HDAC3 inhibitor RGFP966 mitigates acute kidney injury by modulating RIPK1-mediated programmed necrosis

HDAC3 inhibitor RGFP966 mitigates acute kidney injury by modulating RIPK1-mediated programmed necrosis | 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 HDAC3 inhibitor RGFP966 mitigates acute kidney injury by modulating RIPK1-mediated programmed necrosis Qi Chen, Qi Chen, man-man xie, man-man xie, Ying Chen, Ying Chen, and 18 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4256363/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 Acute kidney injury (AKI) refers to clinical syndromes culminating in sharp reduction in renal function over a short period of time because of various reasons. These syndromes manifest in the form of inflammation and apoptosis of renal tubular epithelial cells via controlled demise. Histone deacetylases are critical in renal physiology and fibrosis. Here, the HDAC3 expression was shown to be upregulated and localized predominantly in the renal tubules in an AKI mouse model. Moreover, the selective HDAC3 inhibitor RGFP966 was found to reduce inflammation and injury caused by cisplatin and hypoxia–reoxygenation in HK2 cells. Importantly, RGFP966 exerted potent protective effects in mouse models of ischemia/reperfusion-induced AKI and cisplatin. Furthermore, RNA sequencing revealed that RGFP966 significantly inhibited the upregulation of RIPK1. Cellular thermal displacement assay and molecular docking demonstrated the physical binding of RGFP966 to HDCA3. In addition, RIPK1 knockdown cell assay signified that RGFP966 directly targeted RIPK1 and inhibited RIPK1 kinase activity. In summary, these findings established the efficacy of the HDAC3 inhibitor RGFP966 in treating AKI. Biological sciences/Genetics/Epigenetics Health sciences/Diseases/Kidney diseases Biological sciences/Drug discovery/Pharmacology Biological sciences/Genetics/Epigenetics Health sciences/Diseases/Kidney diseases Biological sciences/Drug discovery/Pharmacology Acute kidney injury Necroptosis RGFP966 Inflammation HDAC3 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Acute kidney injury (AKI) refers to the rapid deterioration of renal functions; clinically, this condition presents with diversified etiologies that affects patients’ long-term prognosis as well as immediate morbidity and mortality [ 1 , 2 ]. The global incidence of AKI-related mortality is significantly higher than that of diabetes mellitus, heart disease, and breast cancer and has remained high for the past 50 years. This elevated mortality has a considerable negative impact on both the economy and society [ 3 , 4 ]. Although medical care has advanced, the death rate of patients requiring renal replacement therapy remains > 50% because specific medications to prevent or treat AKI are currently not available. Hence, supportive care and dialysis are the mainstay treatments [ 5 – 7 ]. AKI is caused by various pathophysiologic factors, including abrupt decline in renal function, inflammatory reaction, and necrotic demise of proximal renal epithelial cells in the tubule [ 8 , 9 ]. Necroptosis is a type of programmed demise of cells that occurs when the receptor-interacting enzymes RIPK1 and RIPK3 form oligomeric complexes known as “necrosomes,” which are composed of these two proteins. RIPK1 is a crucial molecular player in necrosis initiation [ 10 , 11 ]. The interplay of two enzymes, namely, histone acetyltransferases and histone deacetylases (HDACs), primarily controls acetylation—a very frequent dynamic post-translational modification of histone [ 12 , 13 ]. Several biological functions, including cell cycle regulation, proliferation, and apoptosis, as well as immune system control are influenced by HDAC enzyme inhibitors [ 14 ]. According to the literature, histone deacetylases have a critical function in renal physiology and fibrosis [ 15 – 17 ]. Silencing of HDAC1 or HDAC2 has been reported to block renal fibroblast proliferation by decreasing STAT3 phosphorylation [ 18 ]. Furthermore, the HDAC6 inhibitor tubastatin A has been observed to increase autophagy and reduce renal injury [ 19 ], and the HDAC9-selective inhibitor TMP195 has been found to attenuate renal fibrosis in UUO mice [ 20 ]. The role of HDAC3i in AKI is yet to be determined. Therefore, in this study, the expression and localization of HDAC3 were explored in an AKI model. The findings indicated that the HDAC3 inhibitor RGFP966 alleviated injury and inflammation induced by cisplatin- and hypoxia–reoxygenation (H/R) in HK2 cells. Moreover, the preventive and therapeutic impact of RGFP966 in cisplatin and ischemia/reperfusion (I/R)-induced AKI mouse models were examined using different protocols. The molecular mechanisms by which RGFP966 exerts its effects were also investigated through RNA sequencing (RNA-seq). 2. Materials and Methods 2.1 Reagents and antibodies A specific HDAC3 inhibitor, (2E)-N-(2-amino-4-fluorophenyl)-3-[(2E)-1-(3-phenyl-2-propen-1-yl)-1H-pyrazol-4-yl]-2-propenamide (RGFP966), of 99.77% purity was purchased from TargetMol. Antibodies against KIM-1 (T58586S; Abmart), HDAC3 (10255-1- AP), RIPK3 (ER190127; HUABIO), p65 (WL01273b, Wanleibio), p-p65 (WL012169, Wanleibio), pRIPK3 (ab195117, Abcam), RIPK1 (ET1701-79; HUABIO), and β-actin (GB12044; Servicebio) were procured from Proteintech. The secondary IRDye 800-conjugated antibody was purchased from LI-COR Biosciences (No. C60113-02). Cisplatin was bought from MedChemExpress (MCE). 2.2 Establishing AKI mice models Male C57BL/6J mice (weight: 20–23 g) were obtained from the Hangzhou Ziyuan Laboratory Animal Technology Co. The animals were cared for as per the humane care guidelines affiliated to the 3R principle, and the experiments were approved by the Animal Experimentation Ethics Committee of the Anhui Medical University. Animal testing was performed at the Anhui Medical University. The mice in the prevention group were pretreated with RGFP966 and injected intraperitoneally at doses of 5, 10, and 20 mg/kg body weight 12 h before the intraperitoneal injection of cisplatin (20 mg/kg), followed by the same treatment once a day for 3 consecutive days for the study of its preventive effect against AKI. Three days after receiving the cisplatin injection, the mice received isoflurane anesthesia and their blood and kidney tissues were sampled for the detection of indicators related to kidney injury and the expression of inflammation. In the mouse model of I/R-induced AKI, RGFP966 was administered intraperitoneally 12 h previously at doses of 20 mg/kg body weight. After the administration of anesthesia, the mouse was placed on a thermometer panel that maintained their body temperature at 36.5°C. The mice were subjected to a 40-min clipping of their bilateral renal pedicles using microaneurysm clamps. Following ischemic therapy, the clamps were withdrawn for a 24-h period for reperfusion, and all treated animals were executed. Sham control animals were subjected to a similar procedure but without the renal pedicle clamping. Blood and kidney tissue samples were obtained for additional examination. Paraffin sections of 4-mm thickness were stained using the periodic acid-Schiff (PAS) staining kit (C0142S, Beyotime Biotechnology, Jiangsu, China). No fewer than six distinct investigations were conducted throughout the study. 2.3 Molecular docking To decipher possible binding between RGFP966 and HDAC3, these were linked to each other. The software and instruments used in the experiment were Discovery Studio 2017 R2 (DS, BIOVIA Software, Inc., San Diego, CA, United States). The 3D model of HDAC3 (ID:4A69) was obtained from a unique protein database. 2.4 Cellular thermal shift assay (CETSA) In a 6-well plate, HK2 cells were co-cultured without or with RG (8 µM) for 2 h. After the cells were collected via trypsin digestion, the obtained mixture of trypsin, medium, and cells were aspirated into 1.5-mL centrifuge tubes, labeled with the treatment temperatures, and heated at the corresponding temperatures for 8–10 min. Subsequently, the supernatant was collected after centrifugation at 800 rpm, and the protein was extracted and analyzed by Western blotting. 2.5 Cell culture HK2 cells were purchased from Procell (Wuhan, China). These cells were placed in a DME/F-12 (1:1) medium containing 5% fetal bovine serum (FBS) and 5% CO 2 at 37℃. The cells were pretreated with RGFP966 (2, 4, and 8 µM) and then stimulated with cell starvation for 12 h when their density in the culture plate reached 50%. Next, it was co-incubated with cisplatin (20 µM) for 24 h; a matching control was also set up. The cells were incubated in a thermostat incubator (37℃, 5% CO 2 ) and a low-glucose solution containing 0.5% FBS at 37°C for 12 h to model H/R injury in vitro. The cells were then placed back in their regular environment for 6 h to allow reoxygenation. The H/R damage induction procedure was performed thrice, and the cells were harvested for analysis. 2.6 Western blotting From the crushed tissues or cells, proteins were extracted in a 6-well plate of frigid radioimmunoprecipitation assay buffer (produced by Beyotime Biotechnology, Jiangsu, China) supplemented with phenylmethylsulfonyl fluoride. The samples were transferred to nitrocellulose membranes following 10% SDS-PAGE. The films were sterilized with 5% skimmed milk and cleansed 3–4 times with Tris-buffered saline with 0.1% Tween 20. Subsequently, the selected antibody was overnight incubated with the membrane at 4°C. Next, the membrane was cleaned 3–4 times the next day and subsequently treated for 2 h at room temperature with a matching IRDye 800-coupled secondary antibody. The membranes were then treated with the selected antibody, and the western blotting strips were developed using a LiCor/Odyssey developer (LI-COR Biosciences, Lincoln, NE). 2.7 RNA extraction and real-time PCR AG RNAex Pro RNA Extraction Reagent (Accurate Biology, Hangzhou, China) was used as per the manufacturer’s recommendations for total RNA extraction from tissues or cells. Then, NanoDrop 2000 Spectrophotometer of Thermo Scientific was used to measure the amount of RNA, while RealMasterMix of TOYOBO (Japan) was used to obtain cDNA from the total RNA. Real-time PCR was performed using Bio-Rad (USA) CFX96 real-time reverse transcription-PCR detection equipment. The aforementioned primer sequences were used, and the index ratio of the desired mRNA was normalized to β-actin. 2.8 Determination of serum Cr, aspartate aminotransferase (AST), BUN, and alanine aminotransferase (ALT) levels The Cr, AST, ALT, and BUN levels in mouse blood samples were assessed by using Cr, BUN, AST, and ALT Assay Kit procured from Nanjing Jianjian Bioengineering Institute. The readings were obtained by strictly adhering to the manufacturer's recommendations. 2.9 Cell viability assay The HK2 cells containing 5% FBS DME/F-12 (1:1) digested in a 96-well plate were incubated with different concentration gradients of RG (0.5–128 µM) for 12 h after 24 h, and afterward given without or with cisplatin (10 µM) for 24 h. Next, the cells were extracted with 10 L of CCK-8 liquid to each well. Cell activity was calculated by reading the 450 nm light absorption value with an enzyme marker (Multiskan FC, Thermo) after 1–4 h. 2.10 Renal histology and Immunofluorescent staining The mouse kidney tissues were collected after fixation, dehydrated, sliced, and embedded in paraffin sections by using a fully automated rotary slicer (Micros), followed by the standardized procedure. For immunofluorescence of the kidney tissues, selected paraffin tissue sections were baked in a 65°C oven for 2 h, followed by sequential deparaffinization in xylene and a primary series of decreasing concentrations of anhydrous ethanol, rinsed in running water, and antigenically repaired using a 20-fold dilution of ethylene diamine tetraacetic acid (EDTA) in a microwave oven. After cooling, circles were drawn and closed with BSA for 30 min, after which the sections were incubated with the desired primary antibody at 4°C overnight. On the second day, the primary antibody was removed, the configured secondary antibody was added, and the results were analyzed by fluorescence microscopy (Leica Microsystems GmbH., Wetzlar, Germany) and then photographed. 