3.1 Overexpression of Gremlin mRNA
Induction of Gremlin expression was achieved by exposing mesangial cells to 5.5 mM glucose, 10 mM glucose, 20 mM glucose and 30 mM glucose in DMEM media for 24 hr. Gremlin mRNA expression was determined by qRT-PCR. The expression level of Gremlin increased by about 5.40-fold in 30 mM glucose (Fig. 1).
3.2 Overexpression of Gremlin mRNA under exposure to 30mM glucose
Mesangial cells were cultured in DMEM media containing 30mM glucose for 0 hr, 24 hr, 48 hr and 72 hr. Gremlin mRNA expression was determined by qRT-PCR. The expression level of Gremlin increased by about 4.94-fold after 48 hr (Fig. 2).
3.3 Efficacy of Infection with lentiviral vector carrying green fluorescein protein-labled shRNAs
To determine the efficacy of infection with Gremlin and negative control shRNAs (shNC), green fluorescein protein (GFP)-labeled shRNAs were transfected into RMCs. The cells were then evaluated by fluorescence microscopy. Most of the RMCs exhibited strong fluorescence (Fig. 3).
3.4 Gremlin shRNA Inhibits Gremlin Expression in RMCs
As seen in Figure 4, Gremlin mRNA expression in the high glucose- treated group was about 3.92-fold greater compared with the normal glucose control group (N). Treatment with Gremlin shRNA significantly inhibited Gremlin expression induced by HG conditions, but the negative control shRNA group was no different than N group.
3.5 Effect of Gremlin shRNA on the level of BMP-7, pSmad 1/5/8, total Smad 1/5/8 protein in high glucose (HG)-induced RMCs
We investigated the mechanism of inhibition of Gremlin expression in fibrosis, by analyzing the expression of BMP-7-Smad signal pathway in RMCs cultured under HG conditions. Incubation of cultured cells under HG conditions over 48 hours revealed a gradual increase in Gremlin expression with associated decrease in BMP-7. Similarly, the level of phosphorylated Smad1/5/8, markers of BMP-7 activity, was significantly and gradually decreased while total Smad1/5/8 protein levels remained constant. Significant changes in BMP-7 expression were seen after infection of cells with Gremlin shRNA. Gremlin shRNA prevented the decrease in phosphorylation of Smad1/5/8 (Fig.5). These results suggest that the protective effects of shRNA-induced inhibition of Gremlin expression on RMCs were, at least partially, mediated by BMP-7.
3.6 Transfection with Gremlin shRNA reduces over-expression of FN and Col IV accumulation induced by high glucose
To evaluate the impact of Gremlin inhibition on fibrosis and the underlying molecular mechanisms, cultured RMCs were again infected with Gremlin shRNA or negative control and subjected to stimulation with high glucose. FN and Col IV levels in the culture medium were determined by ELISA (Table 1). Significant accumulation of these fibrotic factors in the culture medium was seen in the HG and HG + shNC groups, while shGremlin infection significantly reduced these changes.