Annexin A2 alleviates renal ischemia/reperfusion injury via regulating membranal ESCRT-III to suppress necroptosis of tubular epithelial cells

Annexin A2 alleviates renal ischemia/reperfusion injury via regulating membranal ESCRT-III to suppress necroptosis of tubular epithelial cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Annexin A2 alleviates renal ischemia/reperfusion injury via regulating membranal ESCRT-III to suppress necroptosis of tubular epithelial cells Xiubin Li, Dong Lai, Jichen Wang, Huayi Feng, Shouqing Cao, Huaikang Li, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6991722/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Renal functional impairment, resulting from nephron-sparing surgery (NSS), is mainly caused by ischemia-reperfusion injury (IRI) and majorly affects kidney function. Necroptosis plays an important role in renal IRI, however its mechanism remains unknown. We used Anxa2 knockout mice to investigate the role and mechanism of Anxa2 in renal IRI. Moreover, We further explored its underlying molecular mechanism by co-immunoprecipitation combined with mass spectrometry (IP-MS). Our results showed that Anxa2 accumulated significantly at the apical membrane of renal tubular epithelial cells of the medulla after renal IRI. In Anxa2 knockout mice, injury was significantly increased in the medulla rather than in the cortex, and necroptosis was found to be aggravated in medullary tubular epithelial cells accompanied by increased neutrophil infiltration in the injured area. Mechanistically, Anxa2 can specifically bind to Chmp2A after renal IR, an important component protein of endosomal sorting complex required for transport III (ESCRT-III). In knockout mice, membranal Chmp2A content in the renal medulla was significantly decreased, but not cytoplasmic Chmp2A. The other component proteins of the ESCRT-III complex, such as Chmp2B, Chmp3, Chmp4B and Vps4B, followed the same trend as Chmp2A. We also found that Cxcl1 and Cxcl2 , which are specific neutrophil chemokines, were significantly upregulated without Anxa2 expression both in vivo and in vitro. Moreover, Necrostatin 1 (Nec1), a specific necroptosis inhibitor, rescued the Anxa2 deficiency-induced necroptosis, neutrophil infiltration and IRI. This study demonstrates that Anxa2 can inhibit necroptosis of renal medullary tubular epithelial cells, reduce local neutrophil infiltration and inflammatory factors, and protect against the renal IRI. More importantly, we describe the molecular mechanism by which Anxa2 inhibits necroptosis by regulating ESCRT-III. Our study clarified the role and mechanism of Anxa2 function in renal IRI, providing new clinical strategies for its prevention and treatment. Biological sciences/Immunology/Cell death and immune response Health sciences/Diseases/Kidney diseases Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Key points Anxa2 is up-regulated after ischemia/reperfusion injury and playing a protective role in its development. Anxa2 is able to reduce infiltration of neutrophils and inflammatory factors by inhibiting necroptosis in renal ischemia/reperfusion injury. We found the molecular mechanism by which Anxa2 inhibits necroptosis by regulating membranal ESCRT-III coplex, providing new clinical strategies for its prevention and treatment. Significance Statement Anxa2 is significantly up-regulated after renal IRI and plays an important role in the occurrence and development of injury. In this study, Anxa2 was found to accumulate on the apical membrane of renal tubular epithelial cells in the renal medulla at the early stage after renal IR and play a protective role in renal IRI. Secondly, we illustrated for the first time that Anxa2 could inhibit necroptosis in renal IRI, thereby affecting the infiltration of neutrophils and inflammatory factors. More importantly, by IP-MS, we further found that Anxa2 specifically binds to Chmp2A, thereby promoting ESCRT-III accumulation at the apical membrane of renal tubular epithelial cells and inhibiting the necroptosis process. The investigation revealed a novel mechanism to explain how Anxa2 protect against renal IRI and provided new clinical ideas for its prevention and treatment of renal IRI caused by NSS. Introduction Renal ischemia-reperfusion injury (IRI) can occur in conditions such as intravascular volume depletion, hypotension, and renal vascular diseases. 1 However, with the widespread implementation of nephron-sparing surgery (NSS) in urology, renal IRI after NSS has increasingly attracted the attention of urologists. 2 To avoid bleeding during surgery, it is necessary to clamp the renal artery during the tumor enucleation process, which leads to warm ischemia and reperfusion of the kidney. Clinical data indicate that the renal function of most patients is restored after surgery. However, 20% of patients experience renal functional impairment after NSS. 3,4 This has become a major obstacle for protecting kidney function, which is the core purpose of NSS. Unfortunately, we lack effective clinical methods for prevention and treatment. Thus, it is particularly important to study the mechanism of renal IRI, explore ways to prevent its occurrence, and treat it. Renal IRI is a complex process involving multiple mechanisms. Currently, the prevailing view is that it is primarily caused by the death of renal tubular epithelial cells and subsequent inflammation, 5-7 which may primarily be caused by necroptosis of renal tubular epithelial cells. 8 Apoptosis causes mild inflammation, whereas necroptosis results in robust inflammation with rupture of the cell membrane and auto-amplification loop of necroinflammation. 9-12 Specific Rip1 inhibitors, such as Necrostatin-1 (Nec-1) and Cpd-71, can protect against kidney injury, suggesting that necroptosis may play an important proinflammatory role in kidney injury. 13-15 However, the molecular mechanisms underlying necroptosis in IRI remain ill-defined. Annexin A2 (Anxa2), an extensively studied member of the annexin superfamily, is a transmembrane protein that connects cell or organelle membranes with other molecules and “anchors” various transmembrane molecules. 16 Anxa2 is involved in autophagy and apoptosis, 17-19 which do not cause severe inflammation. Thus, we speculated that Anxa2 may be involved in necrosis. Determining whether Anxa2 is involved in necroptosis and its role in renal IRI will be beneficial for clinical translation. Using a renal IRI mouse model, through in vivo and in vitro experiments, we explored the function and molecular mechanism of Anxa2 in renal IRI，and provided a new clinical basis for the prevention and treatment of NSS-induced renal IRI caused by NSS. Results Anxa2 is upregulated and accumulates at the apical membrane of the medullary tubular epithelial cells after renal IRI Anxa2 is significantly upregulated after kidney IRI. 20 Kim1 and Lcn2 were significantly upregulated, alongside Anxa2 mRNA and protein levels 24h after reperfusion (Figure 1a and b). Contrary to previous views, growing evidence supports that renal IRI and inflammation may originate in the medulla rather than in the cortex. 21-24 We found that the degree of injury in the medullary fraction was significantly higher than that in the cortical fraction at 1 and 3 days after renal IRI. On the 7th day after IRI, the degree of injury in the cortex was significantly higher than that in the cortex 1 d after IRI (Supplementary Figure S1A and B). This suggests that renal IRI spreads over time from the medulla, especially the outer medulla, to the cortex. Anxa2 expression in the medulla was significantly higher than that in the cortex 1 d after renal IR, but there was no significant difference between the medulla and cortex 3 and 7 days after renal IR. Additionally, Anxa2 expression level was significantly higher on the 7th day in the cortex than on the 1st day after IR (Supplementary Figure S1C and D). This is consistent with the injury progression pattern, indicating that Anxa2 expression occurs during injury and originates from the renal medulla, particularly the outer medulla. PAS and immunohistochemical staining showed that Anxa2 expression was significantly increased in the medulla (especially the outer medulla) after renal IRI, and Anxa2 significantly accumulated on the apical membrane of renal tubular epithelial cells (Figure 1c). To further determine the location of Anxa2 protein, we performed immunofluorescence co-staining for markers of different renal tubular segments and Anxa2 in the medulla. The results showed that Anxa2 mainly accumulated on the apical membrane of renal tubular epithelial cells in the thick ascending limb of Henle-loop (Nkcc2 25 ), part of distal convolulated tubules (Aqp2 26 ) and collecting tubules (Aqp2 and Atp6v1g3 26,27 ) of medulla after renal IR (Figure 1d). Therefore, Anxa2 is localized at the apical membrane of renal tubular epithelial cells, which belongs to the thick ascending limb of Henle- loop, part of the distal convoluted tubules, and the collecting duct, which most likely plays a role in renal IRI. Anxa2 protects against early kidney injury and long-term renal fibrosis of the medulla after renal IRI Previous studies have shown that Anxa2 has diverse functions, including regulating inflammation and cell death. 19,20,28-30 Our data showed that Anxa2 was significantly upregulated in medullary tubular epithelial cells after renal IR. Anxa2 knockout (KO) mice were used to explore the function of Anxa2 in renal tubular epithelial cells. The results showed that at 1 day after renal IR, the injury of renal medulla in KO mice was more severe than that in wild type (WT) mice. However, there were no significant differences in the cortical components between WT and KO mice (Figure 2a and b). This is consistent with previous experimental results showing that Anxa2 is significantly upregulated in renal medulla at the initial stage of renal IRI and plays a protective role against early injury in the medulla. To confirm this conclusion, we isolated the medullary tissue of IR kidneys from WT and KO mice. We found that Kim1 and Lcn2 expression in the medulla of KO mice was significantly higher than that in the medullae of WT mice (Figure 2c). To further characterize the cellular injury of renal medulla and lumen structure, we used Aqp1, a cellular injury assessment marker, 31 in WT and KO mice. The results showed that the collapse of the tubular luminal structure was more severe in KO mice (Figure 2f). This suggests that Anxa2 inhibits the death of renal medullary tubular epithelial cells, thereby preventing the collapse of the renal tubular lumen structure and reducing early injury after IRI. Moreover, the degree of early injury determines long-term kidney fibrosis. 32 Therefore, we evaluated the long-term fibrosis levels after renal IRI, and the results showed that long-term kidney fibrosis in KO mice was significantly higher in the medullary region, but there was no significant difference between WT and KO mice in the cortical region (Figure 2d and e). This suggests that Anxa2 affects long-term renal medullary fibrosis by protecting the medulla against early injury following IR. The mice used in our experiments were genotyped twice using mouse tail DNA to ensure that the control and experimental groups were Anxa2 +/+ and Anxa2 –/– mice (Supplementary Figure S2). Anxa2 upregulation in renal tubular epithelial cells after renal IR suppresses necroptosis of tubular epithelial cells in the renal medulla Our data indicate that Anxa2 can inhibit the death of medullary renal tubular epithelial cells. To clarify the mechanism of cell death specifically regulated by Anxa2, we isolated renal medullary tissue and detected the levels of key proteins related to different death pathways. The results showed that for apoptosis, 33 Caspase 3 and cleaved-Caspase 3 levels were not significantly different between WT and KO mice. For pyroptosis, 34 the Gsdmd and N-Gsdmd levels did not significantly differ between WT and KO mice. For ferroptosis, 35 Gpx4 levels were not significantly different between WT and KO mice (Supplementary Figure S3A). However, pMlkl levels were significantly upregulated in KO mice (Figure 3a). This suggests that Anxa2 upregulation can inhibit the necroptosis in renal medullary tubular epithelial cells. This is consistent with previous reports indicating that necroptosis occurs in a high number of renal tubular epithelial cells in the medulla during kidney injury. 