Gremlin Regulates Podocyte Apoptosis via Transforming Growth Factor-β (TGF-β) Pathway in Diabetic Nephropathy

Abstract

BACKGROUND Gremlin has been reported to be up-regulated in glomerular mesangial cells in diabetic nephropathy (DN). However, the regulation of gremlin in podocytes is still rarely reported. This study aimed to investigate the underlying mechanisms by which gremlin mediates the pathogenesis of DN via transforming growth factor-β (TGF-β) signaling pathways. MATERIAL AND METHODS Lentiviral and RNAi transfection were performed to increase and decrease gremlin expression in high-glucose conditions. Expression at the mRNA and protein level was detected by RT-qPCR and Western blotting. RESULTS The expression of gremlin was significantly higher in high-glucose (HG, 30mM) than normal-glucose (NG, 5.5 mM) conditions. The gremlin overexpression significantly suppressed the expression of nephrin and synaptopodin. The phosphorylation of canonical TGF-b signaling pathway components, including Smad2/3 and MKK, was increased in the gremlin-overexpressing group. In addition, the expression levels of Bax and cleaved caspase-3 were also higher in the gremlin-overexpressing group. TGF-β pathway inhibitor (SB505124) significantly inhibited TGF-β pathway activity and enhanced the expression of nephrin and synaptopodin. CONCLUSIONS These results indicate that gremlin can aggravate podocyte lesions through the TGF-β signaling pathway, providing a novel therapeutic target for DN.

Figure 1

Lentiviral-mediated gremlin expression in podocytes. (A) The expression of gremlin mRNA was detected by RT-qPCR. (B, C) The expression of gremlin protein was analyzed by Western blotting. The data represent the means ±SD of 3 experiments. * P<0.05 compared with the control group. DownReg represents knockdown, upReg represents overexpression.

Figure 2

The expression of nephrin and synaptopodin was detected by Western blotting and RT-qPCR. (A) The expression of nephrin and synaptopodin mRNA was detected by RT-qPCR. (B, C) The expression of nephrin and synaptopodin was analyzed by Western blotting. * P<0.05 compared with the NG group.

Figure 3

The activity of the corresponding TGF-β signaling pathway was detected by Western blotting. (A) The expression of gremlin protein was analyzed by Western blotting. (B–G) The ratio of p-Smad2/Smad2, p-Smad3/Smad3, p-MKK/MKK, p-p38/p38, p-ERK/ERK, and p-JNK/JNK. * P<0.05 compared with the NG group.

Figure 4

The effect of SB505124 on nephrin and synaptopodin expression. (A) nephrin and synaptopodin protein expression levels were detected by Western blotting. (B, C) Quantitated protein levels of nephrin and synaptopodin. * P<0.05 vs. the control group.

Figure 5

The effect of gremlin regulation on podocyte apoptosis. (A) mRNA expression levels were detected by RT-qPCR analysis in different groups. (B, C) The protein levels of Bcl-2 and Bax were quantified by Western blotting. * P<0.05 vs. the NG group.