Mechanism of Claudin-2 in RTECS apoptosis after renal obstruction

Mechanism of Claudin-2 in RTECS apoptosis after renal obstruction | 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 Research Article Mechanism of Claudin-2 in RTECS apoptosis after renal obstruction Dongsheng zhao, Guijiang Tang, Guoqian Hu, Liang Zeng, Wen Su, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8008643/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Background Acute kidney injury (AKI) is a clinical condition characterized by a rapid decline in glomerular filtration function caused by multiple factors. Factors such as stones and tumors can lead to AKI following renal obstruction. Renal tubular epithelial cell injury is a key component of the pathophysiological mechanism of ischemic acute kidney injury after obstruction. Methods Oxygen-glucose deprivation (OGD) in HK-2 cells and a mouse model of unilateral ureteral ligation (UUO) were used to investigate the role of Claudin-2 in renal tubular epithelial cell apoptosis in ischemia-induced AKI. Results In animal experiments, the expression of Claudin-2 protein was decreased, while Bax and Caspase-3 expression were increased, and Bcl-2 expression was decreased in the renal tissue of UUO mice. Similarly, after OGD treatment, Claudin-2 protein expression was decreased, Bax and Caspase-3 expression were increased, and Bcl-2 expression was decreased. Upregulation of Claudin-2 protein expression through lentivirus transfection in OGD-treated HK-2 cells reduced the decline in cell viability and the proportion of apoptotic cells. Additionally, upregulation of Claudin-2 protein expression reduced OGD-induced Caspase-3 expression, while the Bax/Bcl-2 ratio showed no significant change. Conclusions The expression of Claudin-2 is decreased during acute obstructive kidney injury, which leads to changes in Caspase-3 apoptotic protein and activates cell apoptosis. Renal obstruction Renal ischemia Acute kidney injury RTECs Claudin-2 Apoptosis of cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction AKI after renal obstruction caused by stones, tumors and other reasons is common. Renal hypoperfusion is one of the most common causes of AKI after renal obstruction, namely renal ischemia. Renal cells are very sensitive to ischemia, especially proximal renal tubular epithelial cells are easy to be damaged and lost during renal tissue ischemia, and further block renal tubules and aggravate renal injury [ 1 , 2 ]. Studies have found that after ureteral obstruction, renal pelvis enlargement, renal tissue compression, and edema lead to renal ischemia and hypoxia, accumulation of toxic products, and apoptosis of renal tubular epithelial cells [ 3 – 5 ]. Apoptosis plays an important role in various renal ischemia-induced AKI. However, intervention of the classical apoptosis regulatory pathway can only reduce apoptosis to a certain extent, suggesting that there may be other unknown molecular pathways regulating apoptosis [ 3 , 6 , 7 ]. At the same time, in our previous study, we found that there are various forms of regulated cell death in meth-induced AKI, and apoptosis is the main mechanism of early renal tubular cell death in AKI [ 8 , 9 ]. And TMT labeled proteomics technology showed that the tight junction protein Claudin-2 may be involved in this process. Claudin protein is a very important class of tight junction proteins, which plays an important role in the selective transport process of ions and water, cell proliferation and tumorigenesis [ 10 , 11 ]. For example, recent studies have shown that the expression of Claudin-2 and other tight junction proteins changes in AKI caused by multiple causes such as ischemia-reperfusion, sepsis, and drugs, and the intervention of Claudin-2 expression can effectively reduce the damage of renal tubular epithelial cells [ 12 – 14 ]. The aim of this study is to first detect the expression of Claudin-2 and apoptotic proteins (Bax, Bcl-2, and Caspase-3) in an animal model of AKI induced by UUO. Then, the OGD model of renal tubular epithelial cells was constructed at the cellular level, and the apoptosis of renal tubular epithelial cells and the expression of apoptotic proteins and Claudin-2 protein were detected. The correlation between Claudin-2 and renal tubular epithelial cell apoptosis was explored by interfering Claudin-2 protein expression by lentiviral vector. To explore the regulatory mechanism of Claudin-2 in renal tubular epithelial cell apoptosis in ischemia-induced AKI, and to provide a theoretical basis for the subsequent treatment of AKI. 2. Materials and Methods 2.1 Antibodies and reagents β-actin antibodies were purchased from Beyotime, Bax, Bcl-2, and Caspase-3 antibodies were purchased from ZENBIO, and Claudin-2 antibodies was purchased from Thermo Fly. Lentivirus reagents were ordered from Syngentech. 2.2.Cell culture and treatments The human proximal tubular epithelial cell line (HK-2 cells) was obtained from the Cell Bank of Chinese Academy of Sciences. HK-2 cells were cultured in a 5%CO 2 humidified incubator at 37℃ with Pricella-specific or sugar-free medium for HK-2 cells. To simulate the hypoxic environment of renal injury, the OGD model of HK-2 cells was constructed by oxygen deprivation. 2.3.Animal and UUO models C57 mice were purchased from Slake Jingda Co, Hunan. C57 mice were fostered at the Laboratory Animal Center of Central South University. The experimental procedures and protocols were in accordance with the "3R" principle and the Code of experimental ethics of Central South University. Eighteen healthy female C57 mice were randomly divided into 3 groups: normal Control group (Control group), Sham operation group (Sham group) and unilateral ureteral ligation model group (UUO group), 6 in each group.The unilateral ureteral ligation (UUO) model group was established as described in previous studies. 2.4.Renal function, histopathology Serum creatinine and urea nitrogen were measured by sarcosine oxidase method and urease method, respectively. Renal tissues were stained for H-E as described previously. Tubular damage was scored according to the percentage of tubular damage (grade 0, no damage; Grade 1: 75%). 2.5.Lentivirus transfection HK-2 cells were inoculated into 6-well plates during passage, and lentivirus infection (the virus carried GFP fluorescent tag) was performed when the cell growth density reached 30%. 1980µl of cell culture medium containing 5µg/mL polybrene (co-infection reagent) was added, followed by 20µl of virus suspension at MOI = 10 (obtained from the pre-experiment). After 12 hours, the virus suspension was partially changed (the volume was 1000µl), and the fluorescence abundance expression was observed by microscope 72 hours after infection. The infection efficiency was about 85% and the cells grew well enough for OGD treatment and subsequent experiments. 2.6.Cell viability assays The viability of HK-2 cells was analyzed using the CCK-8 kit (Dojindo, CK04) according to the manufacturer's instructions. 2.7.Western Blot analysis Proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 8% to 12% gels. The dissolved proteins were transferred to a polyvinylidene difluoride membrane (0.45 µm) and blocked with 5% skim milk. The membranes were incubated with β-Actin-HRP Rabbit(Beyotime, 1:1000, ab60008), Bax Rabbit pAb (ZENBIO 1:1000,ab380709), Bcl2 Rabbit pAb (ZENBIO 1:1000, ab381702), Caspase-3 pAb(ZENBIO 1:1000, AB380709) ab350192), Claudin-2 pAb(Thermo, 1:500, ab516100), with two resistance (HRP - conjugated anti - Mouse, ZENBIO, 1:50 00, ab511103 / HRP - conjugated anti - Rabbit, Proteintech, 1:10 000, ab00001-2) hybrid 60 min. Blots were visualized by a gel imaging system and analyzed by ImageJ software. Each immunoblot experiment was repeated three times. 2.8.Analysis of apoptosis Apoptosis was analyzed by TUNEL assay and flow cytometry. ANXA5/annexin v positive cells were regarded as apoptotic cells. 2.9.Statistical Analysis All data were obtained from three independent experiments, analyzed by SPSS 19.0 (IBM, Armonk, NY). Qualitative data were expressed as mean ± standard error of the mean (SEM). The differences between two groups were analyzed by t-test, and the differences among multiple groups were analyzed by one-way ANOVA followed by Tukey's multiple comparison test. P < 0.05 was considered statistically significant. 3. Results 3.1.The levels of serum creatinine and urea nitrogen increased significantly after UUO. Eighteen healthy female C57 mice were randomly divided into 3 groups: normal Control group (Control group), Sham operation group (Sham group) and unilateral ureteral ligation model group (UUO group), there were 6 mice in each group. Blood was collected after modeling, and serum creatinine (CRE) and blood urea nitrogen (BUN) were detected by sarcoline oxidase method and urease method. The results showed that the plasma concentrations of CRE and BUN in UUO model group were significantly higher than those in Control group and Sham group, and the difference was statistically significant (P < 0.001). Note Figure 1 A shows that the serum creatinine (CRE) value of the UUO group was significantly higher than that of the normal control group and the sham operation group, ***vs normal control group, P < 0.001; Fig. 1 B showed that the blood urea nitrogen (BUN) value of the UUO group was significantly higher than that of the normal control group and the sham operation group, ***vs normal control group, P < 0.001. 