36 Necroptosis mainly depends on the the perforation of pMlkl on the cell membrane, resulting in the extravasation of cytoplasm and inflammatory response. 37-39 To verify the location of pMlkl expression, immunohistochemical staining of pMlkl was performed, which showed that pMlkl accumulated on the apical membrane of renal tubular epithelial cells in the renal medulla of KO mice, but not in other parts of the cell membrane (Figure 3b). In addition, we performed Transmission Electron Microscopy (TEM) scanning of the kidneys after IRI to further verify whether and to what extent the apical membranes of renal tubular epithelial cells in the medulla of WT and KO mice were damaged. We found that the apical membranes of renal medulla in KO mice were more severely ruptured (Figure 3c and d). The regulatory relationship between Anxa2 and necroptosis was also verified using in vitro experiments. We used Tcmk1 cells, a mouse renal tubular epithelial cell line, to knockdown Anxa2 and treated both control and knockdown cells with the same dose of TSZ to induce necroptosis. We compared the level of necroptosis between knockdown and control cells and observed that it was significantly higher in the Anxa2 knockdown than in the control group (Figure 3e). This suggests that Anxa2 can significantly inhibit necroptosis of renal tubular epithelial cells. Overexpression of Anxa2 is able to reduce cellular necroptosis in Tcmk1 To verify that Anxa2 inhibits necroptosis in renal tubular epithelial cells, we overexpressed and knocked down Anxa2 in Tcmk1. We found that pMlkl levels significantly decreased in Anxa2-overexpressed Tcmk1 to the control and knockdown groups (Figure 4a). This suggests that Anxa2 could reduce the necroptosis caused by TSZ. Cell proliferation was evaluated by a cck8 assay and brightfield imaging. We observed that the cell proliferation ability in OE was significantly higher than control and knockdown groups. The proliferation ability of the knockdown group was lower than control group. The same results were obtained using brightfield imaging (Figure 4b and c). This indicates that Anxa2 effectively inhibits necroptosis of renal tubular epithelial cells. Flow cytometry was used to assess cell necrosis (primarily TSZ-induced necroptosis) and apoptosis. After TSZ addition, the proportion of necroptotic cells in the knockdown group was significantly higher than that in the control group. The proportion of necroptotic cells in OE was significantly decreased. (Figure 4d and e). However, we found that the level of apoptosis in OE was lower than that in siAnxa2, which probably means Anxa2 also has some effects on the early apoptosis of Tcmk1 (Supplementary Figure S3B). The proportion of apoptotic cells increased after the addition of TSZ. A possible reason is that Z-VAD-FMK, an inhibitor of apoptosis included in TSZ Kit, may not completely block apoptosis. Anxa2 upregulation after renal IR inhibits the inflammatory response and neutrophil recruitment in the medulla At present, it is explicitly reported that necroptosis and inflammation are closely related and mutually promote each other, playing an important role in renal IRI. 12,40-44 Thus, we isolated renal medullary tissue and measured the mRNA expression of necroptosis-related inflammatory factors in WT and KO mice to evaluate the level of inflammation. We found that Nlrp3 45 , Tnfα 46 , Il6 and Il1b 47 were significantly upregulated in KO mice after renal IRI compared with WT mice (Figure 5a). This suggests that Anxa2 significantly suppressed the inflammatory response after renal IRI. In addition, we used flow cytometry to examine the infiltration of innate immune cells after renal IRI. We observed that the level of neutrophil infiltration was significantly increased in KO mice after renal IRI compared with WT mice (Figure 5b and c). This indicated that Anxa2 inhibited neutrophil recruitment after renal IRI. However, there is no significant difference in monocyte or macrophage recruitment between WT and KO mice. During the infiltration of innate immune cells after renal IR, the proportion of neutrophils and monocytes was significantly upregulated, and the proportion of macrophages was significantly decreased compared to that in the sham group (Supplementary Figure S4A and B). Immunohistochemical staining for Ly6G was performed to determine the location and number of neutrophil infiltration sites during early renal IRI. The results showed that neutrophils mostly infiltrated the early injury area (outer medulla). The number of neutrophils in the injured area of KO mice was significantly higher than that in WT mice, which was consistent with the flow-cytometry results (Figure 5d and e). Neutrophil infiltration has been suggested to be spatially specific and consistent with the area of injury. According to our data, the loss of Anxa2 led to massive necroptosis of renal tubular epithelial cells in the medulla, accompanied by a significant increase in neutrophil infiltration in the injured area. Here, we aimed to determine whether necroptosis directly or indirectly regulates neutrophil aggregation. We detected the mRNA levels of Cxcl1 and Cxcl2 , 48,49 which are specific neutrophil chemokines, in renal medullary tissue. The results showed that Cxcl1 and Cxcl2 were significantly upregulated in KO mice after renal IRI (Figure 5f). In vitro experiments showed that the protein concentrations of Cxcl1 and Cxcl2 in the cell supernatant of OE Tcmk1 were significantly higher than those in control (Figure 5g). This suggests that necroptosis can cause the renal tubular epithelium to release more Cxcl1 and Cxcl2 to recruit neutrophils, meanwhile Anxa2 can reduce the synthesis and secretion of Cxcl1 and Cxcl2 and mitigate the recruitment of neutrophils by inhibiting necroptosis. Nec1 rescues Anxa2 deficiency-induced injury by reducing necroptosis and neutrophil infiltration in the medulla after renal IRI Nec1 is a specific inhibitor of necroptosis but has no direct inhibitory effects on inflammation. 50 To rescue the Anxa2 deficiency, we performed the Nec1 intervention described in the Methods section during the process of renal IRI. The results showed that the renal IRI level in Nec1-treated mice was significantly ameliorated in WT and KO mice. However, there was no significant effect on the level of injury between WT and KO mice, and the levels of injury did not differ significantly between Nec1-intervention KO mice and non-intervention WT mice (Figure 6a and b). This suggests that the increase in renal IRI caused by loss of Anxa2 can be reversed by Nec1, indicating that Anxa2 protects from the occurrence of IRI by inhibiting necroptosis. To confirm this conclusion, we used medullary kidney tissue to detect Kim1 and Lcn2 expression and found that Nec1 rescues renal IRI in the medulla of KO mice (Figure 6c). In addition, we found that pMlkl was significantly downregulated in Nec1-treated mice both in the WT and KO groups (Figure 6d). Immunofluorescence staining of Aqp1 showed that the renal tubular and epithelial cell structure of Nec1-treated mice was better preserved than that of untreated mice both in the WT and KO groups (Figure 6e). These results suggest that Nec1 could adequately rescue the increase in renal IRI caused by Anxa2 deficiency. Although the related indicators in Nec1-treated KO mice were significantly lower than those in the non-treated group, they were still significantly higher than those in Nec1-treated WT mice. There was no significant difference between Nec1-treated KO and untreated WT mice (Figure 6c and d). Our previous results demonstrated Anxa2 could inhibit neutrophil infiltration after renal IR. However, whether this neutrophil infiltration is due to necroptosis or other factors remains unclear. Thus, in vivo, we found that Nec1-treated mice had significantly reduced neutrophil infiltration compared to untreated mice. Neutrophil infiltration in Nec1-treated KO mice was also significantly higher than that in Nec1-treated WT mice. However, there was no significant difference between Nec1-treated KO mice and untreated WT mice (Figure 6f and g). This is consistent with previous RT-qPCR results for Kim1 and Lcn2 as well as western blotting for pMlkl. Anxa2 can specifically bind to Chmp2A and promote the aggregation of ESCRT-III to protect against necroptosis after renal IRI To explore the molecular mechanism of how Anxa2 affects necroptosis in renal tubular epithelial cells, we obtained the proteins in renal medulla that bind to Anxa2 after renal IR by IP and subsequently analyzed these proteins by MS. We found that the necroptosis-related protein that could specifically bind to Anxa2 was Chmp2A (Supplementary Figure S7A). According to previous reports, Chmp2A is an important component of ESCRT-III complex, and other components include Chmp2B, Chmp3, Chmp4B and Vps4B. Immunofluorescence co-localization staining of Anxa2 and Chmp2A was performed, and the results showed that Chmp2A was not only expressed in the cytoplasm, but also co-expressed in the apical membrane with Anxa2 (Figure 7c). At the same time, we also performed IP experiments by extracting membrane proteins from renal medulla tissue proteins, and we found that Anxa2 could specifically bind to Chmp2A after renal IR, whereas this was not observed in the normal kidney. IP on the other components of ESCRT-III was also performed, and the results showed that Anxa2 did not bind to them specifically (Figure 7a). Therefore, we hypothesized that Anxa2 may bind to Chmp2A at the apical membrane and promote the aggregation of ESCRT-III to inhibit necroptosis. To confirm this hypothesis, we examined the protein levels of ESCRT-III components using extracted membrane proteins before, and the results showed that the expression of ESCRT-III molecules was significantly decreased in Anxa2 knockout mice (Figure 7d). However, a similar trend was not observed for cytosolic proteins (Supplementary Figure S7B). In addition, Chmp2A expression was found to be mainly located in the cytoplasm rather than the apical membrane in Anxa2 knockout mice compared with WT mice after renal IR (Figure 7b). These results suggest that Anxa2 may affect the effect of ESCRT-III on inhibiting necroptosis by affecting the aggregation of Chmp2A at the apical membrane. Notably, the IP result showed that Anxa2 does not bind to Chmp2A in the normal kidney, which is inconsistent with the IP-MS result, indicating that Anxa2 is mainly expressed in the cytoplasm, where it may bind to Chmp2A in normal kidney. The involvement of ESCRT-III in the generation of autophagic vesicles gives a new direction for our future research. In summary, Anxa2 is able to promote ESCRT-III accumulation at the apical membrane of renal tubular epithelial cells and inhibit necroptosis, thereby mitigating the release of Cxcl1 and Cxcl2 and the recruitment of neutrophils to injured areas. We schematized the mechanism of action of Anxa2 protective effect against renal IRI (Figure 8). Discussion Currently, the regulatory mechanism of necroptosis in renal IRI remains poorly understood. Here, we showed that Anxa2 can significantly inhibit necroptosis in renal IRI, suppress inflammation, and alleviate kidney injury. We found that both the abundance and expression pattern of Anxa2 changed during IRI. Anxa2 accumulated significantly at the apical membrane of renal tubular epithelial cells and inhibited cellular necroptosis in the medulla after renal IR. Mechanistically, we also innovatively found that Anxa2 specifically bound to Chmp2A to promote the aggregation of ESCRT-III complex on the cell membrane, thereby protecting against necroptosis. In addition, necroptosis of renal tubular epithelial cells upregulates the expression of neutrophil chemotactic factors and aggravates neutrophil infiltration into the injured area. These results suggest that in NSS-induced renal IRI, the inflammation and tissue injury after IR can be mitigated by the early inhibition of necroptosis in medullary tubular epithelial cells. Increasing evidence suggests that necroptosis plays an important role in kidney injury. 