Targeting Stat3 with conditional knockout or PROTAC technology alleviates renal injury by Limiting pyroptosis

Abstract

Background: Acute kidney injury (AKI) is a critical clinical syndrome with high morbidity, mortality, and no effective treatment in clinical practice. The role of the Signal Transducer and Activator of Transcription 3 (Stat3) in AKI remains controversial, and its complex regulatory mechanisms must be further explored.

Methods: We generated renal tubular epithelial cells Stat3 conditional knockout (cKO) mice and used them in cecal ligation and puncture (CLP) and ischaemia-reperfusion (I/R) induced AKI models. Additionally, proteolysis-targeting chimaera (PROTAC) compound E034 was designed and synthesised. We also utilised human kidney tissues, mouse renal tubular epithelial cells (mTECs) and HK-2 cells for further studies, including immunohistochemistry, Western blot analysis, Real-time PCR, chromatin immunoprecipitation (ChIP) and RNA sequencing, scanning electron microscopy (SEM) and Co-Immunoprecipitation (Co-IP) assay.

Findings: An upregulation of total Stat3 protein was observed in AKI mouse models, which correlated with patient biopsy results. This increase may be attributed to histone H3K27 acetylation. Stat3 knockout in renal tubular epithelial cells significantly reduced AKI injury and inflammation in mice. Mechanistically, Stat3 induces the transcription of tripartite motif-containing protein 21 (Trim21), triggering a cascade that activates gasdermin D (Gsdmd), resulting in pyroptosis. Administration of E034, which selectively targets Stat3 for ubiquitination and degradation, significantly alleviated renal injury in a low-dose, single-dose regimen.

Interpretation: In the context of renal injury, PROTAC emerges as a promising modality by explicitly targeting the Stat3/Trim21/Gsdmd axis, which our study has identified as a potential therapeutic target, potentially endowing clinically significant therapeutic strategies.

Funding: This work was supported by the National Key R&D Program (2022YFC2502503), the National Natural Science Foundation of China (No. 82270738), the National Natural Science Foundation of China (No. 82400806) and the Graduate Research and Practice Innovation Project of Anhui Medical University (YJS20230059).

Keywords: Histone modification; PROTAC; Pyroptosis; Renal injury; Stat3.

Fig. 1

Stat3 and p-Stat3 are markedly induced by H3K27ac modification in renal tubular cells of patients with AKI and CLP- and I/R-induced mouse models. (a) Western blot analysis of Stat3 and p-Stat3 proteins in a CLP-induced AKI mouse model (n = 6). (b) Real-time PCR analysis of Stat3 mRNA levels in the CLP-induced AKI mouse model (n = 6, t-test). (c) Western blot analysis of Stat3 and p-Stat3 proteins in an I/R-induced AKI mouse model (n = 6). (d) Real-time PCR analysis of Stat3 mRNA levels in the I/R-induced AKI mouse model (n = 6, t-test). (e) IHC staining for Stat3 in patients with AKI (Scale bar = 50 μm). (f–g) Immunofluorescence staining for Stat3 and p-Stat3 with LTL, Calbindin, and DBA in the CLP-induced AKI mouse model (Scale bar = 50 μm and 10 μm; n = 6). (h) Western blot analysis of Stat3 and p-Stat3 proteins in mTECs treated with LPS (n = 3). (i) Real-time PCR analysis of Stat3 mRNA levels in LPS-treated mTECs (n = 3, t-test). (j) Western blot analysis of Stat3 and p-Stat3 proteins in mTECs subjected to H/R treatment (n = 3). (k) Real-time PCR analysis of Stat3 mRNA levels in H/R-treated mTECs (n = 3, t-test). (l) Western blot analysis of H3K4me1, H3K4me3, H3K27ac, and Histone 3 proteins in LPS-treated mTECs (n = 3). (m) Real-time PCR analysis of Stat3 mRNA levels after treatment with MM-102 and C646 following LPS induction (n = 3, one-way ANOVA). (n–o) Western blot analysis of H3K4me1, H3K4me2, H3K4me3, and Stat3 proteins after treatment with MM-102 following LPS induction (n = 3). (p–q) Western blot analysis of H3K27ac and Stat3 proteins after treatment with C646 following LPS induction (n = 3). (r–s) ChIP assay for the binding of H3K27ac to Stat3 (n = 3, one-way ANOVA). (t) ChIP assay for the binding of Cbp to Stat3 (n = 3, one-way ANOVA). (u) ChIP assay for the binding of P300 to Stat3 (n = 3, one-way ANOVA). (Data are presented as mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001).

