Figure 7. Knockdown of CNN2 promotes Hmgcs2 desuccinylation to repress tubular cell death in vitro.

(A) ELISA showed CNN2 levels in the conditioned medium (CM) collected from the cultured fibroblasts after knockdown of CNN2 under hypoxic stress. *P < 0.05 (n = 6). (B) Schematic diagram. (C) Western blot assay demonstrated that CNN2-deprived CM enhanced sirt5 and Hmgcs2 expression in NRK-52E cells stimulated with CoCl2 (400 µM). (D and F) Western blot assay demonstrated CNN2-deprived CM decreased the abundance of succinyl-lysine motif (Succ-K) in NRK-52E cells under hypoxia stress (D), but they were increased after knockdown of sirt5 (F). (E and G) Co-immunoprecipitation of endogenous Hmgcs2 from cell lysates of normal control (NC) CM and CNN2-deprived CM. Immunoprecipitation revealed that lysine succinylation on Hmgcs2 is less in CNN2-deprived CM under hypoxia stress (E) but increased after knockdown of sirt5 (G), compared with controls. (H) Western blot assay demonstrated decreased Bax and NGAL levels after incubation with CNN2-deprived CM under hypoxic stress, compared with vehicles. (I–K) After stimulation with staurosporine (STS, 1 μM) for 3 hours, Western blot assay demonstrated the reduced abundance of cleaved caspase-3 (CCP3) in cultured NRK-52E cells incubated with CNN2-deprived CM (I) and immunofluorescence staining showed fewer CCP3⁺ tubular cells after treatment with CNN2-deprived CM (J). Quantitative data are presented (K). Scale bar, 25 μm. *P < 0.05 (n = 3). (L) Western blot assay demonstrated that CNN2-deprived CM reduced abundance of CCP3 in cultured NRK-52E cells, but they were increased after knockdown of Hmgcs2, compared with scramble controls. (M) Western blot assay demonstrated CNN2-deprived CM repressed Bax and NGAL inductions in cultured NRK-52E cells under hypoxia stress, but they were increased after knockdown of sirt5. Graphs are presented as means ± SEM. Differences between groups were analyzed using unpaired t tests or ANOVA followed by the Student-Newman-Keuls test.