3.2.HE staining was used to evaluate the renal injury of C57 mice after UUO. HE staining was used to observe the renal tissue injury after different treatments in the Control group, Sham group, and UUO group. The results of HE staining showed that the renal pathological sections of the UUO group showed obvious dilatation of the renal tubules, flat renal tubular epithelial cells, glomerular atrophy, and local inflammatory cell infiltration compared with the Control group. There were no obvious pathological changes in renal tissue sections of the Control group and the Sham group. Note Figure 2 shows HE staining to evaluate renal injury in mice in the normal control group, sham operation group, and unilateral ureteral ligation group, respectively, scale bar = 100µm. 3.3.TUNEL staining was used to detect the apoptosis of renal tubular epithelial cells. After the UUO acute kidney injury model was successfully constructed, the death form of renal tubular epithelial cells was detected. Through TUNEL staining of renal tissue sections, the results showed that: TUNEL staining was positive in renal pathological sections of UUO group, suggesting that the number of apoptotic cells in acute kidney injury after UUO was significantly higher than that in Control group and Sham group. Combined with previous literature research, we speculated that apoptosis may be the main form of renal tubular epithelial cell death in acute ischemic kidney injury. Note Figure 3 shows apoptosis of renal tubular epithelial cells detected by TUNEL staining; nuclei in blue and positive TUNEL staining in green, scale bar = 100µm. 3.4.The expression of Claudin-2 protein decreased and the expression of apoptotic proteins changed after UUO. Western Blot was used to further detect the changes of apoptosis-related proteins to clarify the regulatory pathway of apoptosis associated with ischemic AKI. The results of Western Blot showed that the expression of Claudin-2 protein in renal tissue of UUO mice decreased, the expression of Caspase-3 increased significantly, the expression of Bax increased, and the expression of Bcl-2 decreased, and the differences were statistically significant. However, there was no significant change in Claudin-2 and apoptotic protein expression between the control group and the sham operation group. Note Figure 4 A shows the changes of Claudin2, Caspase-3, Bcl-2, and Bax related apoptosis indicators in the renal tissue of mice after UUO. Figure 4 B shows the statistical analysis of Claudin2 protein gray values, *vs normal control, p < 0.05; Fig. 4 C shows the statistical analysis of the gray value of Caspase-3 protein, **vs normal control, p < 0.01; Fig. 4 D shows the statistical analysis of Bax protein gray value, ***vs normal control group, p < 0.001; Panel E shows the statistical analysis of Bcl-2 protein gray values, *vs normal control, p < 0.05. 3.5.The viability of HK-2 cells decreased after OGD treatment. In order to understand the role of Claudin-2 in apoptosis induced by ischemia and hypoxia, human HK-2 cells were selected to establish the OGD model. The changes of HK-2 cell status after OGD treatment were observed under inverted microscope, the cell death rate was detected by LDH method, and the cell viability was detected by CCK8. Figure 5 A shows the changes of cell state after OGD treatment in the blank group and different OGD time. It can be observed that HK-2 cells in the blank group had a small gap, a typical cobbling-stone arrangement, and a good cell growth. After oxygen-glucose deprivation treatment, the cell morphology showed shrinkage, irregular, and even cell membrane fragmentation, and the cell gap became wider. Figure 5 B shows the cell death rate measured by LDH method. The results showed that the cell death rate increased with the extension of OGD time when the OGD treatment time was 2h, 4h, 6h, 8h, 12h, 24h, and 48h, and the cell death rate increased to about 90% after 24h-48h of OGD. Figure 5 C shows the cell viability measured by CCK-8 method. The results showed that the cell viability decreased with the increase of OGD time when the OGD treatment time was 2h, 4h, 6h, 8h, 12h, 24h, and 48h, and the cell viability decreased to about 60% after OGD4h (P < 0.05). After OGD6h, the cell viability was reduced to about 40% (P < 0.05). Note Figure 5 A shows the changes of cell state of HK-2 cells treated with different OGD times under an inverted optical microscope, scale bar = 100µm. Figure 5 B shows the curve of cell death rate of HK-2 cells after different OGD time treatment by LDH method. Figure 5 C shows the statistical analysis of cell viability of HK-2 cells treated with different OGD times by CCK-8 method, ****vs normal control group, P < 0.0001. 3.6. The apoptosis of HK-2 cells increased after OGD treatment. Annexin V-FITC/PI double staining was performed on HK-2 cells after OGD treatment, and the cell apoptosis was detected by flow cytometry. Figure 6 A shows that Q1 quadrant region represents mechanically necrotic cells, Q2 quadrant region represents late apoptotic cells, Q3 quadrant region represents early apoptotic cells, and Q4 quadrant region represents normal living cells. Flow cytometry showed that HK-2 cells underwent apoptosis after OGD treatment, and the apoptosis rate increased with the extension of OGD treatment time. Among them, the apoptosis rate was significantly increased at 4h, 6h, and 8h of OGD treatment, and the apoptosis rate increased significantly at 12h of OGD treatment, but the number of living cells decreased sharply. It was not appropriate to select this time point for subsequent experiments. Notes: Fig. 6 A shows the 2D scatter plot of HK-2 cell apoptosis detected by flow cytometry after different OGD treatment durations. Figure 6 B shows statistical analysis of flow cytometry data for apoptosis, *vs normal control, p < 0.05, ***vs normal control, p < 0.001. 3.7.The expression of Claudin-2 protein decreased and the levels of apoptotic proteins changed after OGD treatment. Based on the above experiments, we selected 4h, 6h and 8h of OGD as the appropriate time to establish the model of ischemia and hypoxia for subsequent experiments. Western blot showed that the expression of Claudin-2 protein decreased, the expression of Bax increased, the expression of Bcl-2 decreased, and the expression of Caspase-3 increased in HK-2 cells after OGD4h. Compared with the normal control group, the differences were statistically significant (p < 0.05). Note Figure 7 A shows the expressions of Caspase-3, Claudin-2, Bax, and Bcl-2 detected by WesternBlot after treatment with different OGD times. Figure 7 B shows the statistical analysis of Caspase-3 protein gray value, Fig. 7 C shows the statistical analysis of Bcl-2 protein gray value, Fig. 7 D shows the statistical analysis of Claudin2 protein gray value, Fig. 7 E shows the statistical analysis of Bax protein gray value, *vs normal control, p < 0.05, ***vs normal control, p < 0.001, ****vs normal control, p < 0.0001. The above results proved that HK-2 cells underwent apoptosis after OGD treatment, Claudin-2 protein expression decreased, Caspase-3, Bax, Bcl-2 and other related apoptotic proteins changed, suggesting that Claudin-2 protein may induce cell apoptosis after hypoxia treatment. We further investigated the role of Claudin-2 in cell apoptosis by up-regulating Claudin-2 protein expression by lentivirus. 3.8. Upregulation of Claudin-2 protein expression by lentivirus can reduce cell apoptosis. Combined with the above experiments, we finally selected the model condition of ischemia and hypoxia for 4 hours for subsequent lentivirus transfection experiments. Claudin-2 protein was up-regulated by lentiviral vector transfection, and the apoptosis of cells after OGD treatment was detected by flow cytometry. Figure 8 A-B shows that compared with the NC group, the apoptosis rate of CLAUDIN2 overexpression group decreased by about 8% (p < 0.05). Notes: Fig. 8 A shows 2D scatter plots of apoptosis of HK-2 cells in the blank control group, negative control group, and CLAUDIN2 overexpression group detected by flow cytometry. Figure 8 B shows the statistical analysis diagram of the data of apoptosis detected by flow cytometry, *vs negative control group, p < 0.05, **vs normal control, p < 0.01. 3.9. Changes of apoptosis-related proteins after upregulation of Claudin-2 protein expression by lentivirus. The above results showed that ischemia and hypoxia caused apoptosis of HK-2 cells and decreased Claudin-2 protein, and up-regulation of Claudin-2 protein by lentivirus could reduce the occurrence of apoptosis. We further explored whether Claudin-2 protein was involved in cell apoptosis through Caspase-3, Bax, and Bcl-2. As shown in Fig. 9 A-E, Claudin-2 protein was significantly increased in the OGD4h + Claudin-2 overexpression group, suggesting that the lentiviral overexpression model at the molecular protein level was successfully established. The expression of Caspase-3 in the OGD4h + Claudin-2 overexpression group was significantly lower than that in the blank group and the negative control group (P < 0.05). There was no significant difference in the ratio of Bax/Bcl-2 between the OGD4h + Claudin-2 overexpression group and the NC group. Note Figure 9 A shows the expression of Claudin-2 and apoptosis-related proteins (Caspase-3, Bax, Bcl-2) detected by WesternBlot after upregulation of Claudin-2 protein expression by lentivirus. Figure 9 B shows the statistical analysis of the gray value of Caspase-3 protein, Fig. 9 C shows the statistical analysis of the gray value of Bcl-2 protein, Fig. 9 D shows the statistical analysis of the gray value of Claudin-2 protein, Fig. 9 E shows the statistical analysis of the gray value of Bax protein, Fig. 9 F shows the statistical analysis of the Bax/ Bcl-2 ratio. *vs normal control, p < 0.05, **vs normal control, p < 0.01, ***vs normal control, p < 0.001, ****vs normal control, p < 0.0001, there was no significant difference between the ns vs NC group. 