2.11 Immunofluorescent staining Eight-chamber glass slides were used to culture the HK2 cells, and subsequently, they were fixed in acetone. The cells were then exposed to diluted (1:200) antibodies against KIM-1 or TNF-α overnight. Cow anti-rabbit IgG-rhodamine from Bioss Antibody Inc. was applied for 2 h at ambient temperatures, after which the cells were rinsed with PBS. 4′,6-Diamidino-2-phenylindole was used as a counterstain, and Leica fluorescence microscopy was used to visualize the cells. 2.12 Transcriptome sequencing (RNA-seq) The TRIzol lysate stored in the refrigerator was taken out and added to the cells treated in advance while avoiding light exposure. The dosage of the lysate per well was 0.5–1.0 mL for 20 s. The solution was observed to be clear upon the naked examination eye, and no cells were observed under the microscope. Afterward, the RNA sample was sent to BGI (BGI Genomics, Shengzhen, China) for sequencing and analysis. Furthermore, the expression patterns and pathways of differentially expressed genes were scrutinized. Subsequently, the candidate genes were cherry-picked to corroborate the expression variation. 2.13 Transmission electron microscopy (TEM) Cultured HK2 cells were removed by fixation with 2.5% glutaraldehyde for 4—12 h, after which they were rinsed and immersed thrice for 10 min each time in 0.1 M phosphate buffer at room temperature. Next, dehydration was performed with a primary increasing concentrations of ethanol and then wrapped with LR White resin (manufactured by London Resin Company, Reading, UK). The obtained materials were then observed by TEM (H-7700, Tokyo, Japan). 2.14 Statistical analyses GraphPad Prism 8 (GraphPad, La Jolla, CA, U.S.A.) was applied for data analyses. *P < 0.05; **P < 0.01; ***P < 0.001; # P < 0.05; ## P < 0.01; ### P < 0.001. Data were expressed as either mean ± standard error (SEM) for 6–8 mice or 3–4 independent experiments. 3. Results 3.1 HDAC3 expression is increased in the mouse AKI model and in the in vitro HK2 cell injury The mouse AKI model, which was produced by injecting cisplatin intraperitoneally and achieving I/R, was found to express HDAC3. The normal group received saline injections and underwent sham surgery. HDAC3 protein and mRNA levels were significantly increased in the mouse AKI model relative to that with the control group according to real-time PCR and Western blotting (Figs. 1 A and 1 B). Immunohistochemical analyses further confirmed this finding (Fig. 1 C). Mouse kidney sections were double fluorescently stained with HDAC3 and proximal tubular marker fluorescent labeling (Leptospirillum lucidum, LTL). The results showed the elevated expression of HDAC3 (red staining), and proximal renal tubules were labeled with LTL (green staining) in the mouse AKI model compared with the kidneys of control mice. This observation indicated the localization of HDAC3 in the renal tubules (Fig. 1 C). The in vivo study confirmed that under the action of cisplatin HDAC3 expression and HDAC3 positioning on the proximal renal tubule, so in the in vitro experiments mainly in renal tubular epithelial cells as the research object. Based on the in vivo results, the HK2 cells were cisplatin-treated to produce AKI, while the protein and mRNA levels of HADC3 were elevated in the model group. This observation was in contrast to the finding in the untreated group (Figs. 1 D and 1 E). Immunofluorescence staining was further used to establish that the HK2 cells in the model group had higher levels of HDAC3 expression than those in the normal group. (Fig. 1 F). A second model of AKI cell injury was also constructed, that is, the H/R-induced AKI model. Western blotting, real-time PCR, and immunofluorescence results showed that H/R-induced AKI increased the expression of HDAC3 (Figs. 1 D, 1 E and 1 F). 3.2 RG reduces cisplatin-induced HK2 cell damage and inflammation The CCK8 assay was performed to assess the protective effect of RG against cisplatin-induced AKI. Initially, the effect of RG on HK2 cell viability was investigated (Fig. 2 B). CCK-8 results indicated that RG alone did not damage HK2 cells, and different concentrations of RG (2, 4, and 8 µM) restored cisplatin-induced inhibition of cell growth (Fig. 2 C). This finding suggests that RG can restore cisplatin-induced inhibition of cell proliferation. Next, KIM-1, a marker for assessing renal tubular injury, was detected in RG-treated cisplatin-induced HK2 cells. Western blotting and real-time PCR results signified that the KIM-1 expression in cisplatin-induced HK2 cells was reduced by all three doses of RG. (Figs. 2 D and 2 E). Furthermore, immunofluorescence analysis confirmed the effective inhibition of KIM-1 by RG (Fig. 2 F). In addition, Western blotting results indicated that RG could decrease the expression of the cisplatin-induced p-p65 protein in HK2 cells (Fig. 2 G). According to real-time PCR, RG dramatically reduced the inflammation-related gene expression for monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β (Fig. 2 H). Cumulatively, based on our results, RG alleviates HK2 inflammatory response induced by cisplatin. 3.3 RG reduces HK2 cell damage and inflammation induced by H/R According to the results of real-time PCR and Western blotting, the expression of KIM-1 protein and mRNA were significantly upregulated in H/R-induced HK2 cells, but this upregulation was prevented via RG pretreatment (Figs. 3 A and 3 B). Moreover, the results of real-time PCR demonstrated RG-assuaged cellular inflammation induced by H/R (Fig. 3 B). Following RG pretreatment, the results of Western blot indicated decreased p-p65 protein levels in H/R-induced HK2 cells (Fig. 3 A). In addition, immunofluorescence staining suggested that RG dramatically decreased the levels of KIM-1 in H/R-induced HK2 cells (Fig. 3 C). 3.4 RG alleviates AKI and inflammation in cisplatin-induced mice Further investigations were conducted using an animal model to examine how RG affected cisplatin-induced AKI. Before cisplatin injection, mice in the treatment group were pretreated with RG (5, 10, and 20 mg/kg) for 12 h. The same treatment was then administered once daily for 3 d. Detection of the Cr and BUN levels in the animal model indicated the effective therapeutic benefits of RG against AKI (Figs. 4 A and 4 B). Hematoxylin–eosin (H&E) and PAS staining of renal tissue sections demonstrated that RG attenuated cisplatin-induced renal pathologies, such as tubular dilatation, in mice. (Figs. 4 C, 4 D, and 4 E). In addition, immunofluorescence, real-time PCR, and Western blot results suggested that RG dramatically decreased the expressions of mRNA and KIM-1 protein (Figs. 4 F, 4 G, and 4 H). Moreover, the anti-inflammatory effect of RG was assessed in cisplatin-induced animal models of inflammation. Western blot findings suggested that RG decreased the upregulation of p-p65 protein expression induced by cisplatin in vivo . Real-time PCR indicated that RG decreased the expressions of numerous markers linked to inflammation, including the mRNA levels of MCP-1 and TNF-α (Figs. 4 F and 4 G). Moreover, immunofluorescence analysis of paraffin sections alluded that RG suppressed the positive TNF-α signal in damaged kidneys (Fig. 4 H). 3.5 RG attenuates the acute renal damage and inflammation caused by IRI in vivo In mice, RG attenuated the increase in serum Cr and BUN levels caused by IRI (Figs. 5 A and 5 B). PAS staining of renal tissue sections indicated tubular dilatation and necrosis in the renal tissues of IRI-treated mice, which were alleviated by RG pretreatment (Fig. 5 E). According to the results of Western blotting and real-time PCR, RG pretreatment in the AKI mouse model inhibited the KIM-1 upregulation caused by IRI (Figs. 5 C and 5 D). These findings confirmed the previous results. Western blot analyses established that RG reduced the p-p65 protein expressions in the IRI model (Fig. 5 C). According to real-time PCR, RG might decrease the mRNA expressions of inflammation-related molecules, such as MCP-1 (Fig. 5 D). Furthermore, immunofluorescence analysis of paraffin sections confirmed these observations (Fig. 5 F). 3.6 RG attenuates cisplatin-induced programmed necrosis of HK2 cells According to the results of RNA-seq-based analysis and KEGG pathway enrichment analysis, the programmed necrosis pathway ranked high (Fig. 6 B). When this pathway was analyzed, the heatmap showed a significant alteration in classical RIPK1 signaling (Fig. 6 C). Moreover, real-time PCR suggested that RG prevented cisplatin from inducing RIPK1 and RIPK3 signaling (Fig. 6 F). In addition, molecular docking and CETSA analysis established that RG enhanced the stability of HDAC3 in vivo (Figs. 6 D and 6 E). Western blotting results indicated that RG pretreatment reduced the expressions and activation status (phosphorylation) of RIPK1 and RIPK3 in cisplatin-induced HK2 cells (Fig. 6 H). Immunofluorescence staining of P-RIPK1 and P-MLKL confirmed this finding (Figs. 6 J). In addition, immunohistochemical analysis of renal tissues from the IRI model showed that RG pretreatment reduced phospho-RIPK1 (pRIPK1) signaling in the IRI-induced mouse model (Fig. 6 G). Moreover, TEM results indicated the appearance of programmed necrotic features in HK2 cells, such as nuclear membrane crumpling, widening, cell membrane dehiscence, mitochondrial swelling, and deformation, in the group treated with cisplatin. However, these changes were alleviated in the group pretreated with RG (Fig. 6 I). 3.7 RG exerts a protective effect by regulating RIPK1-mediated programmed necrosis To verify whether RG exerts a protective effect via RIPK1 production, RIPKI expression was knocked down in HK2 cells. Moreover, according to the results of real-time PCR and Western blotting, small-interfering RNA (siRNA) significantly reduced RIPK1 protein and mRNA levels (Figs. 7 A and 7 B). Additionally, real-time PCR, immunofluorescence, and Western blotting findings suggested that RIPK1 knockdown abolished the impact of RG (Figs. 7 D, 7 E, and 7 F). These results allude that RG controls RIPK1-mediated programmed necrosis to reduce acute kidney damage. 3.8 Effects of RG on the heart, liver, spleen, and lungs of normal mice Mice were administered RG (20 mg/kg) or saline (medium control) to test the effects of the toxin on different organs. Variations were not detected in serum ALT or AST levels between the groups receiving RG and saline (Figs. 8 A and 8 B). H&E staining was performed to assess the impact on various other organs, including the liver, heart, spleen, and lungs. In contrast to the mediator group, the organs obtained from mice receiving RG did not exhibit any variations, thus validating its safety at the prescribed dosage of 20 mg/kg (Fig. 8 C). 