9 , 12 , 51 The mechanism remains unclear. Our results suggest that Anxa2 suppresses necroptosis in renal tubular epithelial cells mainly by regulating membranal ESCRT-III. Anxa2 was significantly upregulated and accumulated at the apical membrane, the main site of cell membrane rupture in the medulla as shown by TEM, of renal tubular epithelial cells after IR. However, how Anxa2 affects necroptosis requests more in-depth studies. Therefore, we surprisingly identified the specific binding molecules of Anxa2 by IP-MS and found that the membrane protein associated with necroptosis was Chmp2A. According to previous reports, Champ2A is an important component of ESCRT-III. Other constituents of such complex include Chmp2B, Chmp3, Chmp4B and Vps4B 52 , 53 . The ESCRT-III complex was identified in early studies as an important functional component for the formation of autophagic vesicles with its main role being phagophore closure 54 . However, in recent years, a large number of studies have confirmed that this complex plays an important protective role in the process of necroptosis 55 – 57 . In necroptosis, pMLKL punches holes in the cell membrane, thereby causing cytoplasmic extravasation and inducing cell necroptosis. However, in this process, ESCRT-III complex can accumulate on the cell membrane and excrete the perforated cell membrane through the form of vesicles, thereby protecting the integrity of the cell membrane and preventing necroptosis 57 , 58 . According to our data, ESCRT-III complex was significantly reduced at the apical membrane of renal tubular epithelial cells without Anxa2, which suggests that Anxa2 is indeed able to promote ESCRT-III accumulation at the apical membrane of renal tubular epithelial cells and inhibit necroptosis. However, how Anxa2 bound with Chmp2A and stabilizes the ESCRT-III complex at the cell membrane still needs to be further studied. Our results suggest that Nec1 rescues renal IRI caused by Anxa2 deficiency and inhibits neutrophil infiltration into the injured areas. Interestingly, regarding the degree of kidney injury, Nec1-treated WT and KO mice did not differ, whereas Kim1 and Lcn2 were upregulated in Nec1-treated KO mice, and necroptosis was increased. This may be caused by the hysteresis of the phenotype; Kim1 and Lcn2 are both sensitive biomarkers that are upregulated even upon mild injury. Alternatively, necroptosis may need to accumulate to a certain extent before it can manifest itself phenotypically. In addition, the infiltration level of neutrophils was increased in Nec1-treated KO mice, indicating that necroptosis of renal tubular epithelial cells may be the primary factor contributing to renal IRI, and inflammation may be a secondary factor. There are increasing reports on the role of Nec1 in the treatment of various necroptosis-related diseases. 50 , 59 However, it also has some limitations such as off-target effects and unstable metabolism. 60 , 61 Therefore, we suggest a synergistic effect between Anxa2 and Nec1. However, further studies are required to test this hypothesis. In addition, Nec1-treated WT mice with IRI had lower levels of injury and inflammation than non-treated WT mice with IRI in the aforementioned experiments. This indicates that necroptosis indeed occurs after renal IRI under normal conditions, which indicates that increasing Anxa2 levels before NSS may better protect kidney function. This study had some limitations. First, we used only male animals for the in vivo experiments, and female animals were not included. Second, clinical resources were unavailable to further validate our hypotheses. Third, we used conventional knockout mice rather than conditional knockout mice, which may have overlooked the function of Anxa2 in other cells or tissues. Thus, further experiments are required to comprehensively verify the protective effect of Anxa2 in renal IRI and the feasibility of a clinical application of Anxa2. In summary, our study clarified the role of Anxa2 upregulation in renal IRI. Anxa2 is able to promote ESCRT-III accumulation at the apical membrane of renal tubular epithelial cells and inhibit necroptosis, reducing local neutrophil infiltration and playing a protective role in renal IRI. These findings provide a new clinical basis for the prevention and treatment of NSS-induced renal IRI caused by NSS. Methods Mice The study has been performed in accordance with the EU Directive 2010/63/EU for animal experiments. The animal experiments were approved by the Ethics Committee of the General Hospital of the People's Liberation Army. (S2013-115-01) Male C57BL/6 wild-type (WT) mice aged 6–8 weeks were purchased from Sinogenetic Biotechnology (Beijing, China). Male and female Anxa2 −/− mice (in a C57BL/6 background) were produced by Cyagen Biosciences Inc (Beijing, China). Male WT and Anxa2 −/− mice were used for experiments. Mice were housed in a pathogen-free, constant temperature with a 12 h light/dark cycle and allowed to acclimatize for a week in the animal facility before the operation. Mouse model of renal IRI and in vivo drug treatment The mice were subjected to unilateral renal IRI. Briefly, the mice were anesthetized with 0.3% lumbarbarbituric sodium (40 mg/kg, i.p.) on a thermostatic blanket, which maintains body temperature at approximately 37 ℃ during surgery. The left renal pedicle was clamped with an atraumatic vascular clip for 30 min, after which the clamp was released. For Nec-1 treatments, we administered intraperitoneal injections of Nec-1 (1.65 mg/kg in PBS) (S8037, Selleck) to WT and Anxa2 −/− mice at two different time points: 15 min before and 1 h after IR surgery. 15,62-64 24h after reperfusion, samples were collected for the experiments. We used a double-blind method for the experiments and data analysis. The allocation and the conduct of the experiment were performed by Dong Lai and Jichen Wang. The outcome assessment and the data analysis were performed by Huayi Feng. RT-qPCR RT-qPCR was performed according to previously reported standard techniques. 65 The primer sequences can be found in the supplementary file. Western blotting Western blots were performed in accordance with standard techniques as reported previously. 66 Antibodies against Anxa2 (D11G2, CST), Rip1 (29932-1-AP, Proteintech), pRip1 (28252-1-AP, Proteintech), Rip3 (17563-1-AP, Proteintech), pRip3(ab222320, Abcam), Mlkl (66675-1-Ig, Proteintech), pMlkl (ab196436, Abcam), Gapdh (60004-1-Ig, Proteintech) and β-tubulin (BE0025, EASYBIO) were used. We used the Mem-PER Plus kit (89842Y, Thermo Scientific) to isolate the membrane and cytoplasmic proteins of kidney tissue for subsequent experiments. Periodic Acid-Schiff (PAS) and Masson stain Renal IRI was evaluated using a PAS kit (C0142M, Beyotime Biotechnology) following the manufacturer’s instructions. Fibrosis of the kidney after IRI was evaluated using Masson’s kit (C0189S, Beyotime Biotechnology). Kidney long-term fibrosis levels were scored based on the proportion of the total area occupied by collagen deposition. Immunohistochemistry and immunofluorescence Antibodies against Ly6G (GB11229-100; Servicebio), F4/80 (GB113373-50; Servicebio), aSMA(14395-1-AP; Proteintech), Nkcc2 (18970-1-AP; Proteintech), Aqp2(29386-1-AP; Proteintech), Atp6v1g3(19523-1-AP; Proteintech), and Aqp1(20333-1-AP; Proteintech) were used. We followed the standard protocols described previously. 67 Flow cytometry Flow cytometry assays were performed in accordance with standard techniques as reported previously. 68 Brilliant Violet 421™ anti-mouse CD45 Antibody (103133, Biolegend), Alexa Fluor® 488 anti-mouse/human CD11b Antibody (101217, Biolegend), APC anti-mouse F4/80 Recombinant Antibody (157306, Biolegend), APC/Cyanine7 anti-mouse Ly-6G Antibody (127624, Biolegend), and PE anti-mouse Ly-6C Antibody (128008, Biolegend) were used. Transmission electron microscope Transmission electron microscopy was performed in accordance with standard techniques, as reported previously. 69 Glutamate fixative was purchased from Beijing LABGIC (BL911A). Cell culture Tcmk1 was obtained from the National Platform of Experimental Cell Resources for Sci-Tech (Beijing, China). Tcmk1 were cultured and maintained in (Minimum Essential Medium MEM (Pricella), supplemented with 10% fetal bovine serum (Hangzhou Lvyuan Biological Technology Co., Ltd.) and 1% penicillin-streptomycin solution. Cell line was passaged <20 times and cultured at 37 ℃ in 5% CO2 under mycoplasma-free conditions. A necroptosis inducer kit containing TSZ (TNF-α, SM-164, and Z-VAD-FMK) was purchased from Beyotime Biotechnology (C1058S). Nec-1 (150 mM in DMSO) was added to the medium at a dilution of 1:1000. 70 Plasmid construction and transfection The full-length coding sequences of Anxa2 was cloned into pcDNA3.1-Flag vector, and the Tcmk1 cell line was transfected with pcDNA3.1-Flag-Anxa2 plasmid. siRNA targeting Anxa2 and negative control siRNA were purchased from Genepharma (Suzhou, PR China). Transient transfection was accomplished using jetPRIME (Polyplus-transfection, Illkirch Graffenstaden, France), following the manufacturer’s instructions. Cell counting kit-8 Cell proliferation was evaluated using a cell counting kit-8 assay (CK001, LABLEAD BIOTECH) following the manufacturer’s instructions. FITC Annexin V Apoptosis Detection Kit I The degree of cell necroptosis was evaluated using an FITC Annexin V Apoptosis Detection Kit (556547, BD Pharmingen) following the manufacturer’s instructions. Statistical analyses Data were analyzed using GraphPad Prism version 10 and expressed as mean ± standard deviation of independent replicates. Dot plots, line plots, and histograms were constructed using the GraphPad Prism software, version 10. Differences between the two groups were analyzed using an unpaired Student’s t-test (two-tailed). Comparisons between multiple groups were performed using one- or two-way ANOVA. P was set at 0.05. Declarations Disclosure All the authors declared no competing interests. Acknowledgments This work was supported by the National Natural Science Foundation of China (grant numbers 81802804), the Fostering Fund of Chinese PLA General Hospital for National Excellent Young Scholar Science Fund (2020-YQPY-006), the National Key Research and Development Program (2022YFB4701700), and the Growth Project of the Youth Independent Innovation Science Fund of Chinese PLA General Hospital (22QNCZ029). Author Contribution DL, CJW and YHF contributed equally to this research. DL, CJW, YHF and BXL contributed to the hypothesis, study design, data analysis, and experiments. CJW, YHF and QSC contributed to experiment assistance. KHL, QB, MWF, YH and LLB contributed to the construction of Anxa2 knockout mice. XZ, XM, NJX and BXL conceived and supervised the study; DL wrote this article, all authors reviewed and approved the final version of the article. Data Sharing Statement The data deposition process is in progress. References Ronco, C., Bellomo, R. & Kellum, J. A. 