4. Discussion Studies have found that apoptosis and necrosis of renal tubular epithelial cells occur in UUO model mice under renal ischemia, accumulation of toxic products and other factors, resulting in renal injury [ 15 , 16 ]. In the UUO rat model, some scholars have found that thymosin may reduce the apoptosis of renal tubular epithelial cells and the process of renal fibrosis in UUO rats by inhibiting the TGF-β pathway [ 17 ]. However, the specific mechanism of renal tubular epithelial cell apoptosis after UUO is still unclear, which needs to be further explored. The effect of OGD on cell apoptosis was detected after Claudin-2 protein was up-regulated by lentivirus transfection. The results showed that after the up-regulation of Claudin-2 protein by lentivirus vector transfection, the apoptosis of renal tubular epithelial cells still existed after OGD treatment, but the proportion of apoptosis decreased compared with NC (negative control group), and the difference was statistically significant. These results indicate that Claudin-2 is associated with apoptosis of renal tubular epithelial cells after OGD treatment, and up-regulation of Claudin-2 can reduce the occurrence of apoptosis. Previous studies have shown that the expression of Claudin-2 is down-regulated and related oxidative stress markers are increased when cisplatin-induced acute kidney injury occurs. Cisplatin may cause kidney injury by changing the localization of tight junction proteins such as Claudin-2 related to oxidative stress [ 18 ], and oxidative stress can exacerbate the occurrence of apoptosis. In addition, Claudin-2, as a key molecule regulating calcium reabsorption, downregulation of Claudin-2 can lead to calcium homeostasis imbalance and hypercalciuria in tubular epithelial cells, and increased calcium levels can further induce apoptosis [ 19 ]. Therefore, the mechanism by which Claudin-2 affects renal tubular epithelial cell apoptosis after acute kidney injury may be related to oxidative stress and calcium imbalance. The reasons why the apoptosis of renal tubular epithelial cells still occurs after OGD treatment with up-regulation of Claudin-2 protein in this study may be related to the following: first, the mechanism of apoptosis of renal tubular epithelial cells after OGD treatment is very complex, and Claudin-2 may be only one of the key molecules involved in the apoptosis pathway. Apoptosis is jointly mediated by mitochondrial pathway, T cell-mediated perforin/granzyme pathway, death receptor pathway and endoplasmic reticulum stress (ERS) pathway [ 20 ], and these apoptotic pathways are not completely independent. For example, endoplasmic reticulum stress pathway and mitochondrial pathway jointly affect BCL2 family proteins to regulate apoptosis [ 21 ]. Mitochondrial membrane permeability is regulated by BCL2 family proteins, and the increase of Bax/Bcl2 ratio can lead to further apoptosis. In this study, the ratio of Bax/Bcl2 did not change significantly after up-regulation by lentiviral vector, suggesting that Claudin-2 may not regulate apoptosis through Bax, Bcl2 and other proteins. In addition, there are a large number of procaspase forms in cells, which can trigger each other once activated, and eventually start the Caspase cascade reaction leading to subsequent apoptosis. Caspase enzymes are mainly divided into the initiation factors of caspase-2, 8, 9, 10, and the execution factors of caspase-3, 6, and 7, and the inflammatory mediators of caspase-1, 4, and 5. Caspase-3 activation is a key step in initiating apoptosis. We found that after upregulation of Claudin-2 protein by lentivirus, Caspase-3 protein decreased compared with NC group and the difference was statistically significant, indicating that Claudin-2 may induce apoptosis through Caspase-3 pathway. Secondly, lentiviral vectors are obtained by the modification of HIV carrying target genes, which can be carried into cells and expressed, but the transduction efficiency is limited. How to improve the transduction efficiency has always been a problem waiting to be solved. Moreover, there are some problems such as the risk of insertion mutation and affecting cell differentiation in subsequent cell growth [ 22 , 23 ]. Therefore, the effect of upregulation of Claudin-2 protein by the lentiviral vector in this experiment was not complete. This study also has some shortcomings: although it has been proved that up-regulation of Claudin-2 protein can inhibit the apoptosis of renal tubular epithelial cells, OGD treatment only using HK-2 cells cannot fully reflect the complete pathophysiological process of acute kidney injury after renal obstruction, and lacks the influence of different types of intercellular interaction and intracellular environment regulation. In addition, the human proximal tubular epithelial cell line HK-2 does not fully reflect the common apoptotic characteristics of other renal tubular epithelial cells. Similarly, overexpression of lentiviral vectors cannot completely replace the compensatory effect of the normal physiological pathway, and thus may have some influence on the experimental results. In addition, this experiment demonstrated that Claudin-2 may induce apoptosis through Caspase-3, but because after the initial apoptotic signal stimulation, The activation of the precursors of caspases (such as Caspase-8 and Caspase-9) during proteolysis leads to the subsequent enzyme cascade of caspase-3 [ 24 ]. Caspase-3 is the central link in the initiation of multiple apoptotic signaling pathways, and the connection between Caspase-3 and Claudin-2 may act through other unknown apoptotic molecules or non-single regulatory pathways. The specific regulatory mechanism of caspase-3 and Claudin-2 remains to be further studied. Finally, apoptosis is only one form of regulated cell death in renal tubular epithelial cells after acute kidney injury. The Caspase family also plays an important role in other regulated cell death such as pyroptosis [ 25 , 26 ]. Further study on the mechanism of Claudin-2 related to other regulated cell death such as pyroptosis may also be one of the future research directions. 5. Conclusions In conclusion, the present study suggests that Claudin-2 protein is involved in the apoptosis of renal tubular epithelial cells after acute obstructive kidney injury, which provides a theoretical basis for further investigation of the molecular mechanism of Claudin-2 in the apoptosis of rtecs in obstructive kidney injury. Claudin-2 may be one of the targets of rtec apoptosis in the treatment of obstructive acute kidney injury. Abbreviations RTECs:Renal epithelial cells;UUO:Unilateral ureteral obstruction;OGD:Oxygen-glucose deprivation;AKI:Actue kidney injury;CRE:Creatinine; TGF-β:Transforming growth factor-β;BUN:Blood urea nitrogen;RCD:Regulated cell death; TMT:Tandem mass tag; MOI:Multiplicity Of Infection; GFP:Green fluorescent protein; Declarations Disclosure statement No potential conflict of interest was reported by the authors. Contributions from authors Jin Tang conceived and designed the experiment. Dongsheng Zhao performed most of the experiments. Zhao Dongsheng wrote the original manuscript, which was substantially revised by Tang Jin. Guijiang Tang, Guoqian Hu, Liang Zeng, and Wen Su performed the collection and data collection. Zhao Dongsheng performed the statistical analysis. All the authors read and approved the final manuscript. Ethical Approval This study was approved by the Experimental Ethics Committee of Central South University. 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Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 01 Dec, 2025 Reviews received at journal 25 Nov, 2025 Reviewers agreed at journal 19 Nov, 2025 Reviewers invited by journal 04 Nov, 2025 Editor assigned by journal 03 Nov, 2025 Submission checks completed at journal 03 Nov, 2025 First submitted to journal 01 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8008643","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":541575476,"identity":"67c2103a-40f5-4df4-854a-7df3c24f878a","order_by":0,"name":"Dongsheng zhao","email":"","orcid":"","institution":"The Third Hosptial, Central South University","correspondingAuthor":false,"prefix":"","firstName":"Dongsheng","middleName":"","lastName":"zhao","suffix":""},{"id":541575477,"identity":"d2d7d675-19b4-41e2-aab5-9b08c98497ca","order_by":1,"name":"Guijiang Tang","email":"","orcid":"","institution":"The Third Hosptial, Central South University","correspondingAuthor":false,"prefix":"","firstName":"Guijiang","middleName":"","lastName":"Tang","suffix":""},{"id":541575478,"identity":"da8a23dc-0fbb-4a7a-bb8a-86101445bca3","order_by":2,"name":"Guoqian Hu","email":"","orcid":"","institution":"The Third Hosptial, Central South University","correspondingAuthor":false,"prefix":"","firstName":"Guoqian","middleName":"","lastName":"Hu","suffix":""},{"id":541575479,"identity":"0495c412-ee04-4fca-9ee3-787ac4dcf660","order_by":3,"name":"Liang Zeng","email":"","orcid":"","institution":"The Third Hosptial, Central South University","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Zeng","suffix":""},{"id":541575480,"identity":"3f4209dd-9d0a-4427-a4cf-242f6a782c4b","order_by":4,"name":"Wen Su","email":"","orcid":"","institution":"The