| Objective To observe pathologic changes of renal fibrosis and dynamic changes of renal integrated backscatter parameters in the diabetic rats at different stages,to explore the relationship between both of them,and to provide reliable experiment evidence for the correct evaluation and earlier detection in diabetic nephropathy by Integrated BackscatterMethods1 Set up model of diabetic nephropathy rats:Fifty 8-week-old SPF grade Male Spregue-Dawley (SD)rats weighing 180~210g were housed in groups of 3~4 in plastic cages with soft bedding , maintained constant temperature of (22±20C) ,humidity(50±10%),and given free access to food and water.Experimental rats were adjusted to the environment for 7 days before conducting this experiment. All rats were randomly divided into control groups(n=18) and experiment groups (n= 32). The diabetic rats model was induced by a single injection of streptozotocin dissolved in 0.1M citrate-buffered saline (pH=4.5) into intraperitoneal at a dose of 65mg/kg body weight. Control rats received vehicle only. About 3, 7 day after the STZ treatment, blood samples were taken from the caudal vein, and glucose levels were measured with a glucose meter. Rats whose blood glucose levels >16.7 mmol/l and urine glucose lasts masculine were considered diabetes. Dead and with glucose level <16.7 mmol/l animals were excluded from the study. Evaluation were performed after 4,12,24 weeks of diabetes inducation.2 Integrated Backscatter:The rats were anesthetized with 10% hydrated chloral(350mg/kg), in the prone position and images were obtained by using a S8 prode of standard commercial ultrasound machine (HP- SONOS - 5500) . coronal section of the kidney were obtained , after the Acoutic Densitometry-Integrated backscatter (AD-IBS) was activated. 62 continuous images within 2.48 second were stored by Loop during the whole check to avoid the interference of breathing. A 11×11 pixel sample volume of ellipse shape was put renal contains ,renal medulla and renal sinus.The position of the sample volume was checked and adjusted in each frame to keep the sample volume within the same region Peak to peak intensity( PPI, that is cyclic variation of integrate backscatter , CVIB) and average image intensity(AII, that is IBS) were measured . The ratio of renal cortex and renal sinus expressed as calibrated IBS of renal cortex (that is IBS% of renal cortex) and the ratio of renal cortex and renal sinus expressed as calibrated IBS of renal medulla (that is IBS% of renal cortex medulla).3 Histopathology:All rats were sacrificed after their kidneys were carefully removed from the abdominal cavity under anesthesiat. The kidneys were weighted, and kidney weight indexes (KWIs) were measured. Two piece of renal tissues at the renal cortex were obtained rapidly and stored under 2.5% glutaraldehyde for transmission electron microscope study. Then kidney was sliced into 2 longitudinal sections from renal hilum,the one fixed with 10% buffered formaldehyde ,another frozen in liquid nitrogen then stored at -700C. Pasin staining were used in the study of glomerular mesangial matrix index(Ms/Gs)and Masson's trichrome staining were used in the study of perivascular collagen area(PVCA),glomerular collagen deposition score (GCDS), tubular interstitial disease score (TIDS) in renal interstitial tissue. Connective tissue growth factor (CTGF) protein expression was evaluated by qualitative and semiquantitative immunohistochemical staining. The renal hydroxylproline concentration(HPC) was measured by using a spectrophotometer, while the age-matched normal rats served as control group(n=6 for each group).4 Statistical analyses :All datas were analysised under SPSS13.0 statistical package: measurement data was express with x±s, One-Way ANOVA method were used in the analyses of interclass differential comparison,and linear correlation was calculated. Probabilility values <0.05 were considered to denote statistical significance.Results1 Weight and blood glucose level:Compared with age-matched normal, rats injected with STZ had lost weight, KWIs were increased(4wk, 4.11±0.24 vs 4.74±0.31, 12 wk , 4.26±0.21vs 7.49±0.13, 24 wk, 4.01±0.08vs 6.95±0.21,P<0.05),the average fasting blood glucose levels were >24mmol/L, and remained substantially constant during the experimental period of 24 weeks (P<0.05).2 Integrated Backscatter:The IBS% of renal cortex was significantly higher in diabetic rats than those in the control group(4wk, 0.33±0.06 vs 0.51±0.04, 12 wk , 0.52±0.06 vs 0.73±0.05, 24 wk, 0.65±0.10 vs 0.95±0.11,P<0.05), The IBS% of renal medulla was significantly higher in diabetic rats than those in the control group(4wk, 0.17±0.03vs 0.31±0.07, 12 wk , 0.34±0.05 vs 0.58±0.03, 24 wk, 0.51±0.06 vs 0.87±0.12,P<0.05). 3 Histological examination and morphometry:As early as at 4th week,the renal pathological changes in DM group were observed by light microscope which were characterized by the renal glomerulus becomes hypertrophic, mesangial matrix increased ,mesangium widened and the collagen microfibril in peripheral area of renal tubule and blood vessel gathered, while arrangement disorder , fusion and the number reduction of foot process,more but vacuolar degenerated mitochondria were revealed under the transmission eletron microscope in DM group.As time going,the progress is aggravating trend.Compared with the control group, HPC,GCDS,PVCA,TILS,Ms/Gs,CTGF which has been demonstrated to reflect the degree of renal fibrosis were significantly higher(P<0.05)in renal tissue's expression of the diabetic rats than age-matched control ones. As the disease progresses, the above indexes also is increasing gradually . HPC (4周123.00±16.00vs 216.50±10.05,12 weeks 158.50±8.89 vs 267.67±11.29,24 weeks 201.00±7.69vs 340.67±39.22, P <0.05) , PVCA (4周0.45±0.07vs 0.96±0.11,12 weeks 0.60±0.06 vs 1.65±0.18,24 weeks 0.72±0.07 vs2.63±0.0.40, P <0.05), GCDS (4周0.14±0.01 vs 0.34±0.03,12 weeks, 0.26±0.04 vs 0.49±0.03,24 weeks 0.33±0.03 vs 0.62±0.06, P <0.05), TILS (4周0 vs 1.5, 12周0vs 3, 24周1 vs 4.5), Ms / Gs (4周0.18±0.01 vs 0.25±0.02,12 weeks 0.21±0.04 vs 0.47±0.05,24 weeks 0.23±0.03 vs 0.56±0.04, P <0.05) and the expression of CTGF (4 weeks 0.41±0.06 vs 0.88±0.05 12 weeks, 0.60±0.05vs1.29±0.05,24 weeks 1.50±0.05 vs3.13±0.08, P <0.05). 4 The correlations between IBS% of renal cortex and KWI (r = 0.74), HPC (r = 0.93), GCDS (r = 0.95), PVCA (r = 0.89), TILS (r = 0.85), Ms / Gs (0.89), CTGF (r = 0.89) were significant(P<0.01). The correlations between IBS% of renal medulla and KWI (r = 0.74), HPC (r = 0.94), GCDS (r = 0.94), PVCA (r = 0.91), TILS (r = 0.83), Ms / Gs (0.90), CTGF (r = 0.93) (P <0.01) were significant(P<0.01).Conclusions1 There is an obvious renal fibrosis in diabetic rats.2 the IBS% increased of renal cortex and renal medulla can be detected early by IBS in diabetic rats ,As the disease progresses, IBS % of renal cortex and renal medulla also is increasing remarkably.3 There is a positive correlation between IBS % of renal cortex and renal medulla and fibrosis indices (HPC, PVCA ,GCDS and TILS, Ms/Gs, CTGF) in diabetic rats. |
