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In vivo delivery of Gremlin siRNA plasmid reveals therapeutic potential against diabetic nephropathy by recovering bone morphogenetic protein-7.

Zhang Q, Shi Y, Wada J, Malakauskas SM, Liu M, Ren Y, Du C, Duan H, Li Y, Li Y, Zhang Y - PLoS ONE (2010)

Bottom Line: The decreased matrix metalloprotease level was partially normalized by transfection with gremlin siRNA plasmid.Additionally, we observed recovery of bone morphogenetic protein-7 signaling activity, evidenced by increases in phosphorylated Smad 5 protein levels.We conclude that inhibition of Gremlin exerts beneficial effects on the diabetic kidney mainly through maintenance of BMP-7 activity and that Gremlin may serve as a novel therapeutic target in the management of diabetic nephropathy.

View Article: PubMed Central - PubMed

Affiliation: Department of Nephrology, Third Hospital, Hebei Medical University, Shijiazhuang, China.

ABSTRACT
Diabetic nephropathy is a complex and poorly understood disease process, and our current treatment options are limited. It remains critical, then, to identify novel therapeutic targets. Recently, a developmental protein and one of the bone morphogenetic protein antagonists, Gremlin, has emerged as a novel modulator of diabetic nephropathy. The high expression and strong co-localization with transforming growth factor-beta1 in diabetic kidneys suggests a role for Gremlin in the pathogenesis of diabetic nephropathy. We have constructed a gremlin siRNA plasmid and have examined the effect of Gremlin inhibition on the progression of diabetic nephropathy in a mouse model. CD-1 mice underwent uninephrectomy and STZ treatment prior to receiving weekly injections of the plasmid. Inhibition of Gremlin alleviated proteinuria and renal collagen IV accumulation 12 weeks after the STZ injection and inhibited renal cell proliferation and apoptosis. In vitro experiments, using mouse mesangial cells, revealed that the transfect ion of gremlin siRNA plasmid reversed high glucose induced abnormalities, such as increased cell proliferation and apoptosis and increased collagen IV production. The decreased matrix metalloprotease level was partially normalized by transfection with gremlin siRNA plasmid. Additionally, we observed recovery of bone morphogenetic protein-7 signaling activity, evidenced by increases in phosphorylated Smad 5 protein levels. We conclude that inhibition of Gremlin exerts beneficial effects on the diabetic kidney mainly through maintenance of BMP-7 activity and that Gremlin may serve as a novel therapeutic target in the management of diabetic nephropathy.

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Gremlin interacts with BMP-7 and regulates BMP-7 activity in mesangial cells.Mouse mesangial cells were cultured in RPMI 1640 and collected 6 h, 12 h, 24 h and 48 h after HG stimulation. (A) Co-immunoprecipitation demonstrates an interaction between BMP-7 and Gremlin in mesangial cells. (B) mRNA levels of gremlin and BMP-7 are detected by RT-PCR. After HG stimulation, a significant increase in Gremlin mRNA level is observed after 6 hours incubation in high glucose, and the expression gradually increases with the culture duration. (C) The expression of BMP-7 mRNA dramatically decreases 48 hours later. Accordingly, increased Gremlin protein levels are observed in the cultured cells. Corresponding to a decrease in the protein level of BMP-7, the level of Smad-5 remained constant, whereas phosphorylated Smad-5 significantly and gradually decreases from 12 h to 48 h (* p<0.05, ** p<0.01 vs. the value of NG group).
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pone-0011709-g007: Gremlin interacts with BMP-7 and regulates BMP-7 activity in mesangial cells.Mouse mesangial cells were cultured in RPMI 1640 and collected 6 h, 12 h, 24 h and 48 h after HG stimulation. (A) Co-immunoprecipitation demonstrates an interaction between BMP-7 and Gremlin in mesangial cells. (B) mRNA levels of gremlin and BMP-7 are detected by RT-PCR. After HG stimulation, a significant increase in Gremlin mRNA level is observed after 6 hours incubation in high glucose, and the expression gradually increases with the culture duration. (C) The expression of BMP-7 mRNA dramatically decreases 48 hours later. Accordingly, increased Gremlin protein levels are observed in the cultured cells. Corresponding to a decrease in the protein level of BMP-7, the level of Smad-5 remained constant, whereas phosphorylated Smad-5 significantly and gradually decreases from 12 h to 48 h (* p<0.05, ** p<0.01 vs. the value of NG group).

Mentions: To investigate the mechanism by which the inhibition of gremlin expression mediates its renal protective effects, the expression and activity of BMP-7 was examined in mouse mesangial cells cultured under HG conditions. Co-immunoprecipitation revealed a physical interaction between BMP-7 and Gremlin (Figure 7A). Incubation of cultured cells under HG conditions over 48 hours revealed a gradual increase in Gremlin expression with associated decrease in BMP-7 at both the mRNA level and protein level (Figure 7B & C). Similarly the level of phosphorylated Smad-5, a marker of BMP-7 activity, significantly and gradually went down while total Smad-5 protein levels remained constant (Figure 7C). No significant changes in BMP-7 expression were seen after transfection of cells with gremlin siRNA plasmid (Figure 8A, B, C & E), whereas the decrease in phosphorylation of Smad-5 was prevented by gremlin siRNA plasmid transfection (Figure 8C & G). These results suggest that the protective effects of siRNA-induced inhibition of gremlin expression on DN were, at least partially, through the recovery of BMP-7 activity.


