Cloning Of Human TIMP-1 CDNA And Its Expression In Renal Cells And In Transgenic Mice

The balance between the synthesis and degradation of extracellular matrix (ECM) in kidney and the proliferation, differentiation and apoptosis of renal cells play important roles in normal development of kidney and various kidney disorders. Tissue inhibitor of metalloproteinase- 1 (TIMP-1) is a major inhibitor of ECM degradation and has been generally accepted as a key factor in ECM remodeling and in the pathogenesis of progressive renal diseases. Interestingly, besides its MMP-inhibiting activity, TIMP- 1 also has some other effects, such as erythroid potentiating activity and cell growth promoting activity in a wide range of cells, suppressing apoptosis in B cells and breast epithelial cells. This study was designed to examine the role of TJMP-lin ECM degradation and whether TIMP-1 have effects on the apoptosis and differentiation of renal cells except its function of MMPs' inhibition.TIMP-1 full-length cDNA containing the signal peptide was amplified by reverse transcription and polymerase chain reaction from total RNA of human normal renal tissues. Confirmed by the DNA sequence analysis, the TIMP-1 cDNA fragments were then inserted to mammalian expression plasmid pCDNA3 respectively. Thus pcDNA3-TIMP-l (PCT) encoding sense TJMP-l and pcDNA3-ATJMP-1 (PCA) encoding antisense TIMP-1 recombinant eukaryotic expression vectors were constructed. Moreover, PCT was transfected into COS-7 cells with lipofectin DOTAP. Northern blot analysis and in situ hybridization demonstrated that no TIMP- 1 mRNA was detected in normal COS-7 cells, while TIMP-1 mRNA was detected in pCDNA3/TIMP-1 －4．transfected COS-7 cells. Further study indicated that TIMP-l overexpressioncould induce the pro1iferation of COS-7 cells.We constructed retroviral vector expressing TIMP-1 sense and antisenseRNA using recombining techniques. These tWo vectors were introduced into thePA3 l7 packaging celline and the high-titer retroviral suPematans were obtained.The rat glomerular mesangial cells were infected with retroviral suPematamscontaining sense or anisense TIMP-l, and both sense and anisense TIMP-lmRNA were successfully expressed in rat mesangial cells. Overexpression ofTIMP-1 induced by sense TIMP-l caused uPregulation of FN and IV typecollagen in protein level, in colltrast, suppression of TIMP-l expression inducedby anisense TlMP- 1 caused decrease of FN and IV tyPe collagen proteinexpression, but neither sense nor amisense TIMP-l infection had no effects onthe RNA level of FN and IV type collagen. These results indicated that TlMP-lsuppresses the degradation of ECM in rat glomerular mesangial cells.The twO vectors, i.e., PCT and PCA were transfected into rat mesangialcells (RMC) Wth lipofectin DOTAP RT-PCR, Northem Bloting and Westembloting were used to detect the expressions of sense and amisense TlMP-l.Overexpression of TIMP-l and suPpression of TIMP-l expression wereachieved in RMC by transfection of PCT and PCA, respectively. Apoptosis wasinduced in normal RMC, RMC transfected with pCDNA3, PCT and PCA byserum deprivation. PCA-induced the suPpression of TIMP-1 coincided with anearlier onset of aPoptosis and PCT-induced TIMP-1 overexpression coincidedwith a much delayed onset of aPoptosis. The PCA-transfected RMC underwentaPoptosis 12 hours after serum deprivation, in conirast to normal RMC andANC transfected with colltrol vectof, Which underwent aPoptosis 48 hours afterserum deprivation, and RMC transfected with PCT did not undergo aPoptosisuntil 4 days after serum deprivation. Furthermore, TIMP-1 over-expressioninhibited the expression of Bax but had no effect on the expression of Bcl-2.This result indicated that TIMP-1 can suPpress aPoptosis of RMC and its effectson RMC apoptosis may partially correlate with inhibition ofbax expression.TO further investigate the effects of overexpression of TIMP-1 on kidney invivo, we constructed mice transgenic for TIMP-l by microinjection. TheTIMP-l transgenic mice were confirmed by...