Isolation of Glomerular Podocytes by Cationic Colloidal Silica-coated Ferromagnetic Nanoparticles

Abstract

Background: Podocyte homeostasis plays a crucial role for the maintenance of physiological glomerular function and podocyte injury is regarded as a major determinant of development and progression of renal disease. Objective: Investigation of podocytes requires appropriate methods for their isolation. Previously reported methods use podocyte specific antibodies or transgenic mice with podocyte specific expression of fluorescent markers for isolation of podocytes by magnetic or fluorescence activated cell sorting. Method: Here, a novel, antibody-free method for isolation of podocyte protein and RNA from mouse glomeruli is described. Preparations of isolated glomeruli were added to a suspension of cationic silica-coated colloidal ferromagnetic nanoparticles. The nanoparticles bound to the negatively charged cell surfaces of podocytes residing on the outer surface of the isolated glomeruli. After enzymatic and mechanical dissociation of glomerular cells, nanoparticle-coated podocytes were isolated in a magnetic field. The method was tested in adult wild-type mice without renal lesions and in mice of two nephropathy models (Growth hormone (GH)-transgenic mice and transgenic mice expressing a dominant negative receptor for the glucose dependent insulinotropic polypeptide, GIPRdn) displaying albuminuria, glomerular hypertrophy and evidence for a reduced negative cell surface charge of podocytes. Results: The isolated cells displayed typical morphological and ultrastructural properties of podocytes. On average, 182,000 ± 37,000 cells were counted in the podocyte isolates harvested from ~10,000-12,000 glomeruli per mouse. On the average, the purity of podocyte isolates of these mice accounted for ~63 ± 18 % and the podocyte isolates displayed high mRNA and protein expression abundances of podocyte markers (nephrin and WT1), whereas the expression of endothelial (Cd31) and mesangial markers (Serpinb7) was significantly decreased in podocyte isolates, as compared to samples of isolated glomeruli. The numbers of cells isolated from GH- transgenic and GIPRdn-transgenic mice were not markedly different from that of wild-type mice. Conclusion: The described method represents an alternative for podocyte isolation, particularly in experiments where podocyte specific antibodies or transgenic animals with podocyte specific expression of fluorescent markers are not applicable.