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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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Collagen type IVand TGF-βexpression and MMP-2 activity in mouse mesangial cells cultured under high glucose conditions.Mouse mesangial cells were cultured in RPMI 1640 and transfected with pBAsi mU6 Neo or pBAsi mU6 Neo gremlin siRNA plasmid as described in the methods. Culture medium was collected for measurement of collagen type IV concentration by radio-immunoassay, and cells were subjected to Western blot analysis to determine MMP-2 and TGF-βexpression levels 48 hours after glucose stimulation. (A) Increased collagen type IV accumulation is observed in the HG group, and gremlin siRNA plasmid transfection significantly inhibits collagen type IV secretion. (B) Compared to the normal glucose control group (NG), TGF-β expression is significantly increased under high glucose conditions, and the HG stimulated TGF-β expression remains the same after gremlin siRNA transfection. (C) Compared with the NG group, MMP-2 activity in culture medium is significantly decreased in the HG and HG+V groups, and this is prevented by transfection with gremlin siRNA plasmid. (* p<0.05, ** p<0.01). Six independent experiments were repeated.
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pone-0011709-g006: Collagen type IVand TGF-βexpression and MMP-2 activity in mouse mesangial cells cultured under high glucose conditions.Mouse mesangial cells were cultured in RPMI 1640 and transfected with pBAsi mU6 Neo or pBAsi mU6 Neo gremlin siRNA plasmid as described in the methods. Culture medium was collected for measurement of collagen type IV concentration by radio-immunoassay, and cells were subjected to Western blot analysis to determine MMP-2 and TGF-βexpression levels 48 hours after glucose stimulation. (A) Increased collagen type IV accumulation is observed in the HG group, and gremlin siRNA plasmid transfection significantly inhibits collagen type IV secretion. (B) Compared to the normal glucose control group (NG), TGF-β expression is significantly increased under high glucose conditions, and the HG stimulated TGF-β expression remains the same after gremlin siRNA transfection. (C) Compared with the NG group, MMP-2 activity in culture medium is significantly decreased in the HG and HG+V groups, and this is prevented by transfection with gremlin siRNA plasmid. (* p<0.05, ** p<0.01). Six independent experiments were repeated.

Mentions: To evaluate the influence of Gremlin inhibition on collagen type IV synthesis and possible mechanisms of interaction, cultured mouse mesangial cells were again transfected with control or gremlin siRNA plasmid and then subjected to stimulation with high glucose. Collagen type IV levels in the culture medium were determined by radio-immunoassay, and cells were collected for Western blot analysis of TGF-β, and matrix metalloprotease-2 (MMP-2) activity in culture medium was determined by zymography (Figure 6). Significant accumulation of collagen type IV in the culture medium was seen in the HG and HG+V groups, while gremlin siRNA plasmid transfection significantly reduced the collagen type IV accumulation (Figure 6A). TGF-β expression significantly increased under high glucose conditions, and no obvious effect was observed after gremlin siRNA transfection. On the other hand, MMP-2 activity was significantly decreased in the HG and HG+V groups compared to control. The glucose-induced suppression of MMP-2 activity was inhibited by transfection with gremlin siRNA plasmid (Figure 6B).


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)

Collagen type IVand TGF-βexpression and MMP-2 activity in mouse mesangial cells cultured under high glucose conditions.Mouse mesangial cells were cultured in RPMI 1640 and transfected with pBAsi mU6 Neo or pBAsi mU6 Neo gremlin siRNA plasmid as described in the methods. Culture medium was collected for measurement of collagen type IV concentration by radio-immunoassay, and cells were subjected to Western blot analysis to determine MMP-2 and TGF-βexpression levels 48 hours after glucose stimulation. (A) Increased collagen type IV accumulation is observed in the HG group, and gremlin siRNA plasmid transfection significantly inhibits collagen type IV secretion. (B) Compared to the normal glucose control group (NG), TGF-β expression is significantly increased under high glucose conditions, and the HG stimulated TGF-β expression remains the same after gremlin siRNA transfection. (C) Compared with the NG group, MMP-2 activity in culture medium is significantly decreased in the HG and HG+V groups, and this is prevented by transfection with gremlin siRNA plasmid. (* p<0.05, ** p<0.01). Six independent experiments were repeated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908623&req=5

pone-0011709-g006: Collagen type IVand TGF-βexpression and MMP-2 activity in mouse mesangial cells cultured under high glucose conditions.Mouse mesangial cells were cultured in RPMI 1640 and transfected with pBAsi mU6 Neo or pBAsi mU6 Neo gremlin siRNA plasmid as described in the methods. Culture medium was collected for measurement of collagen type IV concentration by radio-immunoassay, and cells were subjected to Western blot analysis to determine MMP-2 and TGF-βexpression levels 48 hours after glucose stimulation. (A) Increased collagen type IV accumulation is observed in the HG group, and gremlin siRNA plasmid transfection significantly inhibits collagen type IV secretion. (B) Compared to the normal glucose control group (NG), TGF-β expression is significantly increased under high glucose conditions, and the HG stimulated TGF-β expression remains the same after gremlin siRNA transfection. (C) Compared with the NG group, MMP-2 activity in culture medium is significantly decreased in the HG and HG+V groups, and this is prevented by transfection with gremlin siRNA plasmid. (* p<0.05, ** p<0.01). Six independent experiments were repeated.
Mentions: To evaluate the influence of Gremlin inhibition on collagen type IV synthesis and possible mechanisms of interaction, cultured mouse mesangial cells were again transfected with control or gremlin siRNA plasmid and then subjected to stimulation with high glucose. Collagen type IV levels in the culture medium were determined by radio-immunoassay, and cells were collected for Western blot analysis of TGF-β, and matrix metalloprotease-2 (MMP-2) activity in culture medium was determined by zymography (Figure 6). Significant accumulation of collagen type IV in the culture medium was seen in the HG and HG+V groups, while gremlin siRNA plasmid transfection significantly reduced the collagen type IV accumulation (Figure 6A). TGF-β expression significantly increased under high glucose conditions, and no obvious effect was observed after gremlin siRNA transfection. On the other hand, MMP-2 activity was significantly decreased in the HG and HG+V groups compared to control. The glucose-induced suppression of MMP-2 activity was inhibited by transfection with gremlin siRNA plasmid (Figure 6B).

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