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www.nature.com/cddiscovery ARTICLE OPEN XBP1 modulates endoplasmic reticulum and mitochondria crosstalk via regulating NLRP3 in renal ischemia/reperfusion injury 1,5 1,5 1,5 2,5 3,4 3,4 3,4 1 1 Haiqiang Ni , Zhiyu Ou , Yuchen Wang , Yanna Liu , Kailun Sun , Ji Zhang , Jiasi Zhang , Wenfeng Deng , Wenli Zeng , 1 1 3,4✉ 1✉ Renfei Xia , Jian Xu , Nianqiao Gong and Yun Miao © The Author(s) 2023 The functional status of mitochondria and the endoplasmic reticulum are central to renal ischemia/reperfusion injury (IRI). X-box binding protein 1 (XBP1) is an important transcription factor in endoplasmic reticulum stress. NLR family pyrin domain containing- 3(NLRP3) inﬂammatory bodies are closely related to renal IRI. In vivo and in vitro, we examined the molecular mechanisms and functions of XBP1-NLRP3 signaling in renal IRI, which inﬂuences ER-mitochondrial crosstalk. In this study, mice were subjected to 45 min of unilateral renal warm ischemia, the other kidney resected, and reperfusion was performed for 24 h in vivo. In vitro, murine renal tubular epithelial cells (TCMK-1) were exposed to hypoxia for 24 h and reoxygenation for 2 h. Tissue or cell damage was evaluated by measuring blood urea nitrogen and creatinine levels, histological staining, ﬂow cytometry, terminal deoxynucleotidyl transferase-mediated nick-end labeling, diethylene glycol staining, and transmission electron microscopy (TEM). Western blotting, immunoﬂuorescence staining, and ELISA were used to analyze protein expression. Whether XBP1 regulates the NLRP3 promoter was evaluated using a luciferase reporter assay. Kidney damage was reduced with decreasing blood urea nitrogen, creatinine, interleukin-1β, and interleukin-18 levels. XBP1 deﬁciency reduced tissue damage and cell apoptosis, protecting the mitochondria. Disruption of XBP1 was associated with reduced NLRP3 and cleaved caspase-1 levels and markedly improved survival. In vitro in TCMK-1 cells, XBP1 interference inhibited caspase-1-dependent mitochondrial damage and reduced the production of mitochondrial reactive oxygen species. The luciferase assay showed that spliced XBP1 isoforms enhanced the activity of the NLRP3 promoter. These ﬁndings reveal that XBP1 downregulation suppresses the expression of NLRP3, a potential regulator of endoplasmic reticulum mitochondrial crosstalk in nephritic injury and a potential therapeutic target in XBP1- mediated aseptic nephritis. Cell Death Discovery (2023) 9:69 ; https://doi.org/10.1038/s41420-023-01360-x INTRODUCTION unfolded protein response signaling pathway [7–9]. Low levels of Acute kidney injury (AKI) is associated with severe morbidity and the unfolded protein response protect the kidneys from renal IRI mortality and is prevalent globally. However, the mechanisms . However, severe ERS induces the activation of cell death underlying renal dysfunction and tissue damage remain to be fully cascades [11, 12]. ERS leads to the opening of the mitochondrial elucidated . Ischemia/reperfusion, a common cause of AKI , permeability transition pore (mPTP), which then depletes the involves inﬂammation, hemodynamic alterations, and epithelial mitochondrial membrane potential (MMP) and triggers the release and endothelial cell death [3, 4]. Accumulating evidence reveals of mitochondrial pro-apoptotic signals , reduction in MMP, and that ischemia/reperfusion injury (IRI) is a multifactorial process in release of cytochrome c (Cyt-c) and other apoptosis-related which the mitochondria and endoplasmic reticulum (ER) play factors. In particular, AKI-induced mitochondrial reactive oxygen central roles . Recent studies have shown that injury to the ER or species (mROS) promote the activation of the NLRP3 inﬂamma- mitochondria could disrupt ER-mitochondrial crosstalk in the some, leading to caspase 1-dependent production of gasdermin D kidneys, leading to renal injury. Thus, ER-mitochondria crosstalk (GSDMD)-mediated pyroptosis and the maturation of the pro- may be a potential therapeutic target in AKI . inﬂammatory cytokines interleukin (IL)-1β and IL-18 [14–16]. A dysfunctional ER may contribute to the accumulation of Recent studies have examined the regulatory functions of the unfolded or misfolded proteins during IRI, causing ER stress (ERS). mitochondria-associated ER membranes (MAMs). Aside from ER Upon the detection of unfolded proteins, three transmembrane chaperones, other inﬂammatory factors such as sigma-1 receptor receptors in the ER, namely ATF6, IRE1α, and PERK, activate the , IP (3) receptor , GRP78 , calnexin , IRE1α, and PERK 1 2 Department of Transplantation, Nanfang Hospital, Southern Medical University, 510515 Guangzhou, China. Department of Gastroenterology and Hepatology, Beijing Youan Hospital, Capital Medical University, 100069 Beijing, China. Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 430030 Wuhan, China. Key Laboratory of Organ Transplantation of Ministry of Education, National Health Commission and Chinese Academy of Medical Sciences, 430030 Wuhan, China. These authors contributed equally: Haiqiang Ni, Zhiyu Ou, Yuchen Wang, Yanna Liu. email: firstname.lastname@example.org; email@example.com Received: 24 June 2022 Revised: 1 February 2023 Accepted: 2 February 2023 Ofﬁcial journal of CDDpress 1234567890();,: H. Ni et al. can also modulate ER-mitochondrial crosstalk [21–23]. When the was 0.82 ± 0.41 vs. 19.49 ± 5.41. Meanwhile, the protein expression mitochondrial ﬁssion protein, ﬁssion 1 homolog (Fis1), binds to B of XBP1u was 0.47 ± 0.06 vs. 0.74 ± 0.06, and that of XBP1s was cell receptor-associated protein 31 (BAP31) located in the MAMs 0.20 ± 0.04 vs. 0.45 ± 0.06 (all P < 0.05). Further, increased XBP1 and forms the Fis1-BAP31 complex, cell death signals are expression enhanced NLRP3 expression (mRNA, 1.01 ± 0.01 vs. conveyed to the ER lumen, initiating the cell apoptosis pathway 1.18 ± 0.04; protein, 0.37 ± 0.06 vs. 0.72 ± 0.10; both P < 0.05). For . Under ERS, unspliced X-box-binding protein 1 (XBP1u) mRNA TCMK-1 cells transfected with XBP1-targeting siRNA, the values (a non-transcriptional form) is activated by the RNase activity of relative RNA expression levels were as follows: for XBP1u, IRE1α, which excises 26 nucleotides, causing a frameshift and 0.84 ± 0.03 vs. 0.03 ± 0.01, P < 0.01; for XBP1s, 1.19 ± 0.09 vs. forming a transcriptionally spliced XBP1 (XBP1s) mRNA . The 0.02 ± 0.01, P < 0.001. The relative protein expression values were transcription factor XBP1s then regulates a series of important 0.48 ± 0.05 vs. 0.05 ± 0.01, P < 0.001 for XBP1u and 0.17 ± 0.03 vs. target genes to determine cell fate . XBP1 downregulation has 0.03 ± 0.01, P < 0.05 for XBP1s. Relative to the empty-vector group, been found to reduce renal IRI by inhibiting HRD1-mediated NRF2 NLRP3 expression was lower in the si-XBP1 group (mRNA: ubiquitination . Although XBP1 downregulation under severe 1.02 ± 0.01 vs. 0.54 ± 0.04, P < 0.001; protein: 0.40 ± 0.07 vs. stress is known to protect tissues and organs, how (or even 0.10 ± 0.01, P < 0.05) (Fig. 3A, B). Moreover, XBP1s production whether) XBP1 participates in the ER-mediated regulation of was inhibited upon treatment with 4μ8c, an IRE1α inhibitor, mitochondria or MAMs in renal IRI remains unknown. To address further reducing NLRP3 expression (Supplementary Fig. S1). this, we examined how the XBP1-NLRP3 axis modulates ER- To further verify the effects of XBP1 on NLRP3 under stress mitochondrial crosstalk during renal IRI in vivo and in vitro. conditions, H/R was used to simulate renal IRI in vitro. Our experiments revealed that XBP1 downregulation inhibited NLRP3 expression (XBP1u: 1.24 ± 0.15 vs. 0.34 ± 0.13, P < 0.05; XBP1s: RESULTS 0.27 ± 0.03 vs. 0.03 ± 0.01, P < 0.01; NLRP3: 0.97 ± 0.07 vs. IRI triggers severe ERS and markedly increases XBP1s 0.22 ± 0.02, P < 0.001; Fig. 3C). expression We then examined the mechanisms through which XBP1 To detect ER morphology in IRI kidney sections, ultrastructural regulates NLRP3. Based on bioinformatics analysis, we predicted analysis was performed using TEM. Abnormal ER morphology, that XBP1 might regulate NLRP3 promoter activity via XBP1s. We manifested as distinct vacuolation and swelling (characteristic of conducted a dual-luciferase reporter assay to conﬁrm if XBP1s ERS), was observed (Fig. 1A). To further evaluate ERS, western binds to the NLRP3 promoter. First, we constructed an XBP1s- blotting was used to analyze the expression of key ERS markers in overexpression plasmid containing the NLRP3 promoter. This, the BIP and IRE1α pathway (IRE1α, XBP1u, XBP1s, and p-ASK1), along with a luciferase reporter plasmid, was transfected into PERK pathway (PERK, p-PERK, CHOP, ATF4, and NRF2), and the 293T cells. Before interaction with the XBP1s transcription factor, ATF6 pathway. The fold-changes in expression relative to the NLRP3 promoter activity was 13.52 times higher in the XBP1- control were 1.24 ± 0.08 (P < 0.05) for BIP, 3.14 ± 0.73 (P < 0.05) for overexpression group than in the control group (P < 0.05), IRE1α, 2.25 ± 0.29 (P < 0.05) for XBP1u, 6.04 ± 1.36, (P < 0.05) for indicating activation of the NLRP3 gene promoter. After interact- XBP1s, 2.53 ± 0.12 (P < 0.001) for p-ASK1, 1.04 ± 0.01 (P < 0.05) ing with the XBP1s transcription factor, NLRP3 gene promoter for PERK, 5.31 ± 0.57 (P < 0.01) for p-PERK, 4.29 ± 1.03 (P < 0.05) for activity was 1.81 times higher than in the control group, indicating CHOP, 2.69 ± 0.60 (P < 0.05) for ATF4, 3.89 ± 0.90 (P < 0.05) for that the XBP1s transcription factor enhances NLRP3 gene NRF2, and 5.11 ± 1.00 (P < 0.05) for ATF6. Notably, the fold-change promoter activity (P < 0.05; Fig. 3D). Unfortunately, we obtained was signiﬁcantly greater for XBP1s than for the other proteins, negative results for our three predicted binding sites. Therefore, indicating that this activated transcription factor may play a key the binding sites require further investigation. role in response to IR-induced kidney injury. XBP1 downregulation reduced the production of NLRP3 IRI induces mitochondrial injury and NLRP3 inﬂammasome downstream effectors, mitochondrial damage, and apoptosis activation in TCMK-1 cells exposed to H/R Mitochondrial damage and the NLRP3 inﬂammasome play After elucidating the possible mechanism whereby XBP1 regulates important roles in ischemia-reperfusion injury [27–29]. TEM NLRP3, we examined whether XBP1 downregulation affects the conﬁrmed mitochondrial damage, characterized by mitochondrial expression of the downstream effector molecules of NLRP3 swelling, cristae fracture, membrane damage, and abnormal ER in in vitro. We then veriﬁed the changes in NLRP3 localization the vicinity of the MAMs (Fig. 2A). The relative expressions of the following XBP1 downregulation using a mitochondrial probe mitochondrial damage markers total Cyt-c and caspase-9 (Casp9) (MitoTracker), ER probe (ER Tracker Blue White DPX dye), and anti- were signiﬁcantly elevated relative to the control (Cyt-c: NLRP3 antibodies and observed them via confocal microscopy. 