Combination of different drugs can enhance prevention of CIAKI through inhibition of endoplasmic reticulum stress-induced apoptotic pathway

Combination of different drugs can enhance prevention of CIAKI through inhibition of endoplasmic reticulum stress-induced apoptotic pathway | 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 Combination of different drugs can enhance prevention of CIAKI through inhibition of endoplasmic reticulum stress-induced apoptotic pathway Xuan Wang, Shan Han, Hongliang Cong This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4572737/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Dec, 2024 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Introduction : Contrast-induced acute kidney injury(CIAKI) is an important clinical complication that occurs after the application of contrast in percutaneous coronary intervention. The pathogenesis of CIAKI is complex. Studies have shown that the cell apoptosis induced by endoplasmic reticulum stress (ERS) play an important role in the renal tubular injury of CIAKI. It was suggested that atorvastatin, probucol and alprostadil can inhibit renal tubular cell apoptosis to prevent CIAKI. However, there is no specific research about the above effect of the drug combination. Therefore, this study intends to establish the rat CIAKI model by meglumine diatrizoate and add drug intervention, to compare the influence of combined drugs with the single atorvastatin for CIAKI by inhibiting ERS specific molecular chaperone. Method and Result : 50 Wistar rats were randomly divided into 5 groups: Group A (atorvastatin group, n=10); Group P (probucol + atorvastatin group, n=10); Group Q (alprostadil + atorvastatin group, n=10); Group NC (contrast group, n=10); Group N (control group, n=10). After treated with meglumine diatrizoate and different drugs, the kidneys were obtained for HE, TUNEL staining and real-time RT-PCR, western blot, immunohistochemitry to detect the expression of nucleic acid and protein levels of GRP78, GADD153/CHOP, Caspase-12 in the ERS pathways. Group NC has the significantly highest creatinine rise rate, expression level of protein and nucleic acid in the five groups, with the most serious cell injury and apoptosis in HE and TUNEL staining. Compared with the single atorvastatin group, creatinine rise rate and expression of protein in the combined medication groups were decreased to some extent, whose histological morphology was also improved. This was especially evident in the group Q. Conclusion : The renal cell apoptosis induced by ERS pathway may play an important role in pathogenesis of CIAKI induced by meglumine diatrizoate. Atorvastatin, probucol and alprostadil can prevent the occurrence of CIAKI, may through the inhibition of cell apoptosis induced by ERS pathway. The protective effect for CIAKI of probucol or alprostadil combined with atorvastatin may be stronger than the single atorvastatin through ERS pathway, with the more effect of the combination of alprostadil and atorvastatin. Biological sciences/Drug discovery Health sciences/Cardiology Health sciences/Diseases Health sciences/Medical research Health sciences/Nephrology Contrast-induced acute kidney injury endoplasmic reticulum stress apoptosis atorvastatin probucol alprostadil Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Contrast-induced acute kidney injury(CIAKI), 1 is defined as an absolute increase in serum creatinine (Scr) of more than 0.5 mg/dL or a 25% rise from baseline within 48–72 hours following intravenous contrast administration excluding other causes affecting renal function. 2 CIAKI has become the third major complication after percutaneous coronary intervention (PCI) following in-stent thrombosis and in-stent restenosis. 3 The pathogenesis of CIAKI is very complicated, which may be probably related to the combined effects of direct nephrotoxic injury of contrast agents, vasoconstriction and ischemic injury, decreased renal perfusion blood flow, increased oxidative stress, inflammatory immune response, apoptosis and so on. 4–5 Endoplasmic reticulum stress (ERS)-induced apoptosis through the associated unfolded protein response (UPR) is important in renal tubular injury of CIAKI. 5–8 The GRP78/PERK/eIF2α signaling pathway is involved in the adaptive UPR in animal experiments, 9 and it has been hypothesized that Caspase-12 and GADD153/ CHOP are involved in the development of CIAKI. 10–11 Currently, for the prevention of CIAKI, hydration therapy is recognized as the most effective method. 12 Statins were recommended to prevent CIAKI in patients with moderate to severe renal insufficiency in the 2014 ESC/EACTS guidelines on revascularization. 13 Previously, atorvastatin was hypothesized to inhibit the ERS pathway and thus inhibit renal tubular cell apoptosis, thereby prevent the development of CIAKI. The protective effect of atorvastatin on CIAKI depends on the dose. 14–15 But high dose of statins have side effects such as myolysis and liver damage, so we need to combine some drug with atorvastatin to prevent CIAKI. Both clinical studies 16–20 and animal experiments 21–22 have demonstrated that probucol or alprostadil can reduce renal damage by contrast agents and reduce the incidence of CIAKI. Combination of atorvastatin with probucol has been also suggested to be superior to single-agent atorvastatin 23 in the prevention of contrast-induced nephropathy by a clinical study. However, there are fewer research on the combination of atorvastatin, probucol and alprostadil for CIAKI, 24–25 and the link between probucol, alprostadil and ERS-induced apoptotic pathway has not been investigated. This study established a rat CIAKI model using meglumine diatrizoate, then compared the combination of atorvastatin with probucol or alprostadil with single application of atorvastatin by observing the expression of markers such as GRP78, CHOP/GADD153, Caspase-12 in apoptosis pathway induced by ERS at protein and nucleic acid levels. The aim of this study was to determine whether the combination of these drugs could further inhibit ERS pathway and protect renal function after CIAKI. 2. Materials and method 2.1 Animals and groups Fifty healthy, clean, male Wistar rats, aged about 6-8 weeks old and weighing about 200-250g, were purchased from the Chinese People's Liberation Army Academy of Medical Sciences. After 7 days of adaptive feeding at room temperature, they were randomly divided into 5 groups: Group A (atorvastatin group, n=10); Group P (probucol + atorvastatin group, n=10); Group Q (alprostadil + atorvastatin group, n=10); Group NC (contrast group, n=10); Group N (control group, n=10). The treatments of these groups are shown in the table 1. 2.2 CIAKI modeling and judgment On Day 22, the rats were anesthetized with isoflurane inhalation, then subclavian intravenous injection of meglumine diatrizoate (13 ml/kg, group A, P, Q, NC) or equal dose of normal saline (13 ml/kg, group N) was given. A greater than 25% increase of serum creatinine at 48 or 72 hours after injecting contrast compared with the baseline is considered as a successful rat CIAKI model. 26 2.3 Materials obtaining 72 hours after modeling, blood samples were taken from subclavian vein. The rats were killed by air embolism after drawing subclavian venous blood, whose kidneys were removed. The right kidney was cryopreserved in -80℃ liquid nitrogen for real-time polymerase chain reaction (PCR) and western blot semi-quantitative analysis, and left kidney was placed in 10% neutral formalin solution for morphological and immunohistochemical study. 2.4 Detection of biochemical indexes The serum was obtained by centrifugation of blood sample in each group. The BUN and Scr was detected by enzyme method, to calculate the creatinine rise rate, which is the ratio of creatinine difference before and after modeling to basal creatinine. 2.5 Expression of GRP78, Caspase-12 and CHOP mRNA detected by Real-time PCR Total RNA was extracted from the kidney using Trizol reagent. Real-time PCR was performed by SYBR Premix Ex TaqTM kit. GAPDH was used as an internal control, and the analysis was performed with the relative quantitative method of 2-ΔΔCt. The primer sequences are shown in table 2. 2.6 Expression of GRP78, Caspase-12 and CHOP protein detected by western blot Total protein (1.3 mg/ml) was extracted from frozen crushed kidney tissue by adding cryo-radioimmunoprecipitation (Ripa) lysate. In Western blot analysis, 150ul denatured protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane (NC membrane) . After blocked with skim milk, antigen-antibody reaction was performed and image was developed with Bio-Rad CHEMIDOC XRS imaging system. β-actin was used as internal reference and the gray value of target protein/β-actin was taken as the statistical data of relative protein expression abundance, which was estimated by Total Lab software. Specific antibodies and working concentration are shown in the table 3. 2.7 Histomorphologic examination The rat kidneys were placed in 10% neutral formalin solution, then immediately paraffin-embedded and sliced to specimens of 5 μm thickness. Hematoxyxin-eosin staining (HE) was applied to observe the morphology of glomerular and tubular cells under a light microscope with a field of view of 400 times. 2.8 Detection of apoptosis in kidney cells The specimen was stained by terminal deoxynucleotidyl transferase-mediated dUTP Nick-End Labeling (TUNEL). Normal cells were blue, while those with brown granules in the nucleus were apoptotic cells. In each specimen, 5 non-repeated fields of view were randomly extracted at 400x field of view, and the ratio of positive nuclei/total nuclei was calculated and averaged, which was recorded as the apoptotic index (AI). 