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Additional Declarations There is no duality of interest Supplementary Files SFigure.docx supplementary.zip Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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12:40:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6991722/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6991722/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87373433,"identity":"3a2a8183-9d26-4682-9fee-f8a6455f20ed","added_by":"auto","created_at":"2025-07-23 07:29:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":834617,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnxa2 is upregulated and accumulates at the apical membrane of the medullary tubular epithelial cells after renal IRI. \u003c/strong\u003eAll model mice were clamped for 30 minutes, and tissue samples were collected 24 hours after reperfusion.\u003cstrong\u003e (a) \u003c/strong\u003eThe relative mRNA expression levels of Kim1, Lcn2, and Anxa2 in mouse kidney tissue of sham and IR groups. Data are expressed as the mean±SEM. n=6, ***P \u0026lt; 0.001. \u003cstrong\u003e(b)\u003c/strong\u003e The protein expression level of Anxa2 in mouse kidney tissue of sham and IR group. \u003cstrong\u003e(c) \u003c/strong\u003ePeriodic Acid-Schiff (PAS) staining was used to evaluate the pathology\u003c/p\u003e\n\u003cp\u003eafter renal I/R. Anxa2 immunohistochemical staining was used to evaluate the expression pattern of Anxa2 in the kidney after IR. Low power field: Bar=1mm; High power field: Bar=50μm.\u003cstrong\u003e (d) \u003c/strong\u003eNkcc2 and Anxa2, Aqp2 and Anxa2, and Atp6v1g3 and Anxa2 were co-stained with immuno-fluorescence to clarify the explicit cell with changes in Anxa2 after renal IRI. Bar=20μm.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6991722/v1/df9854a2fc2401cd3fe5c1f3.png"},{"id":87373434,"identity":"57f3c45f-e759-437c-9897-aca6b4c91f8b","added_by":"auto","created_at":"2025-07-23 07:29:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":841241,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnxa2 plays a protective role in kidney early injury and long-term renal fibrosis of the medulla after renal IR. \u003c/strong\u003eAll model mice were clamped for 30 minutes, and tissue samples were collected 1d and 28d after reperfusion. The acute injury score is based on the proportion of the PAS damaged area to the total area (a score of 0 corresponds to no injury, 1 to 0–25% injury, 2 to 26–50% injury, 3 to 51–75% injury, and 4 to 76–100% injury). \u003cstrong\u003e(a) \u003c/strong\u003ePAS staining was used to evaluate the degree of kidney injury in Anxa(+/+) and Anxa2(-/-) mice. Bar=50μm. \u003cstrong\u003e(b) \u003c/strong\u003eAnxa(+/+) and Anxa2(-/-) mice were scored for early renal injury after IR with acute kidney scoring method. Data are expressed as the mean±SEM. n=6, ***P \u0026lt; 0.001.\u003cstrong\u003e (c) \u003c/strong\u003eThe medullary kidney tissue was specifically isolated from the clamped kidney for qPCR to evaluate the relative mRNA expression levels of \u003cem\u003eKim1\u003c/em\u003e and \u003cem\u003eLcn2\u003c/em\u003eafter 1d reperfusion in Anxa(+/+) and Anxa2(-/-) mice. Data are expressed as the mean±SEM. n=6, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003cstrong\u003e (d) \u003c/strong\u003eMasson and αSMAimmunohistochemical staining were used to evaluate the degree of kidney fibrosis in Anxa(+/+) and Anxa2(-/-) mice after 28d reperfusion. Masson staining: Bar=50μm; αSMA immunohistochemical staining: Bar=20μm. \u003cstrong\u003e(e) \u003c/strong\u003eEach sample is assigned a Masson staining score for the medulla and cortex of the kidney, respectively, using the method mentioned before. The count of αSMA-positive cells was averaged by taking 3 fixed fields in the cortex and medulla, respectively. Data are expressed as the mean±SEM. n=5, **P \u0026lt; 0.01. \u003cstrong\u003e(f) \u003c/strong\u003eImmunofluorescence staining of Aqp1 was used to evaluate the structural damage of renal tubules and the death of renal tubular epithelial cells in renal IRI after 1d reperfusion in Anxa(+/+) and Anxa2(-/-) mice. Bar=50μm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6991722/v1/c2ff8c5a69837295003e008b.png"},{"id":87373981,"identity":"13608a65-eabd-46e2-9857-c134e0af0b2d","added_by":"auto","created_at":"2025-07-23 07:37:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":461963,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUp-regulation of Anxa2 in renal tubular epithelial cells after renal IR suppresses necroptosis of tubular epithelial cells in renal medulla. \u003c/strong\u003eAll model mice were clamped for 30 minutes, and tissue samples were collected 24 hours after reperfusion. Small interfering RNA (siRNA) was used to knock down Anxa2 in Tcmk1 cells. \u003cstrong\u003e(a) \u003c/strong\u003eThe medullary kidney tissue was specifically isolated from the clamped kidney for western-blotting to evaluate protein levels associated with necroptosis after renal IR in Anxa(+/+) and Anxa2(-/-) mice. \u003cstrong\u003e(b) \u003c/strong\u003eImmunofluorescence staining of pMlkl was used to assess the location of upregulated pMLKL expression in the renal medulla of Anxa2(-/-) mice. Low power field: Bar=100μm; High power field: Bar=50μm. \u003cstrong\u003e(c) \u003c/strong\u003eTransmission electron microscopy (TEM) scanning was used to evaluate the destruction of apical membrane of medullary tubular epithelial cells after renal IR, reflecting the degree of cellular necroptosis. Bar=1μm. \u003cstrong\u003e(d) \u003c/strong\u003eFor each sample, 4 fixed fields were taken in the injured fraction (outer medulla), and the ratio of cells with severely ruptured apical membrane of renal tubular epithelial cells in each field to the total cells in the field was calculated and averaged. Data are expressed as the mean±SEM. n=3, *P \u0026lt; 0.05. \u003cstrong\u003e(e) \u003c/strong\u003eTcmk1 cells were used to verify the regulatory effect of Anxa2 on necroptosis in renal tubular epithelial cells. After siRNA knockdown of Anxa2 in cells, TSZ (TNF-α, SM-164 and Z-V AD-FMK) was added to induce cellular necroptosis, and the expression level of necroptosis-related protein in control and knockdown group was evaluated.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6991722/v1/2989911f0f5f8b653949be8d.png"},{"id":87373437,"identity":"03efcd94-1bf6-44e8-87fa-f48cbccb3b09","added_by":"auto","created_at":"2025-07-23 07:29:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":292997,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of Anxa2 is able to reduce cellular necroptosis in Tcmk1. \u003c/strong\u003eTSZ reagent was added to the medium at a ratio of 1:1000 to induce necroptosis. \u003cstrong\u003e(a) \u003c/strong\u003eThe\u003cstrong\u003e \u003c/strong\u003eprotein expression levels of Anxa2, Mlkl and pMlkl in NC, si-Anxa2 and Overexpression (OE) in Tcmk1. \u003cstrong\u003e(b) \u003c/strong\u003eProliferation curves of NC, si-Anxa2 and OE in Tcmk1 obtained by CCK8 assay. 3 independent replicates were performed for each group of cells. Data are expressed as the mean±SEM. \u003cstrong\u003e(c) \u003c/strong\u003eThe bright field of NC, si-Anxa2 and OE in Tcmk1 was photographed to evaluate the level of necroptosis in each group. Transfection was performed at 40% cell density, and TSZ was added 24 h after transfection. Data collection was performed at 72 h after transfection. \u003cstrong\u003e(d) \u003c/strong\u003eFlow cytometry was used to determine the proportion of necrotic (mainly TSZ-induced necroptosis) and apoptotic cells in each group.Transfection was performed at 40% cell density, and TSZ was added 24 h after transfection. Data collection was performed at 72 h after transfection. \u003cstrong\u003e(e) \u003c/strong\u003eThe proportion of cells with necroptosis to all cells in each group was counted to evaluate the level of necroptosis. Annexin V+/PI+ and Annexin V-/PI+ cells were considered as cells of necroptosis. 3 independent replicates were performed for each group of cells. Data are expressed as the mean±SEM. **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6991722/v1/f9ea6d6e3149ae42fb3765cf.png"},{"id":87373436,"identity":"a222bcf0-f593-460d-bbc4-53c1afd0d8b5","added_by":"auto","created_at":"2025-07-23 07:29:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":392940,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUp-regulation of Anxa2 after renal IR inhibits the inflammatory response and neutrophil recruitment in the kidney. \u003c/strong\u003eAll model mice were clamped for 30 minutes, and tissue samples were collected 24 hours after reperfusion. \u003cstrong\u003e(a) \u003c/strong\u003eThe medullary kidney tissue was specifically isolated from the clamped kidney for RT-qPCR to evaluate mRNA levels of necroptosis-related inflammatory factors after renal IR in Anxa(+/+) and Anxa2(-/-) mice. Data are expressed as the mean±SEM. n=6, ***P \u0026lt; 0.001. \u003cstrong\u003e(b) \u003c/strong\u003eThe\u003cstrong\u003e \u003c/strong\u003ewhole kidney was collected for flow cytometry to examine the infiltration of neutrophils after renal IR. CD45+CD11b+\u003c/p\u003e\n\u003cp\u003eLy6G+Ly6C\u003csup\u003ehigh\u003c/sup\u003e cells were considered as neutrophils. \u003cstrong\u003e(c) \u003c/strong\u003eThe percent of neutrophils in CD45+CD11b+ cells was counted to evaluate the level of renal inflammation after IR in Anxa(+/+) and Anxa2(-/-) mice. Data are expressed as the mean±SEM. sham, n=5; IR, n=4 (One mouse in each of the Anxa(+/+) and Anxa2(-/-) died of anesthetic overdose). *P \u0026lt; 0.05, **P \u0026lt; 0.01. \u003cstrong\u003e(d) \u003c/strong\u003eImmunohistochemical staining of Ly6G was used to determine the location and number of neutrophil infiltration during early injury after renal IR. Low power field: Bar=200μm; High power field: Bar=50μm. \u003cstrong\u003e(e) \u003c/strong\u003eThe count of Ly6G-positive cells was averaged by taking 3 fixed fields in the injured area of kidney (mainly outer medulla). Data are expressed as the mean±SEM. sham, n=5; IR, n=4 (same as (c)). *P \u0026lt; 0.05. \u003cstrong\u003e(f) \u003c/strong\u003eThe medullary kidney tissue was isolated for RTqPCR to evaluate mRNA levels of neutrophils’ chemokines after renal IR in Anxa(+/+) and Anxa2(-/-) mice. Data are expressed as the mean±SEM. n=6, ***P \u0026lt; 0.001. \u003cstrong\u003e(g) \u003c/strong\u003eThe Protein concentration of cell supernatant were detected in control, TSZ-treated and OE TSZ-treated. 