Regional podocyte isolation using laser capture microdissection for molecular profiling of glomerular disease
Al-Sowaimel, Lujain Fawzi
MetadataShow full item record
FSGS (focal segmental glomerulosclerosis) is a pathologic pattern that affects the glomerulus of the kidneys and is associated with progression toward end-stage kidney disease. It is characterized by destruction of the glomerular filtration system and replacement by scar tissue, which leads to chronic renal failure. An increase in glomerular capillary pressure may be an important factor in glomerulosclerosis. The podocyte is a major structural component of the filtration barrier. Since podocytes have an important role in maintaining the structural and functional integrity of glomerular filtration, a chronic increase in glomerular filtration pressure could ultimately damage and compromise the podocyte filtration apparatus. The segmental nature of FSGS suggests that podocyte damage may only occur at specific regions in the glomerulus. This poses the question as to whether differences in podocyte function, assessed by gene expression, may be dependent on their location in the glomerulus, and these regional differences might be responsible for FSGS development. Consequently, in order to evaluate this hypothesis there is a need to selectively isolate podocytes from various regions in a glomerulus. Therefore, the objective of this study was to determine the feasibility of selectively isolating podocyte cells from other renal cells using laser capture microdissection (LCM), and analyzing gene expression in these isolated cells using quantitative real-time polymerase chain reaction (qRT-PCR). If feasible, this method could be used to examine isolated cells for unique podocyte gene expression patterns that vary regionally throughout the glomerulus, and identify potential molecular and cellular mechanisms responsible for the initiation and progression of FSGS. Such information could then be used to identify new and pharmacologically accessible molecular targets. The specific goals of this project were to: 1) determine the feasibility of employment of the LCM system in the Boston University Department of Pathology for the capture of isolated mouse podocytes. 2) determine optimal tissue preservation and preparation methods for laser capture and mRNA analysis of isolated podocytes; and 3) determine the minimum number of isolated podocytes required to analyze gene expression using qRT-PCR. The results show that 1) liquid nitrogen was the preferred method of tissue freezing; 2) the use of Histogene stain improved cell identification during laser capture; 3) the LCM instrument parameters required for selective podocyte capture were identified; and 4) RNA quality obtained from the LCM samples was suboptimal. These results indicate that the possibility of using LCM for regional podocyte isolation and gene expression analysis is quite promising, and further optimization of the technique will likely yield an important new method for the study of kidney disease pathogenesis.