Cold‐Inducible RNA‐Binding Protein Alleviates Hypertensive Renal Injury Through the KEAP1‐NRF2 Pathway

Figures

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  FIGURE 1. Decrease of Cirbp expression during Ang II‐mediated renal injury. (A) Schematic illustration depicting the renal RNA‐seq analysis strategy. GSE37463 includes patients with hypertensive nephropathy (n = 20) and healthy kidneys (n = 21). GSE207102 includes Ang II‐induced mice (n = 6) and controls (n = 6). GSE311806 includes rats receiving low‐ (n = 12) and high‐salt water (n = 12). Con, control; HTN, hypertension; HS, high‐salt drinking water; LS, low‐salt water. (B) GO analysis of 112 genes shared by the three datasets (p < 0.05). (C) Circular heatmap of 19 genes differentially expressed in all three datasets (FDR < 0.05). (D) Transcriptional expression of Cirbp in the three RNA‐seq datasets. (E) Single‐cell transcriptomic analysis of mouse kidney samples in GSE107585. Unsupervised analysis identified 16 cell populations displayed on a UMAP plot. (F) Cirbp staining in (E). (G) Cluster‐based distribution of Cirbp expression. (H, I) Western blotting assessment of CIRBP protein abundance in kidneys from WT mice and in NRK‐52E receiving saline or Ang II (n = 6). (J, K) Quantification of protein levels in (H, I). (L) qRT‐PCR quantification of Cirbp transcripts in kidney obtained from…

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  FIGURE 2. Differential consequences of Cirbp knockout and overexpression during Ang II‐mediated renal injury and dysfunction. (A) Flowchart of the mice experimental procedures. (B) ELISA of BUN levels in serum from WT, Cirbp‐OE, and Cirbp‐KO mice given saline or treated with Ang II (n = 6). BUN, blood urea nitrogen. (C) ELISA measuring serum creatinine levels in WT, Cirbp‐OE, and Cirbp‐KO mice receiving Ang II (n = 6). (D, E) H&E‐stained kidneys from WT, Cirbp‐OE, and Cirbp‐KO mice receiving saline or Ang II and measurement of tubulointerstitial injury (n = 6). (F, G) Images from laser speckle blood flow analysis of kidneys in living mice. Darker red indicates greater blood flow (n = 6). (H, I) qRT‐PCR quantification of Ngal transcripts in kidneys from WT, Cirbp‐OE, and Cirbp‐KO mice (n = 6). (J) Blood pressure measurements in WT, Cirbp‐OE, and Cirbp‐KO mice (n = 6). *WT + Ang II versus WT + Saline; †Cirbp‐KO + Saline versus WT + Saline; #Cirbp‐OE + Ang II versus WT + Ang II; ‡Cirbp‐KO + Ang II versus WT + Ang II. (K) Flowchart of the cell experimental procedures. (L, M) qRT‐PCR quantification of Ngal transcripts in NRK cultures transfected with negative control, Cirbp‐OE, or si‐Cirbp…

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  FIGURE 3. Cirbp modulates Ang II‐associated renal fibrosis. (A, B) Masson trichrome staining of cortical and medullary fibrosis in WT, Cirbp‐OE, and Cirbp‐KO mice (n = 6). (C, D) qRT‐PCR‐based quantification of Col I and α‐sma transcripts in kidneys from WT and Cirbp‐OE or Cirbp‐KO mice (n = 6). (E, F) Immunoblot analysis of COL III, COL Ia1, and α‐SMA protein levels in kidneys from WT and Cirbp‐OE kidneys given saline or Ang II treatment (n = 6). (G, H) Western blotting analysis of COL III, COL Ia1, and α‐SMA protein levels in kidneys from WT and Cirbp‐KO mice (n = 6). (I, J) Western blotting analysis of COL III, COL Ia1, and α‐SMA abundance in NRK cells with Cirbp‐OE after PBS or Ang II stimulation (n = 6). (K, L) Western blotting assessment of COL III, COL Ia1, and α‐SMA abundance in NRK cells with si‐Cirbp after PBS or Ang II stimulation (n = 6).

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  FIGURE 4. Effects of Cirbp on Ang II‐mediated renal apoptosis. (A, B) TUNEL staining showing apoptosis within kidneys from WT, Cirbp‐OE and Cirbp‐KO mice receiving Ang II treatment (n = 6). (C, D) TUNEL staining showing apoptosis in NRK cells with Cirbp‐OE after Ang II stimulation (n = 6). (E, F) TUNEL staining of NRK with si‐Cirbp after Ang II (n = 6). (G, H) Western blotting assessment of apoptosis‐related protein abundance in kidneys from WT and Cirbp‐OE mice kidney (n = 6). (I, J) Corresponding Western blotting analysis in WT and Cirbp‐KO kidneys (n = 6). (K, L) Western blotting assessment of apoptosis‐related protein expression in NRK cells with Cirbp‐OE after PBS or Ang II stimulation (n = 6). (M, N) Corresponding Western blotting analysis in si‐Cirbp NRK cells (n = 6).