0.56 ± 0.04 vs. 0.84 ± 0.08, P < 0.05; Casp9, p39: 0.06 ± 0.01 vs. Under hypoxia, colocalization with NLRP3 was enhanced in the 0.83 ± 0.12, P < 0.01; Casp9, p37: 0.12 ± 0.02 vs. 2.53 ± 0.33, mitochondria and ER; colocalization at all three sites was reduced P < 0.01; Fig. 2B). Western blot analysis revealed a signiﬁcantly after XBP1 downregulation (Fig. 4A). Based on western blot elevated expression of NLRP3 molecules and caspase-1 activators analysis, XBP1 downregulation inhibited inﬂammation in TCMK-1 in the IRI group relative to the control (0.10 ± 0.01 vs. 0.14 ± 0.01 cells after 24 h of hypoxia followed by 2 h of reoxygenation, which and 1.02 ± 0.12 vs. 1.50 ± 0.03, respectively,P < 0.01; Fig. 2C), was not exhibited in the siRNA-treated control (Casp1, p20: indicating inﬂammasome activation. In addition, immunoelectron 1.31 ± 0.10 vs. 0.10 ± 0.01, P < 0.01; IL-1β: 0.99 ± 0.10 vs. 0.26 ± 0.07, microscopy (IEM) revealed an enhanced NLRP3 colocalization with P < 0.05; IL-18: 0.54 ± 0.06 vs. 0.16 ± 0.03, P < 0.05; Fig. 4B). mitochondria under IRI, and that NLRP3 may even be able to We then examined the protective effect of XBP1 inhibition on translocate to the MAMs (Fig. 2D). the mitochondria using an in vitro H/R model. To do this, we used a DCFH-DA probe to stain the total reactive oxygen species (tROS) The molecular mechanism of the crosstalk between ERS and and the mitochondrial superoxide indicator MitoSOX red to stain mitochondrial dysfunction in response to IRI is mediated by mitochondrial ROS (mROS). ROS were detected via ﬂow cytometry. the XBP1-NLRP3 axis MMP was detected via staining with the ﬂuorescent dye JC-1, To further analyze the effect of XBP1 modulation on NLRP3 followed by ﬂow cytometry. The results showed that siRNA- expression and activity, we transfected TCMK-1 cells with an XBP1- induced XBP1 downregulation reduced ROS production (tROS: overexpression lentivirus. In these cells, the relative RNA expres- 221 ± 20.11 vs. 97.60 ± 8.17, P < 0.01; mROS: 56.27 ± 2.21 vs. sion of XBP1u was 1.21 ± 0.11 vs. 3.25 ± 0.48, and that of XBP1s 43.77 ± 0.98, P < 0.01; Fig. 4C), and elevated the MMP Cell Death Discovery (2023) 9:69 H. Ni et al. Fig. 1 IRI triggers severe ERS and noticeable XBP1s expression. To construct the mouse IRI model, we used microvascular clips to block the left renal pedicle of the mouse for 45 min and cut off the right kidney after opening the bloodstream. A TEM analysis showed ER damage was induced by IRI. The white arrow indicates the normal ER, while the red arrow indicates swollen ER lumens. Scale bar = 1 μm; ×5000 magniﬁcation. B The expression levels of ER stress markers were detected using western blotting. The protein level of XBP1s was considerably higher than that of other molecules after IRI, as shown by the fold-changes. *P < 0.05, **P < 0.01, ***P < 0.001 vs. The normal control group (n = 3 samples/group). IRI ischemia/reperfusion injury, ER endoplasmic reticulum. +/− (72.77 ± 0.91 vs. 45.53 ± 1.29, P < 0.0001; Fig. 4D). These changes Genotyping of Xbp1 mice was conﬁrmed via PCR using Ef and did not occur in the si-negative control (NC)-transfected cells. Kr primers (Fig. 5B). These results indicate that XBP1 inhibition can protect mitochon- drial function in an in vitro H/R cell model. Downregulation of XBP1-NLRP3 signaling reduced the Western blot analysis of Cyt-c and Casp9 expression revealed crosstalk between ERS and mitochondrial damage and +/− that the rates of mitochondrial damage were lower in the si-XBP1- protected the kidneys of Xbp1 mice from IRI treated group than in the control (Casp9, p39: 1.38 ± 0.07 vs. To evaluate the importance of the XBP1-NLRP3 axis in the 0.09 ± 0.01, P < 0.05; Casp9, p37: 0.65 ± 0.19 vs. 0.05 ± 0.02, crosstalk between ERS and mitochondrial damage in IR-induced +/− P < 0.0001; Cyt-c: 1.03 ± 0.14 vs. 0.30 ± 0.08, P < 0.05; Fig. 4E). renal inﬂammation, we studied heterozygous Xbp1 mice To detect apoptosis, we conducted Annexin V/PI analysis generated using the knockout-ﬁrst strategy. XBP1 expression +/− (22.01 ± 1.56 vs. 17.03 ± 0.36, P < 0.05; Fig. 4F) and used western was observed to be somewhat lower in the kidneys of Xbp1 blotting to measure the expression of caspase-3 (Casp3), a key mice subjected to ischemia-reperfusion than in the control group, apoptosis marker (Casp3, p19: 0.59 ± 0.14 vs. 0.16 ± 0.07, P < 0.05; resulting in reduced NLRP3 expression (XBP1u: 1.09 ± 0.039 vs. Casp3, p17: 2.15 ± 0.04 vs. 1.33 ± 0.22, P < 0.05; Fig. 4G). The 0.70 ± 0.04, P < 0.01; XBP1s: 0.29 ± 0.01 vs. 0.14 ± 0.01, P < 0.001; proportion of apoptotic cells was lower in the si-XBP1 group than NLRP3: 0.14 ± 0.01 vs. 0.05 ± 0.01, P < 0.01; Fig. 6A). IEM revealed in the control group. the reduced localization of NLRP3 in the mitochondria or MAMs of +/− +/− Xbp1 mice. TEM revealed that the Xbp1 mice had more complete mitochondrial structures than the control mice (Fig. 6B). +/− +/− Genotyping of Xbp1 mice In Xbp1 mice, following mitochondrial damage, there was +/− The heterozygous murine Xbp1 allele contains a trapping lower production of ROS (76.07 ± 3.19 vs. 36.88 ± 1.60; P < 0.001) cassette, the splice acceptor-β-geo-polyA (SA-βgeo-pA), which is and Cyt-c (0.84 ± 0.08 vs. 0.26 ± 0.03; P < 0.01), and caspase-9 ﬂanked by the ﬂippase recombination target of the upstream activation was inhibited (Casp9, p39: 0.83 ± 0.12 vs. 0.16 ± 0.01, exon. This causes the truncation of endogenous transcripts and P < 0.01; Casp9, p37: 2.53 ± 0.33 vs. 0.40 ± 0.07, P < 0.01) +/− constitutive null mutations that reduce XBP1 expression (Fig. 5A). (Fig. 6C, D). Xbp1 mice also exhibited a reduced expression of Cell Death Discovery (2023) 9:69 H. Ni et al. Fig. 2 IRI induces mitochondrial injury and NLRP3 inﬂammasome activation. To construct the mouse IRI model, we used microvascular clips to block the left renal pedicle of the mouse for 45 min and then cut off the right kidney after opening the bloodstream. A ERS and mitochondrial damage developed simultaneously. TEM analysis showed that the ER maintained an appropriate distance or formed a direct connection with the mitochondria. The white arrow indicates normal ER, the black arrow indicates normal mitochondria, the red arrow indicates ER swelling, the orange arrow indicates damaged mitochondria, and the blank rectangle indicates MAMs. Scale bar = 1 μm; ×5000 magniﬁcation. B The protein expression of caspase-9 and total Cyt-c were increased in the IRI group. Western blot; *P < 0.05, **P < 0.01 vs. the normal control group (n= 3 samples/group). C The expressions of NLRP3 and caspase-1 were increased in the IRI group. Western blot; *P < 0.05 vs. the normal control group (n = 3 samples/group). D IEM analysis showed more NLRP3 translocated to the mitochondria or MAMs after renal IRI. The red arrow indicates the ER, the orange arrow indicates the mitochondria, the blank rectangle indicates the MAMs, and the small black dots indicate 10-nm colloidal gold particles. Scale bar = 0.5 μm; ×10,000 magniﬁcation. IRI ischemia/reperfusion injury, ER endoplasmic reticulum, MAMs mitochondria-associated endoplasmic reticulum membranes. Cell Death Discovery (2023) 9:69 H. Ni et al. Fig. 3 The XBP1-NLRP3 axis forms a molecular mechanism involved in the crosstalk between ERS and mitochondrial dysfunction in response to IRI. TCMK-1 cells were transfected with lenti-Xbp1, siRNA-Xbp1, and corresponding control vectors, respectively. For the cell H/R model, hypoxia was performed in a tri-gas hypoxia incubator for 24 h, and then reoxygenation was performed for 2 h after applying a fresh medium. A NLRP3 protein expression correlated with XBP1 protein expression following lenti-Xbp1 and siRNA-Xbp1 transfection. Western blot; *P < 0.05, ***P < 0.001 vs. the normal control group (n = 3 samples/group). B NLRP3 mRNA expression correlated with XBP1 mRNA expression following lenti-Xbp1 and siRNA-Xbp1 transfection. Real-time quantitative reverse-transcription PCR; *P < 0.05, **P < 0.01, ***P < 0.001 vs. normal control group (n = 3 samples/group). C TCMK-1 cells were transfected with siRNA- Xbp1 or siRNA-control before H/R. NLRP3 protein expression correlated with XBP1 protein expression after H/R, as shown in the western blot and densitometric analysis. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the normal control H/R group. D The pGL4.10 ﬁreﬂy luciferase reporter plasmids with the wild-type (WT) NLRP3 promoter were transiently transfected into 293T cells together with the pcDNA3.1(+)-XBP1s plasmid or overexpression (OE) negative control and a Renilla luciferase reporter for normalization. Luciferase activities were measured after 48 h. The results showed that the NLRP3 promoter activity of the XBP1 overexpression group was higher than that of the control group; *P < 0.05. Further details are shown in the “Materials and methods” section. LV-NC lentivirus normal control, si-NC siRNA normal control, H/R hypoxia/reoxygenation. Cell Death Discovery (2023) 9:69 H. Ni et al. caspase-3 (Casp3, p19: 0.76 ± 0.14 vs. 0.13 ± 0.02, P < 0.05; Casp3, We then performed ELISA to examine the expression of IL-1β p17: 0.47 ± 0.06 vs. 0.07 ± 0.02, P < 0.01) and caspase-1 (1.50 ± 0.03 (915.50 ± 41.19 vs. 31.57 ± 7.33, P < 0.0001) and IL-18 (723.90 ± 27.08 vs. 0.24 ± 0.16, P < 0.01). These results reveal that inhibiting NLRP3 vs. 303.60 ± 34.05, P < 0.001) in the mouse serum (Fig. 6F). These expression reduced NLRP3-induced apoptosis and inﬂammasome ﬁndings indicate that the NLRP3-inﬂammasome-induced inﬂamma- +/− activation (Fig. 6E). tory response was greatly attenuated in Xbp1 mice. Cell Death Discovery (2023) 9:69 H. Ni et al. Fig. 4 Downregulation of XBP1 ameliorated NLRP3 downstream molecules, mitochondrial damage, and apoptosis in TCMK-1 cells exposed to H/R. TCMK-1 cells were transfected with lenti-Xbp1, siRNA-Xbp1, and their corresponding control vectors. For the cell H/R model, hypoxia was induced in a tri-gas hypoxia incubator for 24 h, and then reoxygenation was performed for 2 h after adding fresh culture medium. A Colocalization of the ER, mitochondria, and NLRP3 after siRNA-Xbp1 transfection in TCMK-1 cells exposed to H/R as analyzed using confocal microscopy. Plot proﬁle: the histograms (right panels) represent densities along the white bars: blue = ERtracker, red = Mitotracker, green = NLRP3, purple = overlap of ERtracker and Mitotracker, yellow = overlap of NLRP3 and Mitotracker, cyan = overlap of NLRP3 and ERtracker. Scale bar = 10 μm. Original magniﬁcation of photograph: ×1000. B Silencing of XBP1 led to the inhibition of inﬂammation (caspase- 1, IL-1β, and IL-18) in TCMK-1 cells exposed to H/R. Western blot; *P < 0.05, **P < 0.01 vs. the siRNA-control H/R group (n= 3 samples/group). C H/R-induced ROS (tROS and mROS) production was signiﬁcantly attenuated in TCMK-1 cells treated with Xbp1 siRNA, as shown using ﬂow cytometry. **P < 0.01 vs. the siRNA-control H/R group (n = 3 samples/group). D Treatment with Xbp1 siRNA dramatically increased the mitochondrial membrane potential in TCMK-1 cells exposed to H/R, as shown using ﬂow cytometry. *P < 0.05 vs. the siRNA-control H/R group (n = 3 samples/group). E Total Cyt-c and cleaved caspase-9 expression were reduced by siRNA-Xbp1 transfection