4. Discussion This study showed that HDAC3 is crucial for the development of AKI. In vivo studies indicated that HDAC3 exacerbates kidney damage in mice with cisplatin-induced AKI. Additionally, the protective effect of the HDAC3 inhibitor RG was established in both laboratory and animal model settings. Our investigations demonstrated that RG acts against cisplatin and H/R-induced AKI in HK2 cells and C57BL/6 mice. Moreover, from RNA-seq results, it was evident that RG is beneficial in attenuating AKI, both in vitro and in vivo , by preventing inflammation and programmed necrosis. Additionally, RG was proven to be an effective modulator of RIPK1-mediated programmed necrosis, thereby ameliorating cisplatin-induced AKI(Figures 9 ). Altogether, these findings implicate that RG may be a potential candidate for treating AKI. First, in this research, HDAC3 was found to be significantly upregulated in mice with cisplatin-induced AKI, especially in proximal renal tubular epithelial cells. This finding was confirmed in the in vitro model of cisplatin- and H/R-induced AKI. Histone deacetylases and acetyltransferases are primarily responsible for regulating histone modifications and are vital for cell survival, homeostasis, cell proliferation, and gene expression [ 21 – 22 ]. Inhibition of HDAC enzymes as a therapeutic approach to diseases has attracted immense attention in recent years. Designing small-molecule HDAC inhibitors for numerous disorders, including cancer, is an emerging area of research [ 23 ]. These inhibitors include class I HDAC (1, 2, 3, and 8), class IIa HDAC (4, 5, 7, and 9), and class IIb HDAC (6, 10) [ 24 – 28 ]. Although the role of HDAC3-selective inhibitors in treating renal fibrosis has been explored and appears to be a viable option, their potential role in AKI and the underlying mechanism are yet to be determined [ 29 ]. Second, in animal models with AKI induced by cisplatin and I/R, RG decreased the inflammation dramatically. These findings were further supported by in vitro research. AKI frequently results in inflammation due to triggers such as ischemia and hypoxia, infection, and nephrotoxic medications [ 9 ]. Severe or persistent AKI often leads to chronic inflammation, fibrosis in the kidneys, tubular degeneration, and finally chronic kidney disease (CKD) [ 30 – 31 ]. Therefore, treating AKI and preventing its transition to CKD via anti-inflammatory therapy is a promising strategy to reduce renal damage. By preventing IGFBP7/IGF1-mediated programmed cell death and inflammation, we previously demonstrated that gibberellin combined with saponin may prevent AKI [ 32 ]. Cpd-6c, a rutaecarpine derivative that targets PDE4B, is an important regulator driving irritation in cisplatin-induced kidney disease and has been reported to alleviate AKI [ 8 ]. Protocatechuic aldehyde has been shown to reduce AKI by inhibiting inflammation and oxidative stress in animal models [ 33 ]. This finding confirmed the significance of inflammation in mediating AKI. Here, our findings indicated that RG had a beneficial impact on inflammation both in vitro and in vivo by inhibiting the expression of numerous inflammatory factors, such as MCP-1. Additionally, NF-κB p65 activation, a well-known mechanism in renal inflammation, is drastically inhibited by RG. Third, the present study suggested that RG prevents programmed necrosis both in vivo and in vitro. Necroptosis, also known as programmed cell necrosis, is a regulated inflammatory cell death mechanism mediated by the activation of RIPK1, RIPK3, and MLKL. We noted that the cell swells, its membrane tears, and the cytoplasmic contents are released during this type of cell death, which differs morphologically from apoptosis (cell death) [ 34 ]. The usual trigger for necrotic apoptosis is external stimulation. When a ligand, such as TNF-α, binds to the death receptor on the cell membrane, it results in cell death. The binding proteins TNFR1, Fas/CD95, DR4/TRAIL-R1, and DR5/TRAIL-R2 belong to the TNF superfamily. These receptors, in their active form, bind to articulatory proteins TRADD and TRAF2, thereby delaying the initiation of receptor-interacting protein kinase [ 35 – 36 ]. Pathogen activation of PRRs, such as toll-like receptors, which in turn activate the interacting, RIPK1-independent, but RIPK3-activating articulators TRIF and ZBP-1 are additional causes of necroptotic apoptosis [ 34 , 37 ]. The fact that RIPK1 is tightly controlled and involved in the initiation of NF-κB p65 signaling; moreover, the complexes of survival, apoptosis, or necrotic apoptosis should be recorded [ 34 , 38 ]. Our research team has been investigating RIPK1/RIPK3 and its role in necrotic apoptosis. Our prior study showed that the RIPK1 antagonist Cpd-71 prevents cisplatin-induced renal insufficiency by reducing necrotic apoptosis and inflammation [ 39 ]. The findings of the present study implied that RG has renal protective effects. The mechanism of action was further examined, and RNA-seq analysis showed that the necrotic and inflammatory pathways ranked high in the KEGG pathway enrichment analysis. Therefore, it is hypothesized that RG may exert its protective effect by attenuating programmed necrosis. The results of Western blotting and real-time PCR of RIPK1 and RIPK3 confirmed this hypothesis. In addition, RIPKI-knockdown experiments demonstrated that RG attenuated AKI by regulating RIPK1-mediated programmed necrosis. Overall, RG attenuated cisplatin and IRI-induced AKI in vivo , necroptosis induced by cisplatin and H/R in vitro , and suppressed inflammation, thereby alleviating HK2 injury. Moreover, the administration of RG had no adverse effects on the heart, liver, spleen, or lung tissues of normal C57BL/6 mice. These findings suggest that RG is a potentially safe clinical candidate for treating AKI. However, the specific regulatory mechanism of HDAC3 and RIPK1 needs to be explored further. Abbreviations Cr Creatinine RG RGFP966 IF Immunofluorescence HK2 Human kidney tubular epithelial cells H/R Hypoxia/reoxygenation IRI Ischemia Reperfusion Injury CKD Chronic Kidney Disease I/R Ischemia/reperfusion TNF-α Tumor Necrosis Factor-α CETSA Cellular thermal shift assay Cis Cisplatin RNA-seq RNA Sequencing RIPK1 Receptor-Interacting Protein Kinase1 RIPK3 Receptor-Interacting Protein Kinase3 PAS Periodic acid schiff HE Hematoxylin and eosin BUN Blood urea nitrogen HDAC3i Histone deacetylase 3 inhibitor AST Aspartate aminotransferase ALT Alanine aminotransferase AKI Acute kidney injury . Declarations Author contributions Q.C., S.-f.Z. and M.-m.X. designed the study. M.-m.X. performed most of the experiments. M.-m.X. and Y.C. performed the bioinformatic analysis. M.-m.X., Y.C.,R.-r.S., J.-t.Y.,X.-f.L., R.H., and.Z.-h.D. performed animal studies. M.-m.X., Y.C., M.-m.L., L.G., and Y.-h.D interpreted the results. M.-m.X., and Q.C. wrote the manuscript. All authors read and approved the final manuscript. Acknowledgments We thank the Center for Scientific Research of Anhui Medical University for valuable assistance. Funding This work was supported by the Anhui Provincial Natural Science Foundation (No. 2308085MC68，2208085QH240), National Natural Science Foundation of China(NO. 41806158)and University Natural Science Research Project of Anhui Province (NO. 2022AH050710). Competing interests The authors have no financial conflicts of interest. References Pickkers P, Darmon M, Hoste E, Joannidis M, Legrand M, Ostermann M, et al. Acute kidney injury in the critically ill: an updated review on pathophysiology and management. Intensive Care Med. 2021;47:835–850. Ronco C, Bellomo R, Kellum JA. Acute kidney injury. Lancet. 2019;394:1949–1964. Lewington AJ, Cerdá J, Mehta RL. Raising awareness of acute kidney injury: a global perspective of a silent killer. Kidney Int. 2013;84:457–67. Hoste EAJ, Kellum JA, Selby NM, Zarbock A, Palevsky PM, Bagshaw SM,et al. Global epidemiology and outcomes of acute kidney injury. Nat Rev Nephrol. 2018;14:607–625. Zuk A, Bonventre JV. Acute kidney injury: Can remote ischaemic preconditioning prevent AKI? Nat Rev Nephrol. 2015;11:512–3. Levey AS, James MT. Acute Kidney Injury. Ann Intern Med. 2018;168:837. Kellum JA, Romagnani P, Ashuntantang G, Ronco C, Zarbock A, Anders HJ. Acute kidney injury. Nat Rev Dis Primers. 2021;7:52. Liu XQ, Jin J, Li Z, Jiang L, Dong YH, Cai YT, et al. Rutaecarpine derivative Cpd-6c alleviates acute kidney injury by targeting PDE4B, a key enzyme mediating inflammation in cisplatin nephropathy. Biochem Pharmacol. 2020;180:114132. Gao L, Liu MM, Zang HM, Ma QY, Yang Q, Jiang L, et al. Restoration of E-cadherin by PPBICA protects against cisplatin-induced acute kidney injury by attenuating inflammation and programmed cell death. Lab Invest. 2018;98:911–923. Frank D, Vince JE. Pyroptosis versus necroptosis: similarities, differences, and crosstalk. Cell Death Differ. 2019;26:99–114. Grootjans S, Vanden Berghe T, Vandenabeele P. Initiation and execution mechanisms of necroptosis: an overview. Cell Death Differ. 2017;24:1184–1195. Zhao C, Dong H, Xu Q, Zhang Y. Histone deacetylase (HDAC) inhibitors in cancer: a patent review (2017-present). Expert Opin Ther Pat. 2020;30:263–274. Li Y, Seto E. HDACs and HDAC Inhibitors in Cancer Development and Therapy. Cold Spring Harb Perspect Med. 2016;6:a026831. Cheshmazar N, Hamzeh-Mivehroud M, Nozad Charoudeh H, Hemmati S, Melesina J, Dastmalchi S. Current trends in development of HDAC-based chemotherapeutics. Life Sci. 2022;308:120946. Brilli LL, Swanhart LM, de Caestecker MP, Hukriede NA. HDAC inhibitors in kidney development and disease. Pediatr Nephrol. 2013;28:1909–21. Fontecha-Barriuso M, Martin-Sanchez D, Ruiz-Andres O, Poveda J, Sanchez-Niño MD, Valiño-Rivas L, et al. Targeting epigenetic DNA and histone modifications to treat kidney disease. Nephrol Dial Transplant. 2018;33:1875–1886. Hyndman KA. Histone Deacetylases in Kidney Physiology and Acute Kidney Injury. Semin Nephrol. 2020;40:138–147. Hadden MJ, Advani A. Histone Deacetylase Inhibitors and Diabetic Kidney Disease. Int J Mol Sci. 2018;19:2630. Tang J, Shi Y, Liu N, Xu L, Zang X, Li P, et al.Blockade of histone deacetylase 6 protects against cisplatin-induced acute kidney injury. Clin Sci (Lond). 2018;132:339–359. Zhang Y, Yang Y, Yang F, Liu X, Zhan P, Wu J,et al. HDAC9-mediated epithelial cell cycle arrest in G2/M contributes to kidney fibrosis in male mice. Nat Commun. 2023;14:3007. Amin SA, Adhikari N, Kotagiri S, Jha T, Ghosh B. Histone deacetylase 3 inhibitors in learning and memory processes with special emphasis on benzamides. Eur J Med Chem. 2019;166:369–380. Sarkar R, Banerjee S, Amin SA, Adhikari N, Jha T. Histone deacetylase 3 (HDAC3) inhibitors as anticancer agents: A review. Eur J Med Chem. 2020;192:112171. Amin SA, Adhikari N, Jha T. Is dual inhibition of metalloenzymes HDAC-8 and MMP-2 a potential pharmacological target to combat hematological malignancies? Pharmacol Res. 2017;122:8–19. Li W, Ali T, Zheng C, Liu Z, He K, Shah FA, et al. Fluoxetine regulates eEF2 activity (phosphorylation) via HDAC1 inhibitory mechanism in an LPS-induced mouse model of depression. J Neuroinflammation. 2021;18:38. de la Fuente Revenga M, Ibi D, Saunders JM, Cuddy T, Ijaz MK, Toneatti R,et al. HDAC2-dependent Antipsychotic-like Effects of Chronic Treatment with the HDAC Inhibitor SAHA in Mice. Neuroscience. 2018;388:102–117. Huang C, Lin Z, Liu X, Ding Q, Cai J, Zhang Z,et al. HDAC4 Inhibitors as Antivascular Senescence Therapeutics. Oxid Med Cell Longev. 2022;2022:3087916. Yang J, Gong C, Ke Q, Fang Z, Chen X, Ye M,et al. Insights Into the Function and Clinical Application of HDAC5 in Cancer Management. Front Oncol. 2021;11:661620. Yang W, Feng Y, Zhou J, Cheung OK, Cao J, Wang J,et al. A selective HDAC8 inhibitor potentiates antitumor immunity and efficacy of immune checkpoint blockade in hepatocellular carcinoma. Sci Transl Med. 2021;13:eaaz6804. Chen F, Gao Q, Wei A, Chen X, Shi Y, Wang H,et al. Histone deacetylase 3 aberration inhibits Klotho transcription and promotes renal fibrosis. Cell Death Differ. 2021;28:1001–1012. Sato Y, Yanagita M. Immune cells and inflammation in AKI to CKD progression. Am J Physiol Renal Physiol. 2018;315:F1501-F1512. Ferenbach DA, Bonventre JV. Mechanisms of maladaptive repair after AKI leading to accelerated kidney ageing and CKD. Nat Rev Nephrol. 2015;11:264–76. Yang Q, Zang HM, Xing T, Zhang SF, Li C, Zhang Y,et al.Gypenoside XLIX protects against acute kidney injury by suppressing IGFBP7/IGF1R-mediated programmed cell death and inflammation. Phytomedicine. 