Huang et al. BMC Nephrology (2016) 17:66 DOI 10.1186/s12882-016-0287-6 RESEARCH ARTICLE Open Access RhoA deficiency disrupts podocyte cytoskeleton and induces podocyte apoptosis by inhibiting YAP/dendrin signal Zongshun Huang1,2, Li Zhang1, Yuanhan Chen1, Hong Zhang1, Chunping Yu1, Fangjian Zhou3,4, Zhiling Zhang3,4, Lijuan Jiang3,4, Ruizhao Li1, Jianchao Ma1, Zhuo Li1, Yuxiong Lai1, Ting Lin1, Xinchen Zhao1, Qianmei Zhang1, Bin Zhang1, Zhiming Ye1, Shuangxin Liu1, Wenjian Wang1, Xinling Liang1, Ruyi Liao1 and Wei Shi1* Abstract Background: Podocyte apoptosis is a major mechanism that leads to proteinuria in many kidney diseases. However, the concert mechanisms that cause podocyte apoptosis in these kidney diseases are not fully understood. RhoA is one of Rho GTPases that has been well studied and plays a key role in regulating cytoskeletal architecture. Previous study showed that insufficient RhoA could result in rat aortic smooth muscle cell apoptosis. However, whether RhoA is involved in podocyte apoptosis remains unknown. Methods: Culture podocytes were treated with LPS, ADR or siRNA for 48 h before harvest. Subcellular immunoblotting, qRT-PCR, immunofluorescence and flow cytometry were used to exam the expression and function of RhoA or YAP in podocytes. Results: We found that the expression of RhoA and its activity were significantly decreased in LPS or ADR-injured podocytes, accompanying loss of stress fibers and increased cell apoptosis. Knocking down RhoA or its downstream effector mDia expression by siRNA also caused loss of stress fibers and podocyte apoptosis. Moreover, our results further demonstrated that RhoA deficiency could reduce the mRNA and protein expression of YAP, which had been regarded as an anti-apoptosis protein in podocyte. Silenced dendrin expression significantly abolished RhoA, mDia or YAP deficiency-induced podocyte apoptosis. Conclusion: RhoA deficiency could disrupt podocyte cytoskeleton and induce podocyte apoptosis by inhibiting YAP/dendrin signal. RhoA/mDia/YAP/dendrin signal pathway may potentially play an important role in regulating podocyte apoptosis. Maintaining necessary RhoA would be one potent way to prevent proteinuria kidney diseases. Keywords: RhoA, mDia, Stress fibers, YAP, Dendrin, Apoptosis Background proteinuria. Accumulating evidences show that podocyte Podocytes are highly differentiated cells with complex apoptosis is one of the most important mechanisms in actin cytoskeletal architecture that play a key role in the pathogenesis of many kidney diseases, such as maintaining the integrity of glomerular filtration barrier, chronic kidney disease [4, 5], diabetic nephropathy [6– which is crucial for normal glomerular function [1–3]. 8], focal segmental glomerular sclerosis [9, 10], et al. Therefore, loss of podocytes will surely disturb the func- Thus, preventing podocyte apoptosis will be a promising tion of glomerular. Apoptosis is one of the main reasons therapeutic target for treating these kidney diseases. that causes loss of podocyte and sequentially induces However, the concert mechanisms that cause podocyte apoptosis in these kidney diseases are still far from being fully understood. * Correspondence: [email protected] 1Division of Nephrology, Guangdong General Hospital, Guangdong Academy It has been well known that severe disorders of the kid- of Medical Sciences, 106 Zhongshan No. 2 Road, Guangzhou 510080, China ney that present with proteinuria are associated with re- Full list of author information is available at the end of the article markable cytoskeletal injury and foot process effacement © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Huang et al. BMC Nephrology (2016) 17:66 Page 2 of 12 [11]. The function of cytoskeletal architecture is mainly this study is to test whether RhoA deficiency could in- regulated by small GTPases belonging to the Rho GTPase duce podocyte apoptosis and explore its mechanism, family [12, 13]. Ras homolog gene family, member A which may be one common reason that causes protein- (RhoA) is one of Rho GTPases that has been well studied. uria in many kidney diseases. Active RhoA binds to its downstream effector mammalian diaphanous-related formins (mDias) to relieve mDias’ Methods autoinhibition of the DID/DAD interaction and activates Cell culture and treatment it, thereby promotes stress fibers formation [14, 15]. A The conditionally immortalized mouse podocyte cell line baseline activity of RhoA is essential for the normal func- (MPC) was a