2.9 Immunohistochemical detection of GRP78, caspase-12, GADD153 expression Sections were processed, repaired, and undergoing antigen-antibody reaction to be colored. The darker the yellow coloration, the more positive the immunohistochemistry. The average of the multiplication results of 5 random visual fields was taken as the final histopathological score for semi-quantitative analysis of staining results. The product of the staining intensity and the percentage of positive cells in each field of view was the comprehensive score. The scoring method was shown in table 4. 2.10 Statistical analysis SPSS25.0 statistical software was applied for statistical analysis. Mean ± standard deviation (±s) was used to measure the values of measurement information, and paired sample t-test method was used to compare the data within the group, while one-way ANOVA, Mann-Whitney U test or Kruksal-Wallis test was used to compare the data between the groups. p﹤0.05 indicated that the difference was statistically significant. 3. Results 3.1 Acute renal injury was induced by meglumine diatrizoate There was no statistically significant difference in basal Scr and BUN between each group. After modeling, creatinine values at 48 and 72 hours postoperatively were more than 25% higher than basal values in the group NC (0.33±0.21, 0.85±0.42, p<0.0001), which was significantly higher than that of the group N (p=0.001) (Table 5, Figure 1). The BUN level in the group NC was significantly higher after 48 hours of modeling (p=0.019) and continued to be higher after 72 hours (p=0.013)(Table 6). In the HE staining (Figure 2), compared with the kidneys of group N, which were close to the normal renal pathological manifestations, the specimens of group NC were shown severe swelling and vacuole-like degeneration of the renal tubular epithelial cell. Evenly, some of the cells underwent fragmentation and necrotic detachment that could block the tubular lumen. The renal tubules and interstitium were infiltrated with a large number of inflammatory cells, which caused a large number of tubular patterns and exudates in the tubular lumen, and even led to occlusion. Endothelial cells also became swollen and damaged, causing intracapillary coagulation and microthrombosis. It is evident that the changes in renal pathology caused by meglumine diatrizoate are mainly due to tubular damage, accompanied by changes in the tethered cells and endothelial cells. Comparison of the apoptotic index(Figure 4) by TUNEL staining (Figure 3) showed that it was significantly higher in the group NC to group N (73.60±8.64 vs. 22.37±8.73, p<0.0001), suggesting that pantethine was able to induce significant apoptosis in rat renal tubular cells. 3.2 Meglumine diatrizoate could induce apoptosis in kidney cells through ERS The expression of GRP78 mRNA was increased in the group NC (1.250±0.186) compared to group N (1.020±0.214), but the difference was not statistically significant (p=0.0513) (Figure 5). Whlie for GADD153/CHOP and Caspase-12, the group NC had significantly higher expression compared to the N group (1.771±0.517 vs. 1.108±0.467, p=0.0426; 1.894±0.710 vs. 1.255±0.619, p=0.0472) (Figure 10, Figure 15). In the expression of proteins GRP 78, GADD153/CHOP and Caspase-12 in the apoptosis signaling pathway detected by Western Blot (Figure 6, Figure 11, Figure 16), it was clearly seen that the group NC protein image grayscales were significantly darkest among the groups (0.197±0.026, 2.539±0.270, 1.576±0.635) (Figure 7, Figure 12, Figure 17). Compared to the N group (0.118 ± 0.020, 1.582 ± 0.341, 0.249 ± 0.184), all of the three target proteins were statistically significant (p < 0.0001). Similarly as the results in the immunohistochemistry, the group NC had a significantly higher histopathological score compared to the group N (p < 0.0001) (Figure 9, Figure 14, Figure 19). The contrast agent was demonstrated to up-regulate the expression of endoplasmic reticulum-induced apoptosis signaling pathway in rat renal tubular cells. 3.3 Protective effect of drug combinations on CIAKI in rats 48 hours after modeling, the creatinine elevation rate was significantly lower in group A, P, and Q than that in group NC (p=0.049, p=0.027, p=0.026). The Scr elevation rate in group A was also significantly higher compared to group N (p=0.007), whereas that in group P and Q was similar to that of group N (p=0.159, p=0.447). For the group Q, the creatinine elevation rate was significantly lower than that of group A (p=0.026), while there was no significant difference between group P and group A. But comparing the creatinine elevation rate of group P and group Q, no significant difference was found (p=0.341). 72 hours after modeling, the level of Scr elevation in the group NC was significantly higher than that of group A (p=0.000), group P (p=0.000), and group Q (p =0.000), while there was no statistically significant difference in the level of Scr elevation between the group A, P, Q and N (Table 5, Figure 1). In the pathomorphology of rat kidney, slight swelling of renal tubular epithelial cells occurred in groups A, P and Q, only a few inflammatory cells infiltrated renal tubules and renal interstitium, a small number of tubular patterns appeared in the tubular lumen, and no microthrombus was seen. The trend of the damage in the three groups decreased sequentially(Figure 2). The number of apoptotic cells was significantly reduced in the three medicated groups compared to the group NC (p=0.0012, p<0.0001, p<0.0001), with that in groups P and Q being less than that in group A (30.95±9.61 vs. 52.95±7.67, p=0.0002; 37.61±12.13 vs. 52.95±7.67, p=0.0142). For group P and Q, there was no significant difference between the two with similar expression (p=0.3347) (Figure 4). 3.4 Effects of drug combinations on endoplasmic reticulum chaperonin expression and apoptosis in rat kidney after exposure to meglumine diatrizoate In Real-time PCR(Figure 5, Figure 10, Figure 15), there was a trend of lower expression of the three ER chaperonin mRNA in the combination group than that in group A, but it was not statistically significant. In western blot assay for the three proteins’ expression, it can be clearly seen that the NC group has the darkest protein image grayscale among all the groups, followed by the group A, the group P and Q with the lightest image grayscale, which is comparable to the N group(Figure 6, Figure 11, Figure 16). In the semi-quantitative analysis, protein expression was significantly lower in both medicated groups than in the NC group (p<0.01). As for GADD153/CHOP, the protein expression in group P and Q was significantly lower than that in group A (p<0.0001) (Figure 12). Meanwhile, the same conclusion was obtained for group P and A for expression of GRP78 (p=0.0027) (Figure7), and there was a statistically significant difference between group Q and A in Caspase-12 (p=0.0005) (Figure 17). For the comparison between the combination groups, the expression of GADD153/CHOP and Caspase-12 was significantly lower in group Q than those in group P (p=0.0019, p=0.0244). Immunohistochemical analysis(Figure 8, Figure 13, Figure 18) showed a trend consistent with western blot, with expression decreasing roughly in the order of group NC, group A, co-administration group, and group N, in which the protein expression of all three groups with medication was significantly lower than that of group NC (p<0.05). Among them, there was a statistically significant difference between group Q and group A for GADD153/CHOP (p=0.0005) (Figure 14), and the expression of caspase-12 was significantly higher in group A compared with group P and group Q (p=0.0006, p=0.0180) (Figure 19). There was a trend that the expression of each protein in group Q was lower than that in group P, but it was not statistically significant. 4. Discussion Although the pathogenesis of CIAKI is complex, it is mainly related to direct toxicity of contrast to renal cells and hemodynamic alterations due to tubular ischemia caused by contrast viscosity. 27 Renal tubular injury has been found to originate from apoptosis of renal tubular cells in a number of clinical specimens of acute kidney injury. 28 The pathway can be through either exogenous or endogenous pathways, as well as endoplasmic reticulum stress. 29 The endoplasmic reticulum (ER) is a reticular structure in eukaryotic cells involved in protein synthesis, modification, processing, peptide chain modification, folding, and transport. 30 When ER homeostasis is disturbed, its folding can be increased by increased transcription of ER molecular chaperones (e.g., glucose-regulatory proteins GRP78, GRP94) in conjunction with unfolded protein. 30 This allows up-regulation of protein folding and degradation pathways within the ER to inhibit protein synthesis and relieve ER stress. 31 GADD153 is expressed at low levels during cellular homeostasis. However, when cells are under stress in ERS, GADD153 is activated by ATF4 or ATF6 in the UPR signaling pathway, and its overexpression leads to cell cycle arrest or even apoptosis. 