Third Hosptial, Central South University","correspondingAuthor":false,"prefix":"","firstName":"Wen","middleName":"","lastName":"Su","suffix":""},{"id":541575481,"identity":"2bd8f01f-cfda-44a9-aea7-73f9f2bf82eb","order_by":5,"name":"Jin Tang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYFCCgw3MP35IyLHxNx8gQQtjj40xn8SxBOLtYWZgS0ucx5BjQJxy/sbDbY8LeA6ntzGcMWD4UbGNsBaJAwfbjWdYHM5tY24rYOw5c5sIaw4cbJPg4QFqYTi8gZmxjQgt8mAtbIfT2RgSDIjTYgDUIs3DlpbAxpBCpBZDoBbJmT02hm3AQD5IlF/kbhx/JvHhh4S8fH/zwQc/KojxvsQBBPsALkWogL+BOHWjYBSMglEwggEAAV5AhehAk3EAAAAASUVORK5CYII=","orcid":"","institution":"The Third Hosptial, Central South University","correspondingAuthor":true,"prefix":"","firstName":"Jin","middleName":"","lastName":"Tang","suffix":""}],"badges":[],"createdAt":"2025-11-02 03:38:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8008643/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8008643/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":96244359,"identity":"a28e8137-50dc-40d2-b967-ef99a8b4dd5e","added_by":"auto","created_at":"2025-11-19 07:18:13","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6057301,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-8008643/v1/25e63b59595933505df4d654.docx"},{"id":96242879,"identity":"8872401b-455f-47bb-a923-8605113448d3","added_by":"auto","created_at":"2025-11-19 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09:21:20","extension":"html","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":84268,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8008643/v1/4c1d630d0a4aba9a96fec1b0.html"},{"id":95905219,"identity":"e018d1de-2a16-4b5e-884f-24d6bf2261ca","added_by":"auto","created_at":"2025-11-14 09:21:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":16390,"visible":true,"origin":"","legend":"\u003cp\u003eConcentrations of serum creatinine (CRE) and blood urea nitrogen (BUN) after UUO\u003c/p\u003e\n\u003cp\u003eNote: Figure 1A shows that the serum creatinine (CRE) value of the UUO group was significantly higher than that of the normal control group and the sham operation group, ***vs normal control group, P\u0026lt;0.001; Figure 1B showed that the blood urea nitrogen (BUN) value of the UUO group was significantly higher than that of the normal control group and the sham operation group, ***vs normal control group, P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8008643/v1/f7a1622a0a05fd8c674c204d.png"},{"id":96243099,"identity":"fb9aeb8b-b5f0-496d-8c46-306a0ca8a749","added_by":"auto","created_at":"2025-11-19 07:15:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":195256,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of kidney injury in mice by HE staining\u003c/p\u003e\n\u003cp\u003eNote: Figure 2 shows HE staining to evaluate renal injury in mice in the normal control group, sham operation group, and unilateral ureteral ligation group, respectively, scale bar =100µm.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8008643/v1/bd6c4e4eeacd69fa200bd75f.png"},{"id":96242698,"identity":"fefac123-af24-4773-baa1-f3eef18f3a80","added_by":"auto","created_at":"2025-11-19 07:14:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1395800,"visible":true,"origin":"","legend":"\u003cp\u003eApoptosis of renal tubular epithelial cells detected by TUNEL staining.\u003c/p\u003e\n\u003cp\u003eNote: Figure 3 shows apoptosis of renal tubular epithelial cells detected by TUNEL staining; nuclei in blue and positive TUNEL staining in green, scale bar =100µm.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8008643/v1/1fae765c4910f876996c3b31.png"},{"id":96243007,"identity":"60904439-4601-409f-beae-2574f5fcf068","added_by":"auto","created_at":"2025-11-19 07:15:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":227952,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in Claudin2 and other related proteins in the control, sham, and UUO groups.\u003c/p\u003e\n\u003cp\u003eNote: Figure 4 A shows the changes of Claudin2, Caspase-3, Bcl-2, and Bax related apoptosis indicators in the renal tissue of mice after UUO. Figure 4 B shows the statistical analysis of Claudin2 protein gray values, *vs normal control, p\u0026lt; 0.05; Figure 4 C shows the statistical analysis of the gray value of Caspase-3 protein, **vs normal control, p\u0026lt; 0.01; Figure 4 D shows the statistical analysis of Bax protein gray value, ***vs normal control group, p\u0026lt;0.001; Panel E shows the statistical analysis of Bcl-2 protein gray values, *vs normal control, p\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8008643/v1/1832e6a8f72aa35da19e0071.png"},{"id":95905228,"identity":"ce2bc04c-9b03-4fcf-8214-7bd15c48ca61","added_by":"auto","created_at":"2025-11-14 09:21:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2797047,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in cell state, cell death rate, and cell viability after OGD.\u003c/p\u003e\n\u003cp\u003eNote: Figure 5A shows the changes of cell state of HK-2 cells treated with different OGD times under an inverted optical microscope, scale bar =100µm. Figure 5B shows the curve of cell death rate of HK-2 cells after different OGD time treatment by LDH method. Figure 5C shows the statistical analysis of cell viability of HK-2 cells treated with different OGD times by CCK-8 method, ****vs normal control group, P\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8008643/v1/9e0086d3e722d5d725f49307.png"},{"id":95905224,"identity":"f305e9ea-b31b-4892-aaf3-bcc05d750fe1","added_by":"auto","created_at":"2025-11-14 09:21:20","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":788185,"visible":true,"origin":"","legend":"\u003cp\u003eApoptosis of HK-2 cells treated with different OGD time.\u003c/p\u003e\n\u003cp\u003eNotes: Figure 6A shows the 2D scatter plot of HK-2 cell apoptosis detected by flow cytometry after different OGD treatment durations. Figure 6B shows statistical analysis of flow cytometry data for apoptosis, *vs normal control, p\u0026lt; 0.05, ***vs normal control, p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8008643/v1/b8bb9e364667a30ab71c7cd8.jpeg"},{"id":96242654,"identity":"980acd51-b7c7-4747-966b-64b6e94c4732","added_by":"auto","created_at":"2025-11-19 07:13:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":245595,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of Caspase-3, Claudin-2, Bax, and Bcl-2 after treatment with different OGD times.\u003c/p\u003e\n\u003cp\u003eNote: Figure 7 A shows the expressions of Caspase-3, Claudin-2, Bax, and Bcl-2 detected by WesternBlot after treatment with different OGD times. Figure 7 B shows the statistical analysis of Caspase-3 protein gray value, Figure 7 C shows the statistical analysis of Bcl-2 protein gray value, Figure 7 D shows the statistical analysis of Claudin2 protein gray value, Figure 7 E shows the statistical analysis of Bax protein gray value, *vs normal control, p\u0026lt; 0.05, ***vs normal control, p\u0026lt; 0.001, ****vs normal control, p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8008643/v1/223a5b6ef32a9593894d0d71.png"},{"id":96243632,"identity":"c31f95f0-6ccf-45bd-8981-f35d95e2e249","added_by":"auto","created_at":"2025-11-19 07:16:46","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":273194,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of apoptosis after upregulation of Claudin-2 protein by lentivirus.\u003c/p\u003e\n\u003cp\u003eNotes: Figure 8 A shows 2D scatter plots of apoptosis of HK-2 cells in the blank control group, negative control group, and CLAUDIN2 overexpression group detected by flow cytometry. Figure 8B shows the statistical analysis diagram of the data of apoptosis detected by flow cytometry, *vs negative control group, p\u0026lt; 0.05, **vs normal control, p\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8008643/v1/cad5dc8726d66ec844c1580d.png"},{"id":96242490,"identity":"c955c6bb-ad7a-4862-827d-bdf81fce3a95","added_by":"auto","created_at":"2025-11-19 07:13:14","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":258350,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in apoptosis-related proteins after upregulation of Claudin-2 protein expression by lentivirus.\u003c/p\u003e\n\u003cp\u003eNote: Figure 9 A shows the expression of Claudin-2 and apoptosis-related proteins (Caspase-3, Bax, Bcl-2) detected by WesternBlot after upregulation of Claudin-2 protein expression by lentivirus. Figure 9 B shows the statistical analysis of the gray value of Caspase-3 protein, Figure 9 C shows the statistical analysis of the gray value of Bcl-2 protein, Figure 9 D shows the statistical analysis of the gray value of Claudin-2 protein, Figure 9 E shows the statistical analysis of the gray value of Bax protein, Figure 9 F shows the statistical analysis of the Bax/ Bcl-2 ratio. *vs normal control, p\u0026lt;0.05, **vs normal control, p\u0026lt; 0.01, ***vs normal control, p\u0026lt; 0.001, ****vs normal control, p\u0026lt;0.0001, there was no significant difference between the ns vs NC group.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8008643/v1/2d816d22f6fea772e6afaf85.png"},{"id":96255351,"identity":"7a64ca22-b0ec-4dce-aefa-43a2faa51af3","added_by":"auto","created_at":"2025-11-19 07:48:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7078913,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8008643/v1/216ffce0-600e-4ab1-86d3-cd5406e3addf.