in H/R-exposed TCMK-1 cells. Western blot; *P < 0.05, ****P < 0.0001 vs. the siRNA-control H/R group (n= 3 samples/group). F The apoptotic proportion of H/R-exposed TCMK-1 cells was decreased by siRNA-Xbp1 transduction, as revealed using ﬂow cytometry. *P < 0.05 vs. the siRNA-control H/R group (n = 3 samples/group). G Cleaved caspase-3 (p17 and p19) expression was decreased by siRNA-Xbp1 transfection in TCMK-1 cells exposed to H/R. Western blot; *P < 0.05 vs. the siRNA-control H/R group (n = 3 samples/group). si-NC siRNA normal control, H/R hypoxia/reoxygenation. TUNEL staining revealed renal cell apoptosis was reduced in examined immune cells, whereas we used renal tubular +/− Xbp1 mice (10.65 ± 0.53 vs. 3.10 ± 0.24, P < 0.001; Fig. 6G). epithelial cells. In other words, NLRP3 subcellular localization +/− Compared with the control, XBP1 inhibition in Xbp1 mice may vary among cells. reduced IR-induced kidney damage and histological scores We then examined the speciﬁc regulatory mechanisms under- (0.95 ± 0.21 vs. 3.75 ± 0.35, P < 0.01, Fig. 6H), and serum creatinine lying the interactions between XBP1 and NLRP3. Our lentiviral- (191.70 ± 5.04 vs. 97.33 ± 10.17, P < 0.01) and blood urea nitrogen mediated transduction experiment revealed that XBP1 over- levels (68.27 ± 0.64 vs. 47.86 ± 0.64, P < 0.0001) (Fig. 6I). expression promoted NLRP3 activation in TCMK-1 cells, consistent +/− The 14-day survival analysis revealed that Xbp1 mice with our other ﬁnding that siRNA-mediated XBP1 downregulation displayed signiﬁcantly longer survival than wild-type mice that reduced NLRP3 expression. Moreover, the results of our luciferase received the same treatment (P < 0.001, Fig. 6J). assay revealed that the expression of the spliced XBP1 isoform enhanced NLRP3 gene promoter activity. More research is essential to further elucidate these mechanisms. DISCUSSION XBP1 knockdown inhibited NLRP3 expression and NLRP3 In this study, we examined how the XBP1-NLRP3 axis modulates inﬂammasome activation, thereby protecting the kidneys from ER-mitochondrial crosstalk during renal IRI in vivo and in vitro. To ischemic reperfusion injury to some extent. This is consistent with the best of our knowledge, this is the ﬁrst study to examine the ﬁndings from previous literature showing that the inhibition of pivotal role of the XBP1-NLRP3 signaling pathway in regulating XBP1 activity reduced NLRP3 inﬂammasome assembly and NLRP3-mediated cell injury in renal IRI. XBP1 increased the caspase-1 processing in a model of hepatic ischemia-reperfusion, expression of the NLRP3 promoter and enhanced its transcrip- thus reducing IRI-triggered liver injury [33, 34]. tional activity, which is necessary to induce renal injury. XBP1 Further, Chou et al.  found that XBP1s deacetylation or knockdown inhibited NLRP3-mediated inﬂammation, thus reg- inhibition improved cadmium-induced ERS- and NLRP3-related ulating the interaction between the mitochondria and MAMs, pyroptosis in human renal tubular epithelial cells. In general, thereby reducing apoptosis activation and alleviating IRI-induced under renal IRI in vivo and H/R in vitro, we found that XBP1 AKI. These ﬁndings highlight the importance of the XBP1-NLRP3 downregulation inhibited NLRP3-related inﬂammation and apop- axis in regulating ER-mitochondrial crosstalk in IR-stressed kidneys tosis; this protected the mitochondria by inhibiting the release of (Fig. 6K). mitochondrial pro-apoptotic factors and reducing mitochondrial Accumulating evidence suggests that the structural and damage. Although our approach did not directly regulate the functional crosstalk between the ER and mitochondria contributes NLRP3 inﬂammasome and ROS, ROS are known to be the primary to the underlying mechanism of renal IRI . In our mouse mediators of NLRP3 inﬂammasome activation [28, 36]. Reducing model, TEM revealed that the ER from ischemia-reperfusion the production of mitochondrial ROS during AKI can inhibit NLRP3 injured kidneys were dilated, indicating ERS. We observed that the inﬂammasome activation and reduce renal tubular epithelial cell damaged mitochondria were surrounded by abnormal ER; this death [37–39]. Moreover, NLRP3 inhibition via XBP1 downregula- implies that the mitochondria and ER collaborate in response to tion can reduce mitochondrial damage in AKI and protect the AKI during IRI. In addition, we observed that the expression of renal tubules . Apart from its role in inﬂammasomes, the direct XBP1s, the activated form of XBP1, was higher than that of other or indirect inhibition of NLRP3 could also help repair tubular ERS-associated molecules. epithelial cells and alleviate the pathological processes of renal IRI; Although ER-mitochondrial crosstalk is known to play a crucial this is consistent with the results of our study and of other studies role in the kidneys, it remains unclear how the ER affects the [41, 42]. Our results imply that NLRP3 is a potential target for mitochondria in IR-mediated renal injury. The NLRP3 inﬂamma- clinical interventions to reduce IRI. some is induced under mitochondrial oxidative stress [28–30]. Based on these ﬁndings, we propose a mechanism whereby the Here, consistent with previous studies [28, 31, 32], our results XBP1-NLRP3 axis regulates ER-mitochondrial crosstalk in renal showed that NLRP3 inﬂammasome expression and activity were ischemia/reperfusion (Fig. 6K).InIRI,IRE1α is induced, which in turn elevated in ischemia-reperfused kidneys, which exhibited activates the splicing of XBP1 mRNA, promoting XBP1 activation. increased clustering of NLRP3 inﬂammasomes