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- Sistani, Laleh
(författare)
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Characterization of novel podocyte proteins in the glomerular filtration barrier
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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 Co- immunoprecipitation 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 RT- PCR, 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.
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| 2. |
- Sistani, Laleh, et al.
(författare)
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Pdlim2 is a novel actin-regulating protein of podocyte foot processes
- 2011
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Ingår i: Kidney International. - : Elsevier BV. - 0085-2538 .- 1523-1755. ; 80:10, s. 1045-1054
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Tidskriftsartikel (refereegranskat)abstract
- 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, alpha-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.
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| 3. |
- Xu, Xiangjun, et al.
(författare)
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Expression of novel podocyte-associated proteins sult1b1 and ankrd25.
- 2011
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Ingår i: Nephron. Experimental nephrology. - : S. Karger AG. - 1660-2129. ; 117:2, s. e39-46
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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