Angptl3 Induces Actin Cytoskeleton Rearrangement In Podocytes And Proteinuria

Investigation of the molecular mechanisms in proteinuria disease states toward the development of therapeutic drug interventions remain an area of active research. Previous studies have shown that glomerular filtration barrier dysfunction is the most critical step in the development of proteinuria. Podocyte injury located in the outer barrier layer, is one of primary factors contributing to barrier dysfunction in proteinuria.Podocytes are highly differentiated pericyte-like cells whose dendrite-like foot processes, closely interdigitate with one other to prevent protein filtration.. The effacement of podocyte foot processes is a common pathological change in the majority of proteinuria diseases. Actin cytoskeletal rearrangement promotes the effacement of podocyte foot processes and increases podocyte motility. Integrins are the key signal factors that mediate cell adhesion and modulate changes in cell motility. It has been shown that both integrinαβ1 and integrinαVβ3 are present within podocytes. Activation of integrin (33 subunit on the surface of podocytes can induce intracellular Rho GTP signaling pathways via Cdc42 and racl resulting in increased podocyte motility and foot process effacement. Integrin-related mechanisms of podocyte injury continue to attract attention in pathological states as the integrinβ3 subunit is a major regulator of podocyte morphology and function.Angiopoietin-like protein 3 (ANGPTL3) belongs to the angiopoietin-like protein family. ANGPTL3 is mainly synthesized by liver cells and is only weakly expressed in the kidney. As a lipid regulating reagent, ANGPTL3 has been extensively studied with respect to lipid metabolism. It was recently determined that only the CLD segment of the ANGPTL3 N terminal can inhibit lipase activities. The C-terminal FHD segment can bind to integrinαVβ3 and plays a role in angiogenesis. However, the potential role of ANGPTL3 and integrinαVβ3 in the occurrence of proteinuria has not been addressed. Recently, we confirmed the expression of ANGPTL3 in human and rat glomeruli and found that its expression in the nephrotic kidney was significantly elevated. We demonstrated that increased ANGPTL3 expression was positively correlated with the severity of proteinuria.In the present study, we examined the mechanism of ANGPTL3 mediated integrinβ3 activation in podocytes in the induction of actin cytoskeletal rearrangement in proteinuria disease states. Our results showed that ANGPTL3 was specifically expressed in glomerular podocytes. We demonstrate that ANGPTL3 can directly bind to the integrinβ3 subunit and regulate adriamycin-induced activation of the integrinβ3 subunit. Compared to the wild-type podocytes, we showed that adriamycin-induced cytoskeletal rearrangement in ANGPTL3 gene knockdown cells was significantly reduced. Furthermore, we demonstrated that ANGPTL3 induced cytoskeletal rearrangement in podocytes via activation of Rho GTP signaling pathways mediated by integrin (33 and downstream signaling via FAK, PI3K and RhoA.1. Podocyte-specific expression of ANGPTL3We examined the expression of ANGPTL3 in the renal tissues of normal human subjects and nephrotic kidney samples of minimal change disease. In human and rat glomeruli, the fluorescence intensity of ANGPTL3 under the normal state was significantly lower than that in dysfunctional glomerular.To further characterize the localization of ANGPTL3 in glomerular cells, colloidal gold labeling ANGPTL3 and immuno-electron microscopy analysis were used. In human and rat glomeruli under normal or nephropathy states, the ANGPTL3 colloidal gold particles were concentrated in podocytes but not in the endothelial cells, mesangial cells and glomerular basement membrane (GBM). The sub-cellular distribution of ANGPTL3 in podocytes of rats with nephropathy ANGPTL3 colloidal gold particles were rarely found in the podocyte cell body and were mainly localized in the podocyte foot processes, consistent with the sub-cellular distribution of integrinβ3 in podocytes reported by Wei et al.We used real time-PCR and ELISA to examine ANGPTL3 synthesis and secretion by podocyte in vitro. There was a weak ANGPTL3 mRNA expression in wild-type cells, but ANGPTL3 mRNA expression was significantly elevated in podocytes treated with ADR or puramycin (PAN), suggesting that pathological factors can stimulate ANGPTL3 gene transcription in podocytes. The ANGPTL3 levels in cell supernatants were detected using ELISA analysis. Detection of ANGPTL3 in cell supernatants confirmed ANGPTL3 status as a secreted protein, consistent with previous studies in liver cells. Secretion of ANGPTL3 by non-treated glomerular podocytes was significantly lower than that observed in the ADR or PAN treated podocytes. Real time-PCR and ELISA were also used to measure ANGPTL3 levels after podocyte ANGPTL3 was knocked down by miRNA and ANGPTL3 cDNA was transient transfected into podocytes.2. ANGPTL3 directly binds to integrinβ3 in podocytesIn glomeruli whose podocytes were positive for synaptopodin expression, we found that integrinβ3 (CD61) was mainly localized podocytes with ADR-treated rat and nephrotic human, consistent with previous reports. Image composition analysis showed that ANGPTL3 and integrinβ3 (CD61) were co-localized in podocytes, suggesting that ANGPTL3 was a potential ligand ofβ3 integrin in podocytes. Co-immunoprecipitation (IP) assay was used to investigate possible binding models of ANGPTL3 with integrinβ3 in podocytes. We found that secreted ANGPTL3 from podocytes could directly bind to integrinβ3, demonstrating that ANGPTL3 was a ligand for integrinβ3 in podocytes.3. ANGPTL3 is one of the key ligands to regulate integrinβ3 expression and activationIn vitro studies we firstly investigated ANGPTL3 role's