3 independent replicates were performed for each group of cells. Data are expressed as the mean±SEM. **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6991722/v1/c15b0723ad1aaa50fe95520e.png"},{"id":87373439,"identity":"04156855-410c-4c44-b9af-1580a811e9ed","added_by":"auto","created_at":"2025-07-23 07:29:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":449065,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNec1 rescues Anxa2 deficiency-induced injury by reducing necroptosis and neutrophil infiltration in the medulla after renal IRI. \u003c/strong\u003eAll model mice were clamped for 30 minutes, and tissue samples were collected 24 hours after reperfusion. Nec1 was used to intervene in the process of renal IR as mentioned before. \u003cstrong\u003e(a) \u003c/strong\u003ePAS staining was used to evaluate the degree of kidney injury after renal IR with or without Nec1 intervention in Anxa(+/+) and Anxa2(-/-) mice. Bar=50μm. \u003cstrong\u003e(b) \u003c/strong\u003eEach sample was scored for early renal injury after IR with acute kidney scoring method in the medulla. Data are expressed as the mean±SEM. n=5, *P \u0026lt; 0.05, ***P \u0026lt; 0.001. \u003cstrong\u003e(c) \u003c/strong\u003eThe medullary kidney tissue was specifically isolated from the clamped kidney for qPCR to evaluate the mRNA expression levels of \u003cem\u003eKim1\u003c/em\u003e and \u003cem\u003eLcn2 \u003c/em\u003eafter IR with or without Nec1 intervention in Anxa(+/+) and Anxa2(-/-) mice. Data are expressed as the mean±SEM. n=5, *P \u0026lt; 0.05, ***P \u0026lt; 0.001. \u003cstrong\u003e(d) \u003c/strong\u003eThe medullary kidney tissue was used for western-blotting to evaluate protein level associated with necroptosis after IR with or without Nec1 intervention in Anxa(+/+) and Anxa2(-/-) mice. \u003cstrong\u003e(e) \u003c/strong\u003eImmuno-fluorescence staining of Aqp1 was used to evaluate the structural damage of renal tubules after IR with or without Nec1 intervention in Anxa(+/+) and Anxa2(-/-) mice. Bar=50μm. \u003cstrong\u003e(f) \u003c/strong\u003eThe\u003cstrong\u003e \u003c/strong\u003ewhole kidney was collected for flow cytometry to examine the infiltration of neutrophils after renal IR. CD45+CD11b+Ly6G+Ly6C\u003csup\u003ehigh\u003c/sup\u003e cells were considered as neutrophils. \u003cstrong\u003e(g) \u003c/strong\u003eThe percent of neutrophils in CD45+CD11b+ cells was counted to evaluate the level of renal inflammation after IR with or without Nec1 intervention in Anxa(+/+) and Anxa2(-/-) mice. Data are expressed as the mean±SEM. n=5, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6991722/v1/10bcba9f950c75919a95d4c6.png"},{"id":87373446,"identity":"21238fed-b3a1-47f7-b2b2-c9b81c155e59","added_by":"auto","created_at":"2025-07-23 07:29:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":387800,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnxa2 can specifically bind to Chmp2A and promote the aggregation of ESCRT-III to protect against necroptosis after renal IRI. \u003c/strong\u003eAll model mice were clamped for 30 minutes, and tissue samples were collected 24 hours after reperfusion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eMembrane proteins were extracted from renal medulla tissue, and the specific binding proteins to Anxa2 were detected by co-immunoprecipitation. \u003cstrong\u003e(b) \u003c/strong\u003eThe distribution of Chmp2A , the specific binding molecule of Anxa2, after renal IR were detected by immunohistochemistry in Anxa(+/+) and Anxa2(-/-) mice. Bar=50μm. \u003cstrong\u003e(c) \u003c/strong\u003eCo-staining of Anxa2 and Chmp2A by Immunofluorescence staining was performed to analyze the relationship between the expression localization of Anxa2 and Chmp2A after renal IR. Bar=50μm. \u003cstrong\u003e(d) \u003c/strong\u003eThe extracted membrane proteins were used to detect the expression of component proteins of ESCRT-III complex, such as Chmp2A, Chmp2B, Chmp3, Chmp4B and Vps4B, by western blotting in Anxa(+/+) and Anxa2(-/-) mice.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6991722/v1/a8750ee5ad9f57998c5cce09.png"},{"id":87373445,"identity":"afc36fb4-ca14-42f5-b148-e46c34b28efa","added_by":"auto","created_at":"2025-07-23 07:29:42","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":206744,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe schematic diagram of the Anxa2 protective effect against renal IRI. \u003c/strong\u003eSchematic diagram of mechanism: Anxa2 is able to promote ESCRT-III accumulation at the apical membrane of renal tubular epithelial cells and inhibit necroptosis, thereby mitigating the release of Cxcl1 and Cxcl2 and the recruitment of neutrophils to injured areas.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6991722/v1/184304e587ef9a00e0fd3ac2.png"},{"id":87716368,"identity":"410e3fde-f79f-49f6-b4ea-5ddfad810f2a","added_by":"auto","created_at":"2025-07-28 09:11:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5217367,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6991722/v1/555fcb9a-be39-4121-8448-b22143e120c8.pdf"},{"id":87375134,"identity":"3e3218b2-20bb-4a0a-9927-51d0e76b552e","added_by":"auto","created_at":"2025-07-23 07:45:42","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3084678,"visible":true,"origin":"","legend":"","description":"","filename":"SFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-6991722/v1/6b035d2b3803550609a96872.docx"},{"id":87373441,"identity":"b183e5b2-b4f0-41c1-b652-f38eb83a2100","added_by":"auto","created_at":"2025-07-23 07:29:42","extension":"zip","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4278673,"visible":true,"origin":"","legend":"","description":"","filename":"supplementary.zip","url":"https://assets-eu.researchsquare.com/files/rs-6991722/v1/bb08f3661b6da2cfde7f4404.zip"}],"financialInterests":"There is no duality of interest","formattedTitle":"Annexin A2 alleviates renal ischemia/reperfusion injury via regulating membranal ESCRT-III to suppress necroptosis of tubular epithelial cells","fulltext":[{"header":"Key points","content":"\u003cul\u003e\n \u003cli\u003eAnxa2 is up-regulated after ischemia/reperfusion injury and playing a protective role in its development.\u003c/li\u003e\n \u003cli\u003eAnxa2 is able to reduce infiltration of neutrophils and inflammatory factors by inhibiting necroptosis in renal ischemia/reperfusion injury.\u003c/li\u003e\n \u003cli\u003eWe found the molecular mechanism by which Anxa2 inhibits necroptosis by regulating membranal ESCRT-III coplex, providing new clinical strategies for its prevention and treatment.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Significance Statement ","content":"\u003cp\u003eAnxa2 is significantly up-regulated after renal IRI and plays an important role in the occurrence and development of injury. In this study, Anxa2 was found to accumulate on the apical membrane of renal tubular epithelial cells in the renal medulla at the early stage after renal IR and play a protective role in renal IRI. Secondly, we illustrated for the first time that Anxa2 could inhibit necroptosis in renal IRI, thereby affecting the infiltration of neutrophils and inflammatory factors. More importantly, by IP-MS, we further found that Anxa2 specifically binds to Chmp2A, thereby promoting ESCRT-III accumulation at the apical membrane of renal tubular epithelial cells and inhibiting the necroptosis process. The investigation revealed a novel mechanism to explain how Anxa2 protect against renal IRI and provided new clinical ideas for its prevention and treatment of renal IRI caused by NSS.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eRenal ischemia-reperfusion injury (IRI) can occur in conditions such as intravascular volume depletion, hypotension, and renal vascular diseases.\u003csup\u003e1\u003c/sup\u003e However, with the widespread implementation of nephron-sparing surgery (NSS) in urology, renal IRI after NSS has increasingly attracted the attention of urologists.\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eTo avoid bleeding during surgery, it is necessary to clamp the renal artery during the tumor enucleation process, which leads to warm ischemia and reperfusion of the kidney. Clinical data indicate that the renal function of most patients is restored after surgery. However, 20% of patients experience renal functional impairment after NSS.\u003csup\u003e3,4\u003c/sup\u003e This has become a major obstacle for protecting kidney function, which is the core purpose of NSS. Unfortunately, we lack effective clinical methods for prevention and treatment. Thus, it is particularly important to study the mechanism of renal IRI, explore ways to prevent its occurrence, and treat it.\u003c/p\u003e\n\u003cp\u003eRenal IRI is a complex process involving multiple mechanisms. Currently, the prevailing view is that it is primarily caused by the death of renal tubular epithelial cells and subsequent inflammation,\u003csup\u003e5-7\u003c/sup\u003e which may primarily be caused by necroptosis of renal tubular epithelial cells.\u003csup\u003e8\u003c/sup\u003e Apoptosis causes mild inflammation, whereas necroptosis results in robust inflammation with rupture of the cell membrane and auto-amplification loop of necroinflammation.\u003csup\u003e9-12\u003c/sup\u003e Specific Rip1 inhibitors, such as Necrostatin-1 (Nec-1) and Cpd-71, can protect against kidney injury, suggesting that necroptosis may play an important proinflammatory role in kidney injury.\u003csup\u003e13-15\u003c/sup\u003e However, the molecular mechanisms underlying necroptosis in IRI remain ill-defined.\u003c/p\u003e\n\u003cp\u003eAnnexin A2 (Anxa2), an extensively studied member of the annexin superfamily, is a transmembrane protein that connects cell or organelle membranes with other molecules and\u0026nbsp;\u0026ldquo;anchors\u0026rdquo;\u0026nbsp;various transmembrane molecules.\u003csup\u003e16\u003c/sup\u003e Anxa2 is involved in autophagy and apoptosis,\u003csup\u003e17-19\u003c/sup\u003e which do not cause severe inflammation. Thus, we speculated that Anxa2 may be involved in necrosis. Determining whether Anxa2 is involved in necroptosis and its role in renal IRI will be beneficial for clinical translation.\u003c/p\u003e\n\u003cp\u003eUsing a renal IRI mouse model, through in vivo and in vitro experiments, we explored the function and molecular mechanism of Anxa2 in renal IRI，and provided a new clinical basis for the prevention and treatment of NSS-induced renal IRI caused by NSS.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnxa2 is upregulated and accumulates at the apical membrane of the medullary tubular epithelial cells after renal IRI\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnxa2 is significantly upregulated after kidney IRI.\u003csup\u003e20\u003c/sup\u003e \u003cem\u003eKim1\u003c/em\u003e and \u003cem\u003eLcn2\u003c/em\u003e were significantly upregulated, alongside Anxa2 mRNA and protein levels 24h after reperfusion (Figure 1a and b). Contrary to previous views, growing evidence supports that renal IRI and inflammation may originate in the medulla rather than in the cortex.