Lipin3 deficiency aggravates cisplatin induced acute kidney injury via activating Sirt1-p21-Caspase 3-GSDME pyroptosis pathway

Figures

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  Figure 1. Link between high expression of Lipin3 and cisplatin-induced AKI. (A) The distribution and relative expression of Lipin3 gene in human injured kidney organoids (From snRNA-Seq database, GSE164647). The Lipin3 gene expression in human kidney organoids (GSE145085 22) (B) and mouse kidneys (GSE106993 21) (C) after cisplatin treatment. (D) WB analysis showing the expression levels of Lipin3 in mice treated with or without cisplatin. (E) WB analysis showing the expression levels of Lipin3 in HK2 cells treated with different concentrations of cisplatin. (F) IHC staining and HE staining showing the representative conditions of Lipin3 expression and renal tubular injury in kidney tissues from healthy control subjects (n = 9) and AKI patients (n = 9). (G) Scatter plots showing the correlation between serum Lipin3 levels and serum creatinine levels (left), the fold increase in serum creatinine (middle) and serum BUN levels (right) in 54 AKI patients. Correlation coefficient r and p value were calculated by the Spearman's rank correlation coefficient test.

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  Figure 2. Overexpression of Lipin3 in mice can alleviate cisplatin-induced AKI. (A) Flow chart of animal study design. (B) WB analysis confirmed successful overexpression of Lipin3 in the mice kidney. (C) Kidney appearance of AAV-Empty mice (n = 5) and AAV-Lipin3 mice (n = 5) treated with or without cisplatin. (D) HE staining analysis showing the renal tubular injury in AAV-Empty mice (n = 5) and AAV-Lipin3 mice (n = 5) treated with or without cisplatin. Peripheral blood UREA (E) and creatinine (F) levels of AAV-Empty mice (n = 5) and AAV-Lipin3 mice (n = 5) treated with or without cisplatin. (G) WB analysis showed the expression of Lipin3, KIM1 and NGAL in AAV-Empty mice (n = 5) and AAV-Lipin3 mice (n = 5) treated with or without cisplatin.

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  Figure 3. Lipin3-KO mice were more sensitive to cisplatin-induced AKI. (A) Flow chart of animal study design. (B) WB analysis verified the successful generation of Lipin3-KO mice. (C) Kidney appearance of WT mice (n = 5) and Lipin3-KO mice (n = 5) treated with or without cisplatin. (D) HE staining analysis showing the renal tubular injury in WT mice (n = 5) and Lipin3-KO mice (n = 5) treated with or without cisplatin. Peripheral blood UREA (E) and creatinine (F) levels of WT mice (n = 5) and Lipin3-KO mice (n = 5) treated with or without cisplatin. (G) WB analysis showed the expression of Lipin3, KIM1 and NGAL in WT mice (n = 5) and Lipin3-KO mice (n = 5) treated with or without cisplatin. (H) Survival curve of each group (n=40).

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  Figure 4. Lipin3 deficiency exacerbated cisplatin induced mitochondrial dysfunction. (A) GSEA analysis for KEGG enrichment in the RNA-seq data. mRNA levels of ND1 (B), COX3 (C), CYTB (D), GPX1 (E), SOD2 (F) and CAT (G) in the kidney of mice in each group. (H) WB analysis revealed the expressions of mitochondrial electronic respiratory chain-related proteins, MFN2 and OPA1 in the kidney of WT mice and Lipin3-KO mice treated with or without cisplatin. ROS levels (I), ATP levels (J) and JC-1 staining (K) in WT and Lipin3-KO PTECs treated with or without cisplatin.

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  Figure 5. Lipin3 deficiency activate the apoptosis and Caspase 3/GSDME‑dependent secondary pyroptosis in cisplatin induced AKI. (A) The TUNEL staining analysis showing the apoptosis in the kidney of WT mice (n = 5) and Lipin3-KO mice (n = 5) treated with or without cisplatin. The fluorescence intensity in the graphs was calculated using Image J software analysis. (B) CCK8 analysis showing the cell viability of WT and Lipin3-KO PTECs treated with or without cisplatin. (C) Flow cytometric analysis of PI- and Annexin V-FITC-stained PTECs treated with or without cisplatin. (D) Hoechst staining (top), AO/EB staining (bottom), and corresponding statistical analysis of apoptosis rates for each group of cells. (E) Phase-contrast images and (F) TEM analysis of WT and Lipin3-KO PTECs were examined after cisplatin treatment. Arrows, pyroptotic cells. The levels of LDH (G), IL-6 (H) and IL-18 (I) in cell supernatant were measured. (J) WB analysis revealed the expressions of Caspase 3, Cleaved-Caspase 3, GSDME, N-GSDME in WT and Lipin3-KO PTECs treated with or without cisplatin.

