MAGI-2 orchestrates the localization of backbone proteins in the slit diaphragm of podocytes
Authors: Hiroyuki Yamada, Naritoshi Shirata, Shinichi Makino, Takafumi Miyake, Juan Alejandro Oliva Trejo, Kanae Yamamoto-Nonaka, Mitsuhiro Kikyo, Maulana A. Empitu, Ika N. Kadariswantiningsih, Maiko Kimura, Koichiro Ichimura, Hideki Yokoi, Masashi Mukoyama, Akitsu Hotta, Katsuhiko Nishimori, Motoko Yanagita, Katsuhiko Asanuma
Journal: Kidney International (2020)

Podocytes are highly specialized cells within the glomerulus that are essential for ultrafiltration. The slit diaphragm between the foot processes of podocytes functions as a final filtration barrier to prevent serum protein leakage into urine. The slit-diaphragm consists mainly of Nephrin and Neph1, and localization of these backbone proteins is essential to maintaining the integrity of the glomerular filtration barrier. However, the mechanisms that regulate the localization of these backbone proteins have remained elusive. Here, we focused on the role of membrane-associated guanylate kinase inverted 2 (MAGI-2) in order to investigate mechanisms that orchestrate localization of slit-diaphragm backbone proteins. MAGI-2 downregulation coincided with a reduced expression of slit-diaphragm backbone proteins in human kidneys glomerular disease such as focal segmental glomerulosclerosis or IgA nephropathy. Podocyte-specific deficiency of MAGI-2 in mice abrogated localization of Nephrin and Neph1 independently of other scaffold proteins. Although a deficiency of zonula occuldens-1 downregulated the endogenous Neph1 expression, MAGI-2 recovered Neph1 expression at the cellular edge in cultured podocytes. Additionally, overexpression of MAGI-2 preserved Nephrin localization to intercellular junctions. Co-immunoprecipitation and pull-down assays also revealed the importance of the PDZ domains of MAGI-2 for the interaction between MAGI-2 and slit diaphragm backbone proteins in podocytes. Thus, localization and stabilization of Nephrin and Neph1 in intercellular junctions is regulated mainly via the PDZ domains of MAGI-2 together with other slit-diaphragm scaffold proteins. Hence, these findings may elucidate a mechanism by which the backbone proteins are maintained. Podocytes are highly specialized cells within the glomerulus that are essential for ultrafiltration. The slit diaphragm between the foot processes of podocytes functions as a final filtration barrier to prevent serum protein leakage into urine. The slit-diaphragm consists mainly of Nephrin and Neph1, and localization of these backbone proteins is essential to maintaining the integrity of the glomerular filtration barrier. However, the mechanisms that regulate the localization of these backbone proteins have remained elusive. Here, we focused on the role of membrane-associated guanylate kinase inverted 2 (MAGI-2) in order to investigate mechanisms that orchestrate localization of slit-diaphragm backbone proteins. MAGI-2 downregulation coincided with a reduced expression of slit-diaphragm backbone proteins in human kidneys glomerular disease such as focal segmental glomerulosclerosis or IgA nephropathy. Podocyte-specific deficiency of MAGI-2 in mice abrogated localization of Nephrin and Neph1 independently of other scaffold proteins. Although a deficiency of zonula occuldens-1 downregulated the endogenous Neph1 expression, MAGI-2 recovered Neph1 expression at the cellular edge in cultured podocytes. Additionally, overexpression of MAGI-2 preserved Nephrin localization to intercellular junctions. Co-immunoprecipitation and pull-down assays also revealed the importance of the PDZ domains of MAGI-2 for the interaction between MAGI-2 and slit diaphragm backbone proteins in podocytes. Thus, localization and stabilization of Nephrin and Neph1 in intercellular junctions is regulated mainly via