The broad long-term objective of this proposal is to study the genetics of a mouse model for polycystic kidney disease to better understand renal cystogenesis.
The specific aims for this proposal are to isolate and characterize the mouse jcpk/bpk gene and its human homologue. When inherited in an autosomal recessive manner, the mouse jcpk gene causes a severe form of early onset polycystic kidney disease (PKD) accompanied by a number of extrarenal effects. The jcpk model is unique from other mouse PKD models and has a phenotype with characteristics resembling both the dominant and recessive forms of PKD in humans. Recent studies have indicated that jcpk and another mouse PKD-causing mutation, bpk, are allelic, despite the different kidney disease phenotypes produced by each. Additionally, jcpk/bpk is a candidate for a locus that modifies the expression of a separate and distinct PKD caused by the jck gene. It is believed that modifying loci play an important role in human PKD based on highly variable intra familial clinical presentation and course of disease. In this grant application, we propose to identify both the mouse jcpk/bpk gene and its human homologue using molecular genetic techniques.
The specific aims of this proposal are 1) to physically map the jcpk gene, 2) to isolate and characterize the jcpk/bpk gene and, 3) to isolate the human homologue of the mouse jcpk/bpk gene. GRANT=R01DK54741 DESCRIPTION (Adapted from Investigator's Abstract): Cytosolic phospholipase A2 (cPLA2) has been implicated in cell injury, however, the mechanisms by which cPLA2 acts are unknown. cPLA2 localizes to an intranuclear site in response to serum deprivation resulting in cell death. Two novel nuclear cPLA2-interacting proteins have been isolated. These cPLA2-interactors are homologous to yeast silencing genes, SIR2 and SAS2/SAS3. One of these interacting proteins, PLIP, (for Phospholipase A2-interacting protein), has an amino acid sequence containing a recently described MYST domain. The MYST domain is made up of an atypical C2HC zinc finger and a signature acetyltransferase sequence. The presence of this domain and homologies of PLIP to yeast drosophila and mammalian proteins suggest a potential role in transcriptional regulation for PLIP and thus cPLA2. The expression of PLIP in renal mesangial cells markedly enhances cell injury due to both serum deprivation and TNFalpha. It is likely that an intranuclear complex containing cPLA2 and the two interactor proteins mediates the mesangial cell response to TNFalpha and to serum deprivation.
The first aim of this proposal is to examine mechanisms by which PLIP modulates cPLA2 activity and contributes to mesangial cell susceptibility to injury. The second specific aim is to characterize the second clone 46, to identify its role in cPLA2 regulation, and to determine its role, if any, in cell susceptibility to injury. To achieve these aims, basic techniques in molecular biology and cellular and lipid biochemistry will be used, specifically including site directed mutagenesis and an adenoviral expression system. Transfected constructs encoding a peroxosomal proliferator activated receptor (PPAR) and containing a reporter gene driven by a PPAR response element will be used for a functional readout of intranuclear arachidonic acid release. The completion of this proposal will greatly enhance our understanding of the role and regulation of cPLA2 in cell injury.
