Effects of HG on Gremlin protein and mRNA in MMCs over time. MMCs were cultured in media containing 5.5 mM glucose (NG), then stimulated with NG plus 24.5 mM mannitol (M) or 30 mM glucose (HG) for 6, 12, 24, and 48 h. At the end of each time interval, total RNA and protein of the cells were extracted for Gremlin mRNA and protein expression. (A) The level of Gremlin mRNA expression was detected using real-time polymerase chain reaction (RT-PCR) analysis. (B) The expression of Gremlin protein was analyzed using western blot. Values are expressed as means ± S.E.M. *P < 0.05, **P < 0.01 vs. NG.