the PDZ domains of MAGI-2 together with other slit-diaphragm scaffold proteins. Hence, these findings may elucidate a mechanism by which the backbone proteins are maintained. Translational StatementTo prevent proteinuria, it is necessary to preserve the slit diaphragm (SD) of podocytes, which functions as a final glomerular filtration barrier. However, what is important to protect nephrin and neph1—the main components of the SD—remains unknown. Investigations using podocyte-specific MAGI-2 knockout mice have shown that MAGI-2 is indispensable for the proper localization of nephrin and neph1. In a human kidney with focal segmental glomerulosclerosis and IgA nephropathy, MAGI-2 downregulation coincides with a reduced expression of the backbone proteins of the SD. Hence, a MAGI-2 protection strategy could be an effective new route to the development of drugs that could prevent proteinuria. To prevent proteinuria, it is necessary to preserve the slit diaphragm (SD) of podocytes, which functions as a final glomerular filtration barrier. However, what is important to protect nephrin and neph1—the main components of the SD—remains unknown. Investigations using podocyte-specific MAGI-2 knockout mice have shown that MAGI-2 is indispensable for the proper localization of nephrin and neph1. In a human kidney with focal segmental glomerulosclerosis and IgA nephropathy, MAGI-2 downregulation coincides with a reduced expression of the backbone proteins of the SD. Hence, a MAGI-2 protection strategy could be an effective new route to the development of drugs that could prevent proteinuria. Podocytes are highly differentiated pericyte-like cells that wrap themselves around the capillary loops in glomeruli.1Kawachi H. Miyauchi N. Suzuki K. et al.Role of podocyte slit diaphragm as a filtration barrier.Nephrology (Carlton). 2006; 11: 274-281Crossref PubMed Scopus (85) Google Scholar The differentiated cells consist of a cell body, major processes, and many foot processes. In the intercellular space between foot processes, there is a fine membrane structure called a slit diaphragm (SD).2Grahammer F. Schell C. Huber T.B. 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Membrane-associated guanylate kinase inverted 2 (MAGI-2) is also 1 of the scaffold proteins identified in neuronal spines and podocytes.26Hirao K. Hata Y. Ide N. et al.A novel multiple PDZ domain-containing molecule interacting with N-methyl-D-aspartate receptors and neuronal cell adhesion proteins.J Biol Chem. 1998; 273: 21105-21110Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 27Lehtonen S. Ryan J.J. Kudlicka K. et al.Cell junction-associated proteins IQGAP1, MAGI-2, CASK, spectrins, and alpha-actinin are components of the nephrin multiprotein complex.Proc Natil Acad Sci U S A. 2005; 102: 9814-9819Crossref PubMed Scopus (138) Google Scholar, 28Empitu M.A. Kadariswantiningsih I.N. Aizawa M. Asanuma K. MAGI-2 and scaffold proteins in glomerulopathy.Am J Physiol Renal Phys. 2018; 315: F1336-F1344Crossref PubMed Scopus (7) Google Scholar It has already been established that MAGI-2 mediates the signaling of G-protein–coupled receptors, Ras homolog family member A, and Ras-associated protein 1.29Hammad M.M. Dunn H.A. Ferguson S.S.G. MAGI proteins can differentially regulate the signaling pathways of 5-HT2AR by enhancing receptor trafficking and PLC recruitment.Cell Signal. 2018; 47: 109-121Crossref PubMed Scopus (6) Google Scholar, 30Ashraf S. Kudo H. Rao J. et al.Mutations in six nephrosis genes delineate a pathogenic pathway amenable to treatment.Nat Commun. 