Objective To observe the expressive changes of angiopoietins(Ang)/Tie-2 axis and vascular endothelial growth factor(VEGF) in kidney tissue and examine the correlation of Ang/Tie-2 axis and VEGF with renal microvascular disease in diabetic rats. Methods SD male rats were randomly divided into streptozotocin-induced diabetes group and control group. The mRNA and protein expression of Ang-1， Ang-2， Tie-2， VEGF and thrombomodulin-1(TM-1)in kidney tissue of two groups was examined by RT-PCR and immunohistochemistry at week 2， 4， 8， 12， 16， 20， and 24 respectively. Results (1) Ang-1 mRNA in diabetic group was up-regulated significantly at week 4 and 8(A，83.58±10.23，88.59±6.97)， but down-regulated significantly at week 24(47.13±8.02 vs 64.53±8.77)， as compared with other time points of diabetic group and the same time points of control group. Ang-1 was expressed outstandingly in glomeruli. From week 4 to week 24， the protein expression of Ang-1 in diabetic glomeruli increased significantly(lgA，1.64±0.12 vs 1.08±0.16，at week 4，1.24±0.11 vs 1.11±0.17 at week 24). (2) From week 4 to week 16， Tie-2 mRNA in diabetic group was up-regulated obviously with the peak between week 8 and week 12(A，87.31±11.69 vs 63.62±5.61 at week 4，81.75±8.58 vs 60.15±2.66 at week 16). The protein expression of Tie-2 was found mainly in glomeruli. Throughout experimental period， the expression of Tie-2 staining in diabetic glomeruli increased apparently with the peak between week 4 and week 8. (3) Ang-2 mRNA in diabetic group was found only at week 16 and week 20. The expression of Ang-2 in peritubular microvessel of diabetic renal cortex was found from week 12 to week 24 with the peak at week 16.(4)compared with control group，TM-1 staining in diabetic glomeruli is much more from week 2 to week 20(lgA，0.99±0.15 vs 0.68±0.17 at week 2，1.03±0.17 vs 0.74±0.13 at week 20)，but a little lower at week 24. (5) The expression of VEGF increased significantly in diabetic group. (6) In diabetic group， correlations were significantly positive among Ang-1， Tie-2， VEGF and TM-1. Meanwhile，they were positively correlated with kidney/body weight， glomerular volume， glomerular area， and urine protein excretion respectively. Conclusions (1) Change of Ang/Tie-2 axis exists in diabetic kidney， which Ang-1 and Tie-2 expression is up-regulated in early stage and down-regulated along with Ang-2 up-regulation in later stage.(2) In diabetic kidney， the change of Ang/Tie-2 axis is partly associated with the renal angiogenesis which is mainly responsible for Ang-1 down-regulation and VEGF， Ang-2 up-regulation. (3) There is a formation of new peritubular microvessels at diabetic renal cortex in middle and later stages.

Objective To get more insight into progression of diabetic nephropathy (DN) and select individual genes associated with the progression of the disease. Methods Ten patients diagnosed as type 2 DN were included in the study. They were divided based on their clinical phenotype. Five donor kidneys served as normal controls. Glomeruli were dissected out from each individual patient abundant residual biopsy sample under microscope. Total RNA was extracted and biotin-labeled cRNA was hybridized to each GeneChip^®Human Genome U133 Plus 2.0(Affymetrix， Santa Clara， CA， USA). Statistical analysis was performed with the GeneSpring 7.2 software (Silicon Genetics， Redwood City， CA， USA) and the further bioinformatic analysis was carried out. The alteration of candidate genes was further confirmed using real-time PCR. Results The glomerular gene profiling of DN was markedly distinct from the normal controls. Different genotypes were given based on the different clinical phenotypes. Combined analyzing the changed gene expression in the development of proteinuria， histology lesions and serum creatinine level， the gene profiling linked to the progression of DN was found， in which there were a lot of genes related to the metabolism. And some molecules related to inflammation response were also included. Conclusions Phenotype-based analysis of glomerular gene profiling using biopsy tissues displayed the gene profiling of patients with progressive DN. Disordered metabolism and the following triggered inflammation response may play an important role in progression of DN.

Objective To investigate the effects of antisense p21 oligodeoxynucleotide (p21 ASODN) on the expression of p21 protein and extracellular matrix in cultured human glomerular mesangial cells (HGMC) under high glucose medium. Methods HGMCs were transfected with 50 nmol/L or 100 nmol/L p21 ASODN or scrambled control oligodeoxynucleotide (SCODN) using lipofectamine 2000. After incubation under normal (5.5 mmol/L) or high (30 mmol/L) glucose media and different times， HGMCs lysates were collected and the expression of p21， fibronectin and laminin was examined by Western blot. The secretion of fibronectin and laminin by HGMCs in supernatants of culture media was also detected with ELISA. Results High glucose media significantly stimulated the expression of p21， increased the syntheses and secretion of fibronectin and laminin in cultured HGMCs in a time-dependent manner. Transfection of HGMCs with p21 ASODN not only decreased p21 protein level caused by high glucose media， but also attenuated the expression and secretion of fibronectin and laminin in supernatants of HGMCs lysates and culture media. Meanwhile， SCODN had no significant effects on the expression of p21， fibronectin and laminin. Conclusions High glucose promotes the expression of p21， fibronectin and laminin in cultured HGMCs. Transfection of HGMCs with p21 ASODN can decrease p21 protein level caused by high glucose media， and attenuate the expression of fibronectin and laminin in supernatants of HGMCs lysates and culture media. Modulation of p21 expression in HGMCs may work as an effective way to mitigate the progression of diabetic nephropathy.

