DEPARTMENT OF MEDICAL BIOCHEMISTRY AND BIOPHYSICS Karolinska Institutet, Stockholm, Sweden CHARACTERIZATION OF NOVEL PODOCYTE PROTEINS IN THE GLOMERULAR FILTRATION BARRIER

The kidney is considered the most important organ of excretion eliminating the body from metabolic waste products. The nephron is the functional unit of the kidney, composed of a cluster of capillaries named glomerulus, and a tubular system. The blood is filtered in the glomerular capillaries across the glomerular filtration barrier. The barrier is an intricate biological structure composed of the inner capillary endothelial cells, the glomerular basement membrane, and the podocytes. The podocyte is phenotypically and functionally a sophisticated cell governed by a highly organized cytoskeleton. Podocytes play a critical role in the initiation and progression of proteinuric kidney diseases. The reorganization of podocyte cytoskeleton causing foot process effacement is a key factor in the development of proteinuria. Better understanding of the molecular composition and the biological interactions in these cells is a necessity for future specific targeted therapies. This study is based on a previous genome-wide approach aimed to characterize the transcriptome of the renal glomerulus through large-scale sequencing and microarray profiling. In reference to other well characterized podocyte-specific proteins, the rationale was to identify and characterize novel podocyte-specific proteins that show a similar restricted expression profile outside the glomerulus. In the papers, several such proteins are described. In paper I, sult1b1 was localized exclusively in the podocyte Golgi apparatus and ankrd25 was localized to podocyte foot processes as shown by RT-PCR, Western blotting and immunofluorescence stainings. In Paper II, pdlim2 was localized to podocyte foot processes as shown by RT-PCR, Western blotting, immunofluorescence and immunoelectron microscopy. In cultured podocytes, pdlim2 seemed to regulate actin dynamics. In addition, using Coimmunoprecipitation experiments, pdlim2 was shown to interact with α-actinin-4 and amotl1. In semi-quantitative immunoelectron microscopy, there was a reduced expression of pdlim2 in podocytes of patients with certain proteinuric renal diseases. In Paper III: hip1, nfasc and olfml2a were localized exclusively in podocyte major processes as shown with RT-PR, Western blotting, immunofluorescence stainings and immunoelectron microscopy. During glomerulogenesis, the proteins were colocalized with a major processes marker vimentin as shown by immunofluorescence double stainings. The expression of hip1, olfml2a and pdlim2 was observed in glomerular crescents indicating the presence of podocytes in these lesions. In PaperIV, gprc5b was expressed exclusively by podocytes as shown with RTPCR, Western blotting, immunofluorescence and immunoelectron microscopy. Gprc5b knockdown in zebrafish exhibited classical features associated with loss of glomerular function such as pericardial edema and dilated Bowman’s capsule. Thus, gprc5b is essential for an intact glomerular filtration barrier. LIST OF PUBLICATIONS