| BackgroundChronic Kidney diseases,(CKD) has become a serious threat to human health, consuming large amounts of health resources. CKD is a progressive disease. Renal fibrosis is the critical path of CKD progression to end-stage renal disease(ESRD). Renal fibrosis is a dynamic process. The core of renal fibrosis is the hyperplasia of fibroblasts and myofibroblasts as soon as the excessive accumulation of extracellular matrix (ECM), which can lead to glomerular fibrosis and renal interstitial fibrosis.Transforming Growth Factor-beta (TGF-β) and Epithelial to Mesenchymal Transition (EMT) play an important role in the renal interstitial fibrosis.Exploring suitable drug to give an effective intervention on the above mechanism have a great significance. In recent years, some studies show that active vitamin D3 in addition to regulating metabolism of calcium, phosphorus, etc, also has a regulation to control cell growth, anti-inflammatory and anti fibrosis effect and other study concerning renal tubular epithelial cells in vitro found that active vitamin D3 can delay EMT induced by the PTH, albumin, suggesting vitamin D3 may have potential ability to alleviate renal tubular interstitial fibrosis. UUO (unilateral ureteral obstruction, UUO) model is a kind of mature renal interstitial fibrosismodel.This research aims to through the animal experiment to research the effect of active vitamin D3 on renal interstitial fibrosis and relevant mechanisms.Hoping provide useful guidance for the treatment of CKD.PurposeWe use unilateral ureteral ligation method to establish UUO rat model and to investigate the effect of activity of vitamin D3 on UUO rat renal pathology and the related mechanism.MethodsBy ligation of the left ureter for two weeks,we tried to establish the animal model of UUO.The 40 SD rats were divided into four groups randomly, including Group A (sham operation group), Group B (UUO model group), Group C (UUO+low doses of vitamin D3 group), Goup D (UUO+large doses of vitamin D3 group). Each group have 10 rats.Since the operation day,A and B group were given peanut oil 0.3 ml by intraperitoneal injection, once a day. Since the operation day group C were given vitamin D30.03ug/kg.day, by intraperitoneal injection,once a day,which Soluble in the peanut oil and also it volume is 0.3 ml. Since the operation day group D were given vitamin D30.06ug/kg.day, by intraperitoneal injection,once a day,which Soluble in the peanut oil and also it volume is 0.3 ml.14 days later, the rats were sacrificed. Blood samples and kidney tissues were collected from the rats.Blood serum was tested by biochemical assay for serum creatinine, serum calcium ion solubility, serum phosphorus solubility. Blood serum was tested by ABC-with double antibody sandwich ELISA method for serum Parathyroid hormone(PTH). Renal tissue was observed to assess glomerular sclerosis, glomerular basement membrane lesions, renal interstitial fibrosis degree of half quantitative analysis by HE stain,Masson stain. Immunohistochemical technique was used to determine the position and quantity of Transforming Growth Factor-beta (TGF-beta)、alpha smooth muscle actin (alpha SMA), and E calcium adhesion protein expression-cadherin (E-cadherin) in Kidney tissue.Results1.Each group have no death2. After the experiment, compared with the rats of group A, the serum creatinine level (43.1±5.55 umol/1 VS 28.0±5.68 umol/l,P<0.05) of group B is increased, blood calcium (2.44±0.15 VS 2.58±0.05, P<0.05) of group B is decreased. Parathyroid hormone and blood phosphorus between the two groups had no significant difference (P>0.05). Compared with group B, the serum creatinine level (39.0±1.83umol/l VS 43.1±5.55 umol/1, P<0.05) of group C is decreased. The blood calcium level (2.75±0.08 VS2.44±0.15, P<0.05) of group C is increased. Parathyroid hormone and blood phosphorus between the two groups had no significant difference. Compared with group B, the serum creatinine level (36.0±2.11umol/l VS 43.1±5.55 umol/1, P<0.05)、Parathyroid hormone level (0.132±0.015mmol/l VS 0.142±0.016mmol/l, P<.05) of group D is decreased. The blood calcium level (2.78±0.14 VS2.44±0.15, P<.05)、blood phosphorus level (2.930±0.30mmol/l VS 2.50±0.31mmol/l, P<0.05) of group D is increased. Compared with group C, the serum creatinine level (36.0±2.11umol/l VS 39.0±1.83umol/1, P<0.05)、 Parathyroid hormone