Basement membranes play important roles in the development and function of the kidney. Major components of basement membranes are laminins, cruciform heterotrimers of related alpha, beta, and gamma chains. Once thought to be comprised of only 3 chains (alpha1, beta1, gamma1), the laminin family has now grown to at least 10 members that can assemble to produce at least 10 heterotrimers. In previous studies we showed that during nephron development, the major laminin beta chain of the glomerular basement membrane switches from beta1 to beta2, as does the collagen IV alpha chain composition, from alpha1/2 to alpha3/4/5. In mutant mice lacking laminin beta2 or collagen alpha3(IV), the respective """"""""early"""""""" isoforms were retained, and the mice exhibited nephrotic syndrome and nephritis, respectively. These results suggest that the individual laminin beta and collagen IV alpha chains have distinct roles. Studies in vivo and in vitro suggest that the laminin alpha chains are also essential, but progress in elucidating their roles has been hampered by uncertainty as to which alpha chains are present in developing and adult kidney. We recently isolated the mouse ortholog of the human laminin alpha4 chain and cloned the novel alpha5 chain, and showed that both are expressed in a defined sequence, along with alpha1 but not alpha2 or alpha3, in developing kidney. In order to elucidate the role of laminins alpha4 and alpha5, as well as other laminins, in the kidney, the following aims will he pursued: I) Document the distribution of the laminin alpha1-alpha5 chains in developing and adult murine kidney, using RNase protection, in situ hybridization, and immunohistochemistry. 2) Assess the functions of the laminin alpha5 chain in vivo and in vitro, using gene targeting in mice and antibody inhibition in metanephric organ culture. 3) Assess the functions of the laminin alpha4 chain in vivo and in vitro, using similar methods. 4) Determine whether the levels or distribution of laminin chains are perturbed in mutant or diseased kidneys, using existing mutant mice, experimentally induced renal disease in rats, and renal biopsy material from human patients exhibiting signs of a kidney disease or disorder. Taken together, these studies will lead to a better understanding of how the deposition of specific basement membrane components is related to proper kidney development and function, and, in addition, may shed some light on whether aberrant localization of basement membrane components is a recurring feature in diverse human kidney diseases.
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