32-35 Caspase-12 is an enzyme in the caspase family that is specifically expressed at ERS. During ERS, IRE1 recruits TRAF2, which forms a complex with procaspase-12 (ER apoptosome) to activate Caspase-12, 36 then activates Caspase-9/3 leading to apoptosis. In this study, the successful modeling of CIAKI was measured by creatinine, and severe damage in the renal tubular epithelial cells observed by HE staining and a significant increase of apoptotic index showed by TUNEL in the rats after applied contrast agent, suggested that renal tubular cell damage in the mechanism of the development of CIAKI, and apoptotic cell death may be involved. For GRP78, CHOP/GADD153, and Caspase-12, which are important targets in the ERS process, the group NC had significantly higher expression in PCR, Western Blot, and immunohistochemistry assays, which further confirmed the importance of ERS-induced apoptosis in the pathogenesis of CIAKI. 37 A series of studies in recent years have confirmed that atorvastatin can inhibit ERS-induced apoptotic pathways thereby protecting cells. Some studies found that atorvastatin decreased the expression of GRP 78, CHOP, Caspase-12 or ATF-4, eIF-2α in cardiomyocytes of alcohol-induced or myocardial ischemia/reperfusion injury models, and in pancreatic islet β-cells of mouse obesity models. 38-43 Moreover, atorvastatin may play a renoprotective role by ameliorating ERS and inflammatory reaction. 44-46 Probucol can inhibit activation of the macrophage ERS-CHOP-associated apoptotic pathway by significantly suppressing ox-HDL-induced upregulation of GRP78 and CHOP. 47 Recent studies have also suggested that probucol improves erectile function by decreasing endothelial dysfunction and inhibiting the PERK/ATF4/CHOP pathway in streptozotocin-induced diabetic rats. 48 And combination of atorvastatin and probucol inhibits ERS and increases the levels of TLR-4, which decreases NF-κB and leads to recovery of atherosclerosis in the ApoE-/- mouse model. 49 A number of studies have demonstrated that probucol and alprostadil or prostaglandin are effective in reducing contrast-induced elevated creatinine 18, 19 and inhibiting apoptosis in renal cells. 50-51 Moreover, alprostadil reduces the occurrence of ERS to protect hepatocytes or brain cells by regulating the expression of protein levels such as GRP78, CHOP, Caspase-3, and Bcl-2. 52-54 Thus, in summary, atorvastatin in combination with probucol or alprostadil may theoretically have a dual role in counteracting renal tubular cell apoptosis and inhibiting ERS-induced apoptosis, which may further protect renal tubular cells. The present study was done to compare the effects between the combination and single agent. The results showed a significant decrease in creatinine elevation in group Q compared to group A at 48 hours after contrast injection (p=0.026), which tentatively suggests that the effect of the combination of alprostadil and atorvastatin is better than that of atorvastatin alone for the protection of renal function in CIAKI. In HE staining, it was seen that the damage severity of renal cells in specimens of groups P and Q was slightly lower than that in group A. In terms of apoptosis, TUNEL staining revealed that the number of apoptotic cells in renal tubular epithelial cells and glomerular endothelial cells in group P and Q was significantly lower than that in group A (p=0.0002, p=0.0142). This result suggests that the combination of probucol, alprostadil and atorvastatin is more effective than single atorvastatin in inhibiting contrast-induced renal apoptosis. For the nucleic acid and protein expression of ERS pathway related factors GRP78, GADD153, Caspase-12 are summarized as Table 7. In our experiment, we found that the protein expression of group P and Q in GADD153 was even lower than that of group N(Figure 12, Figure 14). It has been found that probucol and prostaglandin can delay cellular aging and necrosis, and reduce the expression of CHOP. 55-56 In this study near a period of 1 month, there was senescence and natural necrosis of renal cells in the blank group, which could be inhibited by probucol and alprostadil, and thus may explain significantly lower CHOP expression in P and Q group than that of the group N. From the above, it can be seen that the renoprotective effect of probucol and alprostadil in combination with atorvastatin was superior to that of single atorvastatin in CIAKI, and the mechanism may be the inhibition of the ERS-induced apoptotic pathway which exerts the above effect. For the comparison of probucol with alprostadil, the study draws a preliminary conclusion that alprostadil and atorvastatin are more effective in nephroprotection of CIAKI from the inhibition of ERS pathway. 5. Conclusion Apoptosis may involved in the development of CIAKI induced by meglumine diatrizoate, and the increased expression of GRP-78, Gadd153/CHOP and Caspase-12 in ERS pathway was induced by contrast medium, which indicated that apoptosis pathway induced by ERS possibly involved in the mechanism of CIAKI. Atorvastatin, probucol, and alprostadil were nephroprotective in preventing meglumine diatrizoate-induced CIAKI in rats and this protective effect may be realized by inhibiting the ERS-induced apoptotic pathway and attenuating apoptosis. In this experiment, it was preliminarily demonstrated that the combination of atorvastatin with probucol or alprostadil had a superior nephroprotective effect to single atorvastatin in the prevention of CIAKI, whereas the effect of combination of atorvastatin and alprostadil was more pronounced in CIAKI by the inhibition of the ERS pathway. Declarations Author Declarations: Ethics Approval and Consent to participate: All experimental procedures were approved by the Animal Care and Use Committee of Yantai Yuhuangding Hospital（No. 2023-396） and performed in accordance with ARRIVE guidelines . Consent for publication: All authors read, contributed, and approved the fnal version of the manuscript. Availability of data and material (data transparency): The authors retained full control over the content of the manuscript. Hongliang Cong has to be contacted in case of any queries or requirement of data. Funding : We have no funding. All the authors have no financial interests to declare. Conflicts of interest/Competing interests: The authors declare no competing interests. Code availability: The authors had access to data and analysis and that no sponsors participates in the research. Authors' contributions: X Wang completed the experiment, collected statistical data and wrote the manuscript. S Han guided the completion of experiment and modification of the manuscript. HL Cong contributed to the direction of the research, provision of experimental materials and modification of the manuscript and approved its submission. References Lawton JS, Tamis-Holland JE, Bangalore S, et al . 2021 ACC/AHA/SCAI Guideline for Coronary Artery Revascularization: Executive Summary: A Report of the American College of Cardiology/American Heart Association Joint Committee on Clinical Practice Guidelines [published correction appears in Circulation. 2022 Mar 15;145(11):e771]. Circulation . 2022;145(3):e4-e17. Modi K, Padala SA, Gupta M . Contrast-Induced Nephropathy. In: StatPearls . 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In vivo and in vitro impact of atorvastatin against myocardial ischaemia-reperfusion injury by upregulation of silent information regulator l and attenuation of endoplasmic reticulum stress-induced apoptosis. J Drug Target . 2022;30(10):1076-1087. Li N, Wen C, Huang P, et al . Atorvastatin reduces alcohol-induced endoplasmic reticulum stress in AC16 cardiomyocytes. Scand Cardiovasc J . 2019;53(1):42-47. Wu H, Tang Q, Yang J, et al . Atorvastatin ameliorates myocardial ischemia/reperfusion injury through attenuation of endoplasmic reticulum stress-induced apoptosis. Int J Clin Exp Med . 2014;7(12):4915-4923. Published 2014 Dec 15. Xia JG, Xu FF, Qu Y, et al . Atorvastatin post-conditioning attenuates myocardial ischemia reperfusion injury via inhibiting endoplasmic reticulum stress-related apoptosis. Shock . 2014;42(4):365-371. Chen ZY, Liu SN, Li CN, et al . Atorvastatin helps preserve pancreatic β cell function in obese C57BL/6 J mice and the effect is related to increased pancreas proliferation and amelioration of endoplasmic-reticulum stress. Lipids Health Dis . 2014;13:98. Published 2014 Jun 21. Song XJ, Yang CY, Liu B, et al . Atorvastatin inhibits myocardial cell apoptosis in a rat model with post-myocardial infarction heart failure by downregulating ER stress response. Int J Med Sci. 2011;8(7):564-572. Jaikumkao K, Pongchaidecha A, Thongnak LO, et al . Amelioration of Renal Inflammation, Endoplasmic Reticulum Stress and Apoptosis Underlies the Protective Effect of Low Dosage of Atorvastatin in Gentamicin-Induced Nephrotoxicity. PLoS One . 2016;11(10):e0164528. Published 2016 Oct 11. Kang J, Sun Y, Deng Y, et al . Autophagy-endoplasmic reticulum stress inhibition mechanism of superoxide dismutase in the formation of calcium oxalate kidney stones. Biomed Pharmacother . 2020;121:109649. Thongnak L, Chatsudthipong V, Lungkaphin A . Mitigation of renal inflammation and endoplasmic reticulum stress by vildagliptin and statins in high-fat high-fructose diet-induced insulin resistance and renal injury in rats. Biochim Biophys Acta Mol Cell Biol Lipids . 2020;1865(9):158755. Yao S, Tian H, Zhao L, et al . Oxidized high density lipoprotein induces macrophage apoptosis via toll-like receptor 4-dependent CHOP pathway. J Lipid Res . 2017;58(1):164-177. Ruan Z, Wang H, Zhang K, et al . Probucol improves erectile function by regulating endoplasmic reticulum stress in rats with streptozotocin-induced diabetes. Andrologia . 2021;53(4):e13999. Guo X, Wang L, Xia X, et al . Effects of atorvastatin and/or probucol on recovery of atherosclerosis in high-fat-diet-fed apolipoprotein E-deficient mice. Biomed Pharmacother . 2019;109:1445-1453. Zhang YH, Zhang YQ, Guo CC, et al . Prostaglandin E1 attenuates high glucose-induced apoptosis in proximal renal tubular cells by inhibiting the JNK/Bim pathway. Acta Pharmacol Sin . 