\u003csup\u003e21-24\u003c/sup\u003e We found that the degree of injury in the medullary fraction was significantly higher than that in the cortical fraction at 1 and 3 days after renal IRI. On the 7th day after IRI, the degree of injury in the cortex was significantly higher than that in the cortex 1 d after IRI (Supplementary Figure S1A and B). This suggests that renal IRI spreads over time from the medulla, especially the outer medulla, to the cortex. Anxa2 expression in the medulla was significantly higher than that in the cortex 1 d after renal IR, but there was no significant difference between the medulla and cortex 3 and 7 days after renal IR. Additionally, Anxa2 expression level was significantly higher on the 7th day in the cortex than on the 1st day after IR (Supplementary Figure S1C and D). This is consistent with the injury progression pattern, indicating that Anxa2 expression occurs during injury and originates from the renal medulla, particularly the outer medulla. PAS and immunohistochemical staining showed that Anxa2 expression was significantly increased in the medulla (especially the outer medulla) after renal IRI, and Anxa2 significantly accumulated on the apical membrane of renal tubular epithelial cells (Figure 1c). To further determine the location of Anxa2 protein, we performed immunofluorescence co-staining for markers of different renal tubular segments and Anxa2 in the medulla. The results showed that Anxa2 mainly accumulated on the apical membrane of renal tubular epithelial cells in the thick ascending limb of Henle-loop (Nkcc2\u003csup\u003e25\u003c/sup\u003e), part of distal convolulated tubules (Aqp2\u003csup\u003e26\u003c/sup\u003e) and collecting tubules (Aqp2 and Atp6v1g3\u003csup\u003e26,27\u003c/sup\u003e) of medulla after renal IR (Figure 1d). Therefore, Anxa2 is localized at the apical membrane of renal tubular epithelial cells, which belongs to the thick ascending limb of Henle- loop, part of the distal convoluted tubules, and the collecting duct, which most likely plays a role in renal IRI.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnxa2 protects against early kidney injury and long-term renal fibrosis of the medulla after renal IRI\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies have shown that Anxa2 has diverse functions, including regulating inflammation and cell death.\u003csup\u003e19,20,28-30\u003c/sup\u003e Our data showed that Anxa2 was significantly upregulated in medullary tubular epithelial cells after renal IR. Anxa2 knockout (KO) mice were used to explore the function of Anxa2 in renal tubular epithelial cells. The results showed that at 1 day after renal IR, the injury of renal medulla in KO mice was more severe than that in wild type (WT) mice. However, there were no significant differences in the cortical components between WT and KO mice (Figure 2a and b). This is consistent with previous experimental results showing that Anxa2 is significantly upregulated in renal medulla at the initial stage of renal IRI and plays a protective role against early injury in the medulla. To confirm this conclusion, we isolated the medullary tissue of IR kidneys from WT and KO mice. We found that \u003cem\u003eKim1\u003c/em\u003e and \u003cem\u003eLcn2\u003c/em\u003e expression in the medulla of KO mice was significantly higher than that in the medullae of WT mice (Figure 2c). To further characterize the cellular injury of renal medulla and lumen structure, we used Aqp1, a cellular injury assessment marker,\u003csup\u003e31\u003c/sup\u003e in WT and KO mice. The results showed that the collapse of the tubular luminal structure was more severe in KO mice (Figure 2f). This suggests that Anxa2 inhibits the death of renal medullary tubular epithelial cells, thereby preventing the collapse of the renal tubular lumen structure and reducing early injury after IRI. Moreover, the degree of early injury determines long-term kidney fibrosis.\u003csup\u003e32\u003c/sup\u003e Therefore, we evaluated the long-term fibrosis levels after renal IRI, and the results showed that long-term kidney fibrosis in KO mice was significantly higher in the medullary region, but there was no significant difference between WT and KO mice in the cortical region (Figure 2d and e). This suggests that Anxa2 affects long-term renal medullary fibrosis by protecting the medulla against early injury following IR. The mice used in our experiments were genotyped twice using mouse tail DNA to ensure that the control and experimental groups were Anxa2\u003csup\u003e+/+\u003c/sup\u003e and Anxa2\u003csup\u003e\u0026ndash;/\u0026ndash;\u003c/sup\u003e mice (Supplementary Figure S2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnxa2 upregulation in renal tubular epithelial cells after renal IR suppresses necroptosis of tubular epithelial cells in the renal medulla\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e Our data indicate that Anxa2 can inhibit the death of medullary renal tubular epithelial cells. To clarify the mechanism of cell death specifically regulated by Anxa2, we isolated renal medullary tissue and detected the levels of key proteins related to different death pathways. The results showed that for apoptosis,\u003csup\u003e33\u003c/sup\u003e Caspase 3 and cleaved-Caspase 3 levels were not significantly different between WT and KO mice. For pyroptosis,\u003csup\u003e34\u003c/sup\u003e the Gsdmd and N-Gsdmd levels did not significantly differ between WT and KO mice. For ferroptosis,\u003csup\u003e35\u003c/sup\u003e Gpx4 levels were not significantly different between WT and KO mice (Supplementary Figure S3A). However, pMlkl levels were significantly upregulated in KO mice (Figure 3a). This suggests that Anxa2 upregulation can inhibit the necroptosis in renal medullary tubular epithelial cells. This is consistent with previous reports indicating that necroptosis occurs in a high number of renal tubular epithelial cells in the medulla during kidney injury.\u003csup\u003e36\u003c/sup\u003e Necroptosis mainly depends on the the perforation of pMlkl on the cell membrane, resulting in the extravasation of cytoplasm and inflammatory response.\u003csup\u003e37-39\u003c/sup\u003e To verify the location of pMlkl expression, immunohistochemical staining of pMlkl was performed, which showed that pMlkl accumulated on the apical membrane of renal tubular epithelial cells in the renal medulla of KO mice, but not in other parts of the cell membrane (Figure 3b). In addition, we performed Transmission Electron Microscopy (TEM) scanning of the kidneys after IRI to further verify whether and to what extent the apical membranes of renal tubular epithelial cells in the medulla of WT and KO mice were damaged. We found that the apical membranes of renal medulla in KO mice were more severely ruptured (Figure 3c and d). The regulatory relationship between Anxa2 and necroptosis was also verified using in vitro experiments. We used Tcmk1 cells, a mouse renal tubular epithelial cell line, to knockdown Anxa2 and treated both control and knockdown cells with the same dose of TSZ to induce necroptosis. We compared the level of necroptosis between knockdown and control cells and observed that it was significantly higher in the Anxa2 knockdown than in the control group (Figure 3e). This suggests that Anxa2 can significantly inhibit necroptosis of renal tubular epithelial cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOverexpression of Anxa2 is able to reduce cellular necroptosis in Tcmk1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo verify that Anxa2 inhibits necroptosis in renal tubular epithelial cells, we overexpressed and knocked down Anxa2 in Tcmk1. We found that pMlkl levels significantly decreased in Anxa2-overexpressed Tcmk1 to the control and knockdown groups (Figure 4a). This suggests that Anxa2 could reduce the necroptosis caused by TSZ. Cell proliferation was evaluated by a cck8 assay and brightfield imaging. We observed that the cell proliferation ability in OE was significantly higher than control and knockdown groups. The proliferation ability of the knockdown group was lower than control group. The same results were obtained using brightfield imaging (Figure 4b and c). This indicates that Anxa2 effectively inhibits necroptosis of renal tubular epithelial cells. Flow cytometry was used to assess cell necrosis (primarily TSZ-induced necroptosis) and apoptosis. After TSZ addition, the proportion of necroptotic cells in the knockdown group was significantly higher than that in the control group. The proportion of necroptotic cells in OE was significantly decreased. (Figure 4d and e). However, we found that the level of apoptosis in OE was lower than that in siAnxa2, which probably means Anxa2 also has some effects on the early apoptosis of Tcmk1 (Supplementary Figure S3B). The proportion of apoptotic cells increased after the addition of TSZ. A possible reason is that Z-VAD-FMK, an inhibitor of apoptosis included in TSZ Kit, may not completely block apoptosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnxa2 upregulation after renal IR inhibits the inflammatory response and neutrophil recruitment in the medulla\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt present, it is explicitly reported that necroptosis and inflammation are closely related and mutually promote each other, playing an important role in renal IRI.\u003csup\u003e12,40-44\u003c/sup\u003e Thus, we isolated renal medullary tissue and measured the mRNA expression of necroptosis-related inflammatory factors in WT and KO mice to evaluate the level of inflammation. We found that \u003cem\u003eNlrp3\u003c/em\u003e\u003cem\u003e\u003csup\u003e45\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e, Tnf\u0026alpha;\u003c/em\u003e\u003cem\u003e\u003csup\u003e46\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e, Il6 \u003c/em\u003eand\u003cem\u003e Il1b\u003c/em\u003e\u003cem\u003e\u003csup\u003e47\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003ewere significantly upregulated in KO mice after renal IRI compared with WT mice (Figure 5a). This suggests that Anxa2 significantly suppressed the inflammatory response after renal IRI. In addition, we used flow cytometry to examine the infiltration of innate immune cells after renal IRI. We observed that the level of neutrophil infiltration was significantly increased in KO mice after renal IRI compared with WT mice (Figure 5b and c). This indicated that Anxa2 inhibited neutrophil recruitment after renal IRI. However, there is no significant difference in monocyte or macrophage recruitment between WT and KO mice. During the infiltration of innate immune cells after renal IR, the proportion of neutrophils and monocytes was significantly upregulated, and the proportion of macrophages was significantly decreased compared to that in the sham group (Supplementary Figure S4A and B). Immunohistochemical staining for Ly6G was performed to determine the location and number of neutrophil infiltration sites during early renal IRI. The results showed that neutrophils mostly infiltrated the early injury area (outer medulla). The number of neutrophils in the injured area of KO mice was significantly higher than that in WT mice, which was consistent with the flow-cytometry results (Figure 5d and e). Neutrophil infiltration has been suggested to be spatially specific and consistent with the area of injury. According to our data, the loss of Anxa2 led to massive necroptosis of renal tubular epithelial cells in the medulla, accompanied by a significant increase in neutrophil infiltration in the injured area. Here, we aimed to determine whether necroptosis directly or indirectly regulates neutrophil aggregation. We detected the mRNA levels of\u003cem\u003e Cxcl1\u003c/em\u003e and \u003cem\u003eCxcl2\u003c/em\u003e,\u003csup\u003e48,49\u003c/sup\u003e which are specific neutrophil chemokines, in renal medullary tissue. The results showed that \u003cem\u003eCxcl1\u003c/em\u003e and \u003cem\u003eCxcl2\u003c/em\u003e were significantly upregulated in KO mice after renal IRI (Figure 5f). In vitro experiments showed that the protein concentrations of Cxcl1 and Cxcl2 in the cell supernatant of OE Tcmk1 were significantly higher than those in control (Figure 5g). This suggests that necroptosis can cause the renal tubular epithelium to release more Cxcl1 and Cxcl2 to recruit neutrophils, meanwhile Anxa2 can reduce the synthesis and secretion of Cxcl1 and Cxcl2 and mitigate the recruitment of neutrophils by inhibiting necroptosis.