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mechanism of Claudin-2 in RTECS apoptosis after renal obstruction","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAKI after renal obstruction caused by stones, tumors and other reasons is common. Renal hypoperfusion is one of the most common causes of AKI after renal obstruction, namely renal ischemia. Renal cells are very sensitive to ischemia, especially proximal renal tubular epithelial cells are easy to be damaged and lost during renal tissue ischemia, and further block renal tubules and aggravate renal injury [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Studies have found that after ureteral obstruction, renal pelvis enlargement, renal tissue compression, and edema lead to renal ischemia and hypoxia, accumulation of toxic products, and apoptosis of renal tubular epithelial cells [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Apoptosis plays an important role in various renal ischemia-induced AKI. However, intervention of the classical apoptosis regulatory pathway can only reduce apoptosis to a certain extent, suggesting that there may be other unknown molecular pathways regulating apoptosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. At the same time, in our previous study, we found that there are various forms of regulated cell death in meth-induced AKI, and apoptosis is the main mechanism of early renal tubular cell death in AKI [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. And TMT labeled proteomics technology showed that the tight junction protein Claudin-2 may be involved in this process.\u003c/p\u003e\u003cp\u003eClaudin protein is a very important class of tight junction proteins, which plays an important role in the selective transport process of ions and water, cell proliferation and tumorigenesis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. For example, recent studies have shown that the expression of Claudin-2 and other tight junction proteins changes in AKI caused by multiple causes such as ischemia-reperfusion, sepsis, and drugs, and the intervention of Claudin-2 expression can effectively reduce the damage of renal tubular epithelial cells [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe aim of this study is to first detect the expression of Claudin-2 and apoptotic proteins (Bax, Bcl-2, and Caspase-3) in an animal model of AKI induced by UUO. Then, the OGD model of renal tubular epithelial cells was constructed at the cellular level, and the apoptosis of renal tubular epithelial cells and the expression of apoptotic proteins and Claudin-2 protein were detected. The correlation between Claudin-2 and renal tubular epithelial cell apoptosis was explored by interfering Claudin-2 protein expression by lentiviral vector. To explore the regulatory mechanism of Claudin-2 in renal tubular epithelial cell apoptosis in ischemia-induced AKI, and to provide a theoretical basis for the subsequent treatment of AKI.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Antibodies and reagents\u003c/h2\u003e\u003cp\u003eβ-actin antibodies were purchased from Beyotime, Bax, Bcl-2, and Caspase-3 antibodies were purchased from ZENBIO, and Claudin-2 antibodies was purchased from Thermo Fly. Lentivirus reagents were ordered from Syngentech.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2.Cell culture and treatments\u003c/h2\u003e\u003cp\u003eThe human proximal tubular epithelial cell line (HK-2 cells) was obtained from the Cell Bank of Chinese Academy of Sciences. HK-2 cells were cultured in a 5%CO\u003csub\u003e2\u003c/sub\u003e humidified incubator at 37℃ with Pricella-specific or sugar-free medium for HK-2 cells. To simulate the hypoxic environment of renal injury, the OGD model of HK-2 cells was constructed by oxygen deprivation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3.Animal and UUO models\u003c/h2\u003e\u003cp\u003eC57 mice were purchased from Slake Jingda Co, Hunan. C57 mice were fostered at the Laboratory Animal Center of Central South University. The experimental procedures and protocols were in accordance with the \"3R\" principle and the Code of experimental ethics of Central South University.\u003c/p\u003e\u003cp\u003eEighteen healthy female C57 mice were randomly divided into 3 groups: normal Control group (Control group), Sham operation group (Sham group) and unilateral ureteral ligation model group (UUO group), 6 in each group.The unilateral ureteral ligation (UUO) model group was established as described in previous studies.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4.Renal function, histopathology\u003c/h2\u003e\u003cp\u003eSerum creatinine and urea nitrogen were measured by sarcosine oxidase method and urease method, respectively. Renal tissues were stained for H-E as described previously. Tubular damage was scored according to the percentage of tubular damage (grade 0, no damage; Grade 1: \u0026lt;25%; Grade 2 :25\u0026ndash;49%; Grade 3, 50\u0026ndash;75%; Grade 4:\u0026gt;75%).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5.Lentivirus transfection\u003c/h2\u003e\u003cp\u003eHK-2 cells were inoculated into 6-well plates during passage, and lentivirus infection (the virus carried GFP fluorescent tag) was performed when the cell growth density reached 30%. 1980\u0026micro;l of cell culture medium containing 5\u0026micro;g/mL polybrene (co-infection reagent) was added, followed by 20\u0026micro;l of virus suspension at MOI\u0026thinsp;=\u0026thinsp;10 (obtained from the pre-experiment). After 12 hours, the virus suspension was partially changed (the volume was 1000\u0026micro;l), and the fluorescence abundance expression was observed by microscope 72 hours after infection. The infection efficiency was about 85% and the cells grew well enough for OGD treatment and subsequent experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6.Cell viability assays\u003c/h2\u003e\u003cp\u003eThe viability of HK-2 cells was analyzed using the CCK-8 kit (Dojindo, CK04) according to the manufacturer's instructions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7.Western Blot analysis\u003c/h2\u003e\u003cp\u003eProteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 8% to 12% gels. The dissolved proteins were transferred to a polyvinylidene difluoride membrane (0.45 \u0026micro;m) and blocked with 5% skim milk. The membranes were incubated with β-Actin-HRP Rabbit(Beyotime, 1:1000, ab60008), Bax Rabbit pAb (ZENBIO 1:1000,ab380709), Bcl2 Rabbit pAb (ZENBIO 1:1000, ab381702), Caspase-3 pAb(ZENBIO 1:1000, AB380709) ab350192), Claudin-2 pAb(Thermo, 1:500, ab516100), with two resistance (HRP - conjugated anti - Mouse, ZENBIO, 1:50 00, ab511103 / HRP - conjugated anti - Rabbit, Proteintech, 1:10 000, ab00001-2) hybrid 60 min. Blots were visualized by a gel imaging system and analyzed by ImageJ software. Each immunoblot experiment was repeated three times.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8.Analysis of apoptosis\u003c/h2\u003e\u003cp\u003eApoptosis was analyzed by TUNEL assay and flow cytometry. ANXA5/annexin v positive cells were regarded as apoptotic cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9.Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll data were obtained from three independent experiments, analyzed by SPSS 19.0 (IBM, Armonk, NY). Qualitative data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). The differences between two groups were analyzed by t-test, and the differences among multiple groups were analyzed by one-way ANOVA followed by Tukey's multiple comparison test. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.1.The levels of serum creatinine and urea nitrogen increased significantly after UUO.\u003c/h2\u003e\u003cp\u003eEighteen healthy female C57 mice were randomly divided into 3 groups: normal Control group (Control group), Sham operation group (Sham group) and unilateral ureteral ligation model group (UUO group), there were 6 mice in each group. Blood was collected after modeling, and serum creatinine (CRE) and blood urea nitrogen (BUN) were detected by sarcoline oxidase method and urease method. The results showed that the plasma concentrations of CRE and BUN in UUO model group were significantly higher than those in Control group and Sham group, and the difference was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eNote\u003c/strong\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA shows that the serum creatinine (CRE) value of the UUO group was significantly higher than that of the normal control group and the sham operation group, ***vs normal control group, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB showed that the blood urea nitrogen (BUN) value of the UUO group was significantly higher than that of the normal control group and the sham operation group, ***vs normal control group, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.2.HE staining was used to evaluate the renal injury of C57 mice after UUO.\u003c/h2\u003e\u003cp\u003eHE staining was used to observe the renal tissue injury after different treatments in the Control group, Sham group, and UUO group. The results of HE staining showed that the renal pathological sections of the UUO group showed obvious dilatation of the renal tubules, flat renal tubular epithelial cells, glomerular atrophy, and local inflammatory cell infiltration compared with the Control group. There were no obvious pathological changes in renal tissue sections of the Control group and the Sham group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eNote\u003c/strong\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows HE staining to evaluate renal injury in mice in the normal control group, sham operation group, and unilateral ureteral ligation group, respectively, scale bar =\u0026thinsp;100\u0026micro;m.