Mechanisms Of Age-related Renal Fibrosis Promoted By TIMP-1 Through Inflammatory Pathway

Backgrounds and Objective: Morphological changes of kidney with aging are characterized by focal segmental glomerulosclerosis,tubular atrophy, and interstitial fibrosis. Extracellular matrix (ECM) accumulation is the final pathway to renal fibrosis. Matrix metalloproteinases and tissue inhibitors of metalloproteinases (MMPs/ TIMPs) are main regulators of ECM degradation. Imbalance of MMPs/TIMPs induced by upregulation of TIMP-1 could promote ECM accumulation during age-related renal fibrosis, however, whether TIMP-1 promotes age-related renal fibrosis through other pathways become noticeable. It has been shown by some studies that some inflammatory mediators, such as intercellular adhesion molecule-1 (ICAM-1), are substrates of MMPs, therefore, TIMP-1 might promote age-related renal fibrosis through influencing inflammation. In present study, targeting ICAM-1, we observed the inflammatory changes with aging in kidneys of human TIMP-1 transgenic mice and the effect of sense and antisense human TIMP-1 gene transfection on the expression of ICAM-1 in human proximal tubular epithelial cells (HKC) for exploring the mechanisms of age-related renal fibrosis promoted by TIMP-1.Methods: At the age of 3, 12, 24 months, samples of serum, urine, and kidneys of human TIMP-1 transgenic mice (each group, n = 8) and wild type mice (each group, n = 8) were harvested. Serum creatinine (Scr) and urine albumin excretion rate (UAER) were measured. Paraffin sections of renal tissues were stained by PAS. F4/80 positive cells were detected by indirect immunofluorescence. The mRNA and protein expressions of TIMP-1, TIMP-2, MMP-9, MMP-2, ICAM-1, transforming growth factor-pl (TGF-pl), collagen III, and collagen IV were examined by Northern Blot and Western Blot respectively. The activities of MMP-2 and MMP-9 were detected by gelatin zymogrophy, and the activity of TIMP-1 was detected by reverse zymography. HKCs weretransfected with vectors containing sense and antisense human TIMP-1 cDNA respectively, then were stimulated with 10~7mol/L angitensin II for 24 hours. The mRNA and protein expressions of TIMP-1, TIMP-2, MMP-9, MMP-2, and ICAM-1 were examined by Northern Blot and Western Blot respectively. Soluble ICAM-1 (sICAM-1) in supernatant was detected by Western Blot. Co-immunoprecipitation was applied to detect interaction between gelatinases and ICAM-1. The activities of gelatinases and TIMP-1 in supernatant were detected by gelatin zymography and reverse zymography respetively.Results: Renal fibrosis was found in two genotype mice with aging. At the age of 24 months, compared with wild type mice, Scr and UAER of transgenic mice were increased (P < 0.05), and in kidneys of transgenic mice, the expression and activity of TIMP-1 were upregulated (P < 0.05), the expressions and activities of gelatinases were downregulated (P < 0.05), the expressions of ICAM-1, TGF-pi, collagen III, and collagen IV were upregulated, and F4/80 positive cells were increased (P < 0.05). It was shown by results in vitro that after stimulated by Ang II , compared with parental/HKC and vector/HKC, in TIMP-1 S/HKC, ICAM-1 was increased {P < 0.05), activities of gelatinases were downregulated (P < 0.05), sICAM-1 in supernatant was decreased (P < 0.05), which were reverse in TIMP-1AS/HKC (P < 0.05). There was an interaction between ICAM-1 and MMP-9, demonstrated by immunoprecipitation.Conclusion: Imbalance of MMPs/TIMPs induced by overexpression of TIMP-1 promoted age-related renal fibrosis not only through inhibiting the ECM degradation, but also through enhancing inflammation, which supplied theoretical basis to prevent renal senescence by interfering TIMP-1.