| [Background and Objective]Renal tubulointerstitial lesions is one of most important factors affecting the prognosis of reanl diseases which is characterized morphologically by the infiltration of interstitial inflammatory cells , interstitial fibrosis and tubular atrophy. Renal interstitial fibrosis is the final result of progressive renal interstitial injury and the main pathologic change of chronic renal dysfunction. The imbalance of matrix metalloproteinases (MMPs) / tissue inhibitors of metalloproteinase (TIMPs) induced by overexpression of TIMP-1 plays a key role in renal tubulointerstitial injury. The continuous inflammation is an important factor in renal tubulointerstitial injury. Recent study indicates that some inflammatory cytokines such as ICAM-1 are also substrates of MMPs. Our research is to observe the effect of MMPs/TIMPs imbalance induced by overexpression of TIMP-1 on tubulointerstitial inflammation in human TIMP-1 (hTIMP-1) transgenic mice constructed successfully in our lab after unilateral ureteral obstruction (UUO) operation. [Methods]Wild-type NIH mice and hTIMP-1 transgenic mice were randomly divided into UUO groups and sham groups respectively and sacrificed at 1, 3, 7, 14 days after UUO or sham operation (n=6,each group).Indirect immunofluorescence was apllied to detect the expression of F4/80 (the marker of mouse monocyte/macrophage), Northern blot and Western blot were applied to analyze the mRNA and protein expressions of COLⅢ, COLⅣ, ICAM-1 , TIMP-1 , MMP-9, TIMP-2 and MMP-2; Gelatin zymography was used to detect the proteolytic activityof MMP-2 and MMP-9 and reverse zymography to analyze the activity of TIMP-1. [Results]1. The histologic changes of tubulointerstitium: PAS and MASSON staining showed that the degree of renal tubulointerstitial injury were increased significantly after UUO compared with sham groups (p<0.05 ) .In comparing with wild-type groups, the degree of renal tubulointerstitial injury were increased more significantly at 7d and 14d post UUO (P<0.05) .The area of tubulointerstitial fibrosis (wild-type mice vs transgenic mice): sham group, 0.81±0.23 vs 1.26±0.35 ; post UUO 1d, 0.89±0.12 vs 1.5 ± 0.24; post UUO 3d, 1.82±0.69 vs 3.2±0.76; post UUO 7d, 6.74±0.89 vs 11.3±0.97: post UUO 14d, 13.19 ± 1.08 vs 19.23 ± 1.23.2. The influx of inflammatory cells in renal tubulointerstitium: Indirect immunofluorescence showed that the numbers of positive F4/80 cells (the marker of monocyte/morphage) were increased significantly after UUO compared with sham groups (P<0.05). The numbers of positive F4/80 cells in renal tubulointerstitial were more than wild-type mice at 7d and 14d post UUO (P<0.05) . The number of positive F4/80 cells (wild-type mice vs transgenic mice): sham operation, 5.2 ± 1.24 vs 5.9±1.35; post UUO 1d, 8.4 ± 2.8 vs 10.2±3.1; post UUO 3d, 13.8±3.9vs 15.6 ± 4.7; post UUO 7d, 30.2±5.8 vs 45.6 ± 7.1; post UUO 14d, 51.8±8.9 vs 68.5 ± 9.2.3. The elements of extracellular matrix: The mRNA expressions of COLIII and COLIV detected by Northern blot were increased significantly in both transgenic and wild-type groups after UUO with a maximum at 14 day. Compared with wild-type groups, the mRNA expressions of COLIII and COLIV in transgenic groups were increased more significantly at 7d and 14d post UUO (P<0.05) .4. The expressions of inflammatory cytokine ICAM-1: The mRNA and protein expressions of ICAM-1 detected by Northern blot and Western blot were increased gradually after UUO with a maximum on day 14 post UUO. Compared with wild-type groups, the mRNA and ICAM-1 protein expressions in transgenic groups were increased more significantly at 7d and 14d after UUO operation (P<0.05) .5. MMPs/TIMPs:(1) The expressions and activity of TIMP-1: The mRNA expressions, protein expressions and activity of TIMP-1 were increased gradually after UUO with a maximum on day 14 postUUO, and there are significant differences between UUO groups and sham groups (P<0.05 ) In comparing with wild-type groups the mRNA expressions, protein expressions and activities of TIMP-1 in transgenic groups were increased more significantly (P<0.05)(2) The expressions and activity of MMP-9: Northern blot showed that the mRNA expressions of MMP-9 were increased gradually with a maximum on day 7 after UUO, The mRNA expressions in transgenic mice are lower than in wild-type mice (P<0.05) . Western blot and zymography showed that the protein expressions and activities of MMP-9 were declined gradually after UUO with a minimum at 14d . There are significant differences between UUO groups and sham groups (P<0.05 ) . In comparing with wild-type groups the protein expressions and activities of MMP-9 in transgenic groups were decreased more significantly at 7d and 14d post UUO(P<0.05).(3) The expressions of TIMP-2: Northern blot and Western blot showed that the mRNA and protein expressions were increased gradually after UUO with a maximum on day 14 post UUO. The differences between UUO groups and sham groups are significant (P<0.05). There are no significant differences between transgenic mice and wild-type mice (p>0.05)(4) The expressions and activities of MMP-2: Northern blot showed that the mRNA expressions of MMP-2 were increased gradually after UUO with a maximum at 7d post UUO , there are significant differences between UUO groups and sham groups.while there are no significant differences between transgenic mice and wild-type mice (P>0.05).Western blot and zymography showed that the protein expressions and activities of MMP-2 were decreased gradually after UUO with a minimum at 14d post UUO. There are significant differences between UUO groups and sham groups (P<0.05). In comparing with wild-type groups the protein expressions and activities of MMP-9 in transgenic groups were decreased more significantly at 7d and 14d post UUO (P<0.05 ) .[Conclusion]1. overexpression of TIMP-1 in transgenic mice could upregulate the expression of ICAM-1 and aggravate renal tubulointerstitial inflammation which leading to renal tubulointerstitial fibrosis.2. The possible mechanism is that overexpressed TIMP-1 inhibit the degradation of ICAM-1 by MMP-9. |
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