on the mitochon- Subsequently, XBP1s becomes translocated into the nucleus to dria and the MAMs. Other studies have reported that NLRP3 is regulate the NLRP3 promoter, promoting NLRP3 expression. NLRP3 localized primarily in the ER in the resting state and only localization in the MAMs or mitochondria contributes to NLRP3- translocates to the mitochondria or MAMs under stress. In this mediated inﬂammation and activation of apoptosis, and ROS study, however, IEM analysis revealed that NLRP3 is localized in derived from damaged mitochondria activates the NLRP3 inﬂam- both the mitochondria and the ER in the resting state. This masome. Hence, the suppression of XBP1 activity may inhibit NLRP3 discrepancy may arise from the fact that the other studies and its downstream pathways to prevent IRI-triggered renal injury. Cell Death Discovery (2023) 9:69 H. Ni et al. +/− +/− Fig. 5 Identiﬁcation of Xbp1 mouse genotypes. A Schematic representation of the heterozygous Xbp1 mouse model. B Genotyping +/− of Xbp1 mice was authenticated via PCR using Ef and Kr primers. XBP1 has distinctive functions in different tissue types. Mice Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (China) Arthur et al. reported that XBP1 deﬁciency induces a pro- +/− provided the C57BL/6 mice used in this study. Heterozygous Xbp1 inﬂammatory response and ERS in intestinal epithelial cells, C57BL/6 mice (C57BL/6NTac-XBP1 < tmla<EUCOMM > Wtsi > /1cs0r1) were inducing apoptosis . In renal tubule epithelial cells, Silvia generated by the European Mouse Mutant Cell Repository (Phil Avner, et al. found that XBP1s overexpression contributed to renal Paris, France). All animals were raised in speciﬁc pathogen-free rooms (at inﬂammation and injury, and promoted septic AKI , 22 °C, 55% humidity, and under a 12/12 h light/dark cycle) at Tongji consistent with our ﬁndings. Conversely, Claudio et al. found Hospital. The primers used to identify the mouse genotypes are listed in that XBP1 deletion in the nervous system improves the survival Supplementary Table S1. of mSOD1G86R transgenic mice by increasing autolysosome production and reducing apoptosis . Further research is Establishment of a renal IRI mouse model required to examine the roles of XBP1 in different tissue types Brieﬂy, 8–10 week-old male mice weighing 22 ± 2 g were anesthetized via and microenvironments. intraperitoneal injection of pentobarbital (50 mg/kg) and placed on a These ﬁndings have important potential clinical and transla- heating pad at 32 °C. A midline abdominal incision was made to expose tional applications. In the short term, the most promising the left kidney, and a noninvasive arterial clamp was used to clamp the left translational strategy may be to examine whether the XBP1- renal pedicle to cause renal ischemia. Afterward, the mice were placed in a NLRP3 signaling pathway drives AKI in patients and whether it is a constant temperature incubator at 32 °C. The vascular clamp was released druggable target for clinical treatment. In the long term, further after 45 min to simulate reperfusion. Meanwhile, the right kidney was removed, and the incision was sutured layer by layer. Blood and kidney research is required to develop a comprehensive understanding of samples were collected after 24 h. The control group was fed normally to XBP1 as a predictive biomarker for early IR-related AKI diagnosis observe their survival rate. and as a blockade target to improve the overall survival of patients with AKI. Histopathological analysis For each slide, 10 ﬁelds of vision were randomly selected under a light MATERIALS AND METHODS microscope (at ×200 magniﬁcation) to observe the shape, nuclear staining state, and lumen dilation of the renal tubular epithelial cells in each visual Ethics approval ﬁeld. The results were graded from 0 to 3 with the following criteria: 0, no The study was approved by the Animal Care and Use Committee of Tongji injury; 1, mild injury, presence of round epithelial cells and tubule lumen; 2, Medical College and Nanfang Hospital, Southern Medical University. The severe injury, epithelial cells ﬂattened, nuclear staining no longer experiments were performed in accordance with the relevant guidelines detectable, lumenal dilation, and lumenal hyperemia; 3, renal tubule and regulations of this committee. Cell Death Discovery (2023) 9:69 H. Ni et al. destruction, epithelial cells ﬂattened, nuclear staining no longer detectable, a graded ethanol series. After penetration, embedding, and polymeriza- and lumenal hyperemia. tion, ultrathin sections (60–80 nm) were processed using a Leica EM UC7 ultramicrotome (Leica, Wetzlar, Germany) followed by lead and uranium staining for electron microscopy (Tecnai G2 F20 TWIN; FEI Company, ROS detection Hillsboro, OR). Renal tissue was prepared as frozen sections. An ROS staining working solution was added to the marked area, and the sections were incubated at 37 °C for 30 min, protected from light. The sections were then washed Treatment with 4μ8C thrice with PBS on a shaker. The sections were then incubated with a DAPI The IRE1α inhibitor 4μ8C was purchased from MedChemExpress (CAS- solution at room temperature for 10 min to stain the nuclei. After washing, number 14003-96-4; Monmouth Junction, NJ, USA), dissolved in DMSO, samples were observed under a ﬂuorescence microscope (Nikon Eclipse and added to the culture medium for 24 h (ﬁnal concentration, 30 μM). C1, Tokyo, Japan) and photographed. Immunoelectron microscopy Transmission electron microscopy Fresh mouse renal tissue samples (1 mm ) were ﬁxed