| Background Acute renal failure (ARF) secondary to renal ischemia/ reperfusion (I/R) injury is a frequent clinical problem, and tubular cell apoptosis accounts for a significant component Tissue inhibitor of metalloproteinase-1 (TIMP-1) has anti-apoptotic activity in some kinds of cells, however the mechanisms by which TIMP-1 exerts its anti-apoptotic effect are not understood. Most studies demonstrated that TIMP-1 protect cells against intrinsic apoptotic, but others suggested its anti-extrinsic apoptotic effect. Whether the anti-apoptotic effect of TIMP-1 depend on its MMPs inhibiting activity is unclear. By far, there is no report about whether TIMP-1 can inhibit tubular cell apoptosis induced by various kinds of causes include ischemia/reperfusion injury. Objective We established a renal ischemia/reperfusion injury model with human TIMP-1 transgenic mice (TG) and compared the characteristics of tubular cell apoptosis with the wild mice (WT) control. To explore the related mechanisms of TIMP-1 against apoptotic induced by renal ischemia/reperfusion injury as well as the effects of PI3K/AKT and MAPK pathways involved in, we determined caspase-3, 8, 9, T-ERK, p-ERK, p-P38, p-JNK, T-AKT, p-AKT, Bc12 and Bax proteins expression. Using the MMPs inhibitor doxycycline and GM6001, we investigated the relationship between the anti-apoptotic effect and MMPs inhibiting activity of TIMP-1 in vivo and in vitro respectively. Methods 1. The hTIMP-1 transgenic and wild male mice (3 months old) underwent bilateral kidney pedicle occlusion for 30 min to induce renal ischemia. Age-matched SHM-operated mice served as controls. Mices were sacrificed at 24h ofreperfusion. Renal morphology changes, tubular cell apoptosis, protein expression of caspase-3, 8, 9, T-ERK, p-ERK, p-P38, p-JNK, T-AKT, p-AKT, Bcl2, Bax, as well as caspase-3 activity were measured. 2. Wild male mice (3 months) were randomly divided into SHAM, I/R, DOX+SHM and DOX+I/R groups. The methods establishing I/R models is the same as above. The post two groups were administed doxycycline 30mg/kg/d via drinking water. Renal morphology changes, tubular cell apoptosis, protein expression of caspase-3, MMP-2, MMP-9 as well as caspase-3, MMP-2, MMP-9 activity were measured. 3. HKC were randomly divided into VC, VC+H/R, GM+SHM and GM+I/R groups. HKC exposed to PBS 60 minutes which contain 10 μ M antimycin A and 10% fetus calf serum DMEM 60 minutes in succession to establish hypoxia/reoxygenation (H/R) models. The post two groups were administed GM6001 (10 μ m). The tubular cell apoptosis, protein expression of caspase-3, MMP-2, MMP-9 as well as caspase-3, MMP-2, MMP-9 activity were measured. Results Renal I/R injury induced a significant increase in tubular cell apoptosis inTG+I/R and WT+I/R group as well as DOX+I/R and I/R group, while the increase was more pronounced in WT mices, being associated with more marked increase of caspase-3 activity, active caspase-3, 9 protein expression, more severe renal morphology changes but less increase of p-ERK, p-AKT, Bcl2 protein expression than in the TG mices (P < 0.05). On the contrary, the increase in tubular cell apoptosis was comparable between the DOX+I/R and I/R group, being associated with comparable renal morphology changes and increase of caspase-3 activity, active caspase-3 protein expression(P >0.05). HKC H/R injury induced a significant increase in tubular cell apoptosis which is comparable between the GM+H/R and VC+H/R group, being associated with comparable increase of caspase-3 activity and active caspase-3 protein expression. Conclusions Our research indicates that TIMP-1 overexpression can inhibit tubular cell apoptosis induced by renal I/R injury, which is probably mainly due to the anti-apoptotic effect of intrinsic pathway. The up-regulation of Bcl2 expression resulted from the increase of p-AKT and p-ERKexpression is probably one of mechanisms by which TIMP-1 inhibit tubular cellapoptotic. The anti-apoptotic effect of TIMP-1 has no relationship with its MMPsinhibiting effects.
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