\u003cem\u003e \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eNec1 rescues Anxa2 deficiency-induced injury by reducing necroptosis and neutrophil infiltration in the medulla after renal IRI\u003c/em\u003e\u003c/strong\u003e \u003c/p\u003e\n\u003cp\u003eNec1 is a specific inhibitor of necroptosis but has no direct inhibitory effects on inflammation.\u003csup\u003e50\u003c/sup\u003e To rescue the Anxa2 deficiency, we performed the Nec1 intervention described in the Methods section during the process of renal IRI. The results showed that the renal IRI level in Nec1-treated mice was significantly ameliorated in WT and KO mice. However, there was no significant effect on the level of injury between WT and KO mice, and the levels of injury did not differ significantly between Nec1-intervention KO mice and non-intervention WT mice (Figure 6a and b). This suggests that the increase in renal IRI caused by loss of Anxa2 can be reversed by Nec1, indicating that Anxa2 protects from the occurrence of IRI by inhibiting necroptosis. To confirm this conclusion, we used medullary kidney tissue to detect \u003cem\u003eKim1\u003c/em\u003e and \u003cem\u003eLcn2\u003c/em\u003e expression and found that Nec1 rescues renal IRI in the medulla of KO mice (Figure 6c). In addition, we found that pMlkl was significantly downregulated in Nec1-treated mice both in the WT and KO groups (Figure 6d). Immunofluorescence staining of Aqp1 showed that the renal tubular and epithelial cell structure of Nec1-treated mice was better preserved than that of untreated mice both in the WT and KO groups (Figure 6e). These results suggest that Nec1 could adequately rescue the increase in renal IRI caused by Anxa2 deficiency. Although the related indicators in Nec1-treated KO mice were significantly lower than those in the non-treated group, they were still significantly higher than those in Nec1-treated WT mice. There was no significant difference between Nec1-treated KO and untreated WT mice (Figure 6c and d). Our previous results demonstrated Anxa2 could inhibit neutrophil infiltration after renal IR. However, whether this neutrophil infiltration is due to necroptosis or other factors remains unclear. Thus, in vivo, we found that Nec1-treated mice had significantly reduced neutrophil infiltration compared to untreated mice. Neutrophil infiltration in Nec1-treated KO mice was also significantly higher than that in Nec1-treated WT mice. However, there was no significant difference between Nec1-treated KO mice and untreated WT mice (Figure 6f and g). This is consistent with previous RT-qPCR results for \u003cem\u003eKim1\u003c/em\u003e and \u003cem\u003eLcn2\u003c/em\u003e as well as western blotting for pMlkl. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnxa2 can specifically bind to Chmp2A and promote the aggregation of ESCRT-III to protect against necroptosis after renal IRI\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the molecular mechanism of how Anxa2 affects necroptosis in renal tubular epithelial cells, we obtained the proteins in renal medulla that bind to Anxa2 after renal IR by IP and subsequently analyzed these proteins by MS. We found that the necroptosis-related protein that could specifically bind to Anxa2 was Chmp2A (Supplementary Figure S7A). According to previous reports, Chmp2A is an important component of ESCRT-III complex, and other components include Chmp2B, Chmp3, Chmp4B and Vps4B. Immunofluorescence co-localization staining of Anxa2 and Chmp2A was performed, and the results showed that Chmp2A was not only expressed in the cytoplasm, but also co-expressed in the apical membrane with Anxa2 (Figure 7c). At the same time, we also performed IP experiments by extracting membrane proteins from renal medulla tissue proteins, and we found that Anxa2 could specifically bind to Chmp2A after renal IR, whereas this was not observed in the normal kidney. IP on the other components of ESCRT-III was also performed, and the results showed that Anxa2 did not bind to them specifically (Figure 7a). Therefore, we hypothesized that Anxa2 may bind to Chmp2A at the apical membrane and promote the aggregation of ESCRT-III to inhibit necroptosis. To confirm this hypothesis, we examined the protein levels of ESCRT-III components using extracted membrane proteins before, and the results showed that the expression of ESCRT-III molecules was significantly decreased in Anxa2 knockout mice (Figure 7d). However, a similar trend was not observed for cytosolic proteins (Supplementary Figure S7B). In addition, Chmp2A expression was found to be mainly located in the cytoplasm rather than the apical membrane in Anxa2 knockout mice compared with WT mice after renal IR (Figure 7b). These results suggest that Anxa2 may affect the effect of ESCRT-III on inhibiting necroptosis by affecting the aggregation of Chmp2A at the apical membrane. Notably, the IP result showed that Anxa2 does not bind to Chmp2A in the normal kidney, which is inconsistent with the IP-MS result, indicating that Anxa2 is mainly expressed in the cytoplasm, where it may bind to Chmp2A in normal kidney. The involvement of ESCRT-III in the generation of autophagic vesicles gives a new direction for our future research. In summary, Anxa2 is able to promote ESCRT-III accumulation at the apical membrane of renal tubular epithelial cells and inhibit necroptosis, thereby mitigating the release of Cxcl1 and Cxcl2 and the recruitment of neutrophils to injured areas. We schematized the mechanism of action of Anxa2 protective effect against renal IRI (Figure 8). \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCurrently, the regulatory mechanism of necroptosis in renal IRI remains poorly understood. Here, we showed that Anxa2 can significantly inhibit necroptosis in renal IRI, suppress inflammation, and alleviate kidney injury. We found that both the abundance and expression pattern of Anxa2 changed during IRI. Anxa2 accumulated significantly at the apical membrane of renal tubular epithelial cells and inhibited cellular necroptosis in the medulla after renal IR. Mechanistically, we also innovatively found that Anxa2 specifically bound to Chmp2A to promote the aggregation of ESCRT-III complex on the cell membrane, thereby protecting against necroptosis. In addition, necroptosis of renal tubular epithelial cells upregulates the expression of neutrophil chemotactic factors and aggravates neutrophil infiltration into the injured area. These results suggest that in NSS-induced renal IRI, the inflammation and tissue injury after IR can be mitigated by the early inhibition of necroptosis in medullary tubular epithelial cells.\u003c/p\u003e\u003cp\u003eIncreasing evidence suggests that necroptosis plays an important role in kidney injury.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e The mechanism remains unclear. Our results suggest that Anxa2 suppresses necroptosis in renal tubular epithelial cells mainly by regulating membranal ESCRT-III. Anxa2 was significantly upregulated and accumulated at the apical membrane, the main site of cell membrane rupture in the medulla as shown by TEM, of renal tubular epithelial cells after IR. However, how Anxa2 affects necroptosis requests more in-depth studies. Therefore, we surprisingly identified the specific binding molecules of Anxa2 by IP-MS and found that the membrane protein associated with necroptosis was Chmp2A. According to previous reports, Champ2A is an important component of ESCRT-III. Other constituents of such complex include Chmp2B, Chmp3, Chmp4B and Vps4B\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. The ESCRT-III complex was identified in early studies as an important functional component for the formation of autophagic vesicles with its main role being phagophore closure\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. However, in recent years, a large number of studies have confirmed that this complex plays an important protective role in the process of necroptosis\u003csup\u003e\u003cspan additionalcitationids=\"CR56\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. In necroptosis, pMLKL punches holes in the cell membrane, thereby causing cytoplasmic extravasation and inducing cell necroptosis. However, in this process, ESCRT-III complex can accumulate on the cell membrane and excrete the perforated cell membrane through the form of vesicles, thereby protecting the integrity of the cell membrane and preventing necroptosis\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. According to our data, ESCRT-III complex was significantly reduced at the apical membrane of renal tubular epithelial cells without Anxa2, which suggests that Anxa2 is indeed able to promote ESCRT-III accumulation at the apical membrane of renal tubular epithelial cells and inhibit necroptosis. However, how Anxa2 bound with Chmp2A and stabilizes the ESCRT-III complex at the cell membrane still needs to be further studied.\u003c/p\u003e\u003cp\u003eOur results suggest that Nec1 rescues renal IRI caused by Anxa2 deficiency and inhibits neutrophil infiltration into the injured areas. Interestingly, regarding the degree of kidney injury, Nec1-treated WT and KO mice did not differ, whereas \u003cem\u003eKim1\u003c/em\u003e and \u003cem\u003eLcn2\u003c/em\u003e were upregulated in Nec1-treated KO mice, and necroptosis was increased. This may be caused by the hysteresis of the phenotype; \u003cem\u003eKim1\u003c/em\u003e and \u003cem\u003eLcn2\u003c/em\u003e are both sensitive biomarkers that are upregulated even upon mild injury. Alternatively, necroptosis may need to accumulate to a certain extent before it can manifest itself phenotypically. In addition, the infiltration level of neutrophils was increased in Nec1-treated KO mice, indicating that necroptosis of renal tubular epithelial cells may be the primary factor contributing to renal IRI, and inflammation may be a secondary factor. There are increasing reports on the role of Nec1 in the treatment of various necroptosis-related diseases.\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e However, it also has some limitations such as off-target effects and unstable metabolism.\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e Therefore, we suggest a synergistic effect between Anxa2 and Nec1. However, further studies are required to test this hypothesis. In addition, Nec1-treated WT mice with IRI had lower levels of injury and inflammation than non-treated WT mice with IRI in the aforementioned experiments. This indicates that necroptosis indeed occurs after renal IRI under normal conditions, which indicates that increasing Anxa2 levels before NSS may better protect kidney function.\u003c/p\u003e\u003cp\u003eThis study had some limitations. First, we used only male animals for the in vivo experiments, and female animals were not included. Second, clinical resources were unavailable to further validate our hypotheses. Third, we used conventional knockout mice rather than conditional knockout mice, which may have overlooked the function of Anxa2 in other cells or tissues. Thus, further experiments are required to comprehensively verify the protective effect of Anxa2 in renal IRI and the feasibility of a clinical application of Anxa2.\u003c/p\u003e\u003cp\u003eIn summary, our study clarified the role of Anxa2 upregulation in renal IRI. Anxa2 is able to promote ESCRT-III accumulation at the apical membrane of renal tubular epithelial cells and inhibit necroptosis, reducing local neutrophil infiltration and playing a protective role in renal IRI. These findings provide a new clinical basis for the prevention and treatment of NSS-induced renal IRI caused by NSS.