| Objectives: Diabetic nephropathy is one of the most common complications of diabetes, which is histologically characterized by overaccumulation of extracellular matrix (ECM) proteins, such as type IV collagen, in glomerular mesangium. Formerly more attentions were paid to factors resulting in the overproduction of ECM. Recently the overaccumulation of extracellular matrix in diabetic glomeruli has been considered to be caused not only by the overproduction of ECM, but also by the decrease in the degradation of matrix proteins. Matrix metalloproteinases and tissue inhibitors of metalloproteinase play a key role in the metabolism of ECM proteins. Chinese medical science thinks that diabetes is belonged to thirst, while puerarin is traditional Chinese medicine to treat thirst, which can decrease blood glucose , dilate blood vessel , improve microcirculation and decrease blood viscosity applicating to cardiovascular and cerebrovascular disease. In this study Wistar rats were used to induce experimental diabetes and the glomerular mesangial cells were cultured in high glucose medium to investigate the expressions of matrix metalloproteinases and their tissue inhibitors and effect of puerarin.Methods:1 Induction of diabetes and examination of matrix metalloproteinases and their tissue inhibitorsUninephrectomized male Wistar rats were divided into two groups: control group and diabetic group. The rats of diabetic group received a single intraperitoneal injection of STZ dissolved in 0.1mol.L-1 sodium citrate (pH 4.5) at a dose of 65mg.kg-1 WT. The rats of control group only received an injection of the same volume of 0.1mol.L-1 sodium citrate. The model of diabetes was considered to be successful when the blood glucose was ≥16.7 mmol.L-1 andthe glucose in urine was +++++++ after 24 hours of the injection. Six rats from each group were respectively sacrificed at weeks 4,8,16 after STZ injection. The deposition of ECM in renal tissues was evaluated by immunohistchemistry. The expressions of MMP-2, MT1-MMP, TIMP-2, MMP-3, MMP-10, TIMP-1 and TGF 3 1 proteins were measured by flow cytometry and immunohistchemistry. The mRNA expressions of MMP-2, TIMP-2, MMP-10 or TIMP-1 were evaluated by in situ hybridization. The activity of MMP-2 was examined by zymography .2 Cell culture and examinatoin of matrix metalloproteinases and their tissue inhibitorsGlomerular mesangial cells were separated from male Wistar rats and were always used between the third and the eighth passages. Cells were cultured separately in normal or high glucose medium for 24,48,72,96 and 120h. Total RNA was extracted with Trizol method and the expression of MMP-2 , TIMP-2, MMP-10 and TIMP-1 mRNA was detected with RT-PCR, result was showed as relative expression levels (ratio of MMP-2 , TIMP-2, MMP-10 or TIMP-1 to GADPH). The expression of MMP-2 or TIMP-2 protein was detected by flow cytometry and immunocytochemical staining; the expression of MT1-MMP, MMP-10 or TIMP-1 protein was evaluated by immunocytochemical staining and western bloting analysis* result was showed as relative expression levels (ratio of MMP-10 or TIMP-1 to ￡ -actin).The mouse monoclonal antibody for IV collagen against rat serum was used to measure quantitatively IV collagen secreted by the mesangial cells using an enzyme-linked immunosorbent assay (ELISA).3 Examination of matrix metalloproteinases and their tissue inhibitors in diabetic rats treated by puerarinUninephrectomized male Wistar rats were used to induce diabetes by injection of STZ. Puerarin was delivered daily by the intraperitoneal injection from the third day of induction to diabetes for 16 weeks at doses of 40mg.kg"1> SOmg.kg"^ 160mg.kg'', while the normal group and diabetic group were injected isodose NS; Urine samples were collected before sacrifice and24-h urinary protein excretion was measured. In situ hybridization , flow cytometry and immunohistochemical staining were used to investigate the expressions of MMPs and TIMPs.Results:1 The expressions of matrix metaIloproteinase-2 and tissue inhibitor of metaIloproteinase-2In vivo results: (D Compared with