Podocyte flattening and disorder of glomerular basement membrane are associated with splitting of dystroglycan-matrix interaction.

The transmembrane component of the dystroglycan complex, a heterodimer of alpha- and beta-dystroglycan, was recently localized at the basal cell membrane domain of podocytes, and it was speculated that it serves as a device of the podocyte for maintaining the complex podocyte foot process architecture, and for regulating the exact position of its ligands, the matrix proteins laminin and agrin, in the glomerular basement membrane (GBM). The redistribution of dystroglycan in two experimental rat models of foot process flattening and proteinuria-i.e., podocyte damage induced by polycationic protamine sulfate perfusion, and reactive oxygen species (ROS)-associated puromycin aminonucleoside nephrosis-was examined. In both experimental diseases, aggregation and reduced density of alpha-dystroglycan by endocytosis by podocytes was observed. In in vitro solid-phase binding assays, protamine and ROS competed with the binding of alpha-dystroglycan with purified laminin and a recombinant C-terminal fragment of agrin that contains the dystroglycan-binding domain. These changes were associated with disorder of the fibrillar components of the lamina rara externa of the GBM, as confirmed quantitatively by fractal analysis. These results indicate that both polycation and ROS induce similar changes in the distribution of podocyte alpha-dystroglycan that involve competitive disruption of alpha-dystroglycan/matrix protein complexes, endocytosis of the liberated receptor by podocytes, and disorganization of the matrix protein arrangement in the lamina rara externa. This links functional damage of the dystroglycan complex with structural changes in the GBM.

Glomerular podocytes possess the synaptic vesicle molecule Rab3A and its specific effector rabphilin-3a.

Several recent studies have focused on similarities between glomerular podocytes and neurons because the two cells share a specialized cytoskeletal organization and several expression-restricted proteins, such as nephrin and synaptopodin. In neurons, the small guanosine triphosphatase Rab3A and its effector rabphilin-3A form a complex required for the correct docking of synaptic vesicles to their target membrane. Because rabphilin-3A binds in neurons to cytoskeletal proteins also important for podocyte homeostasis, and the complex rabphilin-3A-Rab3A has been demonstrated in neurons and neuroendocrine cells, the aim of our work was to investigate their possible expression and regulation in podocytes. Normal kidneys from mouse, rat, and human were studied by immunohistochemistry, Western blotting, and reverse transcriptase-polymerase chain reaction to evaluate the expression of Rab3A and rabphilin-3A. Double-staining immunohistochemistry and immunogold electron microscopy were then used to precisely localize the two proteins at the cellular and subcellular levels. Rab-3A and rabphilin-3A regulations in disease were then analyzed in growth hormone-transgenic mice, a well established model of focal and segmental glomerulosclerosis, and in human biopsies from proteinuric patients. Our results demonstrated that rabphilin-3A and Rab3A are present in normal mouse, rat, and human kidneys, with an exclusively glomerular expression and a comma-like pattern of positivity along the glomerular capillary wall, suggestive for podocyte staining. Co-localization of both molecules with synaptopodin confirmed their presence in podocytes. By immunogold electron microscopy both proteins were found around vesicles contained in podocyte foot processes. Their expression was increased in growth hormone-transgenic mice compared to their wild-type counterpart, and in a subset of biopsies from proteinuric patients. Our data, demonstrating the presence of two synaptic proteins in podocytes, further supports similarities between cytoskeletal and vesicular organization of podocytes and neurons. The altered expression observed in mouse and human proteinuric diseases suggests a possible role for these molecules in glomerulopathies.