| Background:Diabetic nephropathy(DN) is one of the microvascular complications of diabetic mellitus(DM). In recent years, the incidence rate of DN increased year by year, and >25% of patients with Type 1 and 2 diabetes suffered from DN. DN has become the first cause of end stage renal disease(ESRD), which is a serious complication of DM and also the main cause of death. DN not only seriously affects the health and quality of life of patients but also places a major burden on healthcare resources.Recent studies showed that growth factor receptor-binding protein 10(Grb10) as the first key molecule of the upstream of insulin and insulin-like growth factor 1(IGF-1) signaling pathway played an important role in the negative regulation of insulin and insulin-like growth factor 1 receptor(IGF-1R) mediated signaling pathway, and played a key role in these signaling pathway disorder that caused by impaired growth and development. However, the in vivo mechanism and mode of action of Grb10 which is an adjustment factor of IGF-1R signaling pathway is currently rarely reported in the kidney. Objective:This study was to observe changes of Grb10, IGF-1 and IGF-IR expression and renal function in the kidneys of diabetic mice from pathology, gene and protein levels. Then, catalpol treated diabetic mice, and observe the changes of above projects again, so as to explore the potential role of Grb10 and the therapeutic effect of catalpol in the pathogenesis of diabetic nephropathy. Methods:Mice were randomly selected to receive a single injection of STZ. STZ was dissolved in 0.1-M sodium citrate-hydrochloric acid buffer solution(p H 4.5). The remaining mice [the control group(Con)] were injected with an equal volume of buffer solution. Animals with a blood glucose level >16.7 mmol/l at 72 h after STZ injection were considered to be diabetic. The diabetic mice were further randomly divided into two groups, the diabetes mellitus(DM) group and DM treated with catalpol(DM + Cat) group(n = 10 per group). Eight weeks later, the DM + Cat group mice were treated by intraperitoneal(i.p) injection of catalpol(10 mg/kg/day) for 14 consecutive days. DM and control group mice were injected with an equal volume of normal saline(NS). Results:1. Mice that were intraperitoneally injected with STZ(the DM group) had higher blood glucose levels than control mice without STZ administration. The mean blood glucose level was significantly higher in DM mice than in non-DM control mice(P < 0.01).2. Expression of endogenous Grb10 in kidneysUsing q PCR and Western blotting, we examined alterations in the expression of Grb10 in the kidney. The expression levels of Grb10 m RNA and protein in the kidney tissues were significantly higher in the DM group than those in non-diabetic control group(Fig 1A; Fig 2A; P < 0.01).3.The renal function of diabetic mice was severely damaged, which was manifested as proteinuria and elevated serum creatinine levels and blood urea nitrogen levels. Following administration with catalpol, the 24 h urinary protein excretion, serum creatinine levels, and blood urea nitrogen in the DM + Cat group was significantly lower than that in the non-treated DM group(P < 0.05).4. The IGF-1 m RNA level and IGF-1R phosphorylation were lower in the kidney tissues of DM mice than those in non-diabetic control mice(P < 0.01). Additionally, IGF-1 m RNA levels and IGF-1R phosphorylation were higher in kidneys of the DM + Cat group than those in the non-treated DM group(P < 0.05).5. pathological changes and immunohistochemistry results in kidneysGlycoproteins are stained purple by Periodic acid-Schiff(PAS) staining. pathological changes were observed in both DM and DM + Cat groups. However, the degree of staining, representative of pathological severity, was lower in the DM + Cat group than that in the DM group. In addition, the kidney fibrosis observed in DM + Cat group was significantly attenuated as compared with that in the non-treated DM group. Grb10 protein expression, located both on the cell membrane and in the cytoplasm, was mainly distributed in the glomerulus, tubules and interstitial blood vessels. The expression level of Grb10 protein in kidneys of the DM group was significantly higher than that in the DM + Cat group(p < 0.05). Caspase-3 expression was increased in kidneys of non-treated diabetic mice, but significantly decreased in kidneys of catalpol-treated diabetic mice(P < 0.05). Conclusions:STZ intraperitoneal injection successfully established a mouse model of diabetes, and contained a state of chronic hyperglycemia. Chronic hyperglycemia may cause structural and functional changes in the kidney. Abnormal glucose metabolism can cause a series of pathophysiological changes and lead to diabetic nephropathy.Administration of catalpol could reverse, to certain degrees, the impaired renal functions and pathological changes in diabetic kidneys. Meanwhile, Grb10 expression was down-regulated by catalpol treatment in diabetic nephropathy.With the progression of diabetic nephropathy, an elevated expression of Grb10 correlated with a decreased level of IGF-1/IGF-1R signaling in diabetic kidneys. Collectively, our findings support the notion that Grb10 may be negative regulators of IGF-1/IGF-1R mediated signaling pathway in diabetic nephropathy.In conclusion, continuous hyperglycemia might lead to a series of pathophysiological changes, an elevated expression of Grb10 and the subsequent decrease in IGF-1/IGF-1R signaling in kidneys. These results indicate that Grb10 may have a negative regulation of IGF1/ IGF-1R mediated signaling pathway in diabetic nephropathy. Catalpol treatment could improve diabetes-associated impaired renal functions and ameliorate pathological changes in diabetic kidneys, while such beneficial effects correlate with a down-regulation of Grb10 expression and a concomitant up-regulation of IGF-1/IGF-1R signaling in diabetic kidneys. These findings suggest that catalpol has a protective effect on diabetic nephropathy. The above studies suggest that Grb10 plays an important role in the development of diabetic nephropathy, and thereby provides a new way for the prevention and treatment of diabetic nephropathy. |
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