ABSTRACT
BACKGROUND/AIMS: Proteinuria is associated with cardiovascular and chronic kidney disease. Microparticles (MPs) are bioactive vesicles shed from activated cells and also linked to cardiovascular disease. MP-like structures have been identified in the glomerular basement membrane, urinary space and between the glomerular basement membrane and the podocyte. We hypothesised that circulating MPs may provide a link between vascular injury and kidney diseases by inducing podocyte phenotypic alterations, thus propagating glomerular dysfunction and proteinuria. METHODS: Human umbilical vein endothelial cells and U937 monocytes were stimulated with TNF-α to produce MPs. These MPs were confirmed by electron microscopy, and added to differentiated podocyte monolayers to determine effects on podocyte albumin endocytosis and the production of soluble mediators. RESULTS: Monocyte and endothelial MPs upregulated podocyte production of pro-inflammatory mediators monocyte chemoattractant protein-1 (p < 0.001) and interleukin-6 (p < 0.001). Only monocyte MPs upregulated podocyte secretion of VEGF (p < 0.001), known to regulate glomerular permeability. Endothelial MPs decreased podocyte albumin endocytosis by 13% compared to control cells (p < 0.01). CONCLUSION: MPs alter endocytic functions of podocytes and induce secretion of pro-inflammatory cytokines, potentially leading to glomerular inflammation in vivo and the development of proteinuria. This study identifies a potential pathophysiological role for circulating MPs in the kidney through effects on the podocyte.
Subject(s)
Cell-Derived Microparticles/pathology , Cell-Derived Microparticles/physiology , Podocytes/pathology , Podocytes/physiology , Albumins/metabolism , Angiopoietin-2/metabolism , Cells, Cultured , Chemokine CCL2/metabolism , Endothelial Cells/pathology , Endothelial Cells/physiology , Glomerulonephritis/etiology , Glomerulonephritis/pathology , Glomerulonephritis/physiopathology , Human Umbilical Vein Endothelial Cells , Humans , Inflammation/etiology , Inflammation/pathology , Inflammation/physiopathology , Inflammation Mediators/metabolism , Interleukin-6/metabolism , Microscopy, Electron, Transmission , Monocytes/pathology , Monocytes/physiology , Phenotype , Proteinuria/etiology , Proteinuria/pathology , Proteinuria/physiopathology , U937 Cells , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Glomerular podocytes are critical regulators of glomerular permeability via the slit diaphragm and may play a role in cleaning the glomerular filter. Whether podocytes are able to endocytose proteins is uncertain. We studied protein endocytosis in conditionally immortalized mouse and human podocytes using FITC-albumin by direct quantitative assay and by fluorescence microscopy and electron microscopy in mouse podocytes. Furthermore, in vivo uptake was studied in human, rat, and mouse podocytes. Both mouse and human podocytes displayed specific one-site binding for FITC-albumin with K(d) of 0.91 or 0.44 mg/ml and B(max) of 3.15 or 0.81 microg/mg cell protein, respectively. In addition, they showed avid endocytosis of FITC-albumin with K(m) of 9.48 or 4.5 mg/ml and V(max) of 474.3 or 97.4 microg.mg cell protein(-1).h(-1), respectively. Immunoglobulin and transferrin were inefficient competitors of this process, indicating some specificity for albumin. Accumulation of endocytosed albumin could be demonstrated in intracellular vesicles by fluorescence confocal microscopy and electron microscopy. Endocytosis was sensitive to pretreatment with simvastatin. In vivo accumulation of albumin was found in all three species but was most pronounced in the rat. We conclude that podocytes are able to endocytose protein in a statin-sensitive manner. This function is likely to be highly significant in health and disease. In addition, protein endocytosis by podocytes may represent a useful, measurable phenotypic characteristic against which potentially injurious or beneficial interventions can be assessed.
