DETECTION OF MULTIPLE VASCULAR ENDOTHELIAL GROWTH-FACTOR SPLICE ISOFORMS IN SINGLE GLOMERULAR PODOCYTES

Glomerular podocytes are major determinants of filtration permselectiv ity in the glomerulus. Although the molecular mechanisms determining t he characteristics of the glomerular filtration unit are incompletely understood, vascular endothelial growth factor (VEGF) has been implica ted. To analyze this process in situ, we established a method that all ows exploration of in vivo mRNA expression of podocytes using single-c ell reverse transcriptase-polymerase chain reaction (RT-PCR). Microdis sected mouse glomeruli were held in a patch-clamp apparatus, and singl e podocytes were harvested by aspiration. After lysis, the cells were reverse transcribed, and PCR was performed (45 cycles). The podocyte n ature of the material was confirmed by detection of podocyte-specific mRNA (glomerular epithelial protein 1 and Wilms' tumor protein 1). Usi ng specific oligonucleotide primers, VEGF was detected in mRNA obtaine d from renal cortex, single microdissected glomeruli, cultured murine podocytes, and single podocytes in situ. All cells examined expressed three VEGF isoforms (121, 165, and 189). These differ in their capacit y for binding to extracellular matrix and could have different potenci es regulating glomerular endothelial permeability. Our approach should allow a semiquantitative, isoform-specific evaluation of VEGF mRNA ex pression in podocytes during nephrogenesis and in glomerular disease.