| Objective: Diabetic nephropathy (DN) is a serious complication of diabetes mellitus and 10% of the patients may lead to the end-stage of renal failure if there is no effective treatment. Specific therapies of diabetic nephropathy are not available and current treatment strategies are limited to management of controlment of blood glucose levels, hypertension and hyperproteinuria. In recent years researchers pay more attention to finding out the ideal theraputic targets against DN. The re-activation of developmental programs in DN suggests new potential as therapeutic targets. In this study, we focus on Gremlin as a theraputic target. We constructed a gremlin siRNA plasmid and delieved it to the STZ-induced diabetes mouse model, and observed the pathological changes, the cell prolifferationa and apoptosis, the changes of renal function and expression of BMP7 after administration of gremlin siRNA plasmid, to investigate the protective role of inhibition of Gremlin expression in DN. We also examined the effect of Gremlin inhibition in vitro. We obseved the changes of cell prolifferation, collagen type IV accumulation and mesangial expansion, and examined the expression changes of Gremlin, BMP7, TGF-β, MMP2 and phos-Smad5 after tansfection of gremlin siRNA, to discuss the possible mechanism of the protective effect.Methods: Animal experiment: 12-week-old male CD-1 mice were divided into three groups: a non-diabetic control group (N), a diabetic group administered a pBAsi mU6 Neo control plasmid (STZ), and a diabetic group administered a pBAsi mU6 Neo gremlin siRNA plasmid (Gremlin-si) (N=18 per group). After 2 weeks of uninephrectomy, animals received a single intravenous dose of 150 mg/kg streptozotocin in citrate buffer to induce diabetes. N group received same dose of citrate buffer. Hyperglycemia (>15 mmol/L) was confirmed 3 days after STZ administration. Plasmids were then delivered weekly by tail vein injection using the TransIT-EE Hydrodynamic Delivery System each week. Animals were sacrificed at week-1, week-2 and week-12. At each time point, urine and blood samples were collected, and kidney tissues were harvested. Kidneys were fixed in 4% paraformaldehyde and embedded in paraffin for light microscopy and immunohistochemistry. The paraformaldehyde-fixed and paraffin-embedded kidney tissues were cut into sections of 2-4μm thick. 2μm sections were stained with Hematoxylin and Eosin (HE) and periodic acid-Schiff (PAS). 4μm sections were used for immunohistochemistry and immune-double staining. HE and PAS staining were used to observe the pathological changes. Immunohistochemistry was used to analyze the renal expression of Gremlin, PCNA and collagen type IV. Immune-double staining was used to find out the relationship between Gremlin and cell prolifferation. And the measurement of apoptotic cells was performed using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) with the in situ Apoptosis Detection Kit. The expression of BMP7 and Gremlin were also detected by Western blotting. Cell culture: Mouse mesangial cells (MCs) were grown in DMEM containing 5% FBS, penicillin (100 U/ml), streptomycin (100μg/ml) at 37°C and 5% CO2 95% air. The cells were devided into 4 groups: a normal glucose group (NG), a high glucose group (HG), high glucose transfected a pBAsi mU6 Neo control plasmid group (HG+V), and high glucose transfected a pBAsi mU6 Neo gremlin siRNA plasmid group (HG+Gremlin-si). When the cells grown to 80% confluence, tansplanted the cell to 6-well culture plates (2×106 cells per well) and Lab-Tek16 chamber slide (2×105 cells per well). Cells were cultured without antibiotics for 24 hours,then were transfected with pBAsi mU6 Neo gremlin siRNA plasmid or pBAsi mU6 Neo plasmid using lipofectamine 2000 reagent (Invitrogen). After 24 hours, HG group, HG+V group and HG+Gremlin-si goup cells were further cultured in DMEM containing high glucose and NG group cells were cultured in normal glucose for up to 48 hours. Cells in 6-well culture plates were collected for protein extraction. Cells on Lab-Tek16 chamber slides were fixed in 4% paraformaldehyde for immunochemistry, and culture medium was collected for Collagen IV measurement. The results were analyzed semi-quantitatively by the pathological image analysis. All the data were analysed by SPSS13.0 statistic software, P value<0.05 was considered to have statistical significance.Results: Animal experiment: The Gremlin expression was inhibited successfully by delivery of gremlin siRNA plasmid. The glomerular and tubular diameters and cell numbers significantly increased in the STZ group compared with those of the control mice, while the treatment with gremlin siRNA plasmid alleviated these changes. 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. Immunohistochemical staining showed that 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. Delivery of gremlin siRNA plasmid reduced the numbers of PCNA positive cells. 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. There was slightly increased expression of BMP-7 in kidney cortical homogenates from the STZ group compared to control at week-1 and week-2 but markedly decreased expression at week-12. Treatment with gremlin siRNA plasmid resulted in upregulation of BMP-7 expression in the diabetic kidney at week-12, which indicated a potential association between Gremlin inhibition and BMP-7 expression. Cell culture: Gremlin protein expression was efficiently inhibited by transfection with gremlin siRNA plasmid. The immunochemical staining showed that the number of proliferative cells significantly increased in the HG group and the HG+V group. Transfection with gremlin siRNA plasmid into MCs significantly inhibited the HG-induced cell proliferation(p<0.05). Collagen type IV levels in the culture medium were determined by radio-immunoassay. 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. While the MMP-2 expression was significantly decreased in the HG and HG+V groups compared to control. And the glucose-induced suppression of MMP-2 levels was inhibited by transfection with gremlin siRNA plasmid. Co-immunoprecipitation revealed a physical interaction between BMP-7 and Gremlin. Incubation of cultured cells under HG conditions over 48 hours revealed a gradual increase in Gremlin expression with associated decrease in BMP-7. Similarly the level of phosphorylated Smad-5, a marker of BMP-7 activity, significantly and gradually went down. Transfection of cells with gremlin siRNA plasmid prevented the glucoseinduced decreases in BMP-7 and Phos-Smad-5 levels(p<0.05). 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.Conclution: Gremlin protein expression was efficiently inhibited by transfection with gremlin siRNA plasmid. Administration of gremlin siRNA plasmid to diabetic mice alleviated renal hypertrophy, decreased cell proliferation and apoptosis, and subsequently suppressed collagen type IV accumulation and mesangial expansion, which indicate that there are beneficial effects of Gremlin inhibition on diabetic nephropathy. We conclude that inhibition of Gremlin expression exerts beneficial effects on the diabetic kidney mainly through maintenance of BMP-7 expression and activity and that Gremlin may serve as a new therapeutic target in the management of diabetic nephropathy.
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