2021;85:153541 Gao L, Wu WF, Dong L, Ren GL, Li HD, Yang Q,et al. Protocatechuic Aldehyde Attenuates Cisplatin-Induced Acute Kidney Injury by Suppressing Nox-Mediated Oxidative Stress and Renal Inflammation. Front Pharmacol. 2016;7:479. Bertheloot D, Latz E, Franklin BS. Necroptosis, pyroptosis and apoptosis: an intricate game of cell death. Cell Mol Immunol. 2021;18:1106–1121. Lawlor KE, Khan N, Mildenhall A, Gerlic M, Croker BA, D'Cruz AA,et al. RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL. Nat Commun. 2015;6:6282. Ketelut-Carneiro N, Fitzgerald KA. Apoptosis, Pyroptosis, and Necroptosis-Oh My! The Many Ways a Cell Can Die. J Mol Biol. 2022;434:167378. Frank D, Vince JE. Pyroptosis versus necroptosis: similarities, differences, and crosstalk. Cell Death Differ. 2019;26:99–114. Xu D, Zou C, Yuan J. Genetic Regulation of RIPK1 and Necroptosis. Annu Rev Genet. 2021;55:235–263. Wang JN, Liu MM, Wang F, Wei B, Yang Q, Cai YT,et al. RIPK1 inhibitor Cpd-71 attenuates renal dysfunction in cisplatin-treated mice via attenuating necroptosis, inflammation and oxidative stress. Clin Sci (Lond). 2019;133:1609–1627. Additional Declarations (Not answered) Supplementary Files SupplementaryTable.doc 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4256363","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":297513997,"identity":"c37facef-229f-4136-8fd2-7df3f43e61b2","order_by":0,"name":"Qi 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08:20:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4256363/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4256363/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56101629,"identity":"52b5ed50-73fa-4f08-a971-0996efcab7a2","added_by":"auto","created_at":"2024-05-08 14:44:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1934854,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncreased HDAC3 expression in a mouse AKI model showcasing \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e renal tubular epithelial cell injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. The HDAC3 protein levels were determined by Western blotting of cisplatin-induced AKI mice. B. The HDAC3 mRNA levels in mice with cisplatin-induced AKI were determined by real-time PCR. C. The HDAC3 expression and localization were determined through immunofluorescence analyses in AKI mice. D. The HDAC3 protein levels in HK2 cells with cisplatin or H/R-induced AKI by Western blotting. E. The real-time PCR results of HDAC3 in cisplatin or H/R-induced HK2 cell. F. Immunofluorescence of HDAC3 in cisplatin or H/R-induced HK2 cells. Data are presented as the mean ±S.E.M. for at least 3–4 independent experiments or at least 6 mice. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001, relative to the control.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4256363/v1/f4c45cf9248ca0e6293c666d.png"},{"id":56101627,"identity":"cb74d819-bc6b-47cf-99ef-362a2d619946","added_by":"auto","created_at":"2024-05-08 14:44:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":805297,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRG reduces HK2 cell damage and inflammation induced by cisplatin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. The molecular structural formula of RG. B. The effect of different concentrations of RG on HK2 cell viability C. RG restored the cell viability of cisplatin-treated HK2 cells D. Protein blotting analysis for detecting the expression of KIM-1 in HK2 cells E. Real-time PCR for KIM-1 expression analysis in HK2 cells. F. Fluorescent expression of KIM-1. G. Western blotting to determine p-p65 expression in HK2 cells induced by cisplatin. H. Real-time PCR analysis to determine the levels of MCP-1, TNF-α, IL-6, and IL-1β mRNA in cells. Data are presented as the mean ±S.E.M. for at least 3–4 independent experiments. Relative to the normal group: *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001. Relative to the cisplatin group: \u003csup\u003e#\u003c/sup\u003eP \u0026lt; 0.05; \u003csup\u003e##\u003c/sup\u003eP \u0026lt; 0.01; \u003csup\u003e###\u003c/sup\u003eP \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4256363/v1/a925ee95b770c3172213c939.png"},{"id":56101624,"identity":"2bca94b3-ec3f-4ed0-a998-b29c5fc268b5","added_by":"auto","created_at":"2024-05-08 14:44:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":350499,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRG reduces HK2 cell damage and the inflammation induced by H/R.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Western blotting results of KIM-1 and p-p65 in HK2 cells. B. The RNA expression of KIM-1, MCP-1, TNF-α, IL-6, and IL-1β. C. The detection of the KIM-1 expression in HK2 cells by immunofluorescence analysis. Data are presented as the mean ±S.E.M. of at least 3 independent experiments. Relative to the normal group: *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001. Data relative to the H/R-induced group: \u003csup\u003e#\u003c/sup\u003eP \u0026lt; 0.05; \u003csup\u003e##\u003c/sup\u003eP \u0026lt; 0.01; \u003csup\u003e###\u003c/sup\u003eP \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4256363/v1/118faa704945fb36217a6bcf.png"},{"id":56102730,"identity":"546e2a48-25ec-4dc4-a5e6-ff3393157545","added_by":"auto","created_at":"2024-05-08 15:00:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3777680,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRG alleviates AKI and inflammation in cisplatin-induced mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA and B. Creatinine and urea nitrogen assays for renal functions. C, D, and E. The HE and PAS staining outcomes indicated that RG attenuated tubular dilatation and cast formation after cisplatin-induced nephropathy. F. Western blotting to detect the expression of KIM-1 and p-p65. G. Real-time PCR to determine the inflammatory index expression. The mRNA content changes in KIM-1, MCP-1, TNF-α, IL-1β, and IL-6. H. Immunofluorescence in paraffin section analysis revealed that RG decreased the KIM-1 and TNF-α expression in kidney injury. Data are presented as the mean ±S.E.M. of at least 6 independent mice. Data relative to the saline group: *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001. Data relative to the cisplatin group: \u003csup\u003e#\u003c/sup\u003eP \u0026lt; 0.05; \u003csup\u003e##\u003c/sup\u003eP \u0026lt; 0.01; \u003csup\u003e###\u003c/sup\u003eP \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4256363/v1/fcfb9a418caac4e1a472597f.png"},{"id":56102227,"identity":"e19bbba6-0fdf-43e0-bae6-158846274d87","added_by":"auto","created_at":"2024-05-08 14:52:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2275695,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRG attenuates the acute renal damage and inflammation induced by IRI \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA and B. The levels of creatinine and urea nitrogen. C. Western blotting to detect the KIM-1 and p-P65 expression after renal ischemia-reperfusion injury. D. RG significantly blocked IRI-induced upregulation of renal KIM-1, MCP-1, TNF-α, and IL-1β mRNA. E. PAS staining and scoring analysis. F. Immunofluorescence analysis of paraffin section analysis for KIM-1 and TNF-α expression. Data are presented as the mean ±S.E.M. of at least 6 independent mice. Data relative to the saline group: *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001. Data relative to the IRI-induced group: \u003csup\u003e#\u003c/sup\u003eP \u0026lt; 0.05; \u003csup\u003e##\u003c/sup\u003eP \u0026lt; 0.01; \u003csup\u003e###\u003c/sup\u003eP \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4256363/v1/4b1e677ea4c83f465565a007.png"},{"id":56101630,"identity":"05e9d588-d261-4a5b-a4e9-795665cae331","added_by":"auto","created_at":"2024-05-08 14:44:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3615812,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRG attenuates cisplatin-induced programmed necrosis of HK2 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Differential Gene Volcano Maps. B. RNASeq KEGG ENrichment Scattter. C. Heat map of the necrotic pathways. D. Molecular docking analysis revealed the physical binding of RG to HDAC3. E. CETSA analyzed the stability of HDAC3 in HK2 cells without and with RG treatment. Stability of HDAC3 in HK2 cells without and with RG treatment. G. Real-time PCR detection of the RIPK1/RIPK3 RNA expression in the cisplatin-treated HK2 cells. H. The detection of the RIPK1/RIPK3 expression and activation in cisplatin-treated HK2 cells. The levels in cisplatin-treated HK2 cells. G. Immunohistochemical analysis of P-RIPK1 in the kidneys of I/R-induced AKI mice. I. Representative electron micrographs of necrotic HK2 cells are shown. J. Immunofluorescence tests of P-RIPK1 and P-MLKL in cisplatin-treated HK2 cells. \u0026nbsp;Data are presented as the mean ±S.E.M. of at least three independent experiments or at least 6 mice. *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001, relative to the NC group. \u003csup\u003e#\u003c/sup\u003eP \u0026lt; 0.05; \u003csup\u003e##\u003c/sup\u003eP \u0026lt; 0.01; \u003csup\u003e###\u003c/sup\u003eP \u0026lt; 0.001 relative to the treated group.\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4256363/v1/7e08f82a1a8048a0deae05b3.png"},{"id":56101631,"identity":"0d8e37f2-6e59-4eb1-aa57-ad71c44c652d","added_by":"auto","created_at":"2024-05-08 14:44:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":343734,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRG exerts a protective effect by regulating RIPK1-mediated programmed necrosis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA and B. Protein and mRNA levels of RIPK1. C, D, and E. Western blotting, real-time PCR, and immunofluorescence analysis of molecules related to kidney injury.\u003c/p\u003e\n\u003cp\u003eData are presented as the mean ±S.E.M. of at least 3 independent experiments ±S.E.M. Relative to the normal group: *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001. Data relative to the cisplatin group: \u003csup\u003e#\u003c/sup\u003eP \u0026lt; 0.05; \u003csup\u003e##\u003c/sup\u003eP \u0026lt; 0.01; \u003csup\u003e###\u003c/sup\u003eP \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4256363/v1/5ca6d0737cd3f53e477872d7.png"},{"id":56102230,"identity":"d291ee60-c2b1-4f87-9acf-b707da1ad279","added_by":"auto","created_at":"2024-05-08 14:52:01","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2339954,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of RG on the heart, liver, spleen, and lungs of normal mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA and A and B. The AST and ALT levels. C. HE staining. Data are presented as the mean ±SEM for 6–8 independent experiments.\u003c/p\u003e","description":"","filename":"figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4256363/v1/813c337fd62b7dc62c30b92a.png"},{"id":56102229,"identity":"51b81106-cc74-496e-ba97-d9ee51d5c5b8","added_by":"auto","created_at":"2024-05-08 14:52:01","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1065743,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHDAC3 inhibitor RGFP966 mitigates acute kidney injury by modulating RIPK1-mediated programmed necrosis.