Fig. 7

Hypoxic rat mesenchymal stem cells (HMSCs) ameliorated hypoxia-reoxygenation (H/R) injured renal tubular cells through enhancing autophagy. a H/R injury (hypoxia for 24 h followed by reoxygenation for 6 h) significantly decreased cell survival in NRK-52E cells determined by an MTT assay. Either co-culture with HMSCs or addition of HMSC-conditioned medium (CM) decreased H/R-injury-induced cell death. Addition of 3-methyl adenine (3-MA), an autophagy inhibitor, abolished the protective effects of HSMCs and HMSC-CM. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Tukey’s post hoc comparison. ^$p < 0.05 vs H/R group; ^#p < 0.05 vs H/R+HMSC group; ^§p < 0.05 vs H/R+CM group. n = 3 per group. b, c Autophagic flux was assayed by a Premo Autophagy Tandem Sensor RFP-GFP-LC3B kit. Scale = 10 μm. Confocal microscopy showed that the LC3B puncta (yellow color) in the NRK-52E were increased by the co-culture with HMSCs or treatment with HMSC-CM. Addition of 3-MA decreased the formation of LC3B puncta. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Tukey’s post hoc comparison. ^$p < 0.05 vs H/R group; ^#p < 0.05 vs H/R+HMSC group; ^§p < 0.05 vs H/R+CM group. n = 3 per group. d, e Western blot analyses showed that the H/R injury markedly increased the autophagy adaptor protein p62 expression in NRK-52E cells, which was suppressed in the H/R+HMSC and H/R+CM groups. The levels of LC3B, Atg5, and Beclin 1 were significantly increased in the H/R+HMSC group. Addition of 3-MA suppressed the HMSC-upregulated expression of LC3B, Atg5, and Beclin 1. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Tukey’s post hoc comparison. #p < 0.05 vs H/R+HMSC group by Student’s t test. n = 3 per group