kind gift from Dr. Jochen Reiser (Rush tion of glomerular podoctyes, podocytes deficiency in University Medical Center, Chicago, IL, USA), and cul- RhoA will lead to loss of stress fibers and proteinuria [16, tured as described previously [38]. Cells were cultured at 17]. Accumulating evidences have showed that RhoA defi- 33 °C in RPMI-1640 medium (Gibco BRL, Gaithersburg, ciency could result in cell apoptosis [11, 18], maybe by de- MD, USA) supplemented with 10 % fetal bovine serum creasing Yes-associated protein (YAP) expression in both (FBS, Gibco BRL, USA) and recombinant IFN-γ (growth A549 and HepG2 cells [19]. permissive conditions; CYT-358, ProSpec, Tany Techno- YAP is a major downstream cascade of the Hippo gene Ltd, Israel). To induce differentiation, podocytes pathway which controls the expression of genes that were reseeded and cultured at 37 °C in 100 cm2 culture promote cell proliferation and inhibit apoptosis [20, 21]. dish coated with 12 mg/ml type-I collagen (BD Bio- Under normal condition, dephosphorylated YAP local- science, Bedford, MA, USA) and in RPMI-1640 medium izes in the nucleus and functions as a transcriptional co- supplemented with 5 % FBS, deprived of IFN-γ(growth activator that mainly through interacting with TEA do- restrictive conditions) for 10 to 13 days. After differenti- main family member (TEAD) family transcriptional fac- ation, podocytes was confirmed by the identification of tors to induce target gene expression [22]. YAP synaptopodin, a podocyte differentiation marker, 106cells phosphorylation promotes its cytoplasmic sequestration were synchronized into quiescence by growing cells in and inactivation [23, 24]. As loss of stress fibers caused serum-free RPMI-1640 medium for 24 h, and then by insufficient RhoA can result in reducing transcrip- treated with LPS (100 μg/ml), ADR (0.125 μg/ml) or tional activity and nuclear localization of YAP [19, 25, small interfering RNA (siRNA, 50nM) for 48 h. Each re- 26], which has been regarded as an anti-apoptosis pro- action was repeated in triplicates. tein in many kinds of cells [20, 27–29], including podo- cyte [30, 31], it is likely that RhoA deficiency may induce Transfection podocyte apoptosis by reducing nuclear YAP. The siRNAs against RhoA, mDia, YAP, dendrin and control Dendrin is a PPXY motif containing dual compart- were designed and synthesized by RIBOBIO CO., LTD. ment protein [32]. It was originally identified in sleep- (Guangzhou, China), and were transfect into podocytes deprived rats [33], and also detected in human kidney using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, podocytes [34]. Ablation of the podocyte death- CA) following the manufacturer’s protocol. The sequences promoting protein dendrin can delay onset and severity of siRNAs used in this study were as follows: RhoA-siRNA of podocytes loss and proteinuria [35]. In normal podo- 5′-GGUAAGACAUGCUUGCUCA dTdT-3′,mDia-siRN cytes, dendrin is a constituent of the glomerular slit dia- A5′-GCAAGUUCUUGUCCUCUUU dTdT-3′,YAP-siRN phragm, where it binds to nephrin and CD2-associated A5′-CGAGAUG AGAGCACAGACA dTdT-3′,Dendrin- protein [32]. In response to glomerular injury, dendrin siRNA 5′-GGCUCCACCUUCUUAUGAA dTdT-3′. relocates to the podocyte nucleus, thereby promoting cell apoptosis [36]. Previous study [30] showed that YAP Immunofluorescence can bind to dendrin in podocyte nuclei, thereby acting Podocytes were planted on cover slides in six-well plates. as an endogenous inhibitor of proapoptotic dendrin sig- After subjected to various treatments, differentiated naling in podocytes. Thus, it is possible that RhoA defi- podocytes were fixed with 4 % paraformaldehyde at ciency can induce podocyte apoptosis by inhibiting YAP/ room temperature for 10 min, and then treated with dendrin signal. 0.1 % Triton X-100 for 10 min, blocked with 1 % bovine In this study, lipopolysaccharide (LPS) or adriamycin serum albumin for 20 min at room temperature, and in- (ADR) was used to elicit podocyte cytoskeleton disrup- cubated with rabbit anti-YAP (Santa Cruz, USA, 1:250) tion and apoptosis, as described previously [9, 37], then overnight at 4 °C. After incubated with the goat anti- we surprisingly found that RhoA expression and its ac- rabbit Alexa Fluor 488 (Cell Signaling Technology, USA, tivity were decreased in LPS or ADR injured podocytes. 1:1000) for 1 h at room temperature, podocytes were Previous studies have showed that insufficient RhoA stained with phalloidin (Cytoskeleton, USA, 1:500) for could