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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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Delivery of gremlin siRNA plasmid into diabetic CD-1 mice post-uninephrectomy.(A) Gremlin protein expression by western blotting in whole-kidney homogenates at different time points after injection of pBAsi mU6 Neo control vector or pBAsi mU6 Neo gremlin siRNA plasmid, respectively. Compared to those treated with pBAsi mU6 Neo plasmid (STZ group), animals administered pBAsi mU6 Neo gremlin siRNA plasmid (Gremlin siRNA group) show low expression of Gremlin in the kidneys. (B) Immunostaining of kidney sections shows the localization of Gremlin protein after the delivery of plasmids. Marked Gremlin expression is observed in both glomeruli and tubules in the STZ group, which is significantly inhibited by the delivery of gremlin siRNA plasmid. (* p<0.01 vs. non-diabetic control group; #p<0.05 vs. STZ group). Scale bars, 100 µm. N = 6 mice per group.
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pone-0011709-g001: Delivery of gremlin siRNA plasmid into diabetic CD-1 mice post-uninephrectomy.(A) Gremlin protein expression by western blotting in whole-kidney homogenates at different time points after injection of pBAsi mU6 Neo control vector or pBAsi mU6 Neo gremlin siRNA plasmid, respectively. Compared to those treated with pBAsi mU6 Neo plasmid (STZ group), animals administered pBAsi mU6 Neo gremlin siRNA plasmid (Gremlin siRNA group) show low expression of Gremlin in the kidneys. (B) Immunostaining of kidney sections shows the localization of Gremlin protein after the delivery of plasmids. Marked Gremlin expression is observed in both glomeruli and tubules in the STZ group, which is significantly inhibited by the delivery of gremlin siRNA plasmid. (* p<0.01 vs. non-diabetic control group; #p<0.05 vs. STZ group). Scale bars, 100 µm. N = 6 mice per group.

Mentions: As seen in Figure 1A, Gremlin protein expression in the STZ-treated group was about 1.5-fold greater than in the non-diabetic control mice (N). Treatment with gremlin siRNA plasmid significantly inhibited Gremlin expression induced by diabetic conditions (Gremlin-si). Immunostaining (Figure 1B) revealed that, in the non-diabetic control group, Gremlin expression was predominantly detected in glomeruli, while signal was barely seen in tubules and interstitial areas. In the STZ group, Gremlin was highly expressed in glomeruli and also in interstitial areas and part of tubules at week-2. In the Gremlin-si group, Gremlin expression was significantly weaker in both glomeruli and tubular interstitial areas, indicating a successful inhibition of Gremlin expression by gremlin siRNA plasmid (Figure 1B).


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)

Delivery of gremlin siRNA plasmid into diabetic CD-1 mice post-uninephrectomy.(A) Gremlin protein expression by western blotting in whole-kidney homogenates at different time points after injection of pBAsi mU6 Neo control vector or pBAsi mU6 Neo gremlin siRNA plasmid, respectively. Compared to those treated with pBAsi mU6 Neo plasmid (STZ group), animals administered pBAsi mU6 Neo gremlin siRNA plasmid (Gremlin siRNA group) show low expression of Gremlin in the kidneys. (B) Immunostaining of kidney sections shows the localization of Gremlin protein after the delivery of plasmids. Marked Gremlin expression is observed in both glomeruli and tubules in the STZ group, which is significantly inhibited by the delivery of gremlin siRNA plasmid. (* p<0.01 vs. non-diabetic control group; #p<0.05 vs. STZ group). Scale bars, 100 µm. N = 6 mice per group.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2908623&req=5

pone-0011709-g001: Delivery of gremlin siRNA plasmid into diabetic CD-1 mice post-uninephrectomy.(A) Gremlin protein expression by western blotting in whole-kidney homogenates at different time points after injection of pBAsi mU6 Neo control vector or pBAsi mU6 Neo gremlin siRNA plasmid, respectively. Compared to those treated with pBAsi mU6 Neo plasmid (STZ group), animals administered pBAsi mU6 Neo gremlin siRNA plasmid (Gremlin siRNA group) show low expression of Gremlin in the kidneys. (B) Immunostaining of kidney sections shows the localization of Gremlin protein after the delivery of plasmids. Marked Gremlin expression is observed in both glomeruli and tubules in the STZ group, which is significantly inhibited by the delivery of gremlin siRNA plasmid. (* p<0.01 vs. non-diabetic control group; #p<0.05 vs. STZ group). Scale bars, 100 µm. N = 6 mice per group.
Mentions: As seen in Figure 1A, Gremlin protein expression in the STZ-treated group was about 1.5-fold greater than in the non-diabetic control mice (N). Treatment with gremlin siRNA plasmid significantly inhibited Gremlin expression induced by diabetic conditions (Gremlin-si). Immunostaining (Figure 1B) revealed that, in the non-diabetic control group, Gremlin expression was predominantly detected in glomeruli, while signal was barely seen in tubules and interstitial areas. In the STZ group, Gremlin was highly expressed in glomeruli and also in interstitial areas and part of tubules at week-2. In the Gremlin-si group, Gremlin expression was significantly weaker in both glomeruli and tubular interstitial areas, indicating a successful inhibition of Gremlin expression by gremlin siRNA plasmid (Figure 1B).

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