2020;41(4):561-571. Mou Y, Zhang Y, Guo C, et al . Integrated Treatment of Prostaglandin E1 and Angiotensin-Converting Enzyme Inhibitor in Diabetic Kidney Disease Rats: Possible Role of Antiapoptosis in Renal Tubular Epithelial Cells. DNA Cell Biol . 2018;37(2):133-141. Yang FW, Fu Y, Li Y, et al . Prostaglandin E1 protects hepatocytes against endoplasmic reticulum stress-induced apoptosis via protein kinase A-dependent induction of glucose-regulated protein 78 expression. World J Gastroenterol . 2017;23(40):7253-7264. Jia C, Dai C, Bu X, et al . Co-administration of prostaglandin E1 with somatostatin attenuates acute liver damage after massive hepatectomy in rats via inhibition of inflammatory responses, apoptosis and endoplasmic reticulum stress. Int J Mol Med . 2013;31(2):416-422. Sheng R, Zhang LS, Han R, et al . Combined prostaglandin E1 and lithium exert potent neuroprotection in a rat model of cerebral ischemia. Acta Pharmacol Sin . 2011;32(3):303-310. Zhou H, Huang B, Han Y, et al . Probucol inhibits JAK2-STAT pathway activation and protects human glomerular mesangial cells from tert-butyl hydroperoxide induced premature senescence. Can J Physiol Pharmacol . 2013;91(9):671-679. Matsumoto T, Watanabe S, Ando M, et al . Diabetes and Age-Related Differences in Vascular Function of Renal Artery: Possible Involvement of Endoplasmic Reticulum Stress. Rejuvenation Res . 2016;19(1):41-52. Tables Tables 1-7 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files dell201601211423Exposure66.7secACTIN.tif dell201601261433Exposure148.3secGRP78.tif dell201601261433Exposure78.3secGRP78.tif dell201601261433Exposure78.3secGRP78d1.tif dell201601281349Exposure125.0secACTIN.tif dell201601281358Exposure183.3secCHOP.tif dell201601291418Exposure160.0secactin.tif dell201601291427Exposure206.7secCASPASE12.tif Table.docx Cite Share Download PDF Status: Published Journal Publication published 30 Dec, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 27 Sep, 2024 Reviews received at journal 19 Sep, 2024 Reviews received at journal 18 Sep, 2024 Reviews received at journal 15 Sep, 2024 Reviewers agreed at journal 05 Sep, 2024 Reviewers agreed at journal 03 Sep, 2024 Reviewers agreed at journal 03 Sep, 2024 Reviewers invited by journal 03 Jul, 2024 Editor assigned by journal 03 Jul, 2024 Editor invited by journal 24 Jun, 2024 Submission checks completed at journal 21 Jun, 2024 First submitted to journal 12 Jun, 2024 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. 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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-4572737","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":324167736,"identity":"b7331972-78ee-483f-8f75-e84b08a4946d","order_by":0,"name":"Xuan Wang","email":"","orcid":"","institution":"Yantai Yuhuangding Hosptial, Qingdao University Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xuan","middleName":"","lastName":"Wang","suffix":""},{"id":324167737,"identity":"ee35311c-7c99-47e6-a294-1c84587b8511","order_by":1,"name":"Shan Han","email":"","orcid":"","institution":"Tianjin Chest Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shan","middleName":"","lastName":"Han","suffix":""},{"id":324167738,"identity":"6c07bcfd-3436-4603-a9d8-238a0e02f1f8","order_by":2,"name":"Hongliang Cong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAUlEQVRIiWNgGAWjYFCCAzCCuQFEyrGxtx/Aq4EHoYURrMWYj+dMAgEtcKsYG0CsxHkSDgZ4tdgzHn74uODXHTlz/oWNB37uqE1vk2BIYPhRsQ2PLceMjWf2PTO2nPGw4WDvmeO5bdKNBxh7ztzGo+UMmzRvz+HEDTcONhzgbTuW2yZzIIGZsY1ILQf/th1LZ5NIMCCshecHUMv5xobDvG01CYS1HAD6hbfhsLHBDcaGw7JtBwzbgIF8EJ9f2GcAQ4znz2E5g/OHD39821YnL9/efvDBjwrcWhgkDgBjpA3ESABxD4MFD+BWDwT8DUDiD4gBVleHV/EoGAWjYBSMTAAA/PRnbBLKQ9IAAAAASUVORK5CYII=","orcid":"","institution":"Tianjin Chest Hospital","correspondingAuthor":true,"prefix":"","firstName":"Hongliang","middleName":"","lastName":"Cong","suffix":""}],"badges":[],"createdAt":"2024-06-13 00:36:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4572737/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4572737/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-83741-5","type":"published","date":"2024-12-30T15:57:38+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60236316,"identity":"1ddac9e2-1b44-4c4b-ba72-ccce72ec6483","added_by":"auto","created_at":"2024-07-14 03:11:33","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":178670,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of the groups by Scr and histomorphologic examination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003e1\u003c/strong\u003e) Comparison of the rate of creatinine elevation in rats before and after modeling in each group. bScr: basal creatinine; бScr48h: creatinine of 48h after modeling – bScr; бScr72h: creatinine of 72h after modeling – bScr; a: NC vs.N; b: A vs.N; e: A vs.NC; f: P vs.NC; g: Q vs.NC; i: Q vs.A. All the above p \u0026lt;0.05. (\u003cstrong\u003e2\u003c/strong\u003e) HE staining of renal histopathology of rats in each group(*400). Normal pathological features were in group N(\u003cstrong\u003eE\u003c/strong\u003e). Severe damage to renal tubular epithelial cells, glomerular endothelial cells and mesenchyme occurred in group NC (\u003cstrong\u003eD\u003c/strong\u003e)；Renal tubules were less damaged in group A(\u003cstrong\u003eA\u003c/strong\u003e), P(\u003cstrong\u003eB\u003c/strong\u003e), Q(\u003cstrong\u003eC\u003c/strong\u003e) than that in group NC. (\u003cstrong\u003e3\u003c/strong\u003e) TUNEL staining of renal histopathology of rats in each group(*400). A large number of apoptotic cells existed in the tubular epithelium and glomerular endothelium in group NC(\u003cstrong\u003eD\u003c/strong\u003e). The number of apoptotic cells was significantly reduced in A(\u003cstrong\u003eA\u003c/strong\u003e), P(\u003cstrong\u003eB\u003c/strong\u003e), Q(\u003cstrong\u003eC\u003c/strong\u003e) compared with the NC group, with the number of apoptotic cells seen in group P, Q slightly less than that in A; N group had little apoptotic cells (\u003cstrong\u003eE\u003c/strong\u003e). (\u003cstrong\u003e4\u003c/strong\u003e) Comparison of apoptotic index(AI) in each group. The difference in apoptotic index among the five groups was statistically significant(Kruskal-Wallis test), and the trend showed a gradual decrease in group NC\u0026gt;A\u0026gt;Q\u0026gt;P\u0026gt;N. a: p\u0026lt;0.0001, NC vs.N; b: p= 0.0012, A vs.NC; c: p\u0026lt;0.0001, P vs NC; d: p=0.0002, P vs. A; e: p\u0026lt;0.0001, Q vs NC; f: p=0.0142, Q vs A. (Mann-Whitney U test)\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4572737/v1/6d1670b1d19637d2f43c79ea.jpg"},{"id":60236320,"identity":"26042532-53ef-41c8-a965-ccca595014c7","added_by":"auto","created_at":"2024-07-14 03:11:33","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":104013,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtein and nucleic acid expression of GRP78 in each group.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003e5\u003c/strong\u003e) mRNA expression of GRP 78 by PCR in each group. The results showed that group NC\u0026gt;A\u0026gt;P, Q\u0026gt;N, with no statistical difference. (\u003cstrong\u003e6\u003c/strong\u003e) Protein expression of GRP 78 by western blot in each group. The grayscale of GRP 78 bands of group NC is significantly darker than the other four groups, and the grayscale of group A is darker than that of P and Q. (\u003cstrong\u003e7\u003c/strong\u003e) Ratio of GRP 78 protein optical density in each group. GRP 78 protein expression decreased in the order of group NC\u0026gt;A\u0026gt;Q\u0026gt;P\u0026gt;N. a: p\u0026lt;0.0001, NC vs.N; b: p= 0.0036, A vs.NC; c: p=0.0003, P vs NC; d: p=0.0027, P vs. A; e: p=0.0005, Q vs NC (Mann-Whitney U test). (\u003cstrong\u003e8\u003c/strong\u003e) Immunohistochemical results of GRP78 in rat kidney tissues (*400). The intensity of staining tends to decrease in the order of NC(\u003cstrong\u003eD\u003c/strong\u003e)\u0026gt;A(\u003cstrong\u003eA\u003c/strong\u003e)\u0026gt;P(\u003cstrong\u003eB\u003c/strong\u003e), Q(\u003cstrong\u003eC\u003c/strong\u003e)\u0026gt;N(\u003cstrong\u003eE\u003c/strong\u003e). (\u003cstrong\u003e9\u003c/strong\u003e) Comparison of immunohistochemical protein expression of GRP78 in rat kidney cells of each group. GRP 78 protein expression decreased in the order of group NC\u0026gt;A\u0026gt;P\u0026gt;Q\u0026gt;N. The expression of GRP78 in NC group was significantly higher than that in all the other four groups, whereas there was no obvious difference between the other four groups.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4572737/v1/2aa4f6729ec650d031ebf1d7.jpg"},{"id":60236317,"identity":"c9805f9e-9b85-4e73-ba84-7f3fb03d6364","added_by":"auto","created_at":"2024-07-14 03:11:33","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":106387,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtein and nucleic acid expression of GADD153/CHOP in each group.