2018; 9: 1960Crossref PubMed Scopus (57) Google Scholar, 31Zhu B. Cao A. Li J. et al.Disruption of MAGI2-RapGEF2-Rap1 signaling contributes to podocyte dysfunction in congenital nephrotic syndrome caused by mutations in MAGI2.Kidney Int. 2019; 96: 642-655Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar We previously reported that podocyte-specific MAGI-2 knockout (KO) mice exhibit glomerulosclerosis and kidney failure in a manner similar to FSGS.32Shirata N. Ihara K.I. Yamamoto-Nonaka K. et al.Glomerulosclerosis Induced by deficiency of membrane-associated guanylate kinase inverted 2 in kidney podocytes.J Am Soc Nephrol. 2017; 28: 2654-2669Crossref PubMed Scopus (18) Google Scholar Other studies also have revealed that MAGI-2 genetic mutation leads to a severe renal phenotype in mice and humans.30Ashraf S. Kudo H. Rao J. et al.Mutations in six nephrosis genes delineate a pathogenic pathway amenable to treatment.Nat Commun. 2018; 9: 1960Crossref PubMed Scopus (57) Google Scholar,33Balbas M.D. Burgess M.R. Murali R. et al.MAGI-2 scaffold protein is critical for kidney barrier function.Proc Natl Acad Sci U S A. 2014; 111: 14876-14881Crossref PubMed Scopus (27) Google Scholar, 34Lefebvre J. Clarkson M. Massa F. et al.Alternatively spliced isoforms of WT1 control podocyte-specific gene expression.Kidney Int. 2015; 88: 321-331Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 35Bierzynska A. Soderquest K. Dean P. et al.MAGI2 mutations cause congenital nephrotic syndrome.J Am Soc Nephrol. 2017; 28: 1614-1621Crossref PubMed Scopus (41) Google Scholar, 36Zuo Z. Shen J.X. Pan Y. et al.Weighted gene correlation network analysis (WGCNA) detected loss of MAGI2 (CKD) by podocyte damage promotes chronic kidney disease.Cell Physiol Biochem. 2018; 51: 244-261Crossref PubMed Scopus (25) Google Scholar However, an understanding of the mechanism underlying the phenotype has remained elusive. In particular, although the SD in podocytes is crucial as a kidney filtration barrier, it is unclear whether MAGI-2 interacts with the SD. In fact, other groups have also shown that MAGI-2 does not bind to nephrin.37Hirabayashi S. Mori H. Kansaku A. et al.MAGI-1 is a component of the glomerular slit diaphragm that is tightly associated with nephrin.Lab Invest. 2005; 85: 1528-1543Crossref PubMed Scopus (52) Google Scholar,38Weng Z. Shang Y. Ji Z. et al.Structural basis of highly specific interaction between nephrin and MAGI1 in slit diaphragm assembly and signaling.J Am Soc Nephrol. 2018; 29: 2362-2371Crossref PubMed Scopus (3) Google Scholar The question of whether MAGI-2 acts as a scaffold protein to bolster SD proteins such as nephrin and neph1 has been shrouded in controversy for decades. In this study, we describe the function of MAGI-2 to show how the localization of nephrin and neph1 could be stabilized in the plasma membrane. MAGI-2 has already been identified as a component of the SD complex.27Lehtonen S. Ryan J.J. Kudlicka K. et al.Cell junction-associated proteins IQGAP1, MAGI-2, CASK, spectrins, and alpha-actinin are components of the nephrin multiprotein complex.Proc Natil Acad Sci U S A. 2005; 102: 9814-9819Crossref PubMed Scopus (138) Google Scholar To provide precise subcellular localization of MAGI-2, we used post-embedding immunoelectron microscopy of a rat kidney and detected the MAGI-2 signal at the cytoplasmic insertion of the SD into the foot process (Figure 1a). To investigate the dynamic expression of MAGI-2 under pathologic conditions, we established an experimental nephrosis model of doxorubicin (Adriamycin)-induced nephropathy, which is regarded as an equivalent model for human FSGS.39Fogo A.B. Animal models of FSGS: lessons for pathogenesis and treatment.Semin Nephrol. 2003; 23: 161-171Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar Adriamycin mice exhibited massive albuminuria 7 days after Adriamycin injection (Supplementary Figure S1A). Immunofluorescence analysis showed that the expressions of nephrin and neph1, key components of SDs, were reduced in the glomeruli on day 7, along with the expression of MAGI-2 (Supplementary Figure S1B). We then investigated a puromycin aminonucleoside nephropathy rat model, which is another nephrosis model.39Fogo A.B. Animal models of FSGS: lessons for pathogenesis and treatment.Semin Nephrol. 