AR, Bax/Bcl-2, PPARγ Gene Expressions And Their Relationship To Diabetic Nephropathy In Rats With Diabetes Mellitus

AIMs:1 To optimize the fluorimetry method on aldose reductase (AR) activity assay and investigate the dynamical changes of blood and kidney AR activities in diabetic rats.2 To detect the expressions of AR, B-cell lymphoma leukemia 2 (Bcl-2), Bcl associated x protein (Bax), peroxisome proliferator activated receptor gamma (PPAR γ) and discuss theirs relationships with diabetic nephropathy.Methods:1.1 40 rats were randomly divided into four equal groups: control group and diabetic groups after streptozotocin inducing for 20,40,60 days.1.2 Sensitivity and reduplication of two methods (NADP-generating and NADPH-reducing methods) were compared.1.3 Whether the fluorescent value was affected by haemoglobin was Observed.1.4 Conditions of the better method were further optimized.1.5 AR activities were assayed by the improved method.2.1 Tissue sections were observed with the PAS staining method.2.2 The gene expression levels of AR, Bax/Bcl-2, PPAR γ were assayed by the polymerase chain reaction.2.3 Then their expression profiles were further confirmed by Northern blot.2.4 The correlations of these changes were analysed and their relationships with diabetic nephropathy were then discussed.Results:1.1 The NADP-generating method was more sensitive and stable than the NADPH-reducing method on AR activity assay.1.2 Fluorescent value was interfered by haemoglobin and this influence can be removed by perchloric acid precipitation.1.3 The NADP-generating method for AR activity assay was optimized as follows: 135mM PBS, 100mM NH₄SO₄, 0.04mM DL-glyceraldehyde, 150μM NADPH, 5μl hemolysis solution (or 20μl homogenated tissue solution). Both the test and the control tubes were consisted of the above components except that NADPH was replaced with water in the control. Total volume was 200μl. The reaction was performed in 37℃ water bath for 5 minutes, then was terminated by adding 600μl HCl to the reaction mixture in 60℃ water bath for 15 minutes. Haemoglobin was precipitated by perchloric acid. After centrifugation for 10 minutes with 2500rpm the fluorescence was excited by adding 6.67mM NaOH 1.8ml to the supernatant fluid. Then AR activities were assayed.1.4 AR activities both in erythrocyte and kidney were significantly increased in diabetic groups compared with the control (P<0.05) and had an increasing tendency during the course of diabetes. The AR activity both in erythrocyte and kidney were correlated with blood glucose (r=0.995 and 0.983, respectively). The AR activity of erythrocyte was lower than that of kidney for the same sample.2.1 PAS showed that the positive staining materials increased in the area of capillary basement membrane and glomerular mesangium with the course of diabetes. In the DM60 group the extracellular matrix increased and the cells in the glomerular mesangium area induced slightly.