using an IEM ﬁxative Renal tissues were cut into small pieces (1 mm × 1 mm × 2 mm), ﬁxed for at (G124-10ML; Servicebio, Wuhan, China) overnight at 4 °C. After rinsing least 4 h in 2.5% glutaraldehyde at 4 °C, rinsed, and treated with 1% with pre-cooled 0.1 M phosphate buffer (PB) (3 times × 10 min each time), osmium tetroxide for 2 h. After washing, renal tissue was dehydrated using the tissue samples were dehydrated in a graded ethanol series. Cell Death Discovery (2023) 9:69 H. Ni et al. Fig. 6 Downregulation of XBP1-NLRP3 ameliorated the crosstalk between ERS and mitochondrial damage and protected the kidneys +/− affected by IRI in Xbp1 mice. To construct the mouse IRI model, we used microvascular clips to block the left renal pedicle of the mouse for 45 min and then cut off the right kidney after opening the bloodstream. A Downregulation of XBP1 expression signiﬁcantly suppressed the protein levels of NLRP3. Western blot; *P < 0.05, **P < 0.01 vs. the IRI group (n = 3 samples/group per group). B IEM showed that the +/− colocalization of NLRP3 and mitochondria or MAMs induced by IRI was reduced in the kidneys of Xbp1 mice. Blank rectangles indicate MAMs, and the small black dots indicate the 10-nm colloidal gold particles Scale bar = 0.5 μm; ×10,000 magniﬁcation. IRI-induced ER and mitochondrial damage were signiﬁcantly alleviated because of XBP1 downregulation, as shown using TEM. The red arrows indicate ER swelling, while the orange arrows indicate the damaged mitochondria. Scale bar = 1 μm; ×5000 magniﬁcation. C XBP1 downregulation reduced the production of ROS in the kidneys exposed to IRI, as shown via DHE staining. Scale bar = 50 μm; ×200 magniﬁcation. D Cleaved +/− caspase-9 and total Cyt-c expression were reduced in the kidneys of Xbp1 mice after IRI. Western blot; **P < 0.01, ***P < 0.001 vs. the IRI group (n = 3 samples/group). E XBP1 downregulation alleviated NLRP3-mediated apoptosis (cleaved caspase-3 p17 and p19) and inﬂammation (caspase-1) in the kidneys exposed to IRI. Western blot; *P < 0.05, **P < 0.01 vs. the IRI group (n = 3 samples/group). F The ELISA results indicated that the protein level of inﬂammatory factors IL-1β and IL-18 were decreased in the serum. ***P < 0.001, ****P < 0.0001 vs. the IRI group (n = 3 samples/group). G Downregulation of XBP1 expression dramatically reduced the ratio of apoptotic cells, as observed using the TUNEL assay. Scale bar = 50 μm; ×200 magniﬁcation. ***P < 0.001 vs. the IRI group (n = 3 samples/group). H Acute renal damage was +/− alleviated in Xbp1 mice subjected to IRI, as shown using H&E staining and the pathological damage scores. Scale bar = 50 μm; ×200 magniﬁcation. ***P < 0.001 vs. the IRI group (n = 3 samples/group). I Downregulation of XBP1 expression dramatically reduced the levels of CR and BUN in the serum. **P < 0.01, ****P < 0.0001 vs. the IRI group (n = 3 samples/group). J Downregulation of XBP1 expression dramatically +/− improved the survival of Xbp1 mice. * P < 0.05 vs. the IRI group (n = 3 samples/group). K Schematic illustration of the mechanism involving the XBP1-NLRP3 axis in regulating the ER and mitochondrial crosstalk in renal ischemia/reperfusion. IRI ischemia/reperfusion injury. After penetration, embedding, and polymerization, ultrathin sections CMXRos (Life Technologies, Carlsbad, CA, USA), diluted in serum-free (70–80 nm) were processed using a Leica EM UC7 ultramicrotome and medium to 100 nM, was added to the cells, which were then incubated in deposited on a nickel grid with a formvar ﬁlm for immunolabeling. The grids the dark at 37 °C for 30 min. ER Tracker Blue White DPX dye (Life prepared for immunolabeling were washed with Tris-buffered saline (TBS) Technologies, Carlsbad, CA, USA), diluted in serum-free medium to a ﬁnal droplets (room temperature; 3 times × 10 min each time) and blocked with concentration of 200 nM, was then added to the cells, which were 1% bovine serum albumin (BSA) in TBS (room temperature, 30 min). For incubated in the dark at 37 °C for 30 min. Next, the cells were ﬁxed with 4% NLRP3 single-labeling, samples were incubated with 1:50 anti-NLRP3 rabbit paraformaldehyde for 15 min and permeabilized using 0.3% after washing antibody (ab214185; Abcam, Cambridge, UK) in diluent overnight at 4 °C. three times with PBS. Triton for 15 min, and sealed with 5% BSA for 30 min. The grids were returned to room temperature the next day before rinsing Primary antibodies (1:100) were added. The cells were incubated in the with TBS droplets (room temperature, for 3 × 5 min). Next, the samples were dark at 4 °C overnight. DyLight 488 goat anti-rabbit IgG (1:200; A23220; incubated with secondary antibodies conjugated with gold particles (10 nm; Abbkine, Wuhan, China) was then added, and the cells were incubated in G7402; Sigma Aldrich, St Louis, MO), diluted in diluent (room temperature, the dark at 37 °C for 1 h. Images were captured using a TiE confocal for 20 min), incubated for 1 h at 37 °C, and ﬁnally incubated for another microscope (Nikon, Tokyo, Japan). A plot proﬁle was generated using 30 min at room temperature. After immunolabeling, the grids were washed ImageJ software to assess the degree of colocalization. with TBS droplets (room temperature; 5 times × 5 min each time) and rinsed with ultrapure water (room temperature; 5 times × 5 min each time). After XBP1 siRNA knockdown and overexpression rinsing, the samples were stained with a saturated alcohol solution of 2% Lentivirus-overexpressing XBP1 particles were purchased from Hanbio uranium acetate in the dark for 8 min, followed