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-4256363/v1/bd70608939a9befc91c56503.png"},{"id":56104292,"identity":"2bd0ab0b-695c-4240-8ea9-8ccdfcea3f72","added_by":"auto","created_at":"2024-05-08 15:16:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5694987,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4256363/v1/e78a3d99-092b-4a5e-a48c-feab42863f5a.pdf"},{"id":56101623,"identity":"f2dcca56-c986-4cef-ab07-680ad0b54a6e","added_by":"auto","created_at":"2024-05-08 14:44:01","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":35328,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SupplementaryTable.doc","url":"https://assets-eu.researchsquare.com/files/rs-4256363/v1/1c7aab35918e69e02388b17b.doc"}],"financialInterests":"(Not answered)","formattedTitle":"HDAC3 inhibitor RGFP966 mitigates acute kidney injury by modulating RIPK1-mediated programmed necrosis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAcute kidney injury (AKI) refers to the rapid deterioration of renal functions; clinically, this condition presents with diversified etiologies that affects patients\u0026rsquo; long-term prognosis as well as immediate morbidity and mortality [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The global incidence of AKI-related mortality is significantly higher than that of diabetes mellitus, heart disease, and breast cancer and has remained high for the past 50 years. This elevated mortality has a considerable negative impact on both the economy and society [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Although medical care has advanced, the death rate of patients requiring renal replacement therapy remains\u0026thinsp;\u0026gt;\u0026thinsp;50% because specific medications to prevent or treat AKI are currently not available. Hence, supportive care and dialysis are the mainstay treatments [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. AKI is caused by various pathophysiologic factors, including abrupt decline in renal function, inflammatory reaction, and necrotic demise of proximal renal epithelial cells in the tubule [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Necroptosis is a type of programmed demise of cells that occurs when the receptor-interacting enzymes RIPK1 and RIPK3 form oligomeric complexes known as \u0026ldquo;necrosomes,\u0026rdquo; which are composed of these two proteins. RIPK1 is a crucial molecular player in necrosis initiation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The interplay of two enzymes, namely, histone acetyltransferases and histone deacetylases (HDACs), primarily controls acetylation\u0026mdash;a very frequent dynamic post-translational modification of histone [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Several biological functions, including cell cycle regulation, proliferation, and apoptosis, as well as immune system control are influenced by HDAC enzyme inhibitors [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. According to the literature, histone deacetylases have a critical function in renal physiology and fibrosis [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Silencing of HDAC1 or HDAC2 has been reported to block renal fibroblast proliferation by decreasing STAT3 phosphorylation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Furthermore, the HDAC6 inhibitor tubastatin A has been observed to increase autophagy and reduce renal injury [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and the HDAC9-selective inhibitor TMP195 has been found to attenuate renal fibrosis in UUO mice [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The role of HDAC3i in AKI is yet to be determined. Therefore, in this study, the expression and localization of HDAC3 were explored in an AKI model. The findings indicated that the HDAC3 inhibitor RGFP966 alleviated injury and inflammation induced by cisplatin- and hypoxia\u0026ndash;reoxygenation (H/R) in HK2 cells. Moreover, the preventive and therapeutic impact of RGFP966 in cisplatin and ischemia/reperfusion (I/R)-induced AKI mouse models were examined using different protocols. The molecular mechanisms by which RGFP966 exerts its effects were also investigated through RNA sequencing (RNA-seq).\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Reagents and antibodies\u003c/h2\u003e \u003cp\u003eA specific HDAC3 inhibitor, (2E)-N-(2-amino-4-fluorophenyl)-3-[(2E)-1-(3-phenyl-2-propen-1-yl)-1H-pyrazol-4-yl]-2-propenamide (RGFP966), of 99.77% purity was purchased from TargetMol. Antibodies against KIM-1 (T58586S; Abmart), HDAC3 (10255-1- AP), RIPK3 (ER190127; HUABIO), p65 (WL01273b, Wanleibio), p-p65 (WL012169, Wanleibio), pRIPK3 (ab195117, Abcam), RIPK1 (ET1701-79; HUABIO), and β-actin (GB12044; Servicebio) were procured from Proteintech. The secondary IRDye 800-conjugated antibody was purchased from LI-COR Biosciences (No. C60113-02). Cisplatin was bought from MedChemExpress (MCE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Establishing AKI mice models\u003c/h2\u003e \u003cp\u003eMale C57BL/6J mice (weight: 20\u0026ndash;23 g) were obtained from the Hangzhou Ziyuan Laboratory Animal Technology Co. The animals were cared for as per the humane care guidelines affiliated to the 3R principle, and the experiments were approved by the Animal Experimentation Ethics Committee of the Anhui Medical University. Animal testing was performed at the Anhui Medical University. The mice in the prevention group were pretreated with RGFP966 and injected intraperitoneally at doses of 5, 10, and 20 mg/kg body weight 12 h before the intraperitoneal injection of cisplatin (20 mg/kg), followed by the same treatment once a day for 3 consecutive days for the study of its preventive effect against AKI. Three days after receiving the cisplatin injection, the mice received isoflurane anesthesia and their blood and kidney tissues were sampled for the detection of indicators related to kidney injury and the expression of inflammation. In the mouse model of I/R-induced AKI, RGFP966 was administered intraperitoneally 12 h previously at doses of 20 mg/kg body weight. After the administration of anesthesia, the mouse was placed on a thermometer panel that maintained their body temperature at 36.5\u0026deg;C. The mice were subjected to a 40-min clipping of their bilateral renal pedicles using microaneurysm clamps. Following ischemic therapy, the clamps were withdrawn for a 24-h period for reperfusion, and all treated animals were executed. Sham control animals were subjected to a similar procedure but without the renal pedicle clamping. Blood and kidney tissue samples were obtained for additional examination. Paraffin sections of 4-mm thickness were stained using the periodic acid-Schiff (PAS) staining kit (C0142S, Beyotime Biotechnology, Jiangsu, China). No fewer than six distinct investigations were conducted throughout the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Molecular docking\u003c/h2\u003e \u003cp\u003eTo decipher possible binding between RGFP966 and HDAC3, these were linked to each other. The software and instruments used in the experiment were Discovery Studio 2017 R2 (DS, BIOVIA Software, Inc., San Diego, CA, United States). The 3D model of HDAC3 (ID:4A69) was obtained from a unique protein database.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cellular thermal shift assay (CETSA)\u003c/h2\u003e \u003cp\u003eIn a 6-well plate, HK2 cells were co-cultured without or with RG (8 \u0026micro;M) for 2 h. After the cells were collected via trypsin digestion, the obtained mixture of trypsin, medium, and cells were aspirated into 1.5-mL centrifuge tubes, labeled with the treatment temperatures, and heated at the corresponding temperatures for 8\u0026ndash;10 min. Subsequently, the supernatant was collected after centrifugation at 800 rpm, and the protein was extracted and analyzed by Western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Cell culture\u003c/h2\u003e \u003cp\u003eHK2 cells were purchased from Procell (Wuhan, China). These cells were placed in a DME/F-12 (1:1) medium containing 5% fetal bovine serum (FBS) and 5% CO\u003csub\u003e2\u003c/sub\u003e at 37℃. The cells were pretreated with RGFP966 (2, 4, and 8 \u0026micro;M) and then stimulated with cell starvation for 12 h when their density in the culture plate reached 50%. Next, it was co-incubated with cisplatin (20 \u0026micro;M) for 24 h; a matching control was also set up. The cells were incubated in a thermostat incubator (37℃, 5% CO\u003csub\u003e2\u003c/sub\u003e) and a low-glucose solution containing 0.5% FBS at 37\u0026deg;C for 12 h to model H/R injury \u003cem\u003ein vitro.\u003c/em\u003e The cells were then placed back in their regular environment for 6 h to allow reoxygenation. The H/R damage induction procedure was performed thrice, and the cells were harvested for analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Western blotting\u003c/h2\u003e \u003cp\u003eFrom the crushed tissues or cells, proteins were extracted in a 6-well plate of frigid radioimmunoprecipitation assay buffer (produced by Beyotime Biotechnology, Jiangsu, China) supplemented with phenylmethylsulfonyl fluoride. The samples were transferred to nitrocellulose membranes following 10% SDS-PAGE. The films were sterilized with 5% skimmed milk and cleansed 3\u0026ndash;4 times with Tris-buffered saline with 0.1% Tween 20. Subsequently, the selected antibody was overnight incubated with the membrane at 4\u0026deg;C. Next, the membrane was cleaned 3\u0026ndash;4 times the next day and subsequently treated for 2 h at room temperature with a matching IRDye 800-coupled secondary antibody. The membranes were then treated with the selected antibody, and the western blotting strips were developed using a LiCor/Odyssey developer (LI-COR Biosciences, Lincoln, NE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 RNA extraction and real-time PCR\u003c/h2\u003e \u003cp\u003eAG RNAex Pro RNA Extraction Reagent (Accurate Biology, Hangzhou, China) was used as per the manufacturer\u0026rsquo;s recommendations for total RNA extraction from tissues or cells. Then, NanoDrop 2000 Spectrophotometer of Thermo Scientific was used to measure the amount of RNA, while RealMasterMix of TOYOBO (Japan) was used to obtain cDNA from the total RNA. Real-time PCR was performed using Bio-Rad (USA) CFX96 real-time reverse transcription-PCR detection equipment. The aforementioned primer sequences were used, and the index ratio of the desired mRNA was normalized to β-actin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Determination of serum Cr, aspartate aminotransferase (AST), BUN, and alanine aminotransferase (ALT) levels\u003c/h2\u003e \u003cp\u003eThe Cr, AST, ALT, and BUN levels in mouse blood samples were assessed by using Cr, BUN, AST, and ALT Assay Kit procured from Nanjing Jianjian Bioengineering Institute. The readings were obtained by strictly adhering to the manufacturer's recommendations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Cell viability assay\u003c/h2\u003e \u003cp\u003eThe HK2 cells containing 5% FBS DME/F-12 (1:1) digested in a 96-well plate were incubated with different concentration gradients of RG (0.5\u0026ndash;128 \u0026micro;M) for 12 h after 24 h, and afterward given without or with cisplatin (10 \u0026micro;M) for 24 h. Next, the cells were extracted with 10 L of CCK-8 liquid to each well. Cell activity was calculated by reading the 450 nm light absorption value with an enzyme marker (Multiskan FC, Thermo) after 1\u0026ndash;4 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Renal histology and Immunofluorescent staining\u003c/h2\u003e \u003cp\u003eThe mouse kidney tissues were collected after fixation, dehydrated, sliced, and embedded in paraffin sections by using a fully automated rotary slicer (Micros), followed by the standardized procedure. For immunofluorescence of the kidney tissues, selected paraffin tissue sections were baked in a 65\u0026deg;C oven for 2 h, followed by sequential deparaffinization in xylene and a primary series of decreasing concentrations of anhydrous ethanol, rinsed in running water, and antigenically repaired using a 20-fold dilution of ethylene diamine tetraacetic acid (EDTA) in a microwave oven. After cooling, circles were drawn and closed with BSA for 30 min, after which the sections were incubated with the desired primary antibody at 4\u0026deg;C overnight. On