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMice\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study has been performed in accordance with the EU Directive 2010/63/EU for animal experiments. The animal experiments were approved by the Ethics Committee of the General Hospital of the People\u0026apos;s Liberation Army. (S2013-115-01) Male C57BL/6 wild-type (WT) mice aged 6\u0026ndash;8 weeks were purchased from Sinogenetic Biotechnology (Beijing, China). Male and female Anxa2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (in a C57BL/6 background) were produced by Cyagen Biosciences Inc (Beijing, China). Male WT and Anxa2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were used for experiments. Mice were housed in a pathogen-free, constant temperature with a 12 h light/dark cycle and allowed to acclimatize for a week in the animal facility before the operation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMouse model of renal IRI and in vivo drug treatment\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mice were subjected to unilateral renal IRI. Briefly, the mice were anesthetized with 0.3% lumbarbarbituric sodium (40 mg/kg, i.p.) on a thermostatic blanket, which maintains body temperature at approximately 37 ℃ during surgery. The left renal pedicle was clamped with an atraumatic vascular clip for 30 min, after which the clamp was released. For Nec-1 treatments, we administered intraperitoneal injections of Nec-1 (1.65 mg/kg in PBS) (S8037, Selleck) to WT and Anxa2\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice at two different time points: 15 min before and 1 h after IR surgery.\u003csup\u003e15,62-64\u003c/sup\u003e 24h after reperfusion, samples were collected for the experiments. We used a double-blind method for the experiments and data analysis. The allocation and the conduct of the experiment were performed by Dong Lai and Jichen Wang. The outcome assessment and the data analysis were performed by Huayi Feng.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRT-qPCR\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRT-qPCR was performed according to previously reported standard techniques.\u003csup\u003e65\u003c/sup\u003e The primer sequences can be found in the supplementary file.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eWestern blotting\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWestern blots were performed in accordance with standard techniques as reported previously.\u003csup\u003e66\u003c/sup\u003e Antibodies against Anxa2 (D11G2, CST), Rip1 (29932-1-AP, Proteintech), pRip1 (28252-1-AP, Proteintech), Rip3 (17563-1-AP, Proteintech), pRip3(ab222320, Abcam), Mlkl (66675-1-Ig, Proteintech), pMlkl (ab196436, Abcam), Gapdh (60004-1-Ig, Proteintech) and \u0026beta;-tubulin (BE0025, EASYBIO) were used. We used the Mem-PER Plus kit (89842Y, Thermo Scientific) to isolate the membrane and cytoplasmic proteins of kidney tissue for subsequent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePeriodic Acid-Schiff (PAS) and Masson stain\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRenal IRI was evaluated using a PAS kit (C0142M, Beyotime Biotechnology) following the manufacturer\u0026rsquo;s instructions. Fibrosis of the kidney after IRI was evaluated using Masson\u0026rsquo;s kit (C0189S, Beyotime Biotechnology). Kidney long-term fibrosis levels were scored based on the proportion of the total area occupied by collagen deposition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eImmunohistochemistry and immunofluorescence\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAntibodies against Ly6G (GB11229-100; Servicebio), F4/80 (GB113373-50; Servicebio), aSMA(14395-1-AP; Proteintech), Nkcc2 (18970-1-AP; Proteintech), Aqp2(29386-1-AP; Proteintech), Atp6v1g3(19523-1-AP; Proteintech), and Aqp1(20333-1-AP; Proteintech) were used. We followed the standard protocols described previously.\u003csup\u003e67\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFlow cytometry\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlow cytometry assays were performed in accordance with standard techniques as reported previously.\u003csup\u003e68\u003c/sup\u003e Brilliant Violet 421\u0026trade; anti-mouse CD45 Antibody (103133, Biolegend), Alexa Fluor\u0026reg; 488 anti-mouse/human CD11b Antibody (101217, Biolegend), APC anti-mouse F4/80 Recombinant Antibody (157306, Biolegend), APC/Cyanine7 anti-mouse Ly-6G Antibody (127624, Biolegend), and PE anti-mouse Ly-6C Antibody (128008, Biolegend) were used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eTransmission electron microscope\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransmission electron microscopy was performed in accordance with standard techniques, as reported previously.\u003csup\u003e69\u003c/sup\u003e Glutamate fixative was purchased from Beijing LABGIC (BL911A).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCell culture\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTcmk1 was obtained from the National Platform of Experimental Cell Resources for Sci-Tech (Beijing, China). Tcmk1 were cultured and maintained in (Minimum Essential Medium MEM (Pricella), supplemented with 10% fetal bovine serum (Hangzhou Lvyuan Biological Technology Co., Ltd.) and 1% penicillin-streptomycin solution. Cell line was passaged \u0026lt;20 times and cultured at 37 ℃ in 5% CO2 under mycoplasma-free conditions. A necroptosis inducer kit containing TSZ (TNF-\u0026alpha;, SM-164, and Z-VAD-FMK) was purchased from Beyotime Biotechnology (C1058S). Nec-1 (150 mM in DMSO) was added to the medium at a dilution of 1:1000.\u003csup\u003e70\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePlasmid construction and transfection\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe full-length coding sequences of Anxa2 was cloned into pcDNA3.1-Flag vector, and the Tcmk1 cell line was transfected with pcDNA3.1-Flag-Anxa2 plasmid. siRNA targeting Anxa2 and negative control siRNA were purchased from Genepharma (Suzhou, PR China). Transient transfection was accomplished using jetPRIME (Polyplus-transfection, Illkirch Graffenstaden, France), following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCell counting kit-8\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell proliferation was evaluated using a cell counting kit-8 assay (CK001, LABLEAD BIOTECH) following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFITC Annexin V Apoptosis Detection Kit I\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe degree of cell necroptosis was evaluated using an FITC Annexin V Apoptosis Detection Kit (556547, BD Pharmingen) following the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical analyses\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were analyzed using GraphPad Prism version 10 and expressed as mean \u0026plusmn; standard deviation of independent replicates. Dot plots, line plots, and histograms were constructed using the GraphPad Prism software, version 10. Differences between the two groups were analyzed using an unpaired Student\u0026rsquo;s t-test (two-tailed). Comparisons between multiple groups were performed using one- or two-way ANOVA. P was set at 0.05.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDisclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors declared no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (grant numbers 81802804), the Fostering Fund of Chinese PLA General Hospital for National Excellent Young Scholar Science Fund (2020-YQPY-006), the National Key Research and Development Program (2022YFB4701700), and the Growth Project of the Youth Independent Innovation Science Fund of Chinese PLA General Hospital (22QNCZ029).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDL, CJW and YHF contributed equally to this research. DL, CJW, YHF and BXL contributed to the hypothesis, study design, data analysis, and experiments. CJW, YHF and QSC contributed to experiment assistance. KHL, QB, MWF, YH and LLB contributed to the construction of Anxa2 knockout mice. XZ, XM, NJX and BXL conceived and supervised the study; DL wrote this article, all authors reviewed and approved the final version of the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Sharing Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data deposition process is in progress.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRonco, C., Bellomo, R. \u0026amp; Kellum, J. A. Acute kidney injury. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e394\u003c/strong\u003e, 1949-1964, doi:10.1016/S0140-6736(19)32563-2 (2019).\u003c/li\u003e\n\u003cli\u003eCampbell, S. 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Necroptosis plays an important role in renal IRI, however its mechanism remains unknown. We used Anxa2 knockout mice to investigate the role and mechanism of Anxa2 in renal IRI. Moreover, We further explored its underlying molecular mechanism by co-immunoprecipitation combined with mass spectrometry (IP-MS). Our results showed that Anxa2 accumulated significantly at the apical membrane of renal tubular epithelial cells of the medulla after renal IRI. In Anxa2 knockout mice, injury was significantly increased in the medulla rather than in the cortex, and necroptosis was found to be aggravated in medullary tubular epithelial cells accompanied by increased neutrophil infiltration in the injured area. Mechanistically, Anxa2 can specifically bind to Chmp2A after renal IR, an important component protein of endosomal sorting complex required for transport III (ESCRT-III). In knockout mice, membranal Chmp2A content in the renal medulla was significantly decreased, but not cytoplasmic Chmp2A. The other component proteins of the ESCRT-III complex, such as Chmp2B, Chmp3, Chmp4B and Vps4B, followed the same trend as Chmp2A. We also found that \u003cem\u003eCxcl1\u003c/em\u003eand \u003cem\u003eCxcl2\u003c/em\u003e, which are specific neutrophil chemokines, were significantly upregulated without Anxa2 expression both in vivo and in vitro. Moreover, Necrostatin 1 (Nec1), a specific necroptosis inhibitor, rescued the Anxa2 deficiency-induced necroptosis, neutrophil infiltration and IRI. This study demonstrates that Anxa2 can inhibit necroptosis of renal medullary tubular epithelial cells, reduce local neutrophil infiltration and inflammatory factors, and protect against the renal IRI. More importantly, we describe the molecular mechanism by which Anxa2 inhibits necroptosis by regulating ESCRT-III. Our study clarified the role and mechanism of Anxa2 function in renal IRI, providing new clinical strategies for its prevention and treatment.\u003c/p\u003e","manuscriptTitle":"Annexin A2 alleviates renal ischemia/reperfusion injury via regulating membranal ESCRT-III to suppress necroptosis of tubular epithelial cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-23 07:29:37","doi":"10.21203/rs.3.rs-6991722/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"921220de-9a87-4bca-8a14-b1a51826f915","owner":[],"postedDate":"July 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51670777,"name":"Biological sciences/Immunology/Cell death and immune response"},{"id":51670778,"name":"Health sciences/Diseases/Kidney diseases"}],"tags":[],"updatedAt":"2025-07-28T09:02:54+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-23 07:29:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6991722","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6991722","identity":"rs-6991722","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}