by three washes in 70% Biotechnology Co., Ltd. (Shanghai, China). TCMK-1 cells were seeded at a ethanol and three washes in ultrapure water. After drying, the samples were density of 2 × 10 cells/well in six-well plates 12 h before transfection. observed and photographed using an HT7800 transmission electron siRNA transfection was performed using Lipofectamine 2000 (Invitrogen; microscope (HITACHI, Japan). Thermo Fisher Scientiﬁc, Inc., Waltham, MA, USA), and treatments were performed 24 h after transfection. The lentiviral solution (1 × 10 TU/mL) TUNEL assay was added to the culture medium (40 μL/well, 4 × 10 TU) at a 50% Cell death was analyzed via terminal deoxynucleotidyl transferase (TdT)- multiplicity of infection. After 24 h, the spent medium was replaced and mediated dUTP digoxigenin nick-end labeling (TUNEL) in frozen kidney the cells were cultured for another 24 h. Stable XBP1-overexpressing sections using an In Situ Cell Death Detection Kit (11684817910; Roche, strains were screened using a culture medium containing 3 μg/mL Basel, Switzerland). puromycin. The siRNA sequences used in this study can be found in Supplementary Table S2. ELISA Serum IL-1β and IL-18 levels were measured using commercially available Western blotting ELISA kits according to their respective manufacturer’s instructions (Mouse Protein quantiﬁcation was performed using ImageJ software. The IL-18 ELISA Kit, ab216165, Abcam; Mouse IL-1β ELISA kit, DKW12-2012-096, antibodies used are listed in Supplementary Table S3. Dakewe Biotech, China). Flow cytometry Cell culture and hypoxia-reoxygenation (H/R) model TCMK-1 cells were cultured in six-well plates and harvested using trypsin The TCMK-1 cell line (CCL-139; American Type Culture Collection) was without EDTA before and after staining. Cellular ROS, mitochondrial ROS, cultured in RPMI 1640 medium (Hyclone; GE Healthcare, Logan, UT, USA) apoptosis, and MMP were determined using a Reactive Oxygen Species supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Assay kit (Beyotime Institute of Biotechnology, Shanghai, China), MitoSOX Scientiﬁc, Waltham, MA, USA) in a 5% CO atmosphere. For the hypoxia kit (Life Technologies, Carlsbad, CA, USA), Annexin V-PI staining kit, and JC- cell model, TCMK-1 cells were cultured in a pre-hypoxia serum-free 1 detection kit ((Beyotime Institute of Biotechnology, Shanghai, China), medium and incubated in a tri-gas incubator (1% O ,5%CO and 94% N ) respectively. The cells were then stained according to the respective 2 2, 2 at 37 °C for 24 h (Don Whitely Scientiﬁc, Bingley, UK). During the manufacturer’s instructions and analyzed via ﬂow cytometry. Ten thousand reoxygenation process, the spent medium was replaced with fresh events were recorded using the FACSCalibur system (BD Biosciences, CA, complete medium, and the cells were cultured under normoxic (21% O ) USA), and the resulting data were analyzed using FlowJo software. conditions for 2 h. Real-time quantitative reverse-transcription PCR Immunoﬂuorescence staining and confocal microscopy Total cell RNA was extracted, and PCR was conducted using the appropriate The cells were seeded into 15 mm confocal dishes, and immunoﬂuores- reaction systems. The total RNA obtained was reverse-transcribed into cence staining was performed after the intervention. MitoTracker Red cDNA (15 min at 37 °C, 5 s at 85 °C, indeﬁnite hold at 4 °C). The obtained Cell Death Discovery (2023) 9:69 H. Ni et al. cDNA stock solution was diluted to 1/8 of its original concentration and 18. 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Ferrè S, Deng Y, Huen SC, Lu CY, Scherer PE, Igarashi P, et al. Renal tubular cell spliced x-box binding protein 1 (xbp1s) has a unique role in sepsis-induced acute Reprints and permission information is available at http://www.nature.com/ kidney injury and inﬂammation. Kidney Int. 2019;96:1359–73. reprints 45. Hetz C, Thielen P, Matus S, Nassif M, Court F, Kifﬁn R, et al. Xbp-1 deﬁciency in the nervous system protects against amyotrophic lateral sclerosis by increasing Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims autophagy. Genes Dev. 2009;23:2294–306. in published maps and institutional afﬁliations. ACKNOWLEDGEMENTS This study was funded by National Natural Science Foundation of China (Grant No. Open Access This article is licensed under a Creative Commons 82270784, 82070770, 82170772, 81873623, 81570678, 81500573), Guangdong Basic Attribution 4.0 International License, which permits use, sharing, and Applied Basic Research Foundation (Grant No. 2023A1515012276) and adaptation, distribution and reproduction in any medium or format, as long as you give Academician Shusen Lanjuan Talent Foundation (grant to Yuchen Wang). appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless AUTHOR CONTRIBUTIONS indicated otherwise in a credit line to the material. If material is not included in the HN, YM, and NG designed and directed the study. HN, YW, and YL wrote the article’s Creative Commons license and your intended use is not permitted by statutory manuscript. HN, ZO, YW, YL, KS, JZ, JSZ, WD, WZ, RX, and JX performed experimental regulation or exceeds the permitted use, you will need to obtain permission directly work and analyzed the data. HN, YW, NG, and YM revised the manuscript. from the copyright holder. To view a copy of this license, visit http:// creativecommons.org/licenses/by/4.0/. COMPETING INTERESTS The authors declare no competing interests. © The Author(s) 2023 Cell Death Discovery (2023) 9:69
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