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  Figure 6. SP600125 alleviate cisplatin-induced renal pyroptosis in vivo. (A) CCK8 analysis showing the cell viability in Lipin3-KO PTECs treated with DMSO or SP600125 for 1 h, followed by treating with or without cisplatin. (B) The levels of LDH in Lipin3-KO PTECs treated with DMSO or SP600125 for 1 h, followed by treating with or without cisplatin were measured. (C) WB analysis revealed the expressions of Caspase 3, Cleaved-Caspase 3, GSDME, N-GSDME in Lipin3-KO PTECs treated with DMSO or SP600125 for 1 h, followed by treating with or without cisplatin. (D) Phase-contrast images of Lipin3-KO PTECs treated with DMSO or SP600125 for 1 h, followed by treating with or without cisplatin. Arrows, pyroptotic cells. (E) Flow chart of animal study design. (F) Kidney appearance of cisplatin-treated Lipin3-KO mice (n = 5) with or without SP600125 pretreatment. (G) HE staining analysis showing the renal tubular injury in cisplatin-treated Lipin3-KO mice (n = 5) with or without SP600125 pretreatment. Peripheral blood UREA (H) and creatinine (I) levels of cisplatin-treated Lipin3-KO mice (n = 5) with or without SP600125 pretreatment. (J) WB analysis showed the expression of KIM1 and NGAL in…

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  Figure 7. Lipin3 can regulate the Caspase 3/GSDME axis through p21. (A)The Venn analysis provided a schematic representation highlighting the overlap of upregulated (left) and downregulated (right) death/pyroptosis-related genes. (B) Schematic of the filter strategies used in our study. (C) Heat maps illustrate the expression patterns of the selected genes across various mouse groups. (D) mRNA levels of p21 in WT and Lipin3-KO PTECs treated with or without cisplatin. (E) WB analysis revealed the expressions of p21 in the kidney of WT mice (n = 5) and Lipin3-KO mice (n = 5) treated with or without cisplatin. (F) WB analysis revealed the expression of p21 in WT and Lipin3-KO PTECs treated with or without cisplatin. (G) CCK8 analysis showing the cell viability in Lipin3-KO PTECs transfected with scrambled or p21 siRNA followed by treating with or without cisplatin. (H) The levels of LDH in Lipin3-KO PTECs transfected with scrambled or p21 siRNA followed by treating with or without cisplatin were measured. (I) WB analysis revealed the expressions of Caspase 3, Cleaved-Caspase 3, GSDME, N-GSDME in Lipin3-KO PTECs transfected with scrambled or p21 siRNA followed by treating with or without…

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  Figure 8. Lipin3 can regulate the p21-Caspase 3/GSDME pathway by interacting with Sirt1. (A) Co-IP analysis confirmed the interaction between Lipin3 and Sirt1 in HK2 cells. (B) Immunofluorescence staining showing the subcellular localization of Lipin3 and Sirt1 in HK2 cells. (C) Molecular docking results indicate that Sirt1 is a potential interacting protein of Lipin3. (D) WB analysis revealed the expressions of Sirt1 in the kidney of WT mice (n = 5) and Lipin3-KO mice (n = 5) treated with or without cisplatin. (E) mRNA levels of Sirt1 in WT and Lipin3-KO PTECs treated with or without cisplatin. (F) WB analysis revealed the degradation rate of Sirt1 in WT and Lipin3-KO PTECs. (G) ChIP assays were performed in WT and Lipin3-KO PTECs using Sirt1-specific antibody. ChIP-qPCR showed the binding of Sirt1 protein to the p21 gene promoter in WT and Lipin3KO cells treated with cisplatin. IgG was used as a negative control. (H) Enrichment of p21 promoter was measured by qPCR. (I) CCK8 analysis showing the cell viability in Lipin3-KO PTECs transfected with Empty or Sirt1 followed by treating with or without cisplatin. (J) The levels of LDH in Lipin3-KO prim PTECs ary cells transfected with Empty or…

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  Figure 9. The mechanism by which Lipin3 regulates the Sirt1-p21-Caspase 3-GSDME pyroptosis signaling pathway in AKI.