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Typle of the Study:Original Research AritleMechanism of Claudin-2 in RTECS apoptosis after renal obstruction
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Dongshengzhao1Emailzds18670013875@163.com
GuijiangTang1Emailtangguijiang2022@163.com
GuoqianHu1Email2621339985@qq.com
LiangZeng1Email1250956440@qq.com
WenSu1Email102285634@qq.com
JinTang
M.D.
2✉Phone13907316880Emailajin530@139.com1Department of UrologyThe Third Hosptial, Central South UniversityChangshaChina
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138 Tongzibo Road, Yuelu District, Hexi DistrictChangsha CityHunan ProvinceDongsheng zhao1, Guijiang Tang2,Guoqian Hu3, Liang Zeng4, Wen Su5, Jin Tang*.
1Department of Urology,The Third Hosptial, Central South University,Changsha,China;email:zds18670013875@163.com.
2Department of Urology,The Third Hosptial, Central South University,Changsha,China;email:tangguijiang2022@163.com.
3Department of Urology,The Third Hosptial, Central South University,Changsha,China;email:2621339985@qq.com.
4Department of Urology,The Third Hosptial, Central South University,Changsha,China;email:1250956440@qq.com.
5Department of Urology,The Third Hosptial, Central South University,Changsha,China;102285634@qq.com.
*Corresponding author:
Author name:Jin Tang,M.D.
Author affiliation:Department of Urology,The Third Hosptial, Central South University,Changsha,China;
Address:138 Tongzibo Road, Yuelu District, Hexi District, Changsha City, Hunan Province.
Tel:13907316880
Fax:0086-731-13907316880
Email:ajin530@139.com
Mechanism of Claudin-2 in RTECS apoptosis after renal obstruction
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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.
Key words:
Renal obstruction
Renal ischemia
Acute kidney injury
RTECs
Claudin-2
Apoptosis of cells
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;
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1.Introduction
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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%CO2 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
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C57 mice were purchased from Slake Jingda Co, Hunan. C57 mice were fostered at the Laboratory Animal Center of Central South University. A
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: <25%; Grade 2 :25–49%; Grade 3, 50–75%; Grade 4:>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.
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2.7.Western Blot analysisProteins 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).
Fig. 1
Concentrations of serum creatinine (CRE) and blood urea nitrogen (BUN) after UUO
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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 < 0.001; Fig. 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 < 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.
Fig. 2
Assessment of kidney injury in mice by HE staining
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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.
Fig. 3
Apoptosis of renal tubular epithelial cells detected by TUNEL staining.
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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.
Fig. 4
Changes in Claudin2 and other related proteins in the control, sham, and UUO groups.
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Figure 4A shows the changes of Claudin2, Caspase-3, Bcl-2, and Bax related apoptosis indicators in the renal tissue of mice after UUO. Figure 4B shows the statistical analysis of Claudin2 protein gray values, *vs normal control, p < 0.05; Fig. 4C shows the statistical analysis of the gray value of Caspase-3 protein, **vs normal control, p < 0.01; Fig. 4D 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 5A 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 5B 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 5C 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).
Fig. 5
Changes in cell state, cell death rate, and cell viability after OGD.
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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 < 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 6A 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.
Fig. 6
Apoptosis of HK-2 cells treated with different OGD time.
Notes: Fig. 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 < 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).
Fig. 7
Expression of Caspase-3, Claudin-2, Bax, and Bcl-2 after treatment with different OGD times.
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Figure 7A shows the expressions of Caspase-3, Claudin-2, Bax, and Bcl-2 detected by WesternBlot after treatment with different OGD times. Figure 7B shows the statistical analysis of Caspase-3 protein gray value, Fig. 7C shows the statistical analysis of Bcl-2 protein gray value, Fig. 7D shows the statistical analysis of Claudin2 protein gray value, Fig. 7E 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 8A-B shows that compared with the NC group, the apoptosis rate of CLAUDIN2 overexpression group decreased by about 8% (p < 0.05).
Fig. 8
Analysis of apoptosis after upregulation of Claudin-2 protein by lentivirus.
Notes: Fig. 8A 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 < 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. 9A-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.
Fig. 9
Changes in apoptosis-related proteins after upregulation of Claudin-2 protein expression by lentivirus.
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Figure 9A 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 9B shows the statistical analysis of the gray value of Caspase-3 protein, Fig. 9C shows the statistical analysis of the gray value of Bcl-2 protein, Fig. 9D shows the statistical analysis of the gray value of Claudin-2 protein, Fig. 9E shows the statistical analysis of the gray value of Bax protein, Fig. 9F 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.
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
A
This study was approved by the Experimental Ethics Committee of Central South University.A
Funding
support
No.
A
Author Contribution
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.
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