the second day, the primary antibody was removed, the configured secondary antibody was added, and the results were analyzed by fluorescence microscopy (Leica Microsystems GmbH., Wetzlar, Germany) and then photographed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Immunofluorescent staining\u003c/h2\u003e \u003cp\u003eEight-chamber glass slides were used to culture the HK2 cells, and subsequently, they were fixed in acetone. The cells were then exposed to diluted (1:200) antibodies against KIM-1 or TNF-α overnight. Cow anti-rabbit IgG-rhodamine from Bioss Antibody Inc. was applied for 2 h at ambient temperatures, after which the cells were rinsed with PBS. 4\u0026prime;,6-Diamidino-2-phenylindole was used as a counterstain, and Leica fluorescence microscopy was used to visualize the cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Transcriptome sequencing (RNA-seq)\u003c/h2\u003e \u003cp\u003eThe TRIzol lysate stored in the refrigerator was taken out and added to the cells treated in advance while avoiding light exposure. The dosage of the lysate per well was 0.5\u0026ndash;1.0 mL for 20 s. The solution was observed to be clear upon the naked examination eye, and no cells were observed under the microscope. Afterward, the RNA sample was sent to BGI (BGI Genomics, Shengzhen, China) for sequencing and analysis. Furthermore, the expression patterns and pathways of differentially expressed genes were scrutinized. Subsequently, the candidate genes were cherry-picked to corroborate the expression variation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Transmission electron microscopy (TEM)\u003c/h2\u003e \u003cp\u003eCultured HK2 cells were removed by fixation with 2.5% glutaraldehyde for 4\u0026mdash;12 h, after which they were rinsed and immersed thrice for 10 min each time in 0.1 M phosphate buffer at room temperature. Next, dehydration was performed with a primary increasing concentrations of ethanol and then wrapped with LR White resin (manufactured by London Resin Company, Reading, UK). The obtained materials were then observed by TEM (H-7700, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Statistical analyses\u003c/h2\u003e \u003cp\u003eGraphPad Prism 8 (GraphPad, La Jolla, CA, U.S.A.) was applied for data analyses. *P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **P\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; \u003csup\u003e#\u003c/sup\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05; \u003csup\u003e##\u003c/sup\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01; \u003csup\u003e###\u003c/sup\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001. Data were expressed as either mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SEM) for 6\u0026ndash;8 mice or 3\u0026ndash;4 independent experiments.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cb\u003e3.1\u003c/b\u003e \u003cb\u003eHDAC3 expression is increased in the mouse AKI model and in the\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eHK2 cell injury\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe mouse AKI model, which was produced by injecting cisplatin intraperitoneally and achieving I/R, was found to express HDAC3. The normal group received saline injections and underwent sham surgery. HDAC3 protein and mRNA levels were significantly increased in the mouse AKI model relative to that with the control group according to real-time PCR and Western blotting (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Immunohistochemical analyses further confirmed this finding (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Mouse kidney sections were double fluorescently stained with HDAC3 and proximal tubular marker fluorescent labeling (Leptospirillum lucidum, LTL). The results showed the elevated expression of HDAC3 (red staining), and proximal renal tubules were labeled with LTL (green staining) in the mouse AKI model compared with the kidneys of control mice. This observation indicated the localization of HDAC3 in the renal tubules (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The \u003cem\u003ein vivo\u003c/em\u003e study confirmed that under the action of cisplatin HDAC3 expression and HDAC3 positioning on the proximal renal tubule, so in the \u003cem\u003ein vitro\u003c/em\u003e experiments mainly in renal tubular epithelial cells as the research object. Based on the \u003cem\u003ein vivo\u003c/em\u003e results, the HK2 cells were cisplatin-treated to produce AKI, while the protein and mRNA levels of HADC3 were elevated in the model group. This observation was in contrast to the finding in the untreated group (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Immunofluorescence staining was further used to establish that the HK2 cells in the model group had higher levels of HDAC3 expression than those in the normal group. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). A second model of AKI cell injury was also constructed, that is, the H/R-induced AKI model. Western blotting, real-time PCR, and immunofluorescence results showed that H/R-induced AKI increased the expression of HDAC3 (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD,\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.2 RG reduces cisplatin-induced HK2 cell damage and inflammation\u003c/h2\u003e \u003cp\u003eThe CCK8 assay was performed to assess the protective effect of RG against cisplatin-induced AKI. Initially, the effect of RG on HK2 cell viability was investigated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). CCK-8 results indicated that RG alone did not damage HK2 cells, and different concentrations of RG (2, 4, and 8 \u0026micro;M) restored cisplatin-induced inhibition of cell growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). This finding suggests that RG can restore cisplatin-induced inhibition of cell proliferation. Next, KIM-1, a marker for assessing renal tubular injury, was detected in RG-treated cisplatin-induced HK2 cells. Western blotting and real-time PCR results signified that the KIM-1 expression in cisplatin-induced HK2 cells was reduced by all three doses of RG. (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Furthermore, immunofluorescence analysis confirmed the effective inhibition of KIM-1 by RG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). In addition, Western blotting results indicated that RG could decrease the expression of the cisplatin-induced p-p65 protein in HK2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). According to real-time PCR, RG dramatically reduced the inflammation-related gene expression for monocyte chemoattractant protein (MCP)-1, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Cumulatively, based on our results, RG alleviates HK2 inflammatory response induced by cisplatin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.3 RG reduces HK2 cell damage and inflammation induced by H/R\u003c/h2\u003e \u003cp\u003eAccording to the results of real-time PCR and Western blotting, the expression of KIM-1 protein and mRNA were significantly upregulated in H/R-induced HK2 cells, but this upregulation was prevented via RG pretreatment (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Moreover, the results of real-time PCR demonstrated RG-assuaged cellular inflammation induced by H/R (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Following RG pretreatment, the results of Western blot indicated decreased p-p65 protein levels in H/R-induced HK2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In addition, immunofluorescence staining suggested that RG dramatically decreased the levels of KIM-1 in H/R-induced HK2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.4 RG alleviates AKI and inflammation in cisplatin-induced mice\u003c/h2\u003e \u003cp\u003eFurther investigations were conducted using an animal model to examine how RG affected cisplatin-induced AKI. Before cisplatin injection, mice in the treatment group were pretreated with RG (5, 10, and 20 mg/kg) for 12 h. The same treatment was then administered once daily for 3 d. Detection of the Cr and BUN levels in the animal model indicated the effective therapeutic benefits of RG against AKI (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Hematoxylin\u0026ndash;eosin (H\u0026amp;E) and PAS staining of renal tissue sections demonstrated that RG attenuated cisplatin-induced renal pathologies, such as tubular dilatation, in mice. (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). In addition, immunofluorescence, real-time PCR, and Western blot results suggested that RG dramatically decreased the expressions of mRNA and KIM-1 protein (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Moreover, the anti-inflammatory effect of RG was assessed in cisplatin-induced animal models of inflammation. Western blot findings suggested that RG decreased the upregulation of p-p65 protein expression induced by cisplatin \u003cem\u003ein vivo\u003c/em\u003e. Real-time PCR indicated that RG decreased the expressions of numerous markers linked to inflammation, including the mRNA levels of MCP-1 and TNF-α (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Moreover, immunofluorescence analysis of paraffin sections alluded that RG suppressed the positive TNF-α signal in damaged kidneys (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.5 RG attenuates the acute renal damage and inflammation caused by IRI in vivo\u003c/h2\u003e \u003cp\u003eIn mice, RG attenuated the increase in serum Cr and BUN levels caused by IRI (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). PAS staining of renal tissue sections indicated tubular dilatation and necrosis in the renal tissues of IRI-treated mice, which were alleviated by RG pretreatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). According to the results of Western blotting and real-time PCR, RG pretreatment in the AKI mouse model inhibited the KIM-1 upregulation caused by IRI (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These findings confirmed the previous results. Western blot analyses established that RG reduced the p-p65 protein expressions in the IRI model (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). According to real-time PCR, RG might decrease the mRNA expressions of inflammation-related molecules, such as MCP-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Furthermore, immunofluorescence analysis of paraffin sections confirmed these observations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.6 RG attenuates cisplatin-induced programmed necrosis of HK2 cells\u003c/h2\u003e \u003cp\u003e According to the results of RNA-seq-based analysis and KEGG pathway enrichment analysis, the programmed necrosis pathway ranked high (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). When this pathway was analyzed, the heatmap showed a significant alteration in classical RIPK1 signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Moreover, real-time PCR suggested that RG prevented cisplatin from inducing RIPK1 and RIPK3 signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). In addition, molecular docking and CETSA analysis established that RG enhanced the stability of HDAC3 \u003cem\u003ein vivo\u003c/em\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Western blotting results indicated that RG pretreatment reduced the expressions and activation status (phosphorylation) of RIPK1 and RIPK3 in cisplatin-induced HK2 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). Immunofluorescence staining of P-RIPK1 and P-MLKL confirmed this finding (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). In addition, immunohistochemical analysis of renal tissues from the IRI model showed that RG pretreatment reduced phospho-RIPK1 (pRIPK1) signaling in the IRI-induced mouse model (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Moreover, TEM results indicated the appearance of programmed necrotic features in HK2 cells, such as nuclear membrane crumpling, widening, cell membrane dehiscence, mitochondrial swelling, and deformation, in the group treated with cisplatin. However, these changes were alleviated in the group pretreated with RG (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.7 RG exerts a protective effect by regulating RIPK1-mediated programmed necrosis\u003c/h2\u003e \u003cp\u003eTo verify whether RG exerts a protective effect via RIPK1 production, RIPKI expression was knocked down in HK2 cells. Moreover, according to the results of real-time PCR and Western blotting, small-interfering RNA (siRNA) significantly reduced RIPK1 protein and mRNA levels (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Additionally, real-time PCR, immunofluorescence, and Western blotting findings suggested that RIPK1 knockdown abolished the impact of RG (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). These results allude that RG controls RIPK1-mediated programmed necrosis to reduce acute kidney damage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Effects of RG on the heart, liver, spleen, and lungs of normal mice\u003c/h2\u003e \u003cp\u003eMice were administered RG (20 mg/kg) or saline (medium control) to test the effects of the toxin on different organs. Variations were not detected in serum ALT or AST levels between the groups receiving RG and saline (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). H\u0026amp;E staining was performed to assess the impact on various other organs, including the liver, heart, spleen, and lungs. In contrast to the mediator group, the organs obtained from mice receiving RG did not exhibit any variations, thus validating its safety at the prescribed dosage of 20 mg/kg (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study showed that HDAC3 is crucial for the development of AKI. \u003cem\u003eIn vivo\u003c/em\u003e studies indicated that HDAC3 exacerbates kidney damage in mice with cisplatin-induced AKI. Additionally, the protective effect of the HDAC3 inhibitor RG was established in both laboratory and animal model settings. Our investigations demonstrated that RG acts against cisplatin and H/R-induced AKI in HK2 cells and C57BL/6 mice. Moreover, from RNA-seq results, it was evident that RG is beneficial in attenuating AKI, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, by preventing inflammation and programmed necrosis. Additionally, RG was proven to be an effective modulator of RIPK1-mediated programmed necrosis, thereby ameliorating cisplatin-induced AKI(Figures \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Altogether, these findings implicate that RG may be a potential candidate for treating AKI.\u003c/p\u003e \u003cp\u003eFirst, in this research, HDAC3 was found to be significantly upregulated in mice with cisplatin-induced AKI, especially in proximal renal tubular epithelial cells. This finding was confirmed in the \u003cem\u003ein vitro\u003c/em\u003e model of cisplatin- and H/R-induced AKI. Histone deacetylases and acetyltransferases are primarily responsible for regulating histone modifications and are vital for cell survival, homeostasis, cell proliferation, and gene expression [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Inhibition of HDAC enzymes as a therapeutic approach to diseases has attracted immense attention in recent years. Designing small-molecule HDAC inhibitors for numerous disorders, including cancer, is an emerging area of research [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These inhibitors include class I HDAC (1, 2, 3, and 8), class IIa HDAC (4, 5, 7, and 9), and class IIb HDAC (6, 10) [\u003cspan additionalcitationids=\"CR25 CR26 CR27\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Although the role of HDAC3-selective inhibitors in treating renal fibrosis has been explored and appears to be a viable option, their potential role in AKI and the underlying mechanism are yet to be determined [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSecond, in animal models with AKI induced by cisplatin and I/R, RG decreased the inflammation dramatically. These findings were further supported by \u003cem\u003ein vitro\u003c/em\u003e research. AKI frequently results in inflammation due to triggers such as ischemia and hypoxia, infection, and nephrotoxic medications [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Severe or persistent AKI often leads to chronic inflammation, fibrosis in the kidneys, tubular degeneration, and finally chronic kidney disease (CKD) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Therefore, treating AKI and preventing its transition to CKD via anti-inflammatory therapy is a promising strategy to reduce renal damage. By preventing IGFBP7/IGF1-mediated programmed cell death and inflammation, we previously demonstrated that gibberellin combined with saponin may prevent AKI [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Cpd-6c, a rutaecarpine derivative that targets PDE4B, is an important regulator driving irritation in cisplatin-induced kidney disease and has been reported to alleviate AKI [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Protocatechuic aldehyde has been shown to reduce AKI by inhibiting inflammation and oxidative stress in animal models [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. This finding confirmed the significance of inflammation in mediating AKI. Here, our findings indicated that RG had a beneficial impact on inflammation both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e by inhibiting the expression of numerous inflammatory factors, such as MCP-1. Additionally, NF-κB p65 activation, a well-known mechanism in renal inflammation, is drastically inhibited by RG.\u003c/p\u003e \u003cp\u003eThird, the present study suggested that RG prevents programmed necrosis both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro.\u003c/em\u003e Necroptosis, also known as programmed cell necrosis, is a regulated inflammatory cell death mechanism mediated by the activation of RIPK1, RIPK3, and MLKL. We noted that the cell swells, its membrane tears, and the cytoplasmic contents are released during this type of cell death, which differs morphologically from apoptosis (cell death) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The usual trigger for necrotic apoptosis is external stimulation. When a ligand, such as TNF-α, binds to the death receptor on the cell membrane, it results in cell death. The binding proteins TNFR1, Fas/CD95, DR4/TRAIL-R1, and DR5/TRAIL-R2 belong to the TNF superfamily. These receptors, in their active form, bind to articulatory proteins TRADD and TRAF2, thereby delaying the initiation of receptor-interacting protein kinase [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Pathogen activation of PRRs, such as toll-like receptors, which in turn activate the interacting, RIPK1-independent, but RIPK3-activating articulators TRIF and ZBP-1 are additional causes of necroptotic apoptosis [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The fact that RIPK1 is tightly controlled and involved in the initiation of NF-κB p65 signaling; moreover, the complexes of survival, apoptosis, or necrotic apoptosis should be recorded [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Our research team has been investigating RIPK1/RIPK3 and its role in necrotic apoptosis. Our prior study showed that the RIPK1 antagonist Cpd-71 prevents cisplatin-induced renal insufficiency by reducing necrotic apoptosis and inflammation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe findings of the present study implied that RG has renal protective effects. The mechanism of action was further examined, and RNA-seq analysis showed that the necrotic and inflammatory pathways ranked high in the KEGG pathway enrichment analysis. Therefore, it is hypothesized that RG may exert its protective effect by attenuating programmed necrosis. The results of Western blotting and real-time PCR of RIPK1 and RIPK3 confirmed this hypothesis. In addition, RIPKI-knockdown experiments demonstrated that RG attenuated AKI by regulating RIPK1-mediated programmed necrosis.\u003c/p\u003e \u003cp\u003eOverall, RG attenuated cisplatin and IRI-induced AKI \u003cem\u003ein vivo\u003c/em\u003e, necroptosis induced by cisplatin and H/R \u003cem\u003ein vitro\u003c/em\u003e, and suppressed inflammation, thereby alleviating HK2 injury. Moreover, the administration of RG had no adverse effects on the heart, liver, spleen, or lung tissues of normal C57BL/6 mice. These findings suggest that RG is a potentially safe clinical candidate for treating AKI. However, the specific regulatory mechanism of HDAC3 and RIPK1 needs to be explored further.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCr\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCreatinine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eRG\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRGFP966\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eIF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImmunofluorescence\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHK2\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuman kidney tubular epithelial cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eH/R\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHypoxia/reoxygenation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eIRI\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIschemia Reperfusion Injury\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCKD\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChronic Kidney Disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eI/R\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIschemia/reperfusion\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTNF-α\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTumor Necrosis Factor-α\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCETSA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCellular thermal shift assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCis\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCisplatin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eRNA-seq\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRNA Sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eRIPK1\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReceptor-Interacting Protein Kinase1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eRIPK3\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReceptor-Interacting Protein Kinase3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePAS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePeriodic acid schiff\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHE\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHematoxylin and eosin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eBUN\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBlood urea nitrogen\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHDAC3i\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHistone deacetylase 3 inhibitor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eAST\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAspartate aminotransferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eALT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAlanine aminotransferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eAKI\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cb\u003eAcute kidney injury\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e.\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQ.C., S.-f.Z. and M.-m.X. designed the study. M.-m.X. performed most of the experiments. M.