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  FIGURE 5. Cirbp suppresses Ang II‐associated renal inflammation. (A) qRT‐PCR‐based assessment of Tnf‐α and Il‐6 mRNA in kidneys from WT and Cirbp‐OE mice given saline or Ang II (n = 6). (B, C) Western blotting assessment of iNOS and IL‐6 protein abundance in kidney tissue from WT and Cirbp‐OE mice (n = 6). (D) qRT‐PCR‐based quantification of Tnf‐α and Il‐6 transcripts in kidneys from WT and Cirbp‐KO mice (n = 6). (E, F) Western blotting analysis of iNOS and IL‐6 protein abundance in kidney tissue from WT and Cirbp‐KO mice (n = 6). (G, H) Immunofluorescence micrographs showing iNOS signal in kidneys from WT, Cirbp‐OE and Cirbp‐KO mice (n = 6). (I) qRT‐PCR quantification of Tnf‐α and Il‐6 transcripts in NRK cells with Cirbp‐OE after PBS or Ang II stimulation (n = 6). (J, K) Immunoblot assessment of iNOS and IL‐6 protein levels in NRK cells with Cirbp‐OE after PBS or Ang II stimulation (n = 6). (L) qRT‐PCR assessment of Tnf‐α and Il‐6 transcripts in NRK cells with si‐Cirbp after PBS or Ang II (n = 6). (M, N) Western blotting analysis of iNOS and IL‐6 protein levels in NRK‐52E with si‐Cirbp after PBS or Ang II stimulation (n = 6).

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  FIGURE 6. Cirbp regulates renal injury via oxidative stress. (A) KEGG enrichment of DEGs from the RNA‐seq (GSE207102) dataset derived from mice given saline or a continuous Ang II infusion (n = 6; |log2FC| > 1; p value < 0.05). (B) GSEA of ROS generation among mice given saline or infused with Ang II. (C) Heatmap of differentially expressed genes associated with ROS generation in mice administered saline or Ang II. (D, E) DHE labeling of ROS in kidneys from WT, Cirbp‐OE, and Cirbp‐KO mice (n = 6). (F, G) 2′,7′‐DCFH‐DA staining of ROS in NRK cells with Cirbp‐OE (n = 6). (H, I) DHE‐based detection of reactive oxygen species within NRK cultures with Cirbp‐OE (n = 6). (J, K) DCFH‐DA staining of ROS in NRK cells with Cirbp‐OE after PBS or Ang II stimulation (n = 6). (L, M) DHE staining of ROS in NRK cells with si‐Cirbp after PBS or Ang II stimulation (n = 6).

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  FIGURE 7. Overexpression of Cirbp decreases KEAP1 abundance in renal tissue after Ang II infusion. (A) Volcano plot depicting DEGs identified by RNA‐seq (GSE207102) in mice receiving saline or Ang II treatment (n = 6; |log2FC| > 1; p < 0.05). (B, C) Immunoblot assessment of KEAP1, NRF2 and HO‐1 protein abundance in kidneys from WT as well as Cirbp‐OE mice following Ang II infusion (n = 6). (D, E) Immunoblot assessment of KEAP1, NRF2 and HO‐1 protein levels in kidneys from WT and Cirbp‐KO mice administered Ang II (n = 6). (F) Representative immunofluorescence micrographs showing KEAP1 and CIRBP labeling in kidneys from WT, Cirbp‐OE, and Cirbp‐KO mice following Ang II administration (n = 6). (G) Fluorescence colocalization between KEAP1 and CIRBP in (F). (H) Quantification of fluorescence intensity in (F). (I) Co‐IP and Western blotting of CIRBP and KEAP1 in the transfected HEK293 cells. (J) Prediction of spatial configuration and peptide chain conformation of the CIRBP and KEAP1 binding by AlphaFold 3. Pink presents CIRBP (Uniprot ID: P60824) and blue represents KEAP1 (Uniprot ID: Q9Z2X8). Visualization of hydrogen bonds between KEAP1 and CIRBP and the corresponding amino acid residues by…

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  FIGURE 8. CIRBP promotes KEAP1 degradation and NRF2 functional activation. (A) Cycloheximide (CHX) chase assay showing KEAP1 protein stability in Cirbp‐OE NRK‐52E cells treated with CHX (0, 1, 2, 4, 6, 8 h) (n = 6). (B) Quantification of KEAP1 protein in (A). (C) Co‐immunoprecipitation assay detecting KEAP1 ubiquitination in Cirbp‐OE HEK293 cells with MG132 treatment. (D, E) Western blotting analysis of NRF2 protein in cytoplasmic and nuclear in NRK cells with si‐Cirbp or Cirbp‐OE after PBS or Ang II stimulation (n = 6). (F, G) Immunofluorescence staining of NRF2 showing cytoplasmic and nuclear localization in NRK cells with si‐Cirbp or Cirbp‐OE after PBS or Ang II (n = 6). (H, I) Western blotting analysis of proteins of fibrosis, apoptosis and oxidative stress markers in NRK‐52E with Cirbp‐OE or si‐Keap1 after saline or Ang II infusion (n = 6). (J, K) Immunoblot assessment of COL Ia1, α‐SMA, BCL‐2, BAX, caspase 3, cleaved caspase 3, NRF2, and HO‐1 protein abundance in NRK‐52E with si‐Cirbp or si‐Keap1 after PBS or Ang II stimulation (n = 6).

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  FIGURE 9. CIRBP relieves Ang II‐evoked renal damage through the KEAP1‐NRF2 axis. (A, B) Western blotting assessment of COL Ia1, α‐SMA, BCL‐2/BAX, cleaved caspase 3/caspase 3 abundance in NRK with si‐Cirbp and/or RA839 after PBS or Ang II stimulation (n = 6). (C, D) Corresponding Western blotting quantification in Cirbp‐OE NRK cells treated with ML385 (n = 6). (E, F) DCFH‐DA detection of ROS in si‐Cirbp cells treated with RA839 under PBS or Ang II conditions (n = 6). (G, H) DHE labeling of ROS within NRK cultures with si‐Cirbp and/or RA839 after PBS or Ang II stimulation (n = 6). (I, J) DCFH‐DA staining for ROS in NRK cells with Cirbp‐OE and/or ML385 after PBS or Ang II stimulation (n = 6). (K, L) DHE‐based ROS detection in Cirbp‐OE NRK cells with or without ML385 (n = 6).