Fig. 2

Conditional knockout of Stat3 in renal tubular epithelial cells alleviates CLP- and I/R-induced kidney injury and inflammation in mice. (a) The schematic diagram for constructing a mouse model with conditional knockout of Stat3 in renal tubular epithelial cells. (b) Validation of successful construction of Stat3 conditional knockout in renal tubular epithelial cells using agarose gel electrophoresis. (c–d) Serum CRE and BUN in the CLP-induced AKI mouse model with or without conditional knockout of Stat3. (e) Western blot analysis of Kim1 in the CLP-induced AKI mouse model. (f) Real-time PCR analysis of Lcn2 mRNA levels in the CLP-induced AKI mouse model. (g) Western blot analysis of p-P65 and P65 in the CLP-induced AKI mouse model. (h) Real-time PCR analysis of Mcp-1 and Tnf-α mRNA levels in the CLP-induced AKI mouse model. (i) PAS staining of kidney sections from Stat3^flox/flox and Stat3 conditional knockout mice with CLP-induced AKI. (j) IF staining of F4/80+ macrophage infiltration in mice with CLP-induced nephropathy with or without conditional knockout of Stat3. (k–l) Serum CRE and BUN in the I/R-induced AKI mouse model with or without conditional knockout of Stat3. (m) Real-time PCR analysis of Kim1 mRNA levels in I/R-induced AKI. (n) Western blot analysis of p-P65 and P65 in the I/R-induced AKI mouse model. (o) Real-time PCR analysis of Tnf-α mRNA levels in I/R-induced AKI. (p) Real-time PCR analysis of Mcp-1 mRNA levels in the I/R-induced AKI mouse model. (q) IF staining of F4/80+ macrophage infiltration in mice with I/R-induced nephropathy with or without conditional knockout of Stat3. (r) Schematic illustrating the rescue experiment in Stat3 conditional knockout mice. (s) Serum CRE in the CLP-induced AKI mouse model after the rescue of Stat3. (t) Western blot analysis of Kim1 in the CLP-induced AKI mouse model after rescue of Stat3. (u) Real-time PCR analysis of Mcp-1 and Tnf-α mRNA levels in the CLP-induced AKI mouse model after rescue of Stat3. (n = 6; Data are presented as mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, One-way ANOVA; Scale bar = 50 μm).

Fig. 3

Silencing Stat3 in mTECs and HK-2 in vitro alleviates LPS- and H/R-induced cell injury and inflammation, and Stat3 overexpression reverses these effects. (a) Western blot analysis of Kim1 in LPS-induced cell models with or without knockdown of Stat3. (b) IF staining of Kim1 in LPS-induced cell models with or without knockdown of Stat3. (c) Real-time PCR analysis of Kim1 mRNA levels in LPS-induced cell models with or without knockdown of Stat3. (d) Western blot analysis of Kim1 in H/R-induced cell models with or without knockdown of Stat3. (e) Western blot analysis of p-P65 and P65 in LPS-induced cell models with or without knockdown of Stat3. (f) Western blot analysis of p-P65 and P65 in H/R-induced cell models with or without knockdown of Stat3. (g) Real-time PCR analysis of Mcp-1, Tnf-α, and Il-1β mRNA levels in LPS-induced cell models with or without knockdown of Stat3. (h) Real-time PCR analysis of Mcp-1, Tnf-α, and Il-1β mRNA levels in H/R-induced cell models with or without knockdown of Stat3. (i–j) Western blot analysis of Kim1, p-P65, and P65 in LPS-induced cell models with or without overexpression of Stat3. (k) IF staining of Kim1 in LPS-induced cell models with or without overexpression of Stat3. (l–m) Real-time PCR analysis of Kim1, Mcp-1, Tnf-α and Il-1β mRNA levels in LPS-induced cell models with or without overexpression of Stat3. (n–o) Western blot analysis of Kim1, p-P65, and P65 in H/R-induced cell models with or without overexpression of Stat3. (p) Real-time PCR analysis of Mcp-1, Tnf-α, and Il-1β mRNA levels in H/R-induced cell models with or without overexpression of Stat3. (n = 3; Data are presented as mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, one-way ANOVA; Scale bar = 50 μm).