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003e10\u003c/strong\u003e) mRNA expression of GADD153/CHOP by PCR in each group. a: p=0.042, NC vs.N; b: p= 0.0365, A vs.NC; c: p=0.0176, Q vs NC; (\u003cstrong\u003e11\u003c/strong\u003e) Protein expression of GADD153/CHOP by western blot in each group. The grayscale tends to decrease in the order of NC\u0026gt;A\u0026gt;N\u0026gt;P\u0026gt;Q. (\u003cstrong\u003e12\u003c/strong\u003e) Ratio of GADD153/CHOP protein optical density in each group. GADD153/CHOP protein expression decreased in the order of group NC\u0026gt;A\u0026gt;N\u0026gt;P\u0026gt;Q. * p\u0026lt;0.0001; a: NC vs.N; b: A vs.NC; c: P vs NC; d: P vs. A; e: Q vs NC; f: Q vs. A; g: p=0.0019 P vs. Q (Mann-Whitney U test). (\u003cstrong\u003e13\u003c/strong\u003e) Immunohistochemical results of GADD153/CHOP in rat kidney tissues (*400). The intensity of staining tends to decrease in the order of NC(\u003cstrong\u003eD\u003c/strong\u003e)\u0026gt;A(\u003cstrong\u003eA\u003c/strong\u003e)\u0026gt;P(\u003cstrong\u003eB\u003c/strong\u003e), Q(\u003cstrong\u003eC\u003c/strong\u003e)\u0026gt;N(\u003cstrong\u003eE\u003c/strong\u003e). (\u003cstrong\u003e14\u003c/strong\u003e) Comparison of immunohistochemical protein expression of GADD153/CHOP in rat renal cells of each group. GADD153/CHOP protein expression decreased in the order of group NC\u0026gt;A\u0026gt;P\u0026gt;Q\u0026gt;N. a: p\u0026lt;0.0001, NC vs.N; b: p=0.0164, A vs.NC; c: p\u0026lt;0.0001, P vs NC; d: p\u0026lt;0.0001, Q vs. A; e: p=0.0277, P vs. Q (Mann-Whitney U test).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4572737/v1/cd8e55c1118bf737f27a7bb3.jpg"},{"id":60236332,"identity":"555540c1-2bd9-423f-ba6f-86e9b1d49de0","added_by":"auto","created_at":"2024-07-14 03:19:33","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":106774,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtein and nucleic acid expression of Caspase-12 in each group.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003e15\u003c/strong\u003e) mRNA expression of Caspase-12 by PCR in each group. a: p=0.0472, NC vs.N; The rest comparison had no statistical difference. (\u003cstrong\u003e16\u003c/strong\u003e) Protein expression of Caspase-12 by western blot in each group. The grayscale tends to decrease in the order of NC\u0026gt;A\u0026gt;P\u0026gt;Q\u0026gt;N. (\u003cstrong\u003e17\u003c/strong\u003e) Ratio of Caspase-12 protein optical density in each group. Caspase-12 protein expression decreased in the order of group NC\u0026gt;A\u0026gt;P\u0026gt;Q\u0026gt;N. a: p\u0026lt;0.0001, NC vs.N; b: p=0.0056, A vs. NC; c: p\u0026lt;0.0001, P vs. NC; d: p\u0026lt;0.0001, Q vs. NC; e: p=0.0005, Q vs. A; f: p=0.0244, P vs. Q (Mann-Whitney U test). (\u003cstrong\u003e18\u003c/strong\u003e) Immunohistochemical results of Caspase-12 in rat kidney tissues (*400). The intensity of staining tends to decrease in the order of NC(\u003cstrong\u003eD\u003c/strong\u003e)\u0026gt;A(\u003cstrong\u003eA\u003c/strong\u003e)\u0026gt;P(\u003cstrong\u003eB\u003c/strong\u003e), Q(\u003cstrong\u003eC\u003c/strong\u003e), N(\u003cstrong\u003eE\u003c/strong\u003e). (\u003cstrong\u003e19\u003c/strong\u003e) Comparison of immunohistochemical protein expression of Caspase-12 in rat renal cells of each group. Caspase-12 protein expression decreased in the order of group NC\u0026gt;A\u0026gt;P\u0026gt;N\u0026gt;Q. a: p\u0026lt;0.0001, NC vs.N; b: p= 0.0314, A vs.NC; c: p=0.0006, P vs NC; d: p=0.0180, P vs. A; e: p\u0026lt;0.0001, Q vs NC; f: p=0.0004, Q vs A. (Mann-Whitney U test)\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4572737/v1/4d24b4e0b24aa7d5a1371d85.jpg"},{"id":73093339,"identity":"a4c8cfda-c118-45a8-b6ff-b95e6c82ffb4","added_by":"auto","created_at":"2025-01-06 16:13:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1283873,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4572737/v1/a3898455-ea1c-451d-ac01-785e7c860158.pdf"},{"id":60236554,"identity":"c03c85f8-7b04-4432-8fa7-681775d24735","added_by":"auto","created_at":"2024-07-14 03:27:33","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16638110,"visible":true,"origin":"","legend":"","description":"","filename":"dell201601211423Exposure66.7secACTIN.tif","url":"https://assets-eu.researchsquare.com/files/rs-4572737/v1/00c87a835afe36a467940c7f.tif"},{"id":60236321,"identity":"8fbc28cd-0749-4f3a-a951-5248aa5e7412","added_by":"auto","created_at":"2024-07-14 03:11:33","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13472682,"visible":true,"origin":"","legend":"","description":"","filename":"dell201601261433Exposure148.3secGRP78.tif","url":"https://assets-eu.researchsquare.com/files/rs-4572737/v1/d919b6bb2ac6fcb28567c5b8.tif"},{"id":60236324,"identity":"590c844f-a6e5-41ea-a849-a056807aed53","added_by":"auto","created_at":"2024-07-14 03:11:33","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":13472682,"visible":true,"origin":"","legend":"","description":"","filename":"dell201601261433Exposure78.3secGRP78.tif","url":"https://assets-eu.researchsquare.com/files/rs-4572737/v1/5943a30668403424e232c5f0.tif"},{"id":60236333,"identity":"7ebd32cf-10a2-4621-9d05-6987ac35c82c","added_by":"auto","created_at":"2024-07-14 03:19:33","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":13472682,"visible":true,"origin":"","legend":"","description":"","filename":"dell201601261433Exposure78.3secGRP78d1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4572737/v1/0b70340427008ec63dc5db5a.tif"},{"id":60236326,"identity":"8c11a510-5865-4a8e-8dd8-3ea6faa245b8","added_by":"auto","created_at":"2024-07-14 03:11:34","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":14081222,"visible":true,"origin":"","legend":"","description":"","filename":"dell201601281349Exposure125.0secACTIN.tif","url":"https://assets-eu.researchsquare.com/files/rs-4572737/v1/9e8d2416dcd9b0560f23c487.tif"},{"id":60236327,"identity":"6719a3cc-a60e-4a2c-9a27-6e1a44130cc6","added_by":"auto","created_at":"2024-07-14 03:11:34","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":14081222,"visible":true,"origin":"","legend":"","description":"","filename":"dell201601281358Exposure183.3secCHOP.tif","url":"https://assets-eu.researchsquare.com/files/rs-4572737/v1/8cf5c6c3a4db64e185103922.tif"},{"id":60236329,"identity":"95e61e9d-437c-4e06-8614-3b3cae350332","added_by":"auto","created_at":"2024-07-14 03:11:34","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":13472682,"visible":true,"origin":"","legend":"","description":"","filename":"dell201601291418Exposure160.0secactin.tif","url":"https://assets-eu.researchsquare.com/files/rs-4572737/v1/8e5e18c081b971c215090f73.tif"},{"id":60236328,"identity":"85c9062b-d7b3-4326-b990-14b51d5a6142","added_by":"auto","created_at":"2024-07-14 03:11:34","extension":"tif","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":14696582,"visible":true,"origin":"","legend":"","description":"","filename":"dell201601291427Exposure206.7secCASPASE12.tif","url":"https://assets-eu.researchsquare.com/files/rs-4572737/v1/dcd0e4bf8c798d6d90a4f13b.tif"},{"id":60236335,"identity":"ab0ea773-8c46-4624-b772-767075c1f7a8","added_by":"auto","created_at":"2024-07-14 03:19:33","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":28404,"visible":true,"origin":"","legend":"","description":"","filename":"Table.docx","url":"https://assets-eu.researchsquare.com/files/rs-4572737/v1/cc1c2f48e7efc8ca920d9b2a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Combination of different drugs can enhance prevention of CIAKI through inhibition of endoplasmic reticulum stress-induced apoptotic pathway","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eContrast-induced acute kidney injury(CIAKI), \u003csup\u003e1\u003c/sup\u003e is defined as an absolute increase in serum creatinine (Scr) of more than 0.5 mg/dL or a 25% rise from baseline within 48\u0026ndash;72 hours following intravenous contrast administration excluding other causes affecting renal function. \u003csup\u003e2\u003c/sup\u003e CIAKI has become the third major complication after percutaneous coronary intervention (PCI) following in-stent thrombosis and in-stent restenosis. \u003csup\u003e3\u003c/sup\u003e The pathogenesis of CIAKI is very complicated, which may be probably related to the combined effects of direct nephrotoxic injury of contrast agents, vasoconstriction and ischemic injury, decreased renal perfusion blood flow, increased oxidative stress, inflammatory immune response, apoptosis and so on. \u003csup\u003e4\u0026ndash;5\u003c/sup\u003e Endoplasmic reticulum stress (ERS)-induced apoptosis through the associated unfolded protein response (UPR) is important in renal tubular injury of CIAKI. \u003csup\u003e5\u0026ndash;8\u003c/sup\u003e The GRP78/PERK/eIF2α signaling pathway is involved in the adaptive UPR in animal experiments, \u003csup\u003e9\u003c/sup\u003e and it has been hypothesized that Caspase-12 and GADD153/ CHOP are involved in the development of CIAKI. \u003csup\u003e10\u0026ndash;11\u003c/sup\u003e Currently, for the prevention of CIAKI, hydration therapy is recognized as the most effective method. \u003csup\u003e12\u003c/sup\u003e Statins were recommended to prevent CIAKI in patients with moderate to severe renal insufficiency in the 2014 ESC/EACTS guidelines on revascularization. \u003csup\u003e13\u003c/sup\u003e Previously, atorvastatin was hypothesized to inhibit the ERS pathway and thus inhibit renal tubular cell apoptosis, thereby prevent the development of CIAKI. The protective effect of atorvastatin on CIAKI depends on the dose. \u003csup\u003e14\u0026ndash;15\u003c/sup\u003e But high dose of statins have side effects such as myolysis and liver damage, so we need to combine some drug with atorvastatin to prevent CIAKI. Both clinical studies\u003csup\u003e16\u0026ndash;20\u003c/sup\u003e and animal experiments\u003csup\u003e21\u0026ndash;22\u003c/sup\u003ehave demonstrated that probucol or alprostadil can reduce renal damage by contrast agents and reduce the incidence of CIAKI. Combination of atorvastatin with probucol has been also suggested to be superior to single-agent atorvastatin\u003csup\u003e23\u003c/sup\u003ein the prevention of contrast-induced nephropathy by a clinical study. However, there are fewer research on the combination of atorvastatin, probucol and alprostadil for CIAKI, \u003csup\u003e24\u0026ndash;25\u003c/sup\u003e and the link between probucol, alprostadil and ERS-induced apoptotic pathway has not been investigated. This study established a rat CIAKI model using meglumine diatrizoate, then compared the combination of atorvastatin with probucol or alprostadil with single application of atorvastatin by observing the expression of markers such as GRP78, CHOP/GADD153, Caspase-12 in apoptosis pathway induced by ERS at protein and nucleic acid levels. The aim of this study was to determine whether the combination of these drugs could further inhibit ERS pathway and protect renal function after CIAKI.