in integrinβ3. The results showed that integrinβ3 expression was significantly up-regulated in podocytes over-expressing ANGPLT3 compared with control cells (P<0.01). In contrast, in the ANGPTL3 miRNA knockdown cells integrin (33 expression was undetectable. As miRNA gene knockdown technology cannot induce total gene-silencing in all target cells, we separately investigated integrinβ3 expression in ANGPTL3 gene knockdown cells showing detectable gene silencing, and in knockdown cells where ANGPTL3 expression was still detectable. We found that integrinβ3 expression was normal in cells without ANGPTL3 gene silencing, suggesting that ANGPTL3 was one of the key ligands controlling the expression of integrinβ3 in normal podocytes. Quantitative analysis revealed a correlation between ANGPTL3 and integrinβ3 in podocytes. Correlation was significant at 0.01 levels.To further investigate the regulation of podocyte integrinβ3 by ANGPTL3 under pathological states, we treated cells with Adriamycin. Compared with an un-knockdown ANGPTL3 podocyte, ANGPTL3 knockdown cell did not show significant integrinβ3 up-regulation after adriamycin-stimulation, suggesting that ANGPTL3 could also regulate integrinβ3 expression under certain pathological conditions.Increased integrinβ3 activation is an important event mediating podocyte injury whereby AP5 is an indicator of integrinβ3 activation. Therefore, double-labeled immunofluorecence assessed for AP5 in glomerular. We found that in normal rat glomeruli, only weak AP5 fluorescence signals could be detected. In rats with adriamycin-induced nephropathy, the expression of AP5 in glomeruli was significantly up-regulated after 28 days of drug treatment.Moreover, the distribution of ANGPTL3 and AP5 in podocytes was observed under the confocal microscope and the results showed co-localization of ANGPTL3 and AP5. Quantitative analysis of MFI showed that there were extremely low levels of activated AP5 in wild-type podocytes and high expression in cells over-expressing ANGPTL3 under pathological states. Following treatment with Adriamycin for 24 h, cell surface expression of AP5 was detected. Compared with the normal control group, increased AP5 expression levels were only found in wild-type cells but not in ANGPTL3 knockdown cells(P<0.01), suggesting that ANGPTL3 induced integrinβ3 activation via ROS signaling in podocytes.4. ANGPTL3 is important during cytoskeleton rearrangementThe cytoskeletal marker, F-actin, was used to examine the effects of ANGPTL3 mediated integrinβ3 activation on cytoskeletal rearrangement in podocytes. Confocal microscopy showed that ANGPTL3 gene knockdown podocytes did not show a change in F-actin staining pattern compared to wild-type cells. After adriamycin treatment (24 h), wild-type cells showed a significant change in the F-actin staining patterns, In contrast, no change in F-actin staining patterns was observed in ANGPTL3 gene knockdown cells, confirming ANGPTL3 involvement in cytoskeletal rearrangement under pathological conditions. Furthermore, we also examined the expression of a-actinin-4, an important cytoskeletal regulatory protein. Compared with the normal cells, increased a-actinin-4 expression was observed in wild-type podocytes after Adriamycin treatment that was not observed in ANGPTL3 gene knockdown cells, suggesting that ANGPTL3 could regulate cytoskeletal rearrangement via a-actinin-4.Scrape-wound assays are typical method of investigating podocyte motility in vitro. The increasing motility of podocytes in vitro maybe means foot process effacement in vivo. Our results showed that the knockdown of ANGPTL3 did not impair the motility of podocytes in physiology condition. The wound closure and immigrations showed ADR treated podocytes have more active migration than the non-treated cells. The defection of ANGPTL3 expression inhibited the increasing of podocyte motility in ADR induced pathological condition.5. ANGPTL3 primarily activates integrinβ3 signaling pathway and results in cytoskeletal rearrangements of podocytesActivation of cell surface integrinβ3 was blocked in podocytes using the CD61 monoclonal antibody. Podocytes over-expressing ANGPTL3 showed significant cytoskeletal rearrangement. Inhibition of integrinβ3 by CD61 monoclonal antibody resulted in no detectable cytoskeletal rearrangement. These results further confirmed that ANGPTL3 mediated cytoskeletal rearrangement in podocytes occurs via the integrinβ3 signaling pathway.Compared with wild-type podocytes, FAK and RhoA phosphorylation levels were significantly increased in ANGPTL3 over-expressing podocytes. Since PI3K is a major downstream substrate of FAK, a PI3K inhibitor (LY294002), was used to treat podocytes over-expressing ANGPTL3. Compared with the untreated cells, podocytes treated with LY294002 showed significantly decreased RhoA phosphorylation. These results suggest that ANGPTL3 mediated activation of integrinβ3 can regulate cytoskeletal rearrangement via downstream induction of FAK, PI3K and RhoA signaling pathways.This is the first study to demonstrate that ANGPTL3 is specifically expressed in renal podocytes has autocrine mediated signaling effects in podocytes. Specifically, ANGPTL3 can bind to integrinαVβ3 on podocyte surface and activate downstream FAK, PI3K, RhoA, triggering actin cytoskeletal rearrangement to promote foot process effacement, and induced proteinuria in vivo. Our findings highlight the role of angiopoietin-like family proteins in kidney disease.In conclusion, this study demonstrates that ANGPTL3 synthesized by podocytes induced autocrine activation of the integrinβ3 signaling pathway. These findings enhance knowledge regarding the mechanisms regulating podocyte injury and glomerular filtration barrier dysfunction, and suggest a novel molecular mechanism in the induction of proteinuria.