| Background: Diabetic nephropathy is increasing dramatically worldwide and is now the most common cause of end-stage renal failure requiring renal replacement therapy in Europe and America. Glomerular hypertrophy and progressive expansion of extracellular matrix (ECM) have been supposed to be the early features of diabetic nephropathy. It is believed that with the progression of these lesions and persistent proteinuria, glomerular sclerosis occurs, which lead to deterioration of renal function and chronic renal failure. Molecular mechanisms underlying the pathogenesis of glomerular hypertrophy and ECM accumulation have, however, remained incompletely understood. NG2 is a large, integral membrane-spanning chondroitin sulfate proteoglycan (CSPG). It is capable of binding to multiple extracellular ligands and participating in cytoplasmic signal transduction. NG2 plays an important role in mediating the cellular interactions or interactions between cells and ECM. NG2 is expressed not only in central nervous system, but also in a variety of mesenchymal cell types such as chondroblasts, skeletal myoblasts, epidermal stem cells, vascular smooth cells/pericytes and melanoma cells. But the expression and role of NG2 in kidneys has not been clarified.Objective: To investigate the expression of NG2 proteoglycan in the kidneys of normal rats and strepozotocin (STZ)-induced diabetic rats, and observe the role of NG2 in the pathogenesis of diabetic nephropathy.Methods: (1) Sprague-Dawley rats were anaesthetized and perfusion-fixed. Immunohistochemistry and immunoelectron microscopy was performed to examine the location of NG2 in rat kidneys. The expression of mRNA and protein of NG2 was measured by RT-PCR and western blot respectively. (2) To induce diabetes, rats were given a single intraperitoneal injection of 65 mg/kg Streptozocin (STZ). 2, 4 and 8 weeks after injection, 6 animals of each group were sacrificed and kidneys were removed for the further investigations. Biochemical parameters and pathological changes were examined. The expression of NG2 was detected by immunohistochemistry, real-time PCR and western blot. (3) Rat glomerular mesangial cell line HBZY-1 was cultured in vitro. HBZY-1 cells were treated with high glucose (30mmol/L) for 24 hs and the changes of NG2 expression were observed. Two small hairpin RNA (shRNA) constructs targeting NG2 were created, and the interference efficiency was measured. The better construct was selected to perform the next experiments. (4) HBZY-1 cells were transfected with Pgenesil-siNG2 and NG2 expression vectors (pcDNA-NG2) by LipofectamineTM 2000. The effect of NG2 on the proliferation of HBZY-1 cells was examined by MTT method and flow cytometry (FCM), and the effect of NG2 on the synthesis of collagen VI and Laminin was measured by real-time PCR.Results: (1) The location of NG2 proteoglycan in kidneys was investigated by confocal microscopy and immunoelectron microscopy. NG2 was mainly expressed in the mesangial matrix and mesangial cells of glomeruli. The expression of mRNA and protein of NG2 in rat kidneys was detected by RT-PCR and western blot. (2) Rats with blood glucose concentration>16.7mmol/L were accepted as diabetic models 3 days after STZ injection. Hyperglycemia persisted in diabetic animals during the 8 weeks study period before they were sacrificed. The blood glucose level, kidney-to-body weigh ratio and urinary albumin/creatinine of diabetic rats were significantly higher than those of control rats (P<0.05 or P<0.01). Glomerular hypertrophy and mesangial expansion were evident in 8 week diabetic rats. There was a significant increase of mean VG from (1.14±0.12) to (1.29±0.11) x106μm3 (P<0.05). The electron micrographs showed segmental fusion of podocyte foot processes and moderate mesangial matrix expansion. The expression of NG2 was also observed predominantly in glomerular mesangium in diabetic rats. Compared with control rats, the expression of NG2 in renal cortex was significantly higher than that in diabetic rats. The NG2 mRNA levels peaked within 2 weeks (increase by 138±76%, P<0.01) and still remained significantly enhanced 4 weeks (by 74±40%, P<0.05) and 8 weeks (by 73±37%, P<0.01) in diabetic rats compared with time-match control rats. (3) The expression of NG2 in HBZY-1 cells significantly increased (P<0.01) after treated with high glucose (30mmol/L) for 24 hs. Two shRNA vectors targeting NG2 (Pgenesil-siNG21 and Pgenesil-siNG22) were constructed successfully, and their interference efficiency was 54% and 72% respectively. Pgenesil-siNG22 was selected to perform the next experiments. (4) HBZY-1 cells were transfected with Pgenesil-siNG22, Pgenesil-siNC, pcDNA-NG2 and empty vector pcDNA respectively. According to MTT assay, overexpression of NG2 significantly stimulated, while NG2 silencing inhibited the proliferation of mesangial cells. The data of cell-cycle analysis by FCM showed that NG2 increased the cell numbers in the S phase and decreased the cell number in the G0/G1 phase, while silencing NG2 induced decrease of the cell numbers in the S phase and increase of the cell numbers in the G0/G1 phase. The expression of collagen VI and Laminin was significantly increased by overexpressing of NG2 and was decreased by treating with NG2 siRNA.Conclusions: In the kidneys of normal and STZ-induced diabetic rats, the expression of NG2 was observed predominantly in glomerular mesangium. Both mRNA and protein levels were significantly higher in diabetic rats than those in control rats. In cultured rat mesangial cell line HBZY-1, overexpression of NG2 promoted cell proliferation and formation of ECM such as type VI collagen and Laminin. Furthermore, gene silencing targeting NG2 resuLted in decreased cell proliferation and formation of ECM. The observations suggest that NG2 is up-regulated in diabetic nephropathy and actively participates in the development and progression of glomerulosclerosis by stimulating the proliferation of mesangial cells and the deposition of ECM. NG2 is a potential target for prevention and therapy of DN.
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