Objective To investigate the effect of Smad7 on epithelial-myofibroblast transdifferentiation and collagenⅠsynthesis in NRK52E cells stimulated by high glucose. Methods Smad7-expression NRK52E cells were grown in RPMI 1640 medium containing 10% fetal bovine serum. They were cultured for 24 h in free serum medium as well as 60%~80% cells were adhered onto the surface of the flask. Afterwards， Smad7-expression NRK52E cells were incubated in the presence or absence of doxycycline (Dox， 2 μg/ml) for 24 h， then stimulated by high glucose. p-Smad2/3 nuclear translocation induced by high glucose was examined by immunocytochemistry. The expression of Smad7 mRNA was detected by RT-PCR. The expression of Smad7， α-SMA，E-cadherin and Col I protein was detected by western blot. Results Smad7-expression NRK52E cells were regulated by Dox compared with the smad7 expression level in no Smad7-transfected NRK52E cells. Smad7 mRNA and protein were highly expressed in smad7 gene-transfected NRK52E cells induced by high glucose. The expression level of p-Smad2/3 was low in normal NRK52E cells(16.1%). Overexpression of Smad7 markedly suppressed the p-Smad2/3 expression and nuclear translocation (30.3% vs 58.5%，P < 0.01)induced by high glucose. Up-regulated expression of Smad7 inhibited the high expression of α-SMA and Col I protein and reversed E-cadherin protein expression level induced by high glucose.Conclusion Overexpression of Smad7 can suppress epithelial-myofibroblast transdifferentiation and collagenⅠsynthesis induced by high glucose through blocking Smad2/3 activation.

Objective To investigate the protective effect of all-trans retinoic acid (ATRA) on podocyte lesion in diabetic rats. Methods Twenty-four rats injected with streptozotocin(STZ) were randomly divided into model(DM) and therapy group(T). Other 12 normal rats were used as control group(N). The rats in T group were administrated ATRA at a dosage of 20 mg·kg^-1·d^-1， and animals in other groups were treated with normal saline. 24 h urinary protein， urinary podocyte excretion， and renal pathological alteration were determined after 4 and 8 weeks. Results 24 h urinary protein， ratio of kidney weight to body weight， and urinary podocyte excretion were significantly higher in DM group compared to N group[0.84(0.60~1.50) vs 0.03(0~0.15)cell/ml，P < 0.05]. The numbers of glomerular poddocyte were significantly reduced in DM group(11.27±2.15 vs 14.07±2.07). Diabetic rats treated with ATRA (T group) had less urinary podocyte excretion[0.46(0.25~0.70)cell/ml] and more glomerular podocyte numbers compared to DM group. Conclusions ATRA can decrease the excretion of urinary podocyte and proteinuria. ATRA has a protective effect on podocyte lesion in diabetic rats.

Objective To observe the expression of bone morphogenetic protein 7 (BMP-7) in different stages of diabetic rats and investigate the potential role of BMP-7 in diabetic nephropathy(DN). Methods Wistar rats were divided into diabetes mellitus(DM) group induced by intravenous injection of streptozotocin (65 mg/kg)， and normal control group injected with citrate buffer. The rats were sacrificed at day 30，60，90，120，150 and 180 following injection， respectively. The blood glucose，Scr， urinary albumin and urinary creatinine were measured in each rat before sacrifice. The renal pathological changes were examined with hematoxylin and eosin (HE)，periodic acid-silver-methenamine staining (PASM) and protein expression of collagen Ⅳ(Col Ⅳ). The mRNA and protein expression of BMP-7 and TGF-β1 in kidney were detected by reverse transcription-polymerase chain reaction (RT-RCR) and immunohistochemical staining respectively， and were quantified by computer image analysis system. Results The levels of blood glucose and urinary albumin in diabetic group were remarkably higher than those in control group(P < 0.01). With the aggravation of diabetic nephropathy and increasing expression of Col Ⅳ，the expression of mRNA and protein of BMP-7 was progressively down-regulated(A%，30 d 95.87±1.19；180 d 26.43±1.26)(P < 0.01 or P < 0.05)， whereas the expression of TGF-β1 mRNA(A% 30 d 40.26±0.98；180 d 102.12±6.45) and protein was significantly up-regulated in the diabetic rats as compared with that of the controls(A%，30 d 98.64±0.80；180 d 98.80±0.49)(P < 0.01). The protein and mRNAlevels of BMP-7 were negatively correlated with TGF-β1 respectively(P < 0.01). Conclusions Endogenous BMP-7 may be an important molecule for maintenance of renal homeostasis. The decrease of BMP-7 protein and mRNA during the early phase of diabetic nephropathy is possibly involved in the occurrence of diabetic nephropathy. BMP-7 may counteract TGF-β1-mediated profibrotic effects.

