Matrix metalloproteinases (MMP) are a multigenic family of more than 20 proteolytic enzymes involved in the degradation of extracellular matrix (ECM) components. Most MMPs are secreted as soluble enzymes into the extracellular milieu;however some are membrane-bound and called the membrane-targeted metalloproteinases-MMPs. Despite in vitro evidence that soluble MMPs play an important role in renal development, no significant renal phenotypes have been described in mice lacking this class of MMPs. In contrast, we have found that mice null for MT1-MMP have dysplastic dysgenic kidneys due to decreased cleavage of ECM components, particularly laminin 5 (Ln-5). More recently we observed that MT4-MMP null mice have hypoplastic dysgenic kidneys, due to an undetermined mechanism. Based on these data we hypothesize that MT-MMPs, namely MT1- and MT4-MMPs play a critical role in normal renal development. The role of these enzymes in normal renal development will be determined in the following 3 Aims. 1) We will determine whether Ln-5 cleavage is required for normal kidney development by a) crossing the Ln-5-null mouse with the MT1-MMP-null mouse and comparing the phenotype of the double null mutants with the single Ln-5-null and MT1-MMP null mice to see if the renal phenotype of the MT1-MMP-null mouse can be rescued;b) isolating whole embryonic kidneys and collecting duct cells from the Ln- 5, MT1-MMP and Ln-5/MT1-MMP null mice and determine their ability to undergo branching movphogenesis. In the tubulogenesis experiments the gels will be reconstituted with different cleavage products of Ln-5 to determine which specific domains of Ln-5 are critical for branching morphogenesis. 2) We will determine how lack of MT4- MMP results in hypoplastic and dysgenic kidneys by a) analyzing embryonic and adult kidneys from wild type and MT4-MMP-null mice and b) isolating collecting duct cells from wild type and MT4-MMP-null mice to determine the effects of lack of this enzyme on ECM-dependent cellular functions such as migration, adhesion and tubulogeneisis.
In Aim 3 we will determine the relevant ECM substrates for MT1- and MT4-MMP by determining a) in vitro the cleavage products of purified renal basement membrane components by purified MT1- and MT4-MMPs alone or in combination with soluble MMPs and b) in vivo whether there are differences in the composition of the renal basement membranes of the MT 1- and MT4-MMP null mice compared to their wild type counterparts.
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