of HDG-treated UUO and FA-induced mouse models; j. The TCMK-1 cells were treated with HDG at concentrations of 0, 5, 10, 20, 30, 60, 160, 320, and 640 μg/ml for 24 h, followed by assessment of cell vitality using the CCK-8 assay; k, WB analysis monitor the modifications in the expression of fibrotic proteins in TCMK1 cells instigated by TGF-β1 stimulation; l.Molecular docking schematic diagram between HDG and Sost; m. Incubate the cellular proteins of cells transfected with Sost-Flag plasmid with HDG conjugated to Biotin, separate the HDG-bound proteins using streptavidin magnetic beads, and employ WB to detect the Flag protein signal to evaluate the binding between HDG and Sost. Compared to Ctrl group, ∗∗∗P < 0.001; Compared to the model group, ^#P < 0.05, ^###P < 0.001.

Fig. 8

HDG alleviates renal fibrosis by inhibiting aerobic glycolysis in the kidneys. a. In the UUO mouse model subjected to DASA-58 intervention, the renal tissues were examined through H&E, Masson's trichrome, and α-SMA immunofluorescence staining; b-d. WB analysis revealed the effects of DASA-58 treatment on renal fibrosis and alterations in glycolysis-related proteins in UUO mice; e. The assay of lactate levels in the supernatants of various cellular groups; f-h. WB analysis reveals alterations in the expression of fibrosis-associated and glycolysis-related proteins across various cell groups; i, j. The ECAR measures the maximum acid production in different cell groups; k, l. The OCR measures the oxygen consumption in different cell groups. Compared to Ctrl group, ∗∗P < 0.01, ∗∗∗P < 0.001; compared to the model group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001; compared to the TGF-β1+HDG group, ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001.

Fig. 9

HDG mitigates fibrosis by upregulating Sost to inhibit aerobic glycolysis. a, b. The WB analysis was employed to detect the levels of fibrosis and glycolytic-related proteins in each group; c-e. The WB analysis was employed to discern the differential expression of Sost, Isg20, markers of fibrosis, as well as glycolytic-related proteins within various cellular cohorts; f. The assay of lactate concentration in the supernatant of various cell groups; g. The immunofluorescence assay reveals the expression alterations of HK2 across various cell groups. Compared to Sham/Ctrl group, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001; compared to the UUO/TGF-β1 group, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001; compared to the UUO + HDG/TGF-β1+HDG group, ^&P < 0.05, ^&&P < 0.01, ^&&&P < 0.001; compared to the TGF-β1+HDG + DASA-58 group, ^¥P < 0.05, ^¥¥P < 0.01, ^¥¥¥P < 0.001.