KLF2 alleviates sepsis-induced acute kidney injury via the lncRNA GAS6-AS2/GOLPH3 axis

Figures

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  Fig. 1. LPS induces HK2 cell injury and downregulates KLF2 expression. A: Cell viability was detected by CCK-8; B: The LDH kit was used to detect LDH release in cells; C: mRNA levels of KIM-1 and NGAL were detected by RT-qPCR; D: Cell apoptosis was tested by flow cytometry; E: ROS level in cells was detected by the ROS kit; F: MDA level in cells was detected by the MDA kit; G: Total GSH was detected by the GSH kit; H: ELISA was used to detect the release of TNF-α, IL-6 and IL-1β in cells; I-J: KLF2 expression was determined by RT-qPCR and WB. Three independent repeated tests were conducted, and the data were expressed as mean ± standard deviation. Data comparisons among multiple groups in panels A, B, D-G, I-J were analyzed by one-way ANOVA. Data comparisons among multiple groups in panels C and H were analyzed by two-way ANOVA. Tukey’s multiple comparisons test was performed for the post hoc test. * P < 0.05, ** P < 0.01

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  Fig. 2. KLF2 alleviates LPS-induced injury of HK2 cells. A-B: KLF2 expression in cells was detected by RT-qPCR and WB; C: Cell viability was detected by CCK-8; D: LDH release in cells was detected by the LDH kit; E: KIM-1 and NGAL mRNA levels were detected by RT-qPCR; F: Cell apoptosis was tested by flow cytometry; G: The release of TNF-α, IL-6 and IL-1β in cells was tested by ELISA; H: ROS level was detected by the ROS kit; I: MDA level in cells was detected by the MDA kit; J: Total GSH was detected by the GSH kit. Three independent repeated tests were conducted, and the data were expressed as mean ± standard deviation. Comparisons between the two groups in panel A were analyzed using t test. Data comparisons among multiple groups in panels B-D, F, H-J were analyzed by one-way ANOVA. Data comparisons among multiple groups in panels E and G were analyzed by two-way ANOVA. Tukey’s multiple comparisons test was performed for the post hoc test. * P < 0.05, ** P < 0.01

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  Fig. 3. KLF2 inhibits lncRNA GAS6-AS2 expression. A: The binding site of KLF2 and the lncRNA GAS6-AS2 promoter was predicted by JASPAR database; B: Ch-IP was performed to detect the enrichment of KLF2 on the lncRNA GAS6-AS2 promoter; C: The binding of KLF2 to the lncRNA GAS6-AS2 promoter was detected by dual luciferase report assay; D-E: LncRNA GAS6-AS2 expression was detected by RT-qPCR. Three independent repeated tests were conducted, and the data were expressed as mean ± standard deviation. Data comparisons among multiple groups in panels D-E were analyzed by one-way ANOVA. Data comparisons among multiple groups in panels B-C were analyzed by two-way ANOVA. Tukey’s multiple comparisons test was performed for the post hoc test. ** P < 0.01

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  Fig. 4. LncRNA GAS6-AS2 overexpression attenuates the protective role of KLF2 overexpression on LPS-induced HK2 cell injury. A: The transfection efficiency of lncRNA GAS6-AS2 in cells was detected by RT-qPCR; B: Cell viability was tested by CCK-8; C: LDH release in cells was detected by the LDH kit; D: Cell apoptosis was tested by flow cytometry; E: KIM-1 and NGAL mRNA levels were detected by RT-qPCR; F: The release of TNF-α, IL-6 and IL-1β in cells was tested by ELISA; G: ROS level was detected by the ROS kit; H: MDA level in cells was detected by the MDA kit; I: Total GSH was detected by the GSH kit. Three independent repeated tests were conducted, and the data were expressed as mean ± standard deviation. Comparisons between the two groups in panel A were analyzed using t test. Data comparisons among multiple groups in panels B-D and G-I were analyzed by one-way ANOVA. Data comparisons among multiple groups in panels E-F were analyzed by two-way ANOVA. Tukey’s multiple comparisons test was performed for the post hoc test. * P < 0.05, ** P < 0.01

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  Fig. 5. LncRNA GAS6-AS2 binds to ELAVL2 and indirectly stabilizes GOLPH3. A: The binding probability of ELAVL2 and GOLPH3 was predicted by RPISeq database; B-C: The binding of lncRNA GAS6-AS2 to ELAVL2 was analyzed by RNA pull down and RIP; D: The enrichment of ELAVL2 on GOLPH3 was detected by RIP; E: GOLPH3 mRNA stability was detected by RT-qPCR; F-G: The transfection efficiency of ELAVL2 in cells was detected by RT-qPCR and WB; H-I: GOLPH3 expression was determined by RT-qPCR and WB. Three independent repeated tests were conducted, and the data were expressed as mean ± standard deviation. Comparisons between the two groups in panels F-G were analyzed using t test. Data comparisons among multiple groups in panels C and H-I were analyzed by one-way ANOVA. Data comparisons among multiple groups in panels D-E were analyzed by two-way ANOVA. Tukey’s multiple comparisons test was performed for the post hoc test. * P < 0.05, ** P < 0.01

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  Fig. 6. GOLPH3 overexpression attenuates the protective role of KLF2 overexpression on LPS-induced HK2 cell injury. A: The transfection efficiency of GOLPH3 in cells was detected by RT-qPCR; B: GOLPH3 expression was determined by WB; C: Cell viability was detected by CCK-8; D: LDH release in cells was detected by the LDH kit; E: KIM-1 and NGAL mRNA levels were detected by RT-qPCR; F: The release of TNF-α, IL-6 and IL-1β in cells was tested by ELISA; G: Cell apoptosis was tested by flow cytometry; H: ROS level was detected by the ROS kit; I: MDA level in cells was detected by the MDA kit; J: Total GSH was detected by the GSH kit. Three independent repeated tests were conducted, and the data were expressed as mean ± standard deviation. Comparisons between the two groups in panel A were analyzed using t test. Data comparisons among multiple groups in panels B-D, G-J were analyzed by one-way ANOVA. Data comparisons among multiple groups in panels E-F were analyzed by two-way ANOVA. Tukey’s multiple comparisons test was performed for the post hoc test. * P < 0.05, ** P < 0.01