pernatants of PECs treated with normal glucose, high glucose or mannitol for 24 or 48 hrs. MMP-9 protein and activity in culture supernatants of AVE8062A pubmed ID:http://www.ncbi.nlm.nih.gov/pubmed/19770275 PECs treated with recombinant TGF-1 for 24 or 48 hrs. doi:10.1371/journal.pone.0123276.g009 very different glomerular diseases that eventually lead to glomerular scarring. However, the exact mechanisms underlying PEC migration and podocyte dysregulation have not been fully understood. It is well known that MMP-9 plays an important role in the invasion of cancer cells including renal carcinoma. Therefore, an identification of increased MMP-9 in activated PECs may provide an explanation of enhanced PEC proliferation and migration in diabetic kidney disease. Activation of PECs in association with de novo expression of CD44 has been shown in three distinct models of FSGS. Interestingly, docking of MMP-9 at the cell surface by CD44 was reported to promote MMP-9-mediated cell migration. Based on 16 / 20 Glomerular MMP-9 in Diabetic Nephropathy these findings, we further speculate that MMP-9 may promote activated parietal cell migration onto the glomerular tuft via its interaction with CD44. In the present study, we also found that the appearance of MMP-9 positive PECs on the glomerular tuft was frequently associated with the loss of podocyte marker proteins in affected glomeruli in advanced stages of diabetic nephropathy. A decrease in podocyte markers could be due to podocyte dedifferentiation or detachment from the glomerular basement membrane. Increased urinary excretion of podocyte marker proteins in the diabetic rats highlights that detachment of podocytes from the GBM rather than dedifferentiation may contribute to the podocyte loss. This PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19769788 is also supported by the previous finding that podocyte detachment was increased in correlation with GBM thickness, albuminuria and fractional mesangial area in patients with type 2 diabetes. The associated loss of podocytes/glomerular filter integrity in the damaged glomeruli may reflect proteolytic degradation of the GBM by locally increased gelatinases produced by invaded PECs in our diabetic animal model. Therefore, it is likely that podocytes can be lost secondarily because of the invasion of PECs, which express and secret high levels of MMP-9 alone or in combination with other proteases in advanced diabetic kidney disease. So, next question is what stimulates MMP-9 expression and secretion by activated PECs in disease condition. Increased urinary albumin excretion is a hallmark of glomerular disease including diabetic nephropathy. Albumin has been shown to upregulate MMP-9 expression and secretion in cultured podocytes. Therefore, we further hypothesized that PECs are sensitive to an elevation of albumin in the Bowman’s space and can produce MMP-9 upon activation. To test this hypothesis, we determined the effect of albumin overload on MMP-9 expression and secretion by primary cultured rat glomerular PECs expressing high-level of claudin-1 protein. As expected, albumin stimulated MMP-9 production as evidenced by an elevation of MMP-9 protein and activity in culture supernatants of primary glomerular PECs. In contrast, albumin had no effect on MMP-2 expression. Our mechanistic 
studies further demonstrate the involvement of p44/42 MAPK activation in albumin-mediated MMP-9 induction. The phosphorylation of ERK1/2 but not p38 MAPK was significantly increased when the primary PECs were incubated with albumin. Moreover, albumin-induced enzymatic activity and s