In vivo delivery of Gremlin siRNA plasmid reveals therapeutic potential against diabetic nephropathy by recovering bone morphogenetic protein-7.

Zhang Q, Shi Y, Wada J, Malakauskas SM, Liu M, Ren Y, Du C, Duan H, Li Y, Li Y, Zhang Y - PLoS ONE (2010)

Gremlin interacts with BMP-7 and regulates BMP-7 activity in mesangial cells.Mouse mesangial cells were cultured in RPMI 1640 and collected 6 h, 12 h, 24 h and 48 h after HG stimulation. (A) Co-immunoprecipitation demonstrates an interaction between BMP-7 and Gremlin in mesangial cells. (B) mRNA levels of gremlin and BMP-7 are detected by RT-PCR. After HG stimulation, a significant increase in Gremlin mRNA level is observed after 6 hours incubation in high glucose, and the expression gradually increases with the culture duration. (C) The expression of BMP-7 mRNA dramatically decreases 48 hours later. Accordingly, increased Gremlin protein levels are observed in the cultured cells. Corresponding to a decrease in the protein level of BMP-7, the level of Smad-5 remained constant, whereas phosphorylated Smad-5 significantly and gradually decreases from 12 h to 48 h (* p<0.05, ** p<0.01 vs. the value of NG group).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2908623&req=5

pone-0011709-g007: Gremlin interacts with BMP-7 and regulates BMP-7 activity in mesangial cells.Mouse mesangial cells were cultured in RPMI 1640 and collected 6 h, 12 h, 24 h and 48 h after HG stimulation. (A) Co-immunoprecipitation demonstrates an interaction between BMP-7 and Gremlin in mesangial cells. (B) mRNA levels of gremlin and BMP-7 are detected by RT-PCR. After HG stimulation, a significant increase in Gremlin mRNA level is observed after 6 hours incubation in high glucose, and the expression gradually increases with the culture duration. (C) The expression of BMP-7 mRNA dramatically decreases 48 hours later. Accordingly, increased Gremlin protein levels are observed in the cultured cells. Corresponding to a decrease in the protein level of BMP-7, the level of Smad-5 remained constant, whereas phosphorylated Smad-5 significantly and gradually decreases from 12 h to 48 h (* p<0.05, ** p<0.01 vs. the value of NG group).
Mentions: To investigate the mechanism by which the inhibition of gremlin expression mediates its renal protective effects, the expression and activity of BMP-7 was examined in mouse mesangial cells cultured under HG conditions. Co-immunoprecipitation revealed a physical interaction between BMP-7 and Gremlin (Figure 7A). Incubation of cultured cells under HG conditions over 48 hours revealed a gradual increase in Gremlin expression with associated decrease in BMP-7 at both the mRNA level and protein level (Figure 7B & C). Similarly the level of phosphorylated Smad-5, a marker of BMP-7 activity, significantly and gradually went down while total Smad-5 protein levels remained constant (Figure 7C). No significant changes in BMP-7 expression were seen after transfection of cells with gremlin siRNA plasmid (Figure 8A, B, C & E), whereas the decrease in phosphorylation of Smad-5 was prevented by gremlin siRNA plasmid transfection (Figure 8C & G). These results suggest that the protective effects of siRNA-induced inhibition of gremlin expression on DN were, at least partially, through the recovery of BMP-7 activity.

Bottom Line: The decreased matrix metalloprotease level was partially normalized by transfection with gremlin siRNA plasmid.Additionally, we observed recovery of bone morphogenetic protein-7 signaling activity, evidenced by increases in phosphorylated Smad 5 protein levels.We conclude that inhibition of Gremlin exerts beneficial effects on the diabetic kidney mainly through maintenance of BMP-7 activity and that Gremlin may serve as a novel therapeutic target in the management of diabetic nephropathy.

View Article: PubMed Central - PubMed

Affiliation: Department of Nephrology, Third Hospital, Hebei Medical University, Shijiazhuang, China.

ABSTRACT
Diabetic nephropathy is a complex and poorly understood disease process, and our current treatment options are limited. It remains critical, then, to identify novel therapeutic targets. Recently, a developmental protein and one of the bone morphogenetic protein antagonists, Gremlin, has emerged as a novel modulator of diabetic nephropathy. The high expression and strong co-localization with transforming growth factor-beta1 in diabetic kidneys suggests a role for Gremlin in the pathogenesis of diabetic nephropathy. We have constructed a gremlin siRNA plasmid and have examined the effect of Gremlin inhibition on the progression of diabetic nephropathy in a mouse model. CD-1 mice underwent uninephrectomy and STZ treatment prior to receiving weekly injections of the plasmid. Inhibition of Gremlin alleviated proteinuria and renal collagen IV accumulation 12 weeks after the STZ injection and inhibited renal cell proliferation and apoptosis. In vitro experiments, using mouse mesangial cells, revealed that the transfect ion of gremlin siRNA plasmid reversed high glucose induced abnormalities, such as increased cell proliferation and apoptosis and increased collagen IV production. The decreased matrix metalloprotease level was partially normalized by transfection with gremlin siRNA plasmid. Additionally, we observed recovery of bone morphogenetic protein-7 signaling activity, evidenced by increases in phosphorylated Smad 5 protein levels. We conclude that inhibition of Gremlin exerts beneficial effects on the diabetic kidney mainly through maintenance of BMP-7 activity and that Gremlin may serve as a novel therapeutic target in the management of diabetic nephropathy.

Show MeSH
Related in: MedlinePlus