\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.3.TUNEL staining was used to detect the apoptosis of renal tubular epithelial cells.\u003c/h2\u003e\u003cp\u003eAfter the UUO acute kidney injury model was successfully constructed, the death form of renal tubular epithelial cells was detected. Through TUNEL staining of renal tissue sections, the results showed that: TUNEL staining was positive in renal pathological sections of UUO group, suggesting that the number of apoptotic cells in acute kidney injury after UUO was significantly higher than that in Control group and Sham group. Combined with previous literature research, we speculated that apoptosis may be the main form of renal tubular epithelial cell death in acute ischemic kidney injury.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eNote\u003c/strong\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows apoptosis of renal tubular epithelial cells detected by TUNEL staining; nuclei in blue and positive TUNEL staining in green, scale bar =\u0026thinsp;100\u0026micro;m.\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.4.The expression of Claudin-2 protein decreased and the expression of apoptotic proteins changed after UUO.\u003c/h2\u003e\u003cp\u003eWestern Blot was used to further detect the changes of apoptosis-related proteins to clarify the regulatory pathway of apoptosis associated with ischemic AKI. The results of Western Blot showed that the expression of Claudin-2 protein in renal tissue of UUO mice decreased, the expression of Caspase-3 increased significantly, the expression of Bax increased, and the expression of Bcl-2 decreased, and the differences were statistically significant. However, there was no significant change in Claudin-2 and apoptotic protein expression between the control group and the sham operation group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eNote\u003c/strong\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA shows the changes of Claudin2, Caspase-3, Bcl-2, and Bax related apoptosis indicators in the renal tissue of mice after UUO. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB shows the statistical analysis of Claudin2 protein gray values, *vs normal control, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC shows the statistical analysis of the gray value of Caspase-3 protein, **vs normal control, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD shows the statistical analysis of Bax protein gray value, ***vs normal control group, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Panel E shows the statistical analysis of Bcl-2 protein gray values, *vs normal control, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.5.The viability of HK-2 cells decreased after OGD treatment.\u003c/h2\u003e\u003cp\u003eIn order to understand the role of Claudin-2 in apoptosis induced by ischemia and hypoxia, human HK-2 cells were selected to establish the OGD model. The changes of HK-2 cell status after OGD treatment were observed under inverted microscope, the cell death rate was detected by LDH method, and the cell viability was detected by CCK8. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA shows the changes of cell state after OGD treatment in the blank group and different OGD time. It can be observed that HK-2 cells in the blank group had a small gap, a typical cobbling-stone arrangement, and a good cell growth. After oxygen-glucose deprivation treatment, the cell morphology showed shrinkage, irregular, and even cell membrane fragmentation, and the cell gap became wider. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB shows the cell death rate measured by LDH method. The results showed that the cell death rate increased with the extension of OGD time when the OGD treatment time was 2h, 4h, 6h, 8h, 12h, 24h, and 48h, and the cell death rate increased to about 90% after 24h-48h of OGD. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC shows the cell viability measured by CCK-8 method. The results showed that the cell viability decreased with the increase of OGD time when the OGD treatment time was 2h, 4h, 6h, 8h, 12h, 24h, and 48h, and the cell viability decreased to about 60% after OGD4h (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). After OGD6h, the cell viability was reduced to about 40% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eNote\u003c/strong\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA shows the changes of cell state of HK-2 cells treated with different OGD times under an inverted optical microscope, scale bar =\u0026thinsp;100\u0026micro;m. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB shows the curve of cell death rate of HK-2 cells after different OGD time treatment by LDH method. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC shows the statistical analysis of cell viability of HK-2 cells treated with different OGD times by CCK-8 method, ****vs normal control group, P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.6. The apoptosis of HK-2 cells increased after OGD treatment.\u003c/h2\u003e\u003cp\u003eAnnexin V-FITC/PI double staining was performed on HK-2 cells after OGD treatment, and the cell apoptosis was detected by flow cytometry. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA shows that Q1 quadrant region represents mechanically necrotic cells, Q2 quadrant region represents late apoptotic cells, Q3 quadrant region represents early apoptotic cells, and Q4 quadrant region represents normal living cells. Flow cytometry showed that HK-2 cells underwent apoptosis after OGD treatment, and the apoptosis rate increased with the extension of OGD treatment time. Among them, the apoptosis rate was significantly increased at 4h, 6h, and 8h of OGD treatment, and the apoptosis rate increased significantly at 12h of OGD treatment, but the number of living cells decreased sharply. It was not appropriate to select this time point for subsequent experiments.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNotes: Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA shows the 2D scatter plot of HK-2 cell apoptosis detected by flow cytometry after different OGD treatment durations. Figure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB shows statistical analysis of flow cytometry data for apoptosis, *vs normal control, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ***vs normal control, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.7.The expression of Claudin-2 protein decreased and the levels of apoptotic proteins changed after OGD treatment.\u003c/h2\u003e\u003cp\u003eBased on the above experiments, we selected 4h, 6h and 8h of OGD as the appropriate time to establish the model of ischemia and hypoxia for subsequent experiments. Western blot showed that the expression of Claudin-2 protein decreased, the expression of Bax increased, the expression of Bcl-2 decreased, and the expression of Caspase-3 increased in HK-2 cells after OGD4h. Compared with the normal control group, the differences were statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eNote\u003c/strong\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA shows the expressions of Caspase-3, Claudin-2, Bax, and Bcl-2 detected by WesternBlot after treatment with different OGD times. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB shows the statistical analysis of Caspase-3 protein gray value, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC shows the statistical analysis of Bcl-2 protein gray value, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD shows the statistical analysis of Claudin2 protein gray value, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE shows the statistical analysis of Bax protein gray value, *vs normal control, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ***vs normal control, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****vs normal control, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eThe above results proved that HK-2 cells underwent apoptosis after OGD treatment, Claudin-2 protein expression decreased, Caspase-3, Bax, Bcl-2 and other related apoptotic proteins changed, suggesting that Claudin-2 protein may induce cell apoptosis after hypoxia treatment. We further investigated the role of Claudin-2 in cell apoptosis by up-regulating Claudin-2 protein expression by lentivirus.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.8. Upregulation of Claudin-2 protein expression by lentivirus can reduce cell apoptosis.\u003c/h2\u003e\u003cp\u003eCombined with the above experiments, we finally selected the model condition of ischemia and hypoxia for 4 hours for subsequent lentivirus transfection experiments. Claudin-2 protein was up-regulated by lentiviral vector transfection, and the apoptosis of cells after OGD treatment was detected by flow cytometry. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-B shows that compared with the NC group, the apoptosis rate of CLAUDIN2 overexpression group decreased by about 8% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNotes: Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA shows 2D scatter plots of apoptosis of HK-2 cells in the blank control group, negative control group, and CLAUDIN2 overexpression group detected by flow cytometry. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB shows the statistical analysis diagram of the data of apoptosis detected by flow cytometry, *vs negative control group, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **vs normal control, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.9. Changes of apoptosis-related proteins after upregulation of Claudin-2 protein expression by lentivirus.