Overexpression of MMP-7 Increases Collagen 1A2 in the Aging Kidney.

The percentage of the U.S. population over 65 is rapidly increasing, as is the incidence of chronic kidney disease (CKD). The kidney is susceptible to age-dependent alterations in structure, specifically tubulointerstitial fibrosis, that lead to CKD. Matrix metalloproteinases (MMPs) were initially characterized as extracellular matrix (ECM) proteinases; however it is clear that their biological role is much larger. We have observed increased gene expression of several MMPs in the aging kidney, including MMP-7. MMP-7 overexpression was observed starting at 16 months, and over a 500 fold up-regulation in 2 year-old animals. Overexpression of MMP-7 is not observed in age-matched, calorically restricted controls that do not develop fibrosis and renal dysfunction, suggesting a role in the pathogenesis. In order to delineate the contributions of MMP-7 to renal dysfunction, we overexpressed MMP-7 in NRK-52E cells. High-throughput sequencing of the cells revealed that two collagen genes, Col1a2 and Col3a1, were elevated in the MMP-7 overexpressing cells. These two collagen genes were also elevated in aging rat kidneys and temporally correlated with increased MMP-7 expression. Addition of exogenous MMP-7, or conditioned media from MMP-7 overexpressing cells also increased Col1A2 expression. Inhibition of PKA, src, and MAPK signaling at p38 and ERK was able to attenuate the MMP-7 up-regulation of Col1a2. Consistent with this finding, increased phosphorylation of PKA, src and ERK was seen in MMP-7 overexpressing cells and upon exogenous MMP-7 treatment of NRK-52E cells. These data suggest a novel mechanism by which MMP-7 contributes to the development of fibrosis leading to CKD.

Promoter methylation is associated with the age-dependent loss of N-cadherin in the rat kidney.

The cadherins are cell adhesion molecules required for cellular homeostasis, and N-cadherin is the predominant cadherin expressed in proximal tubular epithelial cells in humans and rats. Our laboratory previously reported an age-dependent decrease in renal N-cadherin expression; the levels of N-cadherin mRNA and protein expression decreased in parallel, implicating a transcriptional mechanism in the age-dependent loss of expression (19). In this study, we examined the hypothesis that promoter hypermethylation underlies the loss of N-cadherin expression in aging rat kidney. We cloned the 5' flanking region of the rat N-cadherin gene and observed basic promoter activity in a 3,992-bp region localized immediately upstream of the ATG start site. Nucleotide analysis revealed 87% identity with the human N-cadherin minimal promoter region. Consistent with a role for regulation by DNA methylation, we found that a dense CpG island, which spans 1,104 bp (-1,158 to -55), flanks the rat N-cadherin gene; a similar CpG profile was found in the human N-cadherin 5' flanking region. Methylation-specific PCR analysis demonstrated that the promoter region of N-cadherin is heavily methylated in aged, but not young, rat kidney. Interestingly, the promoter is not methylated in age-matched, calorically restricted animals. In contrast, the promoter region is not methylated in either young or aged rat liver; this corresponds to the finding that aging is not associated with decreased N-cadherin expression in the liver. In addition, N-cadherin expression is markedly induced in NRK-52E cells treated with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine, further suggesting that methylation at CpG in the promoter region may underlie the age-dependent decrease in renal N-cadherin expression.

Ischemia-induced cleavage of cadherins in NRK cells is not sufficient for beta-catenin transcriptional activity.

Although ischemia is associated with disruption of cadherin-mediated adhesion in renal cell lines, the impact of decreased cadherin function on the transcriptional activity of beta-catenin remains poorly defined. In these studies, we used a simulated ischemia model in normal rat kidney (NRK) cells to disrupt cadherin function. Cell viability; cadherin/catenin expression, function, and localization; and beta-catenin-mediated transcriptional activity were assessed during ischemia/reperfusion. Following 6 hr of ischemia, a decrease in the expression of E- and N-cadherin was seen that correlated with altered cell morphology indicative of decreased intercellular adhesion. While ischemia was associated with activation of glycogen synthase kinase 3 beta (GSK-3beta), this did not correlate with increased phosphorylation of beta-catenin as assessed by Western blots using phosphoryl-specific antibodies. beta-Catenin was not localized to the nucleus by immunofluorescence in ischemic NRK cells, but rather a strong perinuclear signal was seen in reperfused cells. This was consistent with the finding that neither ischemia nor reperfusion activated the transcriptional activity of beta-catenin as assessed by the TCF-optimal promoter (TOPFlash) construct. However, NRK cells possess a competent Wnt pathway, as challenge with lithium chloride elicited a ten-fold increase in luciferase activity. These results suggest that ischemia-induced disruption of cadherin/catenin complexes is not sufficient to stimulate beta-catenin transcriptional activity in NRK cells.

Immunohistochemical localization of cadherin and catenin adhesion molecules in the murine growth plate.

Mouse tibial growth plates were examined for the presence of adhesion molecules using immunohistochemistry and RT-PCR. All of the components of the classical cadherin/catenin complex (cadherin, alpha-, beta-, and gamma-catenin), as well as a heavy presence of p120, were identified in the murine growth plate. All of the major cadherins (1-5, 11, 13, and 15) were, for the first time, identified and localized in the murine growth plate. We have demonstrated that most of the cadherins and catenins reside in the zone of hypertrophy. Only alpha-catenin and E-, P-, R-, and VE-cadherin were found in all regions of the growth plate. The results for T-cadherin were inconclusive.