Fig. 4

Combined ChIP-seq and RNA-seq analysis screens Trim21, a target gene that directly binds to its promoter region and regulates transcription. (a) Venn diagram of intersecting genes between ChIP-seq and RNA-seq analysis. (b) Comparison of TSS peak plots for samples undergoing ChIP-seq. (c) Quadrant plot of intersecting genes from ChIP-seq and RNA-seq. (d) Real-time PCR analysis of Mrc2, Ifi44, Trim21, Cxcl10, Xkr6, Baiap3, and Slfn2 mRNA levels in LPS-induced cell models with or without knockdown of Stat3 (n = 3). (e) Real-time PCR analysis of Mrc2, Ifi44, Trim21, Ccxl10, Xkr6, Baiap3, and Slfn2 mRNA levels in CLP-induced AKI models with or without conditional knockout of Stat3 (n = 6). (f) Visualisation of Trim21 ChIP-seq data. (g) GSEA of RNA-seq results for the pyroptosis pathway. (h) Heatmap of genes related to the pyroptosis pathway. (i) Real-time PCR analysis of the dynamic mRNA levels of Stat3 and Trim21 for 0–24 h in LPS-induced cell models with or without knockdown of Stat3 (n = 3). (j) Western blot analysis of Trim21 with or without knockdown of Stat3 in LPS-induced cell models (n = 3). (k) Western blot analysis of Trim21 in Stat3^flox/flox and Stat3 cKO mice with CLP-induced AKI (n = 6). (l) IF staining of Trim21 in LPS-induced cell models with or without knockdown of Stat3 (n = 3). (m) Western blot analysis of Trim21 with or without overexpression of Stat3 in LPS-induced cell models (n = 3). (n) Binding of Stat3 to Trim21 assessed by ChIP assay (n = 3). (o) Schematic illustration of mutations in the Trim21 promoter region. (p) Dual-luciferase reporter assay analysing the effect of mutations at different sites in the Trim21 promoter region on binding affinity (n = 3). (Data are presented as mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, one-way ANOVA; Scale bar = 50 μm).