Objective To investigate the mechanism of ERK1/2 signal pathway in the inhibition of TIMP-1 on apoptosis of rat glomerular mesangial cells induced by high glucose. Methods The human TIMP-1 sense， antisense plasmid were transferred into rat mesangial cells through liposome. The cells were divided into control group， non-transfected group，veotor group，sense group and antisense group. After cultured in high glucose (HG 25 mmol/L) with the presence of PD98059(50 µmol/L)for 24 hours， cell structure was observed by electron microcope. Western blot was performed to test the expression of phospho-ERK1/2， phospho-P90rsk， phospho-Bad， Bad， P90rsk and Bcl-2. Results Electron microcope showed that PD98059 caused a significant increase of apoptosis bodies in every group of rat mesangial cells. In contrast with the control group， the expression levels of phospho-ERK1/2， phospho-P90rsk， phospho-Bad and Bcl-2 were inhibited by antisense TIMP-1 and PD98059(P < 0.05). Among these groups，the expression levels of total Bad and total P90rsk protein were not different. Conclusion TIMP-1 exerts its anti-apoptoic effect， at least in part， by activation of ERK1/2 pathway.

Objective To observe the expression of tissue transglutaminase (tTG) in renal tissues of diabetic nephropathy rats， and to investigate its contribution to the progression of diabetic nephropathy. Methods Streptozotocin-induced diabetic rats were sacrificed at 30 d， 60 d， 90 d， and 120 d respectively. Albuminuria excretion rate (AER)， kidney weight index and serum creatinine (Scr) of the rats were measured. Hematoxylin and eosin (HE) staining and periodic acid-silver-methenamine (PASM) staining were used to observe the renal pathological changes. Collagen Ⅳ(Col Ⅳ) and soluble tTG protein in kidney were examined by immunohistochemistry，insoluble tTG by immunofluorescence. Expression of total tTG mRNA in kidney was detected by reverse transcription-polymerase chain reaction (RT-PCR). Results AER， kidney weight index and Scr of diabetic rats were all progressive during the period of the experiment. Compared with control group， the levels of Col Ⅳ， soluble tTG and insoluble tTG protein in kidneys of hyperglycemic rats were significantly increased at 30 d， 60 d， 90 d and 120 d(P < 0.05，P < 0.01).During the whole course， levels of Col Ⅳ，soluble tTG and insoluble tTG of kidneys from diabetic rats increased gradually.Both soluble tTG expression and insoluble tTG expression were positively correlated with AER( r=0.937，P < 0.01 and 0.809，P < 0.01). The location of increased Col Ⅳ in the glomeruli was the same as that of increased insoluble tTG in the mesangium， and the correlation coefficients of insoluble tTG and Col Ⅳ was 0.831 (P < 0.05). Total tTG mRNA levels started to rise in diabetic kidneys at 30 d， reaching its peak at 90 d， and reduced slightly at 120 d. Conclusion The over-expression of tTG protein and the change of total tTG mRNA levels in the diabetic kidney，and the positive correlation of insoluble tTG and Col Ⅳ in glomeruli demonstrates that tTG may play a role in the progression of early diabetic nephropathy.

Objective To investigate the effect of dehydroascorbate on reactive oxygen species(ROS) in mesangial cell induced by high glucose. Methods Mesangial cells were cultured in RPMI-1640 medium containing 10% newborn calf serum. Intracellular AA and DHA contents were measured with vitamin C assay system. The intracellular formation of ROS was detected with the fluorescent probe CM-H2DCFDA by using confocal microscopy. Activity of AP-1 was detected by EMSA. Results AA entry into cells was not significantly different from background noise. At a DHA concentration of 1 mmol/L， increasing concentrations of glucose competitively inhibited DHA entry into the cells such that the accumulation of DHA was smaller than half maximal at about 22 mmol/L glucose. Cytochalasin B，a kind of hexose transporter inhibitor，inhibited DHA entry into the cells. At a glucose concentration of 25 mmol/L， DHA inhibited intracellular ROS formation in a dose-dependent manner when DHA level was smaller than 4 mmol/L. In addition， the inhibitory effect of DHA on ROS generation was accompanied by lowering AP-1 activity in mesangial cell incubated by high glucose. Conclusions Mesangial cells are DHA dependent. VitC exclusion from mesangial cells through competition of glucose and DHA for common transport mechanism will deprive the cells of the central antioxidant and can lead to ROS accumulation. Proper doses of DHA will protect mesangial cell from injury of high glucose by inhibition on ROS formation and AP-1 activation.