control group, volum of glomeruli, mesangial matrix, thickness of glomerular and tubular basement membrane increased with progression of diabetes; (2) Immunohistochemical staining showed that the positive expressions of collagen IV and Laminin were observed in mesangial area, capillary basement membrane and tubular interstitium. Image analysis showed that the staining intensity for collagen IV and Laminin markedly increased in diabetic kidneys compared with control group and progressively increased with duration of diabetes ; (3) MMP-2,TIMP-2,MT1-MMP and TGF fi 1 were positively expressed in tubules and glomeruli by immunohistochemistry. Compared with control group, fluorescent indexes (FI) of MMP-2 and MT1-MMP was lower in DM group, and progressively decreased with duration of diabetes, while FI of TIMP-2 and TGF B 1 was higher, and progressively increased with the duration of diabetes; @ By in situ hybridization, mRNA of MMP-2 and TIMP-2 was detected in glomeruli and tubules. mRNA of MMP-2 was down-regulated, while mRNA of TIMP-2 was up-regulated in DM group with duration of diabetes; (§) Zymography analysis indicated that there were two pellucid zone respectively in 72KD (non-active level) and 62KD (active level). Compared with control group, the active level of 62KD was higher at the 4th week, while at the 8th week and 16th week the active level was lower in DM, the non-active level of 72KD was lower, and progressively decreased with duration of diabetes. (6) The expression of MMP-2 was negatively correlated with collagen IV and lamnin deposition in the glomeruli (r=-0.543, -0.728, P=0.005, 0.000) , while the expression of TIMP-2 was positively correlated with collagen IV and lamnin deposition in the glomeruli (r=0.616, 0.604,P=0.001, 0.002); the expression of MTl-MMP was positively correlated with the expression of MMP-2 (r=0.731, P=0.000) , while the expression of TGF P 1 was positively correlated with the expression of TIMP-2 (r=0.643, P=0.000) .In vitro results: ? Immunocytochemically, the positive stainings for MMP-2,TIMP-2 and MTl-MMP were observed in cytoplasm of the mesangial cells cultured in the medium with normal level and high level glucose. Positive signal was not observed in the nucleus of the cells. Compared with those in mesangial cells cultured in the medium with normal level glucose, the stainings were strengthened for TIMP-2, weakened for MMP-2 and MTl-MMP in the mesangial cells exposed to high glucose from 48h. (D By FCM , the expression of MMP-2 in mesangial cells cultured in high glucose medium was lower than that in normal glucose medium from 48h; while the increased expression of TIMP-2 was detected in mesangial cells exposed to high glucose from 48h(P<0.05or 0.01). Western bloting analysis showed that the expression of MTl-MMP in mesangial cells cultured in normal glucose medium were maintained on steady levels, the decreased expression of MTl-MMP was detected in mesangial cells exposed to high glucose. (3) RT-PCR showed that the relative expression level of MMP-2 was 2.76, 1.97, 0.84, 0.50, 0, 0 respectively in N, 24h, 48h, 72h, 96h and 120h , while the expression of TIMP-2 was 0.14,0.34,0.41,0.51,0.56 respectively in 24h, 48h, 72h, 96h and 120h ; which indicated that the expression of MMP-2 decreased and the expression of TIMP-2 increased with duration of culture. ? ELISA showed that the level of IV collagen secreted by mesangial cells exposed to high glucose was higher than that in normal glucose from 72h (P=0.034, P=0.006) .