Limits...
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.

Show MeSH

Related in: MedlinePlus

Cell proliferation and apoptosis in diabetic mouse kidneys.(A) Detection of proliferating cell nuclear antigen (PCNA) by immunoperoxidase staining, in the kidneys of non-diabetic control mice (N), streptozotocin-induced diabetic mice treated with pBAsi mU6 Neo control plasmid (STZ) or pBAsi mU6 Neo gremlin siRNA plasmid (Gremlin siRNA). (B and C) PCNA positive cells in kidneys from the STZ group dramatically increase at week-1 and -2, and pBAsi mU6 Neo gremlin siRNA plasmid treatment significantly reduces PCNA positive cells both in glomeruli and tubules. Proliferating cells are barely seen in all three groups at week 12. (D) Co-immunostaining of diabetic kidney sections with antibodies against PCNA and Gremlin. Intensive Gremlin expression is often seen in the cells with PCNA positive signal. (E, F) In situ TUNEL assay. Apoptotic cells are observed predominantly in tubules in the STZ group at week-12. The number of apoptotic cells is significantly reduced by pBAsi mU6 Neo gremlin siRNA plasmid treatment. (* p<0.01 vs. non-diabetic control group, # p<0.01 vs. STZ group). Scale bars, 100 µm (A, B and E), and 10 µm (D). N = 6 mice per group.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2908623&req=5

pone-0011709-g003: Cell proliferation and apoptosis in diabetic mouse kidneys.(A) Detection of proliferating cell nuclear antigen (PCNA) by immunoperoxidase staining, in the kidneys of non-diabetic control mice (N), streptozotocin-induced diabetic mice treated with pBAsi mU6 Neo control plasmid (STZ) or pBAsi mU6 Neo gremlin siRNA plasmid (Gremlin siRNA). (B and C) PCNA positive cells in kidneys from the STZ group dramatically increase at week-1 and -2, and pBAsi mU6 Neo gremlin siRNA plasmid treatment significantly reduces PCNA positive cells both in glomeruli and tubules. Proliferating cells are barely seen in all three groups at week 12. (D) Co-immunostaining of diabetic kidney sections with antibodies against PCNA and Gremlin. Intensive Gremlin expression is often seen in the cells with PCNA positive signal. (E, F) In situ TUNEL assay. Apoptotic cells are observed predominantly in tubules in the STZ group at week-12. The number of apoptotic cells is significantly reduced by pBAsi mU6 Neo gremlin siRNA plasmid treatment. (* p<0.01 vs. non-diabetic control group, # p<0.01 vs. STZ group). Scale bars, 100 µm (A, B and E), and 10 µm (D). N = 6 mice per group.

Mentions: Proliferation of kidney cells was evaluated with PCNA staining. PCNA positive cells were occasionally seen in the non-diabetic control group and were significantly increased in the tubules and glomeruli of the STZ group at week-1 and -2. Delivery of gremlin siRNA plasmid reduced the numbers of PCNA positive cells. By week-12, the numbers of PCNA positive cells returned to basal levels in the STZ and Gremlin-si groups, and there were no differences among the three groups (Figure 3, A, B & C). The kidney tissue of the diabetic mice at week-2 was double stained with antibodies against PCNA and Gremlin. PCNA positive signals were often seen in cells with intense Gremlin expression, both in glomeruli and tubules, as well as in the renal medulla (Figure 3D). No apparent apoptotic cells were seen in the three groups at week-1 and week-2; at week-12, cell apoptosis was barely seen in the non-diabetic control group and in glomeruli from the STZ group. However, there was clustering of apoptotic cells in the tubules of the STZ group. Treatment with gremlin siRNA plasmid significantly reduced the number of apoptotic cells (Figure 3E, F).


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)

Cell proliferation and apoptosis in diabetic mouse kidneys.(A) Detection of proliferating cell nuclear antigen (PCNA) by immunoperoxidase staining, in the kidneys of non-diabetic control mice (N), streptozotocin-induced diabetic mice treated with pBAsi mU6 Neo control plasmid (STZ) or pBAsi mU6 Neo gremlin siRNA plasmid (Gremlin siRNA). (B and C) PCNA positive cells in kidneys from the STZ group dramatically increase at week-1 and -2, and pBAsi mU6 Neo gremlin siRNA plasmid treatment significantly reduces PCNA positive cells both in glomeruli and tubules. Proliferating cells are barely seen in all three groups at week 12. (D) Co-immunostaining of diabetic kidney sections with antibodies against PCNA and Gremlin. Intensive Gremlin expression is often seen in the cells with PCNA positive signal. (E, F) In situ TUNEL assay. Apoptotic cells are observed predominantly in tubules in the STZ group at week-12. The number of apoptotic cells is significantly reduced by pBAsi mU6 Neo gremlin siRNA plasmid treatment. (* p<0.01 vs. non-diabetic control group, # p<0.01 vs. STZ group). Scale bars, 100 µm (A, B and E), and 10 µm (D). N = 6 mice per group.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0011709-g003: Cell proliferation and apoptosis in diabetic mouse kidneys.(A) Detection of proliferating cell nuclear antigen (PCNA) by immunoperoxidase staining, in the kidneys of non-diabetic control mice (N), streptozotocin-induced diabetic mice treated with pBAsi mU6 Neo control plasmid (STZ) or pBAsi mU6 Neo gremlin siRNA plasmid (Gremlin siRNA). (B and C) PCNA positive cells in kidneys from the STZ group dramatically increase at week-1 and -2, and pBAsi mU6 Neo gremlin siRNA plasmid treatment significantly reduces PCNA positive cells both in glomeruli and tubules. Proliferating cells are barely seen in all three groups at week 12. (D) Co-immunostaining of diabetic kidney sections with antibodies against PCNA and Gremlin. Intensive Gremlin expression is often seen in the cells with PCNA positive signal. (E, F) In situ TUNEL assay. Apoptotic cells are observed predominantly in tubules in the STZ group at week-12. The number of apoptotic cells is significantly reduced by pBAsi mU6 Neo gremlin siRNA plasmid treatment. (* p<0.01 vs. non-diabetic control group, # p<0.01 vs. STZ group). Scale bars, 100 µm (A, B and E), and 10 µm (D). N = 6 mice per group.
Mentions: Proliferation of kidney cells was evaluated with PCNA staining. PCNA positive cells were occasionally seen in the non-diabetic control group and were significantly increased in the tubules and glomeruli of the STZ group at week-1 and -2. Delivery of gremlin siRNA plasmid reduced the numbers of PCNA positive cells. By week-12, the numbers of PCNA positive cells returned to basal levels in the STZ and Gremlin-si groups, and there were no differences among the three groups (Figure 3, A, B & C). The kidney tissue of the diabetic mice at week-2 was double stained with antibodies against PCNA and Gremlin. PCNA positive signals were often seen in cells with intense Gremlin expression, both in glomeruli and tubules, as well as in the renal medulla (Figure 3D). No apparent apoptotic cells were seen in the three groups at week-1 and week-2; at week-12, cell apoptosis was barely seen in the non-diabetic control group and in glomeruli from the STZ group. However, there was clustering of apoptotic cells in the tubules of the STZ group. Treatment with gremlin siRNA plasmid significantly reduced the number of apoptotic cells (Figure 3E, F).

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