\u003c/h2\u003e\u003cp\u003eThe above results showed that ischemia and hypoxia caused apoptosis of HK-2 cells and decreased Claudin-2 protein, and up-regulation of Claudin-2 protein by lentivirus could reduce the occurrence of apoptosis. We further explored whether Claudin-2 protein was involved in cell apoptosis through Caspase-3, Bax, and Bcl-2. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-E, Claudin-2 protein was significantly increased in the OGD4h\u0026thinsp;+\u0026thinsp;Claudin-2 overexpression group, suggesting that the lentiviral overexpression model at the molecular protein level was successfully established. The expression of Caspase-3 in the OGD4h\u0026thinsp;+\u0026thinsp;Claudin-2 overexpression group was significantly lower than that in the blank group and the negative control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). There was no significant difference in the ratio of Bax/Bcl-2 between the OGD4h\u0026thinsp;+\u0026thinsp;Claudin-2 overexpression group and the NC group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eNote\u003c/strong\u003e\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA shows the expression of Claudin-2 and apoptosis-related proteins (Caspase-3, Bax, Bcl-2) detected by WesternBlot after upregulation of Claudin-2 protein expression by lentivirus. Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB shows the statistical analysis of the gray value of Caspase-3 protein, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC shows the statistical analysis of the gray value of Bcl-2 protein, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD shows the statistical analysis of the gray value of Claudin-2 protein, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE shows the statistical analysis of the gray value of Bax protein, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eF shows the statistical analysis of the Bax/ Bcl-2 ratio. *vs normal control, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **vs normal control, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***vs normal control, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****vs normal control, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, there was no significant difference between the ns vs NC group.\u003c/p\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eStudies have found that apoptosis and necrosis of renal tubular epithelial cells occur in UUO model mice under renal ischemia, accumulation of toxic products and other factors, resulting in renal injury [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In the UUO rat model, some scholars have found that thymosin may reduce the apoptosis of renal tubular epithelial cells and the process of renal fibrosis in UUO rats by inhibiting the TGF-β pathway [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, the specific mechanism of renal tubular epithelial cell apoptosis after UUO is still unclear, which needs to be further explored.\u003c/p\u003e\u003cp\u003eThe effect of OGD on cell apoptosis was detected after Claudin-2 protein was up-regulated by lentivirus transfection. The results showed that after the up-regulation of Claudin-2 protein by lentivirus vector transfection, the apoptosis of renal tubular epithelial cells still existed after OGD treatment, but the proportion of apoptosis decreased compared with NC (negative control group), and the difference was statistically significant. These results indicate that Claudin-2 is associated with apoptosis of renal tubular epithelial cells after OGD treatment, and up-regulation of Claudin-2 can reduce the occurrence of apoptosis. Previous studies have shown that the expression of Claudin-2 is down-regulated and related oxidative stress markers are increased when cisplatin-induced acute kidney injury occurs. Cisplatin may cause kidney injury by changing the localization of tight junction proteins such as Claudin-2 related to oxidative stress [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and oxidative stress can exacerbate the occurrence of apoptosis. In addition, Claudin-2, as a key molecule regulating calcium reabsorption, downregulation of Claudin-2 can lead to calcium homeostasis imbalance and hypercalciuria in tubular epithelial cells, and increased calcium levels can further induce apoptosis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Therefore, the mechanism by which Claudin-2 affects renal tubular epithelial cell apoptosis after acute kidney injury may be related to oxidative stress and calcium imbalance.\u003c/p\u003e\u003cp\u003eThe reasons why the apoptosis of renal tubular epithelial cells still occurs after OGD treatment with up-regulation of Claudin-2 protein in this study may be related to the following: first, the mechanism of apoptosis of renal tubular epithelial cells after OGD treatment is very complex, and Claudin-2 may be only one of the key molecules involved in the apoptosis pathway.\u003c/p\u003e\u003cp\u003eApoptosis is jointly mediated by mitochondrial pathway, T cell-mediated perforin/granzyme pathway, death receptor pathway and endoplasmic reticulum stress (ERS) pathway [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and these apoptotic pathways are not completely independent. For example, endoplasmic reticulum stress pathway and mitochondrial pathway jointly affect BCL2 family proteins to regulate apoptosis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Mitochondrial membrane permeability is regulated by BCL2 family proteins, and the increase of Bax/Bcl2 ratio can lead to further apoptosis. In this study, the ratio of Bax/Bcl2 did not change significantly after up-regulation by lentiviral vector, suggesting that Claudin-2 may not regulate apoptosis through Bax, Bcl2 and other proteins. In addition, there are a large number of procaspase forms in cells, which can trigger each other once activated, and eventually start the Caspase cascade reaction leading to subsequent apoptosis. Caspase enzymes are mainly divided into the initiation factors of caspase-2, 8, 9, 10, and the execution factors of caspase-3, 6, and 7, and the inflammatory mediators of caspase-1, 4, and 5. Caspase-3 activation is a key step in initiating apoptosis. We found that after upregulation of Claudin-2 protein by lentivirus, Caspase-3 protein decreased compared with NC group and the difference was statistically significant, indicating that Claudin-2 may induce apoptosis through Caspase-3 pathway.\u003c/p\u003e\u003cp\u003eSecondly, lentiviral vectors are obtained by the modification of HIV carrying target genes, which can be carried into cells and expressed, but the transduction efficiency is limited. How to improve the transduction efficiency has always been a problem waiting to be solved. Moreover, there are some problems such as the risk of insertion mutation and affecting cell differentiation in subsequent cell growth [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Therefore, the effect of upregulation of Claudin-2 protein by the lentiviral vector in this experiment was not complete.\u003c/p\u003e\u003cp\u003eThis study also has some shortcomings: although it has been proved that up-regulation of Claudin-2 protein can inhibit the apoptosis of renal tubular epithelial cells, OGD treatment only using HK-2 cells cannot fully reflect the complete pathophysiological process of acute kidney injury after renal obstruction, and lacks the influence of different types of intercellular interaction and intracellular environment regulation. In addition, the human proximal tubular epithelial cell line HK-2 does not fully reflect the common apoptotic characteristics of other renal tubular epithelial cells. Similarly, overexpression of lentiviral vectors cannot completely replace the compensatory effect of the normal physiological pathway, and thus may have some influence on the experimental results.\u003c/p\u003e\u003cp\u003eIn addition, this experiment demonstrated that Claudin-2 may induce apoptosis through Caspase-3, but because after the initial apoptotic signal stimulation, The activation of the precursors of caspases (such as Caspase-8 and Caspase-9) during proteolysis leads to the subsequent enzyme cascade of caspase-3 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Caspase-3 is the central link in the initiation of multiple apoptotic signaling pathways, and the connection between Caspase-3 and Claudin-2 may act through other unknown apoptotic molecules or non-single regulatory pathways. The specific regulatory mechanism of caspase-3 and Claudin-2 remains to be further studied. Finally, apoptosis is only one form of regulated cell death in renal tubular epithelial cells after acute kidney injury. The Caspase family also plays an important role in other regulated cell death such as pyroptosis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Further study on the mechanism of Claudin-2 related to other regulated cell death such as pyroptosis may also be one of the future research directions.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn conclusion, the present study suggests that Claudin-2 protein is involved in the apoptosis of renal tubular epithelial cells after acute obstructive kidney injury, which provides a theoretical basis for further investigation of the molecular mechanism of Claudin-2 in the apoptosis of rtecs in obstructive kidney injury. Claudin-2 may be one of the targets of rtec apoptosis in the treatment of obstructive acute kidney injury.