Mechanisms Of Aging-associated Impaired Renal Angiogenesis Promoted By TIMP-1 Via PTEN Pathway

Backgrounds and objective:Renal aging plays an important role in aging of organisms. Kidney is a highly vascularized organ which with aging, exhibits impaired angiogenesis such as impaired focal peritubular and glomerular capillary, decreased area of glomerular capillary bed and obliteration. Tissue inhibitor of metalloproteinase-1 (TIMP-1) is an endogenous inhibitor of matrix metalloproteinases (MMPs) with multiple functions. TIMP-1 participates in many pathological and physiological progressions through MMP-dependent and/or MMP-independent pathway, including inhibiting angiogenesis. Previously we have found that gene and protein level of TIMP-1 and its enzymetic activity are upregulated with aging, which plays a role in renal aging progression. However, it is still unclear whether overexpression of TIMP-1 contributes to impaired renal angiogenesis with aging. On the other hand, the mechanisms by which effects mediated the functions of TIMP-1 remain to be elucidated. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a gene of tumor suppressor encoding a phosphatase which exhibits activities of lipo-phosphatase and protein phosphatase. Previous studies have shown that angiogenesis increases resulting from mutation or downregulated expression of PTEN gene. With aging, the expression of PTEN is upregulated in human and rat gastric mucosa accompanying decreased mucosa blood flow and tissue hypoxia. In vitro studies have indicated that TIMP-1 can upregulate the protein expression of PTEN through MMP-independent pathway. Based on these facts, we hypothesize that TIMP-1 may affect the impaired renal angiogenesis associated with aging by regulating the expression and activity of PTEN. In this study, we focused on the role of PTEN in renal aging and observed the changes of renal angiogenesis on the kidney with aging in TIMP-1 transgenic mice and investigate the effect of TIMP-1 on both angiogenesis and PTEN expression in human proximal tubular epithelial cells (HKC) and human umbilical vein endothelial cells (ECV304).Methods:Firstly, at the age of 3,12 and 24 months, renal tissues of human TIMP-1 transgenic mice (each group, n=8) and wild type mice (each group, n=8) were harvested. The paraffin sections were stained by PAS. Vascular endothelial growth factor (VEGF) and CD34 were detected by immunohistochemistry. The mRNA and protein expression of TIMP-1, MMP-9, MMP-2, PTEN, VEGF and Flk-1 in renal tissues were examined by RT-PCR and Western Blot, respectively. Secondly, sense or antisense TIMP-1 gene was transfected into HKC and ECV304 cells. In another group, the cells were interfered by MMP-2/MMP-9 InhibitorⅢ(100μM) with similar cellular enzyme activity for sense transfection group. The mRNA and protein expression of TIMP-1, MMP-2, MMP-9, PTEN, VEGF and Flk-1 were examined by RT-PCR and Western Blot, respectively. In each group, the expression of PTEN, VEGF and Flk-1 were also detected by indirect immunofluorescence. The activity of gelatinase in supernatant was measured by gelatin zymography. Thirdly, siRNA expression vector of PTEN was stably transfected into HKC and ECV304 cells. The mRNA and protein expression of TIMP-1, PTEN, VEGF and Flk-1 were examined by RT-PCR and Western Blot, respectively.Finally, we observed the angiogenesis in sense or antisense TIMP-1 transfected and MMP-2/MMP-9 InhibitorⅢ(100μM) treated ECV304 cells by in vitro 3D angiogenesis experiment.Results:With aging, impaired angiogenesis was found in renal tissues of human TIMP-1 transgenic mice and wild type mice, showing reduced number of glomerular capillary loops, reduction of peritubular capillary densities and increased peritubular capillary rarefaction index. At the age of 24 months, the number of glomerular capillary loops in transgenic mice is less than that in wild type mice (18.57±3.52 vs 25.58±4.22, P<0.05), and the peritubular capillary density of transgenic mice is lower than that of wild type mice (peritubular capillary rarefaction index 41.72±8.276% vs 32.96±5.58%, P<0.05). Compared with wild type mice, the expression of TIMP-1 was upregulated in renal tissues of transgenic mice (P<0.05), the expression of gelatinase (MMP-2, 9) was downregulated (P<0.05), the PTEN expression was upregulated (P<0.05), while the VEGF and Flk-1 expression was downregulated (P<0.05). In vitro studies indicated that compared with non-transfected and empty vector transfected groups, PTEN expression was upregulated in sense TIMP-1 transfected group (P<0.05), and the gelatinase activity was decreased (P<0.05). Meanwhile, the expression of VEGF and Flk-1 were downregulated (P<0.05). The antisense TIMP-1 transgenic group showed reverse results (P<0.05). There is no significant difference in expression of PTEN, VEGF and Flk-1 among the enzyme inhibitor group, non-transfected group and empty vector transfected group. Compared with non-tranfected and control plasmid transfected groups, PTEN expression was downregulated in PTEN siRNA expression vector transfected group (P<0.05), and the expression of VEGF and Flk-1 was upregulated (P<0.05). In vitro 3D angiogenesis experiments indicate that the total tubular length is the longest in antisense TIMP-1 transfected group (4.29±0.35mm), the second longest in non-transfected group (2.99±0.33mm) and empty vector transfected group (3.03±0.18mm), the third in enzyme inhibitor group (1.71±0.22mm), and the shortest in sense TIMP-1 transfected group (0.95±0.09mm). The difference among these groups is significant (P<0.05).Conclusions:In renal aging progression, high expression of TIMP-1 upregulates PTEN expression through MMP-independent pathway, and subsequently downregulates the expression of VEGF and Flk-1, which involved in aging-associated impaired renal angiogenesis Our study provides a theoretic base for futher revealing the role of TIMP-1 in renal aging progression.