-m.X. and Y.C. performed the bioinformatic analysis. M.-m.X., Y.C.,R.-r.S., J.-t.Y.,X.-f.L., R.H., and.Z.-h.D. performed animal studies. M.-m.X., Y.C., \u0026nbsp; M.-m.L., L.G., and Y.-h.D interpreted the results. M.-m.X., and Q.C. wrote the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Center for Scientific Research of Anhui Medical University for valuable assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by\u0026nbsp;the\u0026nbsp;Anhui Provincial Natural Science Foundation (No.\u0026nbsp;2308085MC68，2208085QH240),\u0026nbsp;National Natural Science Foundation of China(NO.\u0026nbsp;41806158)and University Natural Science Research Project of Anhui Province (NO. 2022AH050710).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no financial conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePickkers P, Darmon M, Hoste E, Joannidis M, Legrand M, Ostermann M, et al. Acute kidney injury in the critically ill: an updated review on pathophysiology and management. Intensive Care Med. 2021;47:835\u0026ndash;850.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRonco C, Bellomo R, Kellum JA. Acute kidney injury. Lancet. 2019;394:1949\u0026ndash;1964.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLewington AJ, Cerd\u0026aacute; J, Mehta RL. Raising awareness of acute kidney injury: a global perspective of a silent killer. Kidney Int. 2013;84:457\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoste EAJ, Kellum JA, Selby NM, Zarbock A, Palevsky PM, Bagshaw SM,et al. Global epidemiology and outcomes of acute kidney injury. Nat Rev Nephrol. 2018;14:607\u0026ndash;625.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZuk A, Bonventre JV. Acute kidney injury: Can remote ischaemic preconditioning prevent AKI? Nat Rev Nephrol. 2015;11:512\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLevey AS, James MT. Acute Kidney Injury. Ann Intern Med. 2018;168:837.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKellum JA, Romagnani P, Ashuntantang G, Ronco C, Zarbock A, Anders HJ. Acute kidney injury. Nat Rev Dis Primers. 2021;7:52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu XQ, Jin J, Li Z, Jiang L, Dong YH, Cai YT, et al. Rutaecarpine derivative Cpd-6c alleviates acute kidney injury by targeting PDE4B, a key enzyme mediating inflammation in cisplatin nephropathy. Biochem Pharmacol. 2020;180:114132.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao L, Liu MM, Zang HM, Ma QY, Yang Q, Jiang L, et al. Restoration of E-cadherin by PPBICA protects against cisplatin-induced acute kidney injury by attenuating inflammation and programmed cell death. Lab Invest. 2018;98:911\u0026ndash;923.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrank D, Vince JE. Pyroptosis versus necroptosis: similarities, differences, and crosstalk. Cell Death Differ. 2019;26:99\u0026ndash;114.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrootjans S, Vanden Berghe T, Vandenabeele P. Initiation and execution mechanisms of necroptosis: an overview. Cell Death Differ. 2017;24:1184\u0026ndash;1195.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao C, Dong H, Xu Q, Zhang Y. Histone deacetylase (HDAC) inhibitors in cancer: a patent review (2017-present). Expert Opin Ther Pat. 2020;30:263\u0026ndash;274.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Seto E. HDACs and HDAC Inhibitors in Cancer Development and Therapy. Cold Spring Harb Perspect Med. 2016;6:a026831.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheshmazar N, Hamzeh-Mivehroud M, Nozad Charoudeh H, Hemmati S, Melesina J, Dastmalchi S. Current trends in development of HDAC-based chemotherapeutics. Life Sci. 2022;308:120946.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrilli LL, Swanhart LM, de Caestecker MP, Hukriede NA. HDAC inhibitors in kidney development and disease. Pediatr Nephrol. 2013;28:1909\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFontecha-Barriuso M, Martin-Sanchez D, Ruiz-Andres O, Poveda J, Sanchez-Ni\u0026ntilde;o MD, Vali\u0026ntilde;o-Rivas L, et al. Targeting epigenetic DNA and histone modifications to treat kidney disease. Nephrol Dial Transplant. 2018;33:1875\u0026ndash;1886.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHyndman KA. Histone Deacetylases in Kidney Physiology and Acute Kidney Injury. Semin Nephrol. 2020;40:138\u0026ndash;147.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHadden MJ, Advani A. Histone Deacetylase Inhibitors and Diabetic Kidney Disease. Int J Mol Sci. 2018;19:2630.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang J, Shi Y, Liu N, Xu L, Zang X, Li P, et al.Blockade of histone deacetylase 6 protects against cisplatin-induced acute kidney injury. Clin Sci (Lond). 2018;132:339\u0026ndash;359.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Yang Y, Yang F, Liu X, Zhan P, Wu J,et al. HDAC9-mediated epithelial cell cycle arrest in G2/M contributes to kidney fibrosis in male mice. Nat Commun. 2023;14:3007.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmin SA, Adhikari N, Kotagiri S, Jha T, Ghosh B. Histone deacetylase 3 inhibitors in learning and memory processes with special emphasis on benzamides. Eur J Med Chem. 2019;166:369\u0026ndash;380.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarkar R, Banerjee S, Amin SA, Adhikari N, Jha T. Histone deacetylase 3 (HDAC3) inhibitors as anticancer agents: A review. Eur J Med Chem. 2020;192:112171.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmin SA, Adhikari N, Jha T. Is dual inhibition of metalloenzymes HDAC-8 and MMP-2 a potential pharmacological target to combat hematological malignancies? Pharmacol Res. 2017;122:8\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi W, Ali T, Zheng C, Liu Z, He K, Shah FA, et al. Fluoxetine regulates eEF2 activity (phosphorylation) via HDAC1 inhibitory mechanism in an LPS-induced mouse model of depression. J Neuroinflammation. 2021;18:38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede la Fuente Revenga M, Ibi D, Saunders JM, Cuddy T, Ijaz MK, Toneatti R,et al. HDAC2-dependent Antipsychotic-like Effects of Chronic Treatment with the HDAC Inhibitor SAHA in Mice. Neuroscience. 2018;388:102\u0026ndash;117.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang C, Lin Z, Liu X, Ding Q, Cai J, Zhang Z,et al. HDAC4 Inhibitors as Antivascular Senescence Therapeutics. Oxid Med Cell Longev. 2022;2022:3087916.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang J, Gong C, Ke Q, Fang Z, Chen X, Ye M,et al. Insights Into the Function and Clinical Application of HDAC5 in Cancer Management. Front Oncol. 2021;11:661620.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang W, Feng Y, Zhou J, Cheung OK, Cao J, Wang J,et al. A selective HDAC8 inhibitor potentiates antitumor immunity and efficacy of immune checkpoint blockade in hepatocellular carcinoma. Sci Transl Med. 2021;13:eaaz6804.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen F, Gao Q, Wei A, Chen X, Shi Y, Wang H,et al. Histone deacetylase 3 aberration inhibits Klotho transcription and promotes renal fibrosis. Cell Death Differ. 2021;28:1001\u0026ndash;1012.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSato Y, Yanagita M. Immune cells and inflammation in AKI to CKD progression. Am J Physiol Renal Physiol. 2018;315:F1501-F1512.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerenbach DA, Bonventre JV. Mechanisms of maladaptive repair after AKI leading to accelerated kidney ageing and CKD. Nat Rev Nephrol. 2015;11:264\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Q, Zang HM, Xing T, Zhang SF, Li C, Zhang Y,et al.Gypenoside XLIX protects against acute kidney injury by suppressing IGFBP7/IGF1R-mediated programmed cell death and inflammation. Phytomedicine. 2021;85:153541\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao L, Wu WF, Dong L, Ren GL, Li HD, Yang Q,et al. Protocatechuic Aldehyde Attenuates Cisplatin-Induced Acute Kidney Injury by Suppressing Nox-Mediated Oxidative Stress and Renal Inflammation. Front Pharmacol. 2016;7:479.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBertheloot D, Latz E, Franklin BS. Necroptosis, pyroptosis and apoptosis: an intricate game of cell death. Cell Mol Immunol. 2021;18:1106\u0026ndash;1121.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLawlor KE, Khan N, Mildenhall A, Gerlic M, Croker BA, D'Cruz AA,et al. RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL. Nat Commun. 2015;6:6282.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKetelut-Carneiro N, Fitzgerald KA. Apoptosis, Pyroptosis, and Necroptosis-Oh My! The Many Ways a Cell Can Die. J Mol Biol. 2022;434:167378.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrank D, Vince JE. Pyroptosis versus necroptosis: similarities, differences, and crosstalk. Cell Death Differ. 2019;26:99\u0026ndash;114.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu D, Zou C, Yuan J. Genetic Regulation of RIPK1 and Necroptosis. Annu Rev Genet. 2021;55:235\u0026ndash;263.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang JN, Liu MM, Wang F, Wei B, Yang Q, Cai YT,et al. RIPK1 inhibitor Cpd-71 attenuates renal dysfunction in cisplatin-treated mice via attenuating necroptosis, inflammation and oxidative stress. Clin Sci (Lond). 2019;133:1609\u0026ndash;1627.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"Acute kidney injury, Necroptosis, RGFP966, Inflammation, HDAC3","lastPublishedDoi":"10.21203/rs.3.rs-4256363/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4256363/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAcute kidney injury (AKI) refers to clinical syndromes culminating in sharp reduction in renal function over a short period of time because of various reasons. These syndromes manifest in the form of inflammation and apoptosis of renal tubular epithelial cells via controlled demise. Histone deacetylases are critical in renal physiology and fibrosis. Here, the HDAC3 expression was shown to be upregulated and localized predominantly in the renal tubules in an AKI mouse model. Moreover, the selective HDAC3 inhibitor RGFP966 was found to reduce inflammation and injury caused by cisplatin and hypoxia\u0026ndash;reoxygenation in HK2 cells. Importantly, RGFP966 exerted potent protective effects in mouse models of ischemia/reperfusion-induced AKI and cisplatin. Furthermore, RNA sequencing revealed that RGFP966 significantly inhibited the upregulation of RIPK1. Cellular thermal displacement assay and molecular docking demonstrated the physical binding of RGFP966 to HDCA3. In addition, RIPK1 knockdown cell assay signified that RGFP966 directly targeted RIPK1 and inhibited RIPK1 kinase activity. In summary, these findings established the efficacy of the HDAC3 inhibitor RGFP966 in treating AKI.\u003c/p\u003e","manuscriptTitle":"HDAC3 inhibitor RGFP966 mitigates acute kidney injury by modulating RIPK1-mediated programmed necrosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-08 14:43:56","doi":"10.21203/rs.3.rs-4256363/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":"88eeb477-c4e7-453d-a057-c26fbc5fee8e","owner":[],"postedDate":"May 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":31384093,"name":"Biological sciences/Genetics/Epigenetics"},{"id":31384094,"name":"Health sciences/Diseases/Kidney diseases"},{"id":31384095,"name":"Biological sciences/Drug discovery/Pharmacology"},{"id":31384096,"name":"Biological sciences/Genetics/Epigenetics"},{"id":31384097,"name":"Health sciences/Diseases/Kidney diseases"},{"id":31384098,"name":"Biological sciences/Drug discovery/Pharmacology"}],"tags":[],"updatedAt":"2024-05-08T14:43:59+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-08 14:43:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4256363","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4256363","identity":"rs-4256363","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}