Basement membranes are specialized extracellular matrices which play a critical role in differentiation, tissue morphogenesis, and the restoration of normal tissue architecture following injury. In the kidney, both acquired and hereditary disorders lead to pathological accumulations of basement membrane, a process which is widely though to be irreversible and inexorable, leading to permanent organ dysfunction or failure. Despite its apparent importance, little is known about the regulation of basement membrane metabolism. An understanding of the molecular events regulating the synthesis and degradation of basement membrane components, could lead to specific therapeutic strategies designed to restore or minimize the loss of renal function. Collagen IV, the major structural component of basement membranes, can be assembled from at least 5 genetically distinct polypeptide chains, designated alpha1-5(IV). The alpha1(IV) and alpha2(IV) chains are the most abundant and are thought present in all basement membranes. Studies of collagen IV gene transcription in cultured cells have identified two opposing mechanisms of transcriptional control. Transcript initiation is regulated by enhancer elements within the structural genes. Once initiated, transcript elongation can be halted within the gene, a process termed transcriptional attenuation. The balance of these two opposing mechanisms may regulate the expression of these collage IV genes during normal renal development and in pathological conditions. In particular, mice develop an autosomal recessive form of polycystic kidney disease (cpk/cpk) resembling infantile polycystic kidney disease in man. Affected animals demonstrate sustained expression of collagen IV genes while unaffected littermates have a 10-fold fall in alpha1 and alpha2(IV) mRNA. This abnormality is likely due to defect in collagen IV gene transcription initiation and/or attenuation. In order to test this hypothesis, the following studies are proposed: 1) Collagen IV transcript initiation and attenuation will be assessed by nuclear run-on transcription during normal renal development and compared with cpk/cpk mice and unaffected littermates; 2) The function of positive and negative cis-acting sequences will be determined by transfection of chimeric gene constructs in cells derived from normal and cpk/cpk kidneys; 3) Nuclear protein which specifically bind to positive and/or negative cis-acting elements will be identified in crude extracts of renal cell nuclei and their binding site characterized; Unique DNA-binding proteins will be further characterized by cDNA cloning from an expression library using the binding site nucleotide sequences; and 4) The temporal and spatial distribution of the DNA-binding proteins and their mRNA will be studies during renal development in cpk/cpk mice and normal controls by immunohistochemistry, and filter and in situ hybridization.