\u003c/p\u003e"},{"header":"2. Materials and method","content":"\u003cp\u003e2.1 Animals and groups\u003c/p\u003e\n\u003cp\u003eFifty healthy, clean, male Wistar rats, aged about 6-8 weeks old and weighing about 200-250g, were purchased from the Chinese People\u0026apos;s Liberation Army Academy of Medical Sciences. After 7 days of adaptive feeding at room temperature, they were randomly divided into 5 groups: Group A (atorvastatin group, n=10); Group P (probucol + atorvastatin group, n=10); Group Q (alprostadil + atorvastatin group, n=10); Group NC (contrast group, n=10); Group N (control group, n=10). The treatments of these groups are shown in the table 1.\u003c/p\u003e\n\u003cp\u003e2.2 CIAKI modeling and judgment\u003c/p\u003e\n\u003cp\u003eOn Day 22, the rats were anesthetized with isoflurane inhalation, then subclavian intravenous injection of meglumine diatrizoate (13 ml/kg, group A, P, Q, NC) or equal dose of normal saline (13 ml/kg, group N) was given. A greater than 25% increase of serum creatinine at 48 or 72 hours after injecting contrast compared with the baseline is considered as a successful rat CIAKI model.\u003csup\u003e\u0026nbsp;26\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e2.3 Materials obtaining\u003c/p\u003e\n\u003cp\u003e72 hours after modeling, blood samples were taken from subclavian vein. The rats were killed by air embolism after drawing subclavian venous blood, whose kidneys were removed. The right kidney was cryopreserved in -80℃\u0026nbsp;liquid nitrogen for real-time polymerase chain reaction (PCR) and western blot semi-quantitative analysis, and left kidney was placed in 10% neutral formalin solution for morphological and immunohistochemical study.\u003c/p\u003e\n\u003cp\u003e2.4 Detection of biochemical indexes\u003c/p\u003e\n\u003cp\u003eThe serum was obtained by centrifugation of blood sample in each group. The BUN and Scr was detected by enzyme method, to calculate the creatinine rise rate, which is the ratio of creatinine difference before and after modeling to basal creatinine.\u003c/p\u003e\n\u003cp\u003e2.5 Expression of GRP78, Caspase-12 and CHOP mRNA detected by Real-time PCR\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from the kidney using Trizol reagent. Real-time PCR was performed by SYBR Premix Ex TaqTM kit. GAPDH was used as an internal control, and the analysis was performed with the relative quantitative method of 2-\u0026Delta;\u0026Delta;Ct. The primer sequences are shown in table 2.\u003c/p\u003e\n\u003cp\u003e2.6 Expression of GRP78, Caspase-12 and CHOP protein detected by western blot\u003c/p\u003e\n\u003cp\u003eTotal protein (1.3 mg/ml) was extracted from frozen crushed kidney tissue by adding cryo-radioimmunoprecipitation (Ripa) lysate. In Western blot analysis, 150ul denatured protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane (NC membrane) . After blocked with skim milk, antigen-antibody reaction was performed and image was developed with Bio-Rad CHEMIDOC XRS imaging system. \u0026beta;-actin was used as internal reference and the gray value of target protein/\u0026beta;-actin was taken as the statistical data of relative protein expression abundance, which was estimated by Total Lab software. Specific antibodies and working concentration are shown in the table 3.\u003c/p\u003e\n\u003cp\u003e2.7 Histomorphologic examination\u003c/p\u003e\n\u003cp\u003eThe rat kidneys were placed in 10% neutral formalin solution, then immediately paraffin-embedded and sliced to specimens of 5 \u0026mu;m thickness. Hematoxyxin-eosin staining (HE) was applied to observe the morphology of glomerular and tubular cells under a light microscope with a field of view of 400 times.\u003c/p\u003e\n\u003cp\u003e2.8 Detection of apoptosis in kidney cells\u003c/p\u003e\n\u003cp\u003eThe specimen was stained by terminal deoxynucleotidyl transferase-mediated dUTP Nick-End Labeling (TUNEL). Normal cells were blue, while those with brown granules in the nucleus were apoptotic cells. In each specimen, 5 non-repeated fields of view were randomly extracted at 400x field of view, and the ratio of positive nuclei/total nuclei was calculated and averaged, which was recorded as the apoptotic index (AI).\u003c/p\u003e\n\u003cp\u003e2.9 Immunohistochemical detection of GRP78, caspase-12, GADD153 expression\u003c/p\u003e\n\u003cp\u003eSections were processed, repaired, and undergoing antigen-antibody reaction to be colored. The darker the yellow coloration, the more positive the immunohistochemistry. The average of the multiplication results of 5 random visual fields was taken as the final histopathological score for semi-quantitative analysis of staining results. The product of the staining intensity and the percentage of positive cells in each field of view was the comprehensive score. The scoring method was shown in table 4.\u003c/p\u003e\n\u003cp\u003e2.10 Statistical analysis\u003c/p\u003e\n\u003cp\u003eSPSS25.0 statistical software was applied for statistical analysis. Mean \u0026plusmn; standard deviation (\u0026plusmn;s) was used to measure the values of measurement information, and paired sample t-test method was used to compare the data within the group, while one-way ANOVA, Mann-Whitney U test or Kruksal-Wallis test was used to compare the data between the groups. p﹤0.05 indicated that the difference was statistically significant.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1 Acute renal injury was induced by meglumine diatrizoate\u003c/p\u003e\n\u003cp\u003eThere was no statistically significant difference in basal Scr and BUN between each group. After modeling, creatinine values at 48 and 72 hours postoperatively were more than 25% higher than basal values in the group NC (0.33\u0026plusmn;0.21, 0.85\u0026plusmn;0.42, p\u0026lt;0.0001), which was significantly higher than that of the group N (p=0.001) (Table 5, Figure 1). The BUN level in the group NC was significantly higher after 48 hours of modeling (p=0.019) and continued to be higher after 72 hours (p=0.013)(Table 6). In the HE staining (Figure 2), compared with the kidneys of group N, which were close to the normal renal pathological manifestations, the specimens of group NC were shown severe swelling and vacuole-like degeneration of the renal tubular epithelial cell. Evenly, some of the cells underwent fragmentation and necrotic detachment that could block the tubular lumen. The renal tubules and interstitium were infiltrated with a large number of inflammatory cells, which caused a large number of tubular patterns and exudates in the tubular lumen, and even led to occlusion. Endothelial cells also became swollen and damaged, causing intracapillary coagulation and microthrombosis. It is evident that the changes in renal pathology caused by meglumine diatrizoate are mainly due to tubular damage, accompanied by changes in the tethered cells and endothelial cells. Comparison of the apoptotic index(Figure 4) by TUNEL staining (Figure 3) showed that it was significantly higher in the group NC to group N (73.60\u0026plusmn;8.64 vs. 22.37\u0026plusmn;8.73, p\u0026lt;0.0001), suggesting that pantethine was able to induce significant apoptosis in rat renal tubular cells.\u003c/p\u003e\n\u003cp\u003e3.2 Meglumine diatrizoate could induce apoptosis in kidney cells through ERS\u003c/p\u003e\n\u003cp\u003eThe expression of GRP78 mRNA was increased in the group NC (1.250\u0026plusmn;0.186) compared to group N (1.020\u0026plusmn;0.214), but the difference was not statistically significant (p=0.0513) (Figure 5). Whlie for GADD153/CHOP and Caspase-12, the group NC had significantly higher expression compared to the N group (1.771\u0026plusmn;0.517 vs. 1.108\u0026plusmn;0.467, p=0.0426; 1.894\u0026plusmn;0.710 vs. 1.255\u0026plusmn;0.619, p=0.0472) (Figure 10, Figure 15). In the expression of proteins GRP 78, GADD153/CHOP and Caspase-12 in the apoptosis signaling pathway detected by Western Blot (Figure 6, Figure 11, Figure 16), it was clearly seen that the group NC protein image grayscales were significantly darkest among the groups (0.197\u0026plusmn;0.026, 2.539\u0026plusmn;0.270, 1.576\u0026plusmn;0.635) (Figure 7, Figure 12, Figure 17). Compared to the N group (0.118 \u0026plusmn; 0.020, 1.582 \u0026plusmn; 0.341, 0.249 \u0026plusmn; 0.184), all of the three target proteins were statistically significant (p \u0026lt; 0.0001). Similarly as the results in the immunohistochemistry, the group NC had a significantly higher histopathological score compared to the group N (p \u0026lt; 0.0001) (Figure 9, Figure 14, Figure 19). The contrast agent was demonstrated to up-regulate the expression of endoplasmic reticulum-induced apoptosis signaling pathway in rat renal tubular cells.