Neuronal proteins are novel components of podocyte major processes and their expression in glomerular crescents supports their role in crescent formation.

The podocyte has a central role in the glomerular filtration barrier typified by a sophisticated morphology of highly organized primary (major) and secondary (foot) processes. The molecular makeup of foot processes is well characterized, but that of major processes is poorly known. Previously, we profiled the glomerular transcriptome through large-scale sequencing and microarray profiling. Unexpectedly, the survey found expression of three neuronal proteins (Huntingtin interacting protein 1 (Hip1), neurofascin (Nfasc), and olfactomedin-like 2a (Olfml2a)), all enriched in the glomerulus. These proteins were expressed exclusively by podocytes, wherein they localized to major processes as verified by RT-PCR, western blotting, immunofluorescence, and immunoelectron microscopy. During podocyte development, these proteins colocalized with vimentin, confirming their association with major processes. Using immunohistochemistry, we found coexpression of Hip1 and Olfml2a along with the recognized podocyte markers synaptopodin and Pdlim2 in glomerular crescents of human kidneys, indicating the presence of podocytes in these lesions. Thus, three neuronal proteins are highly expressed in podocyte major process. Using these new markers we found that podocytes contribute to the formation of glomerular crescents.

Pdlim2 is a novel actin-regulating protein of podocyte foot processes.

The slit diaphragm and the apical and basal membrane domains of podocytes are connected to each other by an actin-based cytoskeleton critical to the maintenance of the glomerular filtration barrier. In an effort to discover novel regulatory proteins of the podocyte foot process, we identified and characterized pdlim2, a member of the actin-associated LIM protein subfamily of cytosolic proteins typified by an N-terminal PDZ domain and a C-terminal LIM domain. In the kidney, the pdlim2 protein is highly specific for the glomerulus and podocyte foot processes as shown by RT-PCR, western blotting, immunofluorescence, and immunoelectron microscopy. In cultured podocytes, pdlim2 was associated with stress fibers and cortical actin. Pdlim2 seems to regulate actin dynamics in podocytes since stress fibers were stabilized in its presence. Mechanistically, pdlim2 interacts with two actin-associated podocyte proteins, α-actinin-4 and angiomotin-like-1, as shown by immunoprecipitation and yeast two-hybrid analyses. By semi-quantitative immunoelectron microscopy, there was a reduced expression of pdlim2 in podocytes of patients with minimal change nephrotic syndrome and membranous nephropathy, whereas its expression was unchanged in patients with focal segmental glomerulosclerosis. Hence, pdlim2 is a novel actin-regulating protein of podocyte foot processes that may have a role in the pathogenesis of glomerular diseases.

Expression of novel podocyte-associated proteins sult1b1 and ankrd25.

BACKGROUND/AIMS: Podocytes have a unique function in the renal ultrafiltration that is achieved by expressing proteins that are highly specific to podocytes. In this study, we identified two novel podocyte-associated proteins. METHODS: The expression of sult1b1 and ankrd25 in mouse tissues was studied by RT-PCR. The protein expression was studied by generating polyclonal antibodies that were used in Western blotting and immunohistochemistry. RESULTS: By RT-PCR we detected sult1b1 expression only in glomerular, liver and brain tissues. By immunohistochemistry, sult1b1 was detected in the kidney exclusively in the Golgi apparatus of the podocyte. No expression outside the glomerulus was observed in the kidney. The ankrd25 transcript was detected in most mouse tissues analyzed by RT-PCR. In the kidney, however, immunohistochemistry showed that this protein was expressed only by podocyte, mesangial, and smooth muscle cells. In podocytes, ankrd25 was localized to foot processes. CONCLUSIONS: Identification of these two novel glomerulus-associated proteins opens up possibilities to investigate their role in the renal filter physiology and diseases. We speculate that sult1b1 may be involved in the sulfonylation of podocyte protein podocalyxin, whereas ankrd25 may contribute to controlling actin dynamics in podocyte foot processes.