(§) Negative correlation was found between the FI of MMP-2 and IV collagen deposition (r=-0.622, P=0.003) , while there was positive correlation between the FI of TIMP-2 and IV collagen deposition (r=0.753, P=0.000) .2 The expressions of matrix metalloproteinase-3,10 and tissue inhibitor of metalloproteinase-lIn vivo results: (D Immunohistochemically, MMP-3, 10 and TIMP-1 were expressed in tubules and glomeruli. There was weak stainings for MMP-3,10 and TIMP-1 in control group, while enhanced stainings were observed in DM. By FCM, the FI of MMP-3 , MMP-10 and TIMP-1 was higher in DM group compared with control group ( P<0.01) ; the expression of MMP-10 decreased, while the expression of TIMP-1 increased with duration of diabetesC P<0.01), the ratio of MMP-10/TIMP-1 decreasedC 1.45 > 1.31 > 1.29, PO.01 ) ; The expression of MMP-3 protein increased significantly in the kidneys of diabetic rats at weeks 4 and 8, afterward it decreased gradually, but was still higher than that of control group at week 16. (2) By in situ hybridization, mRNA of MMP-10 and TIMP-1 was detected in glomeruli and tubules . Only weakly positive stainings for mRNA of MMP-10 and TIMP-1 were observed in some tubular epithelial cells in control group. The number of positive cells markedly increased in diabetic group. The relative content of MMP-lOmRNA was up-regulated in DM, while the integra-light density (ILD) of MMP-10 was higher in diabetic rats at week 16 compared with control group; the relative content and ILD of TIMP-1 mRNA were up-regulated in DM compared with control group ( PO.Olor 0.05 ) . (3) Positive correlation was found between the expression of TIMP-lmRNA and the deposition of IV collagen and Laminin(r=0.764,0.711,0.547,0.63l,P<0.01) , while the ratio of MMP-10/TIMP-1 was negatively correlated with laminin deposition ( r=-0.592, -0.52, P<0.05 ) .In vitro results: (T) Immunocytochemically, the positive reactions for MMP-2,TIMP-2 and MT1-MMP were observed in cytoplasm of the mesangial cells cultured in the medium with normal level and high level glucose. Positive signal was not observed in the nucleus of the cells. Compared with those in mesangial cells cultured in the medium with normal level glucose, the stainings were strengthened for TIMP-1, weakened for MMP-10 in the mesangial cells exposed to high glucose from 24h . Western bloting analysis showed that the expressions of MMP-10 and TIMP-1 in mesangial cells cultured continuously in normal glucose medium were maintained on steadylevels. The decreased expression of MMP-10 was detected in mesangial cells exposed to high glucose; while increased expression of TIMP-1 was observed from 24h. (2) RT-PCR showed that the relative expression level of MMP-10 was 1.63, 1.49, 1.43, 1.30, 0.72, 0.55, while the expression of TIMP-1 was 0.16, 0.36, 0.25, 0.57, 0.63, 0.80 respectively in N ,24h,48,72h,96h and 120h; which indicated that the expression of MMP-10 decreased and expression of TIMP-1 increased with duration of culture. (3) There was negative correlation between relative expression level of MMP-10 and IV collagen(r=0.735,P=0.000); the relative expression level of TIMP-1 was positively correlated with IV collagen(r=0.698,P=0.000).3 Effects of puerarin on renal function and expressions of MMPs and TIMPs in diabetic kidney(D Compared with control rats, the kidney/body weight ratio was increased at week 16 of diabetes, while body weight was higher and the kidney/body weight ratio was decreased in puerarin treated group ( P<0.01) . (D Compared with that of control rats, the change of renal function in diabetic rats included the increase of blood sugar, blood urine nitrogen and urine protein (PO.05 or 0.01) , whereas blood sugar and urine protein were improved in puerarin treated group (P<0.05 or 0.01), especially in high dose group. (D Compared with those of control rats, the glomerular enlargement and mesangial expansion were observed in diabetic rats, while these parameters were improved in treated group, especially in high dose group. ? Immunohistochemically , the expressions of IV collagen and laminin were lower in treated group compared with DM group, especially in high dose group. ?The mRNA of MMP-2 and TIMP-2 was consistent with protein expression. Compared with DM group, the expressions of MMP-2 and MT1-MMP were increased significantly, while the expressions of TIMP-2 and TGF 0 1 were decreased in puerarin treated group. ?Compared with DM group, the mRNA and protein expressions of MMP-10 and TIMP-1 decreased in puerarin treated group ^specially in high dose group. The protein expression of MMP-3 decreased in puerarin treated group. (7) At week 16 of...
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