CUL4B protects kidneys from acute injury by restraining p53/PAI-1 signaling

Abstract

Acute kidney injury (AKI) caused by nephrotoxins, ischemia reperfusion (IR) or sepsis is associated with high morbidity and mortality. Unveiling new mechanisms underlying AKI can help develop new therapeutic strategy. Cullin 4B (CUL4B) is a scaffold protein in the CUL4B-RING E3 ubiquitin ligase (CRL4B) complex. Here, we demonstrate that CUL4B can protect kidneys from acute injury induced by cisplatin and IR. CUL4B is upregulated in mouse tubular epithelial cells (TECs) after cisplatin treatment or IR. Loss of CUL4B in kidneys exacerbates renal injury, inflammation, and apoptosis of TECs caused by cisplatin and IR. Transcriptome analysis reveals that Cul4b deficiency enhances injury-induced PAI-1 expression. CUL4B suppresses PAI-1 expression by promoting polyubiquitination and degradation of p53. Inhibition of either PAI-1 or p53 can prevent the aggravated renal injury and inflammation caused by loss of CUL4B. Our work has identified the kidney-protective role of CUL4B against acute injury.

Fig. 1. CUL4B is upregulated in kidneys with acute injury.

A The creatinine (Cr) and BUN levels in the serum collected from mice at 24, 48 or 72 h after peritoneal injection of 20 mg/kg cisplatin (Cis) or at 24 h after injection of the vehicle control (Con). N = 5. B Western blots showing the level of CUL4B protein in kidneys of the indicated mice. C Immunohistochemistry staining of CUL4B in kidneys of the indicated mice. The bar graph shows integrated density (intDen) relative to Con. 5 fields were imaged for each mouse and 3 mice were included in each group. Scale bar, 60 μm. D The creatinine (Cr) and BUN levels in the serum collected from mice at 24, 48 or 72 h after IRI or at 24 h after sham operation. N = 5. E Western blots showing the level of CUL4B protein in kidneys of the indicated mice. F Immunohistochemistry staining of CUL4B in kidneys of the indicated mice. The bar graph shows integrated density (intDen) relative to Sham. 5 fields were imaged for each mouse and 3 mice were included in each group. Scale bar, 60 μm. G Immunohistochemistry staining of CUL4B in kidneys from patients with acute renal failure (ARF) or acute nephritic syndrome (ANS) or the paracancerous tissues (Normal) obtained from individuals with kidney cancer and without other renal diseases who underwent nephrectomies. Scale bar, 60 μm. Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 2. Cul4b deficiency exacerbates cisplatin-induced AKI.

A The creatinine (Cr) and BUN levels in the serum collected from the Cul4b^Con or Cul4b^cKO mice at 72 h after injection of cisplatin (Cis) or the vehicle control (Con). N = 5. B Western blots of CUL4B and NGAL in the kidneys from the indicated mice. C The representative images of immunohistochemistry staining of KIM-1 and quantification of the percentage of KIM-1⁺ area in the indicated kidneys. 5 fields were imaged for each mouse and 5 mice were included in each group. Scale bar, 60 μm. D The representative images of Hematoxylin & Eosin (H & E) staining and the tubular damage scores of the kidneys from the indicated mice. 10 fields were imaged for each mouse and 5 mice were included in each group. Scale bar, 60 μm. E The mRNA levels of the indicated inflammatory factors in the kidneys. N = 5. F The representative images of immunohistochemistry staining of the macrophage marker CD68 and quantification of the percentage of CD68⁺ area in kidneys. 5 fields were imaged for each mouse and 5 mice were included in each group. Scale bar, 60 μm. In all bar graphs, the grey bars represent Cul4b^Con mice injected with vehicle control (Cul4b^Con-Con); the white bars represent Cul4b^cKO mice injected with vehicle control (Cul4b^cKO-Con); the red bars represent Cul4b^Con mice injected with cisplatin (Cul4b^Con-Cis); the blue bars represent Cul4b^cKO mice injected with cisplatin (Cul4b^cKO-Cis). Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 3. Loss of CUL4B renders TECs more susceptible to apoptosis.

A, B The representative images of TUNEL staining on kidneys and the quantification of the number of TUNEL⁺ cells in each field. 5 fields were imaged for each mouse and 5 mice were included in each group. Scale bar, 85 μm. C, D Western blots showing the levels of cleaved caspase-3 in kidneys from the indicated groups. E Western blots showing the levels of CUL4B protein in HK2 cells expressing control shRNA (NS) or shRNA against CUL4B (shCUL4B). The representative images of Annexin V/7-AAD analysis of HK2 cells with or without CUL4B knockdown exposed to different doses of cisplatin (F) or cobalt chloride (G) and the quantification of the percentage of Annexin V⁺ cells. N = 3. Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 4. Cul4b deficiency enhances AKI through upregulating PAI-1 expression.

