Journal: Advanced Science

Article Title: Sirtuin1 Suppresses Calcium Oxalate Nephropathy via Inhibition of Renal Proximal Tubular Cell Ferroptosis Through PGC‐1α‐mediated Transcriptional Coactivation

doi: 10.1002/advs.202408945

Figure Lengend Snippet: Sirt1 inhibited CaOx‐induced ferroptosis in tubular epithelium. A) Sirt1 activation and overexpression restored the cell viability of COM‐treated HK‐2 cells evaluated by CCK‐8. B, C) Lipid peroxidation in HK‐2 cells was determined by BODIPY C11 staining and flow cytometry. D) Sirt1 activation and overexpression reduced the level of MDA in COM‐treated HK‐2 cells. Immunoblotting E) and qPCR F) revealed the expression of ferroptosis associated markers in HK‐2 cells. In vivo, mice were pretreated with SRT1720/EX527 and then induced to establish a model of CaOx nephrocalcinosis. Immunoblotting G) and qPCR H) revealed the expression of ferroptosis associated markers in HK‐2 cells. The deposition of CaOx was observed under a polarized light microscopy I) and evaluated by Pizzolato staining J). K) PAS staining was used for the scoring of tubular injury. The blue arrows highlight damaged renal tubules. L) IHC staining was performed to test the level of GPX4 and 4HNE in mouse kidney. M) The changes of mitochondrial morphology in tubular epithelium were observed by TEM. The red arrows indicate the morphologically abnormal mitochondria, characterized by mitochondrial shrinkage and disorganized cristae. N, O) The serum levels of creatinine and BUN were detected to assess kidney function. P) MDA in tissues of mice kidney was quantified. Q) The expression of KIM‐1 mRNA in tissues of mice kidney was determined by qPCR. Data were presented as mean ± SD, n = 3 for in vitro experiments and n = 6 for in vivo experiments. P value was determined by one‐way (C, D, F, H, N‐Q) or two‐way (A) ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Renal tubular epithelium‐specific Sirt1 knockout (Sirt1cKO) mice were obtained by crossing Cdh16‐Cre males (T007046, GemPharmatech Inc., China) with Sirt1 fl/fl females (T006657, GemPharmatech Inc.) on a C57BL/6J background.

Techniques: Activation Assay, Over Expression, CCK-8 Assay, Staining, Flow Cytometry, Western Blot, Expressing, In Vivo, Light Microscopy, Immunohistochemistry, In Vitro

Journal: Advanced Science

Article Title: Sirtuin1 Suppresses Calcium Oxalate Nephropathy via Inhibition of Renal Proximal Tubular Cell Ferroptosis Through PGC‐1α‐mediated Transcriptional Coactivation

doi: 10.1002/advs.202408945

Figure Lengend Snippet: Sirt1 inhibited CaOx‐induced ferroptosis in tubular epithelium dependent on GPX4.HK‐2 cells were treated with increasing concentrations of erastin, along with 5 µM SRT1720 or 1 µM Fer for 24h. A) Cell viability of HK‐2 cells was evaluated by CCK‐8. B, C) Lipid peroxidation in HK‐2 cells was determined by BODIPY C11 staining and flow cytometry. D) Level of MDA in HK‐2 cells treated with erastin, along with SRT1720 or Fer for 24 h. (E) HK‐2 cells were treated with increasing concentrations of RSL3, along with 5 µM SRT1720 or 1 µM Fer for 24 h. Cell viability of HK‐2 cells was evaluated by CCK‐8. F, G) Lipid peroxidation in HK‐2 cells was determined by BODIPY C11 staining and flow cytometry. H) Level of MDA in HK‐2 cells treated with erastin, along with SRT1720 or Fer for 24 h. I) Cell viability of Sh ctrl and GPX4 sh HK‐2 cells treated with increasing concentrations of COM and SRT1720 was evaluated by CCK‐8. J, K) Lipid peroxidation in Sh ctrl and GPX4 sh HK‐2 cells was determined by BODIPY C11 staining and flow cytometry. L) Level of MDA in Sh ctrl and GPX4 sh HK‐2 cells treated with increasing concentrations of COM and SRT1720 for 24 h. Data were presented as mean ± SD, n = 3, and P value was determined by one‐way (C, D, G, H, K, L) or two‐way (A, E, I) ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Renal tubular epithelium‐specific Sirt1 knockout (Sirt1cKO) mice were obtained by crossing Cdh16‐Cre males (T007046, GemPharmatech Inc., China) with Sirt1 fl/fl females (T006657, GemPharmatech Inc.) on a C57BL/6J background.