level (0.132±0.015mmol/l VS 0.135±0.016mmol/l, P<0.05) of group D is decreased. The blood phosphorus levels (2.93±0.30mmol/l VS 2.54±0.23mmol/l, P<0.05) of group D is increased.blood calciumparathyroid and hormone between the two groups had no significant difference (P> 0.05).3. There was no obvious pathological changes in the kidney of Group A. Atrophy of renal tubular epithelial cells, tube cavity expansion, interstitial broadening, interstitial mononuclear cell infiltrates,irregular change of renal tubular basement membrane were observed in the majority view of Group B. Compared with group B the semi quantitative index of renal fibrosis of group C is decreased (P< 0.05). Renal tubular expansion、small amount of fibrous tissue hyperplasia which locating in the interstitial area were observed in Group D. Compared with group C the kidney damage index and the index of renal fibrosis semi-quantitative of Group D is significantly decreased (P< 0.05).4. The kidney tissue immunohistochemical results of TGF-beta showed that four groups’renal tubular epithelial cell cytoplasm, renal interstitial cells, glomerular mesangial cells can see the tan particles which on behalf of TGF-tan beta,and group B dyeing the most. Compared with group A, the average optical density of TGF-beta of group B is significantly increased (0.32±0.04Vs 0.21±0.04, P<0.05). Compared with group B, the average optical density of TGF-beta of group C is significantly decreased (0.26±0.03 Vs 0.32±0.04 P<0.05). Compared with group B, the average optical density of TGF-beta of group D is significantly decreased (0.25±0.03Vs 0.32±0.04 P<0.05).The TGF-beta average optical density had no significant difference between Group C and Group D (P> 0.05).5.The kidney tissue immunohistochemical results of alpha SMA showed that four groups’renal tubular epithelial cell cytoplasm, renal interstitial cells, can see the tan particles,which on the behalf of alpha SMA. and in group B the tan particles dyeing the most. Compared with group A, the group B’s average optical of alpha SMA density in is significantly increased (0.24±0.02 Vs 0.03±0.00, P<0.05). Compared with group B, the group C’s average optical density alpha SMA is significantly reduce (0.22±0.02 Vs 0.24±0.02, P<0.05). Compared with group B, the group D’s average optical density alpha SMA is significantly reduce (0.20±0.03Vs0.24±0.02, P<0.05).alpha SMA average optical density had no significant difference between the C, D group (P>0.05)6.The kidney tissue immunohistochemical results of E-cadherin showed that four groups’s renal tubular epithelial cell cytoplasm, renal interstitial cells, glomerular mesangial cells can see tan particles which on the half of E-cadherin, and in group B the tan particles dyeing the most. Compared with group A, the group B’s average optical density of E-cadherin is significantly decreased (0.22±0.07 Vs 0.45±0.06, P<0.05). Compared with group B, the group C’s average optical density of E cadherin is significantly increased (0.30±0.08Vs 0.22±0.07, P<0.05). Compared with group B, the group D’s average optical density of E cadherin is significantly increased (0.34±0.11 Vs 0.22±0.07, P<0.05). Compared with group C, the group D’s E-cadherin average optical density had no significant difference (P> 0.05)7. The correlation analysis showed that the half quantitative index of renal interstitial fibrosis and TGF-beta expression were positively correlated. TGF-beta expression and alpha SMA expression were positively correlated. TGF-beta is negatively related to the E-cadherin expression. Alpha SMA expression and E-cadherin expression is negative correlation. Renal fibrosis and alpha SMA expression are related. Renal fibrosis is negatively related to the E-cadherin expressionConclusionOur experiment successfully established murine model of renal fibrosis by using ureteral ligation method and we found that the activation of vitamin D3 could alleviate the development of renal fibrosis. Large dose of active vitamin D3 improve the development of renal fibrosis is superior to the conventional dose of active vitamin D3. The activation of vitamin D3 may through inhibiting TGF-βto stop the expression of a-SMA and reduce the loss of E-cadherin to delay the renal interstitial fibrosis, and then further postpone the proceeding of CKD. |
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