Objective To develop a model of type 2 diabetes with early renal injury on spontaneously hypertensive rats (SHR). Methods The 6-week old SHR were fed with the diets enriched with sucrose (20%, W/W), lard (10%, W/W), cholesterol (2.5%, W/W) and chleolate (1%, W/W) to induce insulin resistance. Hyperglycemia was developed by intraperitoneal injection of streptozotocin (STZ, 35 mg/kg). Wistar-Kyoto rats (WKY) were used as normal controls. Rats with plasma glucose (PGL) ≥ 16.7 mmol/L were diagnosed as diabetes. Eight weeks after the induction of diabetes, plasma triglyceride (TG), cholesterol (CHO), glucose, systolic pressure(SP), 24-h urine protein excretion (Upro) were examined in all the rats, and the homeostasis model assessment of insulin resistance (HOMA-IR) was analyzed. Renal pathological changes were studied by immunohistochemical staining and electron microscope. Results After 2 weeks on the high sucrose and fat diets, the model rats exhibited significant increase in basal PGL, TG and CHO levels as compared to control rats (P<0.05, respectively). The insulin resistance was developed in model rats demonstrated by the higher HOMA-IR (5.03±0.38 vs 2.61±0.34, P<0.05). At the end of the experiment, model rats were associated with hypertension. Upro level was significantly increased in model rats compared with that in controls [(57.58±16.54) mg/24 h vs (5.35±1.90) mg/24 h, P<0.01]. The kidney hypertrophy index (KWI) was significantly increased in the model rats compared to controls (P <0.05). Moreover, the diabetic model rats showed glomerular hypertrophy, foot process effacement, micro villous transformation, glomerular basement membrane (GBM) thickening. Conclusion A rat model is successfully established, which presents typical features of human type 2 diabetes and can be served as an ideal model to study the diabetic nephropathy.

Objective To explore the effect of irbesartan on cardiac endothelial-mesenchymal transition (EndMT) in diabetic rats.Methods The model of diabetic rat was induced by intraperitoneal injection with streptozotocin (STZ,35 mg/kg) in spontaneous hypertensive rats (SHR).Diabetic rats were divided into diabetic group and the Irbesartan treated group.The pathological changes were investigated by fluorescence microscope and electron microscope.The EndMT was studied in human aortic endothelial cells (HAEC) exposure to high glucose.The concentration of angiotensin Ⅱ in the supernatant was detected by radioimmunoassay.Immunofluorescence staining was performed to detect the co-localization of CD31 and FSP1.Results The significant myocardial fibrosis was presented in the diabetic group.Endothelial protrusions were prominent feature in myocardial microvascular of diabetic rat compared with the control group rats.Double staining of HAEC showed co-localization of CD31 and FSP1,which was decreased by the treatment of Irbesartan (P ＜ 0.05).When HAEC was exposed to high glucose,it showed some cells acquired spindle-shaped morphology and lost CD31 staining,and FSP1 and α-SMA protein expression levels were markedly upregulated,which attenuated by the treatment of Irbesartan.Conclusion Irbesartan might prevent diabetes from myocardial fibrosis via inhibition of EndMT in diabetic rats.

Objective To explore whether high glucose (HG)-induced endothelial-to-mesenchymal transition (EndMT) could be transitioned into mesenchymal stem cells (MSCs) and further differentiated into chondrocytes.Methods Human aortic endothelial cells (HAECs) were divided into three groups:normal glucose (NG,5.5 mmol/L glucose) group,HG (30 mmol/L glucose) group,and mannitol (5.5 mmol/L glucose + 24.5 mmol/L mannitol) group,and were cultured for 48 h.Immunofluorescence staining was performed to detect the co-expression of CD31 (endothelial markers),and fibroblast-specific protein 1 (FSP1,fibroblast markers).The expression of CD31 and FSP1 mRNA and protein was detected by real-time PCR and Western blotting.When endothelial-derived MSCs were grown in MSC medium for one week,the expression of the MSCs markers CD44,CD10 and the chondrocyte marker SOX9 was detected by Western blotting and RT-PCR.Chondrocyte expression was detected by alcian blue staining.Calcium deposit was analyzed by alizarin red staining.Pathological changes were investigated using electron microscopy.Results The expression of FSP1 mRNA and protein was significantly increased,but the expression of CD31 mRNA and protein was decreased (P ＜0.01),and the cells undergoing EndMT also significantly expressed CD10,CD44 and SOX9 in the HG group compared with those in normal glucose group (P ＜ 0.01).The incubation of HAECs exposed to HG resulted in a fibroblast-like phenotype,wherein increased microfilamentation and a roughened endoplasmic reticulum structure were observed in the cytoplasm.Double staining of the HAECs indicated a co-localization of CD31 and FSP1.After one week culture for chondrocyte medium,the expression of MSCs marker STRO-1 was significantly increased by immunofluorescence staining.Additionally,aleian blue staining in the HG group was positive compared to the NG group.Consistent with the elevation of SOX9 expression,calcium deposit also enhanced in the HG group.Conclusion HG can induce endothelial cells transdifferentiation into chondrocyte-like cells via the EndMT.

