| Abstract:bjective:To observe the expression of alpha-Smooth muscle actin (a-SMA),fibronectin(FN) in podocyte in a rat membranous nephropathy(MN) model induced by cationic bovine serum albumin(C-BSA),also to observe the roles which tetramethylpyrazine(TMP) play on them.To explore the transdifferentiation of podocyte in MN, the possible preventive and therapentic effects and the possible mechanisms of TMP. Methods:96 male Spargue-Dawley (SD) rats were divided into four groups randomly:control group, model group, TMP group,32 for each group. We didn’t preimmuniz or make model in control group.Mean while, we preimmunizd the model group and the TMP group, injected 1 ml normal saline(NS) which replaced C-BSA through the vena caudalis erery other day,and injected 2 ml NS instead of TMP through into abdominal cavity every day. We duplicated MN model in the control group and the TMP group with the improved Border way. From the first week of formal immunization, we injected intraperitoneally TMP in TMP intervene group at a dose of 100 mg·kg-1·day-1,and injected the same amount of normal saline intraperitoneally in model group. After the 1st,2nd, 3th and 4th week of formal immunization respectively,8 rats of each group were killed and their renal tissues were collected. Before the rats were killed, the urine protein in 24 hours (24hUTP) and the concentration of urine creatinine of every rat were detected dynamically. We drew some blood from every rat, detected clinical biochemical indicators, and compared respectively the condition of creatinine (Scr), blood urea nitrogen (BUN), creatinine clearance(Ccr) per minute, plasma albumin (Alb), triglyceride (TG) and total holesterol(TC). The nephridial tissue of each rat was observed by light microscope, electron microscope, immunofluorescence technology respectively in order to identify the condition of model-formating and to observe the conditions of glomerular basement membrane (GBM) and tubular basement membrane (TBM) at each time spot. The indirect immunofluorescence double label(WT-1/a-SMA and WT-1/FN) technique was used to mark and locate podocytes, and the expressions of a-SMA,FN in podocyte in each group were observed dynamically. We made the correlation analysis of a-SMA,FN, GBM,TBM of podocyte in each group. Results:(1) kidney morphology:Light microscope showed:In the control group, the renal color, the renal morphology and the renal pathology were normal. In the model group and the TMP intervene groups, the renal morphology was normal, but mild pathological changes were observed after the 1 st week. With the experiment proceeding, the rat kidneys become larger and pale gradually, the pathological lesions of nephridial tissues aggravated gradually. After the 4th week, kidney enlarged and got pale significantly, the glomcruli intumesced obviously; the basement membrane also got thicken diffusely, parts of the capsular space become angusty, collagen fibers deposited obviously, interstitium widened even more significantly, and fibrosis become heavier. Electron microscopy showed:a great quantity of electrondense materials deposited under the glomerular epithelium. Foot process coalesced, flattened or vanished. Immunofluorescence showed: No IgG deposit was observed in the control group at every time spot. In the model group, IgG deposited at glomerular capillary walls and mesangial regions which were observed after 2 weeks, and the fluorescence intensity was +++to++++, which increased gradually and was most obvious at the end of 4 weeks. Compared with that of model group, the fluorescence intensity at glomerular capillary walls and mesangial regions in TMP intervene group was more lighter at each time spot. (2) The dynamic change of a-SMA, FN in podocytes:In the control group, no a-SMA and only a little FN expressed in podocytes which showed faint red fluorescence. WT-1 was only expressed in the nucleoli of podocytes and showed green fluorescence. But much more a-SMA and FN which showed strong red fluorescence were observed in podocyte in the model group and the TMP intervene group. With the experiment proceeding, the expression of a-SMA and FN increased gradually and the fluorescence intensity enhanced gradually in the two groups.α-SMA and FN expressed much more in the model group and the TMP intervene group than that in the control group, and expressed less in the TMP intervene group than that in the model group, and the difference was significant (P<0.05). WT-1 only expressed in the renal capsule visceral distribution. In the model group and the TMP intervene group, the regions where a-SMA and WT-1 or FN and WT-1 expressed together showed yellow fluorescence in podocyte. Compared with the model group, there were less yellow fluorescence in the TMP intervene group. (3) The changes of clinic indexes:The indexes in the control group were all nomal. Scr, BUN, TG, TC and 24h UTP increased in the model group, while Ccr, Alb decreased, especially the changes of Scr, BUN, Alb and 24h UTP (P<0.05). However, in the TMP intervene group, Scr, BUN, TG, TC and 24h UTP decreased (P<0.05), while Ccr, Alb increased (P<0.05), especially the changes of Scr, BUN, Alb at the end of the 4th week (P<0.05). (4) The correlation of each index at the end of the 4th week:In the control group, FN in glomeruli was positive correlated with the thickness of GBM. But in the model group and the TMP intervene group,.α-SMA was positive correlated with FN and the thickness of GBM significantly and was negative correlated with Ccr. Conclusion:(1)α-SMA can express in podocytes of MN rat models and the expressions ofα-SMA will increase gradually with the experiment proceeding, which shows that transdifferentiation of podocyte exists in the development of MN. (2)The FN in the podocytes of the rat MN model increased with the process of podocyte transdifferentiation and is positive correlated withα-SMA,GBM significantly, which shows that the podocyte transdifferentiation increased synthesis of ECM components leading to ECM accumulation,GBM thickening of one of the reasons. (3) TMP can reduce the expressions ofα-SMA protein in podocytes of MN rats and inhibited podocyte transdifferentiation on a certain extent and reduce the compositions of FN protein, which can reduce the accumulation of extracellular matrix on a certain extent and lessen the kidney pathology harm, and finally plays a role in kidney protection. |
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