\u003c/p\u003e\n\u003cp\u003e3.3 Protective effect of drug combinations on CIAKI in rats\u003c/p\u003e\n\u003cp\u003e48 hours after modeling, the creatinine elevation rate was significantly lower in group A, P, and Q than that in group NC (p=0.049, p=0.027, p=0.026). The Scr elevation rate in group A was also significantly higher compared to group N (p=0.007), whereas that in group P and Q was similar to that of group N (p=0.159, p=0.447). For the group Q, the creatinine elevation rate was significantly lower than that of group A (p=0.026), while there was no significant difference between group P and group A. But comparing the creatinine elevation rate of group P and group Q, no significant difference was found (p=0.341). 72 hours after modeling, the level of Scr elevation in the group NC was significantly higher than that of group A (p=0.000), group P (p=0.000), and group Q (p =0.000), while there was no statistically significant difference in the level of Scr elevation between the group A, P, Q and N (Table 5, Figure 1). In the pathomorphology of rat kidney, slight swelling of renal tubular epithelial cells occurred in groups A, P and Q, only a few inflammatory cells infiltrated renal tubules and renal interstitium, a small number of tubular patterns appeared in the tubular lumen, and no microthrombus was seen. The trend of the damage in the three groups decreased sequentially(Figure 2). The number of apoptotic cells was significantly reduced in the three medicated groups compared to the group NC (p=0.0012, p\u0026lt;0.0001, p\u0026lt;0.0001), with that in groups P and Q being less than that in group A (30.95\u0026plusmn;9.61 vs. 52.95\u0026plusmn;7.67, p=0.0002; 37.61\u0026plusmn;12.13 vs. 52.95\u0026plusmn;7.67, p=0.0142). For group P and Q, there was no significant difference between the two with similar expression (p=0.3347) (Figure 4).\u003c/p\u003e\n\u003cp\u003e3.4 Effects of drug combinations on endoplasmic reticulum chaperonin expression and apoptosis in rat kidney after exposure to meglumine diatrizoate\u003c/p\u003e\n\u003cp\u003eIn Real-time PCR(Figure 5, Figure 10, Figure 15), there was a trend of lower expression of the three ER chaperonin mRNA in the combination group than that in group A, but it was not statistically significant. In western blot assay for the three proteins\u0026rsquo; expression, it can be clearly seen that the NC group has the darkest protein image grayscale among all the groups, followed by the group A, the group P and Q with the lightest image grayscale, which is comparable to the N group(Figure 6, Figure 11, Figure 16). In the semi-quantitative analysis, protein expression was significantly lower in both medicated groups than in the NC group (p\u0026lt;0.01). As for GADD153/CHOP, the protein expression in group P and Q was significantly lower than that in group A (p\u0026lt;0.0001) (Figure 12). Meanwhile, the same conclusion was obtained for group P and A for expression of GRP78 (p=0.0027) (Figure7), and there was a statistically significant difference between group Q and A in Caspase-12 (p=0.0005) (Figure 17). For the comparison between the combination groups, the expression of GADD153/CHOP and Caspase-12 was significantly lower in group Q than those in group P (p=0.0019, p=0.0244). Immunohistochemical analysis(Figure 8, Figure 13, Figure 18) showed a trend consistent with western blot, with expression decreasing roughly in the order of group NC, group A, co-administration group, and group N, in which the protein expression of all three groups with medication was significantly lower than that of group NC (p\u0026lt;0.05). Among them, there was a statistically significant difference between group Q and group A for GADD153/CHOP (p=0.0005) (Figure 14), and the expression of caspase-12 was significantly higher in group A compared with group P and group Q (p=0.0006, p=0.0180) (Figure 19). There was a trend that the expression of each protein in group Q was lower than that in group P, but it was not statistically significant.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAlthough the pathogenesis of CIAKI is complex, it is mainly related to direct toxicity of contrast to renal cells and hemodynamic alterations due to tubular ischemia caused by contrast viscosity.\u003csup\u003e\u0026nbsp;27\u003c/sup\u003e Renal tubular injury has been found to originate from apoptosis of renal tubular cells in a number of clinical specimens of acute kidney injury.\u003csup\u003e\u0026nbsp;28\u003c/sup\u003e The pathway can be through either exogenous or endogenous pathways, as well as endoplasmic reticulum stress.\u003csup\u003e\u0026nbsp;29\u003c/sup\u003e The endoplasmic reticulum (ER) is a reticular structure in eukaryotic cells involved in protein synthesis, modification, processing, peptide chain modification, folding, and transport.\u003csup\u003e\u0026nbsp;30\u003c/sup\u003e When ER homeostasis is disturbed, its folding can be increased by increased transcription of ER molecular chaperones (e.g., glucose-regulatory proteins GRP78, GRP94) in conjunction with unfolded protein.\u003csup\u003e\u0026nbsp;30\u003c/sup\u003e This allows up-regulation of protein folding and degradation pathways within the ER to inhibit protein synthesis and relieve ER stress.\u003csup\u003e\u0026nbsp;31\u003c/sup\u003e GADD153 is expressed at low levels during cellular homeostasis. However, when cells are under stress in ERS, GADD153 is activated by ATF4 or ATF6 in the UPR signaling pathway, and its overexpression leads to cell cycle arrest or even apoptosis.\u003csup\u003e\u0026nbsp;32-35\u003c/sup\u003e Caspase-12 is an enzyme in the caspase family that is specifically expressed at ERS. During ERS, IRE1 recruits TRAF2, which forms a complex with procaspase-12 (ER apoptosome) to activate Caspase-12,\u003csup\u003e\u0026nbsp;36\u003c/sup\u003e then activates Caspase-9/3 leading to apoptosis. In this study, the successful modeling of CIAKI was measured by creatinine, and severe damage in the renal tubular epithelial cells observed by HE staining and a significant increase of apoptotic index showed by TUNEL in the rats after applied contrast agent, suggested that renal tubular cell damage in the mechanism of the development of CIAKI, and apoptotic cell death may be involved. For GRP78, CHOP/GADD153, and Caspase-12, which are important targets in the ERS process, the group NC had significantly higher expression in PCR, Western Blot, and immunohistochemistry assays, which further confirmed the importance of ERS-induced apoptosis in the pathogenesis of CIAKI.\u003csup\u003e\u0026nbsp;37\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eA series of studies in recent years have confirmed that atorvastatin can inhibit ERS-induced apoptotic pathways thereby protecting cells. Some studies found that atorvastatin decreased the expression of GRP 78, CHOP, Caspase-12 or ATF-4, eIF-2\u0026alpha; in cardiomyocytes of alcohol-induced or myocardial ischemia/reperfusion injury models, and in pancreatic islet \u0026beta;-cells of mouse obesity models.\u003csup\u003e\u0026nbsp;38-43\u003c/sup\u003e Moreover, atorvastatin may play a renoprotective role by ameliorating ERS and inflammatory reaction.\u003csup\u003e\u0026nbsp;44-46\u003c/sup\u003e Probucol can inhibit activation of the macrophage ERS-CHOP-associated apoptotic pathway by significantly suppressing ox-HDL-induced upregulation of GRP78 and CHOP.\u003csup\u003e\u0026nbsp;47\u003c/sup\u003e Recent studies have also suggested that probucol improves erectile function by decreasing endothelial dysfunction and inhibiting the PERK/ATF4/CHOP pathway in streptozotocin-induced diabetic rats.\u003csup\u003e\u0026nbsp;48\u003c/sup\u003e And combination of atorvastatin and probucol inhibits ERS and increases the levels of TLR-4, which decreases NF-\u0026kappa;B and leads to recovery of atherosclerosis in the ApoE-/- mouse model.\u003csup\u003e\u0026nbsp;49\u003c/sup\u003e A number of studies have demonstrated that probucol and alprostadil or prostaglandin are effective in reducing contrast-induced elevated creatinine\u003csup\u003e18, 19\u003c/sup\u003e and inhibiting apoptosis in renal cells.\u003csup\u003e\u0026nbsp;50-51\u003c/sup\u003e Moreover, alprostadil reduces the occurrence of ERS to protect hepatocytes or brain cells by regulating the expression of protein levels such as GRP78, CHOP, Caspase-3, and Bcl-2.\u003csup\u003e\u0026nbsp;52-54\u003c/sup\u003e Thus, in summary, atorvastatin in combination with probucol or alprostadil may theoretically have a dual role in counteracting renal tubular cell apoptosis and inhibiting ERS-induced apoptosis, which may further protect renal tubular cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe present study was done to compare the effects between the combination and single agent. The results showed a significant decrease in creatinine elevation in group Q compared to group A at 48 hours after contrast injection (p=0.026), which tentatively suggests that the effect of the combination of alprostadil and atorvastatin is better than that of atorvastatin alone for the protection of renal function in CIAKI. In HE staining, it was seen that the damage severity of renal cells in specimens of groups P and Q was slightly lower than that in group A. In terms of apoptosis, TUNEL staining revealed that the number of apoptotic cells in renal tubular epithelial cells and glomerular endothelial cells in group P and Q was significantly lower than that in group A (p=0.0002, p=0.0142). This result suggests that the combination of probucol, alprostadil and atorvastatin is more effective than single atorvastatin in inhibiting contrast-induced renal apoptosis. For the nucleic acid and protein expression of ERS pathway related factors GRP78, GADD153, Caspase-12 are summarized as Table 7. In our experiment, we found that the protein expression of group P and Q in GADD153 was even lower than that of group N(Figure 12, Figure 14). It has been found that probucol and prostaglandin can delay cellular aging and necrosis, and reduce the expression of CHOP.