The levels of Serpine1 mRNA (A) and PAI-1 protein (B) in kidneys from the indicated groups. N = 3 in (A). C Immunohistochemistry staining of PAI-1 in kidneys from the indicated groups. Scale bar, 60 μm. D The creatinine (Cr) and BUN levels in the serum collected from the Cul4b^Con or Cul4b^cKO mice at 72 h after injection of cisplatin (Cis) with or without pretreatment of PAI-039 (039), an inhibitor for PAI-1. N = 5. E Western blots showing the levels of CUL4B and KIM-1 in the indicated kidneys. F The representative images of H & E staining and the tubular damage scores of the kidneys from the indicated mice. 10 fields were imaged for each mouse and 5 mice were included in each group. Scale bar, 60 μm. G The representative images of immunohistochemistry staining of the macrophage marker CD68 and quantification of the percentage of CD68⁺ area in kidneys. 5 fields were imaged for each mouse and 5 mice were included in each group. Scale bar, 60 μm. H The mRNA levels of the indicated inflammatory factors in the kidneys. N = 5. I The representative images of TUNEL staining on kidneys and the quantification of the number of TUNEL⁺ cells in each field. 5 fields were imaged for each mouse and 5 mice were included in each group. Scale bar, 85 μm. In all bar graphs in (D)–(I), the bars from left to right represent Cul4b^Con mice injected with cisplatin (Cul4b^Con-Cis), Cul4b^Con mice pretreated with PAI-039 before injection with cisplatin (Cul4b^Con-Cis+039), Cul4b^cKO mice injected with cisplatin (Cul4b^cKO-Cis) and Cul4b^cKO mice pretreated with PAI-039 before injection with cisplatin (Cul4b^Con-Cis+039), respectively. J The representative images of Annexin V/7-AAD analysis of the cisplatin-treated HK2 cells expressing control shRNA (NS) or shRNA against CUL4B (shCUL4B) together with or without siRNA against SERPINE1 (siSERPINE1) and the quantification of the percentage of Annexin V⁺ cells. N = 3. Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 5. CUL4B suppresses PAI-1 expression by upregulating p53.

A Western blots showing the level of p53 in the indicated kidneys. B The level of Serpine1 mRNA in kidneys from the cisplatin (Cis)-treated Cul4b^Con or Cul4b^cKO mice with or without pretreatment of PFT-α, an inhibitor for p53. N = 5. C Western blots showing the levels of PAI-1 and NGAL in the indicated kidneys. D The creatinine (Cr) and BUN levels in the serum collected from the indicated mice. N = 5. E The representative images of immunohistochemistry staining of KIM-1 in kidneys and the quantification of the percentage of KIM-1⁺ area. 5 fields were imaged for each mouse and 5 mice were included in each group. Scale bar, 60 μm. F The representative images of H & E staining and the tubular damage scores of the kidneys from the indicated mice. 10 fields were imaged for each mouse and 5 mice were included in each group. Scale bar, 60 μm. G The representative images of immunohistochemistry staining of the macrophage marker CD68 and quantification of the percentage of CD68⁺ area in kidneys. 5 fields were imaged for each mouse and 5 mice were included in each group. Scale bar, 60 μm. H The mRNA levels of the indicated inflammatory factors in the kidneys. N = 5. I The representative images of TUNEL staining on kidneys and the quantification of the number of TUNEL⁺ cells in each field. 5 fields were imaged for each mouse and 5 mice were included in each group. Scale bar, 85 μm. In all bar graphs, the bars from left to right represent Cul4b^Con mice injected with cisplatin (Cul4b^Con-Cis), Cul4b^Con mice pretreated with PFT-α before injection with cisplatin (Cul4b^Con-Cis+PFT-α), Cul4b^cKO mice injected with cisplatin (Cul4b^cKO-Cis) and Cul4b^cKO mice pretreated with PFT-α before injection with cisplatin (Cul4b^Con-Cis+PFT-α), respectively. Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 6. CRL4B complex catalyzes polyubiquitination of p53 at K164 to promote its degradation.

A The level of Trp53 mRNA in kidneys from the indicated mice. N = 5. B The level of TP53 mRNA in the indicated HK2 cells. N = 5. Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. ns: no significance. C Western blots showing the level of p53 protein in HK2 cells with CUL4B stably knocked down (shCUL4B) or control HK2 cells (NS) treated with 20 μg/ml cisplatin (20 μg/ml) or vehicle control (Con). D Western blots showing the level of p53 protein in the cisplatin-treated HK2 cells with or without CUL4B knocked down at different time points after treating with CHX and the quantification of the protein levels. E The co-immunoprecipitation (IP) assay showing p53 interacts with CUL4B and DDB1 in HK2 cells treated with cisplatin and MG132. Ubiquitination assay showing ubiquitination of p53 in HK2 cells stably knocking down CUL4B (F) or overexpressing CUL4B (G). Cells were transfected with HA-Ub plasmid and treated with MG132. The lysates were immunoprecipitated with anti-p53 antibody. H Western blots showing the level of wild type or K164R mutant p53 in HEK293T cells at different time points after treating with CHX and the quantification of the protein levels. Cells were transfected with 5×Myc: p53-WT or 5×Myc: p53-K164R. I Ubiquitination assay showing the levels of polyubiquitination on wild type or K164R mutant p53 in CUL4B-overexpressing HEK293T cells. Cells were transfected with the indicated plasmids and treated with MG132. The lysates were immunoprecipitated with anti-Myc antibody. J Schematic showing the molecular mechanism underlying CUL4B regulation of AKI.