Objective To investigate whether elevated parathyroid hormone (PTH) levels could induce endothelial - to - mesenchymal transition (EndMT) and adipocyte transition in endothelial cells (ECs), and to determine the possible underlying mechanism. Methods (1) A rat model of secondary hyperparathyroidism and chronic kidney disease (CKD) was established. The adiposity in bone marrow was detected by oil red O staining. Immunofluorescence staining was performed to detect the expression and localization of cluster of differentiation 31 (CD31) and fibroblast-specific protein 1 (FSP1). (2) The human umbilical vein ECs were cultured in vitro. Western blotting was performed to detect protein expressions of EndMT-related markers CD31, FSP1 and α-smooth muscle actin (α-SMA) in interference groups with different PTH concentrations (0, 10-11, 10-9, 10-7 mol/L PTH for 48 h) and times (0, 12, 24, 48 h, 10-7 mol/L PTH), as well as the expression of β-catenin in interference groups with different PTH concentrations. The localizations of CD31, FSP1 and β - catenin were observed by cell immunofluorescence. Protein expressions of adipocytes markers peroxisome proliferator - activated receptor-γ (PPAR-γ) and CCAAT/enhancer binding protein-α (C/EBP-α) by Western blotting and the degree of adipogenesis by oil red O staining were detected after transformed ECs were cultured in adipogenic culture medium for one week. Small interfering RNA (siRNA) was performed to silenceβ - catenin expression. ECs were divided into control siRNA group, β - catenin siRNA group, PTH +control siRNA group and PTH+β-catenin siRNA group. Protein expressions of CD31, FSP1 and PPAR-γby Western blotting and the degree of adipogenesis by oil red O staining were determined. Results (1) In vivo, compared with the control, CKD rats had increased adipocytes in bone marrow (P＜0.05), and the co-expression of CD31 and FSP1 in bone marrow ECs. (2) In vitro, PTH significantly inhibited the expression of endothelial marker CD31 and increased the expressions of mesenchymal markers FSP1 and α-SMA in concentration-and time-dependent manners. These indexes in 10-7 mol/L PTH group and 0 mol/L PTH group, in 48 h group and 0 h group showed statistical differences (all P＜0.05). In PTH group ECs with 10-7 mol/L PTH for 48 h showed FSP1 accumulation in the cytoplasm and reduced expressions of CD31, and ECs had higher expressions of PPAR-γ and C/EBP-α as well as the degree of adipogenesis than those in control group (all P＜0.05). Furthermore, PTH enhanced the nuclearβ-catenin protein levels in ECs in concentration-dependent. The expressions of β-catenin in 10-7 mol/L PTH group and 0 mol/L PTH group showed statistical differences (P＜0.05). β - catenin expressed in the cytoplasm in control group, while it enter into the nucleus in PTH group. Compared with those in PTH+control siRNA group, the expressions of CD31 and PPAR-γ as well as the degree of adipogenesis decreased in PTH+β-catenin siRNA group (all P＜0.05), while the expression of FSP1 increased (P＜0.05). Conclusions PTH induces ECs - to - adipocytes transition by the canonical Wnt/β - catenin signaling pathway, which might account for bone loss in CKD. Silenced β - catenin expression can inhibit PTH-induced EndMT and adipogenesis.