\u003csup\u003e\u0026nbsp;55-56\u003c/sup\u003e In this study near a period of 1 month, there was senescence and natural necrosis of renal cells in the blank group, which could be inhibited by probucol and alprostadil, and thus may explain significantly lower CHOP expression in P and Q group than that of the group N. From the above, it can be seen that the renoprotective effect of probucol and alprostadil in combination with atorvastatin was superior to that of single atorvastatin in CIAKI, and the mechanism may be the inhibition of the ERS-induced apoptotic pathway which exerts the above effect. For the comparison of probucol with alprostadil, the study draws a preliminary conclusion that alprostadil and atorvastatin are more effective in nephroprotection of CIAKI from the inhibition of ERS pathway.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eApoptosis may involved in the development of CIAKI induced by meglumine diatrizoate, and the increased expression of GRP-78, Gadd153/CHOP and Caspase-12 in ERS pathway was induced by contrast medium, which indicated that apoptosis pathway induced by ERS possibly involved in the mechanism of CIAKI.\u003c/p\u003e\n\u003cp\u003eAtorvastatin, probucol, and alprostadil were nephroprotective in preventing meglumine diatrizoate-induced CIAKI in rats and this protective effect may be realized by inhibiting the ERS-induced apoptotic pathway and attenuating apoptosis.\u003c/p\u003e\n\u003cp\u003eIn this experiment, it was preliminarily demonstrated that the combination of atorvastatin with probucol or alprostadil had a superior nephroprotective effect to single atorvastatin in the prevention of CIAKI, whereas the effect of combination of atorvastatin and alprostadil was more pronounced in CIAKI by the inhibition of the ERS pathway.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Declarations:\u003c/strong\u003e \u003c/p\u003e\n\u003cp\u003eEthics Approval and Consent to participate: All experimental procedures were approved by the Animal Care and Use Committee of Yantai Yuhuangding Hospital（No. 2023-396）\u0026nbsp;and performed in accordance with \u003cstrong\u003eARRIVE guidelines\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eConsent for publication: All authors read, contributed, and approved the fnal version of the manuscript.\u003c/p\u003e\n\u003cp\u003eAvailability of data and material (data transparency): The authors retained full control over the content of the manuscript. Hongliang Cong has to be contacted in case of any queries or requirement of data.\u003c/p\u003e\n\u003cp\u003eFunding : We have no funding. All the authors have no financial interests to declare.\u003c/p\u003e\n\u003cp\u003eConflicts of interest/Competing interests: The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eCode availability: The authors had access to data and analysis and that no sponsors participates in the research.\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions: X Wang completed the experiment, collected statistical data and wrote the manuscript. S Han guided the completion of experiment and modification of the manuscript. HL Cong contributed to the direction of the research, provision of experimental materials and modification of the manuscript and approved its submission.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u003cstrong\u003eLawton JS, Tamis-Holland JE, Bangalore S, et al\u003c/strong\u003e. 2021 ACC/AHA/SCAI Guideline for Coronary Artery Revascularization: Executive Summary: A Report of the American College of Cardiology/American Heart Association Joint Committee on Clinical Practice Guidelines [published correction appears in Circulation. 2022 Mar 15;145(11):e771]. \u003cem\u003eCirculation\u003c/em\u003e. 2022;145(3):e4-e17.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eModi K, Padala SA, Gupta M\u003c/strong\u003e. Contrast-Induced Nephropathy. \u003cem\u003eIn: StatPearls\u003c/em\u003e. Treasure Island (FL): StatPearls Publishing; July 24, 2023.\u003c/li\u003e\n\u003cli\u003e\u003cstrong\u003eDeng YH, Wang XF, Wu X, et al\u003c/strong\u003e. 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Diabetes and Age-Related Differences in Vascular Function of Renal Artery: Possible Involvement of Endoplasmic Reticulum Stress. \u003cem\u003eRejuvenation Res\u003c/em\u003e. 2016;19(1):41-52.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1-7 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Contrast-induced acute kidney injury, endoplasmic reticulum stress, apoptosis, atorvastatin, probucol, alprostadil","lastPublishedDoi":"10.21203/rs.3.rs-4572737/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4572737/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction\u003c/strong\u003e: Contrast-induced acute kidney injury(CIAKI) is an important clinical complication that occurs after the application of contrast in percutaneous coronary intervention. The pathogenesis of CIAKI is complex. Studies have shown that the cell apoptosis induced by endoplasmic reticulum stress (ERS) play an important role in the renal tubular injury of CIAKI. It was suggested that atorvastatin, probucol and alprostadil can inhibit renal tubular cell apoptosis to prevent CIAKI. However, there is no specific research about the above effect of the drug combination. Therefore, this study intends to establish the rat CIAKI model by meglumine diatrizoate and add drug intervention, to compare the influence of combined drugs with the single atorvastatin for CIAKI by inhibiting ERS specific molecular chaperone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod and Result\u003c/strong\u003e: 50 Wistar rats were randomly divided into 5 groups: Group A (atorvastatin group, n=10); Group P (probucol + atorvastatin group, n=10); Group Q (alprostadil + atorvastatin group, n=10); Group NC (contrast group, n=10); Group N (control group, n=10). After treated with meglumine diatrizoate and different drugs, the kidneys were obtained for HE, TUNEL staining and real-time RT-PCR, western blot, immunohistochemitry to detect the expression of nucleic acid and protein levels of GRP78, GADD153/CHOP, Caspase-12 in the ERS pathways. Group NC has the significantly highest creatinine rise rate, expression level of protein and nucleic acid in the five groups, with the most serious cell injury and apoptosis in HE and TUNEL staining. Compared with the single atorvastatin group, creatinine rise rate and expression of protein in the combined medication groups were decreased to some extent, whose histological morphology was also improved. This was especially evident in the group Q.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: The renal cell apoptosis induced by ERS pathway may play an important role in pathogenesis of CIAKI induced by meglumine diatrizoate. Atorvastatin, probucol and alprostadil can prevent the occurrence of CIAKI, may through the inhibition of cell apoptosis induced by ERS pathway. The protective effect for CIAKI of probucol or alprostadil combined with atorvastatin may be stronger than the single atorvastatin through ERS pathway, with the more effect of the combination of alprostadil and atorvastatin.\u003c/p\u003e","manuscriptTitle":"Combination of different drugs can enhance prevention of CIAKI through inhibition of endoplasmic reticulum stress-induced apoptotic pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-14 03:11:28","doi":"10.21203/rs.3.rs-4572737/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-27T16:04:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-19T19:51:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-18T22:28:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-15T05:59:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"175889969634230142975687649178385819515","date":"2024-09-05T14:34:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"211196565499997864216016122432570346624","date":"2024-09-03T23:35:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"177182940489143542716916627981895566953","date":"2024-09-03T14:27:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-03T12:39:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-03T12:36:24+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-06-24T18:02:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-21T11:46:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-06-13T00:35:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8b375a85-68c2-49e6-8890-87907819e891","owner":[],"postedDate":"July 14th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":34283492,"name":"Biological sciences/Drug discovery"},{"id":34283493,"name":"Health sciences/Cardiology"},{"id":34283494,"name":"Health sciences/Diseases"},{"id":34283495,"name":"Health sciences/Medical research"},{"id":34283496,"name":"Health sciences/Nephrology"}],"tags":[],"updatedAt":"2025-01-06T16:02:51+00:00","versionOfRecord":{"articleIdentity":"rs-4572737","link":"https://doi.org/10.1038/s41598-024-83741-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-12-30 15:57:38","publishedOnDateReadable":"December 30th, 2024"},"versionCreatedAt":"2024-07-14 03:11:28","video":"","vorDoi":"10.1038/s41598-024-83741-5","vorDoiUrl":"https://doi.org/10.1038/s41598-024-83741-5","workflowStages":[]},"version":"v1","identity":"rs-4572737","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4572737","identity":"rs-4572737","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}