Fig. 5

Trim21 bypasses the inflammasome pathway and binds to Gsdmd through the PRY/SPRY domain, promoting LPS-induced cell injury, inflammation, and pyroptosis. (a) Western blot analysis of p-P65 and P65 in LPS-induced cells with or without knockdown of Trim21. (b) Real-time PCR analysis of Mcp-1 and Tnf-α mRNA levels in LPS-induced cells with or without knockdown of Trim21. (c) IF staining of Kim1 in LPS-induced cells with or without knockdown of Stat3 (Scale bar = 50 μm). (d) Western blot analysis of Gsdmd-N and Gsdmd in LPS-induced cells with or without knockdown of Trim21. (e–f) ELISA for determining Il-1β and Il-18 levels in the supernatant of LPS-induced cells with or without knockdown of Trim21. (g) SEM morphology observation of LPS-induced cells with or without knockdown of Trim21 (Scale bar = 10 μm). (h) Western blot analysis of p-P65 and P65 in LPS-induced cells with or without overexpression of Trim21. (i) Real-time PCR analysis of Mcp-1 mRNA levels in LPS-induced cells with or without overexpression of Trim21. (j) IF staining of Kim1 in LPS-induced cells with or without overexpression of Trim21 (Scale bar = 50 μm). (k) Western blot analysis of Gsdmd-N and Gsdmd in LPS-induced cells with or without overexpression of Trim21. (l–m) ELISA for determination of Il-1β and Il-18 levels in the supernatant of LPS-induced cell models with or without overexpression of Trim21. (n) Western blot analysis of Stat3, Nlrp3, caspase1-p20 and caspase1 in LPS-induced cells with or without overexpression of Trim21. (o) Co-IP assay for the binding of Trim21 to Stat3, Nlrp3, Caspase1, and Gsdmd in LPS-induced cells with or without overexpression of Trim21. (p) Schematic illustration of Trim21 and the ΔPRY/SPRY domain. (q) Co-IP assay for Trim21 or ΔPRY/SPRY binding to Gsdmd in LPS-induced cell models. (n = 3; Data are presented as mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, one-way ANOVA).

Fig. 6

Stat3 stimulates LPS-induced pyroptosis by promoting Trim21 transcription. (a) Western blot analysis of Gsdmd-N and Gsdmd in Stat3^flox/flox and Stat3 cKO mice with CLP-induced AKI. (b) IF staining of Gsdmd-N in Stat3^flox/flox and Stat3 cKO mice with CLP-induced AKI(Scale bar = 50 μm). (c–d) ELISA for determining Il-1β and Il-18 levels in the serum from Stat3^flox/flox and Stat3 cKO mice with CLP-induced AKI (n = 6). (e–f) ELISA for the determination of Il-1β and Il-18 levels in the serum from Stat3 cKO mice with CLP-induced AKI with or without Stat3 rescue (n = 6). (g) SEM morphology observation of LPS-induced cells with or without knockdown of Stat3 (Scale bar = 10 μm). (h–i) ELISA for determining Il-1β and Il-18 levels in the supernatant of LPS-induced cells with or without knockdown of Stat3 (n = 3). (j) Western blot analysis of Gsdmd-N and Gsdmd in LPS-induced cells with or without knockdown of Stat3 (n = 3). (k–l) ELISA for determining Il-1β and Il-18 levels in the supernatant of LPS-induced cells with or without overexpression of Stat3 (n = 3). (m) Western blot analysis of Gsdmd-N and Gsdmd in LPS-induced cells with or without overexpression of Stat3 (n = 3). (n) Western blot analysis of Kim1, p-65 and P65 with or without Trim21 rescue after Stat3 knockdown in the LPS-induced cells (n = 3). (o)Real-time PCR analysis of Mcp-1 mRNA levels in the supernatant of LPS-induced cells with or without Trim21 rescue after Stat3 knockdown (n = 3). (p) Western blot analysis of Gsdmd-N and Gsdmd with or without Trim21 rescue after Stat3 knockdown in the LPS-induced cells (n = 3). (q) ELISA for the determination of Il-1β level in the supernatant of LPS-induced cells with or without Trim21 rescue after Stat3 knockdown (n = 3). (r) SEM morphology observation of LPS-induced cells with or without Trim21 rescue after Stat3 knockdown (Scale bar = 5 μm). (Data are presented as mean ± SEM; ∗P < 0.05, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, one-way ANOVA).