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eRTECs:Renal epithelial cells;UUO:Unilateral ureteral obstruction;OGD:Oxygen-glucose deprivation;AKI:Actue kidney injury;CRE:Creatinine; TGF-\u0026beta;:Transforming growth factor-\u0026beta;;BUN:Blood urea nitrogen;RCD:Regulated cell death; TMT:Tandem mass tag; MOI:Multiplicity Of Infection; \u0026nbsp;GFP:Green fluorescent protein;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDisclosure statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo potential conflict of interest was reported by the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions from authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJin Tang conceived and designed the experiment. Dongsheng Zhao performed most of the experiments. Zhao Dongsheng wrote the original manuscript, which was substantially revised by Tang Jin. Guijiang Tang, Guoqian Hu, Liang Zeng, and Wen Su performed the collection and data collection. Zhao Dongsheng performed the statistical analysis. All the authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Experimental Ethics Committee of Central South University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding support\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eEl Gazzar WB, Allam MM, Shaltout SA et al (2023) Pioglitazone modulates immune activation and ameliorates inflammation induced by injured renal tubular epithelial cells via PPARγ/miRNA\u0026ndash;124/STAT3 signaling [J]. Biomedical Rep 18(1):2\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKanagasundaram NS (2015) Pathophysiology of ischaemic acute kidney injury [J]. Ann Clin Biochem 52(Pt 2):193\u0026ndash;205\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePriante G, Gianesello L, Ceol M et al (2019) Cell Death in the Kidney [J]. Int J Mol Sci, 20(14)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTruong LD, Petrusevska G, Yang G et al (1996) Cell apoptosis and proliferation in experimental chronic obstructive uropathy [J]. Kidney Int 50(1):200\u0026ndash;207\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eN\u0026oslash;rregaard R, Mutsaers HAM, Fr\u0026oslash;ki\u0026aelig;r J et al (2023) Obstructive nephropathy and molecular pathophysiology of renal interstitial fibrosis [J]. Physiol Rev 103(4):2827\u0026ndash;2872\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSanz AB, Sanchez-Ni\u0026ntilde;o MD, Ramos AM et al (2023) Regulated cell death pathways in kidney disease [J]. Nat Rev Nephrol 19(5):281\u0026ndash;299\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e倪佳芸 陆利民 (2022) 调节性细胞死亡与急性肾损伤 [J] 生理学报 74(01):4\u0026ndash;14\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHavasi A, Borkan SC (2011) Apoptosis and acute kidney injury [J]. Kidney Int 80(1):29\u0026ndash;40\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDevarajan P (2006) Update on mechanisms of ischemic acute kidney injury [J]. J Am Soc Nephrology: JASN 17(6):1503\u0026ndash;1520\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDan Q, Shi Y, Rabani R et al (2019) Claudin-2 suppresses GEF-H1, RHOA, and MRTF, thereby impacting proliferation and profibrotic phenotype of tubular cells [J]. J Biol Chem 294(42):15446\u0026ndash;15465\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHwang I, An BS, Yang H et al (2013) Tissue-specific expression of occludin, zona occludens-1, and junction adhesion molecule A in the duodenum, ileum, colon, kidney, liver, lung, brain, and skeletal muscle of C57BL mice [J]. J Physiol Pharmacology: Official J Pol Physiological Soc 64(1):11\u0026ndash;18\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e韦伟, 杨乐天 (2022) 赵宇亮. 急性肾损伤中肾小管上皮紧密连接的研究进展 [J]. 肾脏病与透析肾移植杂志 31(06):567\u0026ndash;572\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOtani T, Furuse M (2020) Tight Junction Structure and Function Revisited [J]. Trends Cell Biol 30(10):805\u0026ndash;817\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu ASL (2015) Claudins and the kidney [J]. J Am Soc Nephrology: JASN 26(1):11\u0026ndash;19\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChevalier RL, Forbes MS, Thornhill BA (2009) Ureteral obstruction as a model of renal interstitial fibrosis and obstructive nephropathy [J]. Kidney Int 75(11):1145\u0026ndash;1152\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSaikumar P, Venkatachalam MA (2003) Role of apoptosis in hypoxic/ischemic damage in the kidney [J]. Semin Nephrol 23(6):511\u0026ndash;521\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYuan J, Shen Y, Yang X et al (2017) Thymosin β4 alleviates renal fibrosis and tubular cell apoptosis through TGF-β pathway inhibition in UUO rat models [J]. BMC Nephrol 18(1):314\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTrujillo J, Molina-Jij\u0026oacute;n E, Medina-Campos ON et al (2014) Renal tight junction proteins are decreased in cisplatin-induced nephrotoxicity in rats [J]. 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Eur J Clin Invest 24(11):715\u0026ndash;723\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003e赵晓煜 徐祺玲 (2021) 赵晓东 et al 基因治疗慢病毒载体的转导增强策略 [J] 中国生物工程杂志 41(08):52\u0026ndash;58\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eArsenijevic Y, Berger A, Udry F et al (2022) Lentiviral Vectors Ocular Gene Therapy [J] Pharm, 14(8)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDaemen MARC, de Vries B, Buurman WA (2002) Apoptosis and inflammation in renal reperfusion injury [J]. Transplantation 73(11):1693\u0026ndash;1700\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVan Opdenbosch N, Lamkanfi M (2019) Caspases in Cell Death, Inflammation, and Disease [J]. Immunity 50(6):1352\u0026ndash;1364\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKesavardhana S, Malireddi RKS, Kanneganti T-D (2020) Caspases in Cell Death, Inflammation, and Pyroptosis [J]. Annu Rev Immunol 38:567\u0026ndash;595\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"urolithiasis","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ures","sideBox":"Learn more about [Urolithiasis](http://link.springer.com/journal/240)","snPcode":"240","submissionUrl":"https://submission.nature.com/new-submission/240/3","title":"Urolithiasis","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Renal obstruction, Renal ischemia, Acute kidney injury, RTECs, Claudin-2, Apoptosis of cells","lastPublishedDoi":"10.21203/rs.3.rs-8008643/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8008643/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eAcute kidney injury (AKI) is a clinical condition characterized by a rapid decline in glomerular filtration function caused by multiple factors. Factors such as stones and tumors can lead to AKI following renal obstruction. Renal tubular epithelial cell injury is a key component of the pathophysiological mechanism of ischemic acute kidney injury after obstruction.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eOxygen-glucose deprivation (OGD) in HK-2 cells and a mouse model of unilateral ureteral ligation (UUO) were used to investigate the role of Claudin-2 in renal tubular epithelial cell apoptosis in ischemia-induced AKI.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eIn animal experiments, the expression of Claudin-2 protein was decreased, while Bax and Caspase-3 expression were increased, and Bcl-2 expression was decreased in the renal tissue of UUO mice. Similarly, after OGD treatment, Claudin-2 protein expression was decreased, Bax and Caspase-3 expression were increased, and Bcl-2 expression was decreased. Upregulation of Claudin-2 protein expression through lentivirus transfection in OGD-treated HK-2 cells reduced the decline in cell viability and the proportion of apoptotic cells. Additionally, upregulation of Claudin-2 protein expression reduced OGD-induced Caspase-3 expression, while the Bax/Bcl-2 ratio showed no significant change.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThe expression of Claudin-2 is decreased during acute obstructive kidney injury, which leads to changes in Caspase-3 apoptotic protein and activates cell apoptosis.\u003c/p\u003e","manuscriptTitle":"Mechanism of Claudin-2 in RTECS apoptosis after renal obstruction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-14 09:21:15","doi":"10.21203/rs.3.rs-8008643/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-01T13:43:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-25T22:20:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"64078711715196656370534638573277603868","date":"2025-11-19T14:02:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-04T20:36:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-03T15:20:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-03T15:19:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Urolithiasis","date":"2025-11-02T03:36:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"urolithiasis","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ures","sideBox":"Learn more about [Urolithiasis](http://link.springer.com/journal/240)","snPcode":"240","submissionUrl":"https://submission.nature.com/new-submission/240/3","title":"Urolithiasis","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8144f2f3-47a3-4848-8398-28d5466e818f","owner":[],"postedDate":"November 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-01-23T18:09:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-14 09:21:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8008643","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8008643","identity":"rs-8008643","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}