Techniques: CCK-8 Assay, Staining, Flow Cytometry

Journal: Advanced Science

Article Title: Sirtuin1 Suppresses Calcium Oxalate Nephropathy via Inhibition of Renal Proximal Tubular Cell Ferroptosis Through PGC‐1α‐mediated Transcriptional Coactivation

doi: 10.1002/advs.202408945

Figure Lengend Snippet: Sirt1 inhibited ferroptosis in tubular epithelium through the PGC‐1α/GPX4 pathway.A, B) Sirt1 activation and overexpression increased the protein and mRNA expression of PGC‐1α. C, D) The protein and mRNA levels of GPX4 in Sh ctrl and PGC‐1α sh HK‐2 cells treated by ZLN005. E) Cell viability of Sh ctrl and PGC‐1α sh HK‐2 cells treated with increasing concentrations of COM and SRT1720 was evaluated by CCK‐8. F, G) Lipid peroxidation in Sh ctrl and PGC‐1α sh HK‐2 cells was determined by BODIPY C11 staining and flow cytometry. Immunoblotting H) and qPCR I) revealed the expression of ferroptosis associated markers in Sh ctrl and PGC‐1α sh HK‐2 cells treated by COM and SRT1720. J) Cell viability of Sh ctrl and PGC‐1α sh HK‐2 cells treated with increasing concentrations of erastin and SRT1720 was evaluated by CCK‐8. K, L) Lipid peroxidation in Sh ctrl and PGC‐1α sh HK‐2 cells was determined by BODIPY C11 staining and flow cytometry. Immunoblotting M) and qPCR N) revealed the expression of ferroptosis associated markers in Sh ctrl and PGC‐1α sh HK‐2 cells treated by erastin and SRT1720. Data were presented as mean ± SD, n = 3, and P value was determined by one‐way (B, D, G, I, L, N) or two‐way (E, J) ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Renal tubular epithelium‐specific Sirt1 knockout (Sirt1cKO) mice were obtained by crossing Cdh16‐Cre males (T007046, GemPharmatech Inc., China) with Sirt1 fl/fl females (T006657, GemPharmatech Inc.) on a C57BL/6J background.

Techniques: Activation Assay, Over Expression, Expressing, CCK-8 Assay, Staining, Flow Cytometry, Western Blot

Journal: Advanced Science

Article Title: Sirtuin1 Suppresses Calcium Oxalate Nephropathy via Inhibition of Renal Proximal Tubular Cell Ferroptosis Through PGC‐1α‐mediated Transcriptional Coactivation

doi: 10.1002/advs.202408945

Figure Lengend Snippet: Sirt1 inhibited CaOx‐induced ferroptosis, crystal deposition and kidney injury through PGC‐1α/NRF2 signaling.A) The conditional knockout of Sirt1 in tubular epithelium in mouse kidney. B) Schematic of establishment of CaOx nephrocalcinosis model and treatment of ZLN005 and TBHQ. The deposition of CaOx was observed under a polarized light microscopy C) and evaluated by Pizzolato staining D). E) PAS staining was used for the scoring of tubular injury. The blue arrows highlight damaged renal tubules. F) IHC staining was performed to test the level of GPX4 and 4HNE in mouse kidney. G) The levels of renal ROS were detected by DHE staining. H, I) The serum levels of creatinine and BUN were detected to assess kidney function. J) MDA in tissues of mice kidney was quantified. Data were presented as mean ± SD, n = 6, and P value was determined by one‐way ANOVA (H‐J). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: Renal tubular epithelium‐specific Sirt1 knockout (Sirt1cKO) mice were obtained by crossing Cdh16‐Cre males (T007046, GemPharmatech Inc., China) with Sirt1 fl/fl females (T006657, GemPharmatech Inc.) on a C57BL/6J background.

Techniques: Knock-Out, Light Microscopy, Staining, Immunohistochemistry