Fig. 7

AAV9-mediated in vivo silencing of Stat3 attenuates CLP- and I/R-induced renal injury, inflammatory response, and pyroptosis. (a) Schematic diagram of in vivo AAV9-mediated silencing of Stat3 in mice. (b) Western blot analysis of Stat3 in mice with or without knockdown of Stat3. (c–d) Serum CRE and BUN in CLP-induced AKI mice with or without knockdown of Stat3. (e) PAS staining of kidney sections in CLP-induced AKI mice with or without knockdown of Stat3. (f) IHC staining of F4/80+ macrophage infiltration in mice with CLP-induced nephropathy with or without knockdown of Stat3. (g) Real-time PCR analysis of Mcp-1 and Tnf-α mRNA levels in CLP-induced AKI mice with or without knockdown of Stat3. (h–i) Western blot analysis of Kim1, Gsdmd-N and Gsdmd in CLP-induced AKI mice with or without knockdown of Stat3. (j–k) ELISA for determining Il-1β and Il-18 levels in the serum from CLP-induced AKI mice with or without knockdown of Stat3. (l) Serum CRE in I/R-induced AKI mice with or without knockdown of Stat3. (m) PAS staining of kidney sections in I/R-induced AKI mice with or without knockdown of Stat3. (n) Real-time PCR analysis of Kim1 and Lcn2 mRNA levels in I/R-induced AKI mice with or without knockdown of Stat3. (o) Western blot analysis of p-P65 and P65 in I/R-induced AKI mice with or without knockdown of Stat3. (p) Real-time PCR analysis of Mcp-1 mRNA level in I/R-induced AKI mice with or without knockdown of Stat3. (q) Western blot analysis of Gsdmd-N and Gsdmd in I/R-induced AKI mice with or without knockdown of Stat3. (r) ELISA for determining Il-1β level in the serum from I/R-induced AKI mice with or without knockdown of Stat3. (n = 3; Data are presented as mean ± SEM; ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, one-way ANOVA; Scale bar = 50 μm).

Fig. 8

PROTAC E034 mitigates CLP-induced kidney injury, inflammation, and pyroptosis by degrading Stat3. (a) Structural formula and mechanism of action of E034. (b) Western blot analysis of Stat3 under the influence of different doses of E034. (c) Western blot analysis of Stat3 with pre-treatment of MG132, MLN4924, thalidomide, and warhead. (d) Computational simulation of molecular docking between Stat3 and E034. (e) Western blot analysis of Kim1, p-P65 and P65 under 0.1, 0.2 and 0.4 μM doses of E034 in LPS-induced cells. (f) Real-time PCR analysis of Mcp-1 and Tnf-α mRNA levels in LPS-induced cells (n = 3). (g) Western blot analysis of Trim21, Gsdmd-N and Gsdmd in LPS-induced cells after treatment with 0.1, 0.2 and 0.4 μM E034. (h) ELISA for the determination of Il-1β levels in the supernatant of LPS-induced cells (n = 3). (i) SEM morphology observation of LPS-induced cells with or without 0.4 μM E034 treatment (Scale bar = 3 μm). (j) IF staining of Kim1 in H/R-induced cells after treatment with 0.4 μM E034 (Scale bar = 50 μm). (k) Western blot analysis of Trim21, Gsdmd-N and Gsdmd in H/R-induced cells. (l) Serum CRE in CLP-induced AKI mice after pre-treatment (n = 6). (m) PAS staining of kidney sections in CLP-induced AKI mice after pre-treatment with 0.05, 0.1 and 0.2 mg/kg E034. (n) Western blot analysis of Kim1, p-P65 and P65 in CLP-induced AKI mice after pre-treatment. (o) Real-time PCR analysis of Mcp-1 mRNA levels in CLP-induced AKI mice (n = 6). (p) ELISA for determining Il-1β levels in serum from CLP-induced AKI mice (n = 3). (q) Western blot analysis of Gsdmd-N and Gsdmd in CLP-induced AKI mice. (r) Serum CRE in CLP-induced AKI mice (n = 6). (s–t) Western blot analysis of Kim1, p-P65, P65, Gsdmd-N and Gsdmd in CLP-induced AKI mice after treatment with 0.2 mg/kg E034. (u) ELISA for determining Il-18 levels in the serum from CLP-induced AKI mice (n = 6). (Data are presented as mean ± SEM; ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, one-way ANOVA).