2003; 23: 161-171Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar This model also showed massive proteinuria at days 7 and 14 after Adriamycin injection (Supplementary Figure S1C). MAGI-2, nephrin, and neph1 expressions were also simultaneously downregulated (Supplementary Figure S1D). Thus, these results suggested the possibility that MAGI-2 interacts with nephrin and neph1 in a manner similar to that of other scaffold proteins. We previously generated congenital podocyte-specific MAGI-2 KO (MAGI-2 pdKO) mice.32Shirata N. Ihara K.I. Yamamoto-Nonaka K. et al.Glomerulosclerosis Induced by deficiency of membrane-associated guanylate kinase inverted 2 in kidney podocytes.J Am Soc Nephrol. 2017; 28: 2654-2669Crossref PubMed Scopus (18) Google Scholar,40Ihara K. Asanuma K. Fukuda T. et al.MAGI-2 is critical for the formation and maintenance of the glomerular filtration barrier in mouse kidney.Am J Pathol. 2014; 184: 2699-2708Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar Our data showed that MAGI-2 pdKO mice exhibited massive albuminuria and severe glomerulosclerosis at 8 weeks of age.32Shirata N. Ihara K.I. Yamamoto-Nonaka K. et al.Glomerulosclerosis Induced by deficiency of membrane-associated guanylate kinase inverted 2 in kidney podocytes.J Am Soc Nephrol. 2017; 28: 2654-2669Crossref PubMed Scopus (18) Google Scholar In additional, all MAGI-2 pdKO mice died by 20 weeks due to renal failure. In this study, using MAGI-2 pdKO mice, we intended to investigate whether MAGI-2 could influence the localization of SD proteins. In the control mice, nephrin and neph1 were distributed in the capillary pattern of glomeruli (Figure 1b and 1c). In contrast, as high-magnification images show, the nephrin staining of MAGI-2 pdKO mice exhibited a cytosolic pattern at 4 and 8 weeks (Figure 1b). Also, Western blots of glomerular lysates showed that the expressions of nephrin and neph1 were dramatically decreased in MAGI-2 pdKO mice from 4 weeks of age (Figure 1d). Meanwhile, the expression of podocin, which interacts with nephrin, was not downregulated in MAGI-2 pdKO mice, although its immunostaining pattern was altered (Figure 1c and 1d). Also, the expression difference in ZO-1, a scaffold protein of neph1,23Huber T.B. Schmidts M. Gerke P. et al.The carboxyl terminus of Neph family members binds to the PDZ domain protein zonula occludens-1.J Biol Chem. 2003; 278: 13417-13421Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar was not as apparent as that in neph1 (Figure 1c and 1d). Moreover, our previous study has shown that the expression of CD2-associated protein, a scaffold protein of nephrin, is at the same level in both groups.32Shirata N. Ihara K.I. Yamamoto-Nonaka K. et al.Glomerulosclerosis Induced by deficiency of membrane-associated guanylate kinase inverted 2 in kidney podocytes.J Am Soc Nephrol. 2017; 28: 2654-2669Crossref PubMed Scopus (18) Google Scholar These results show that MAGI-2 deletion altered the expression of nephrin and neph1, although that of other scaffold proteins was sustained. These results also indicate that MAGI-2 is important for the proper localization and steady-state abundance of nephrin and neph1 similar to that of other scaffold proteins. Investigations with MAGI-2 pdKO mice revealed evidence supporting the hypothesis that MAGI-2 regulates the localization of SD key components. However, we could not deny that the reduced SD expression in MAGI-2 pdKO mice simply reflects glomerulosclerosis, because these mice exhibited sclerotic lesions from 8 weeks of age. In additional, Cre/loxP-mediated deletion could have a substantial impact on SD-associated genes during developmental stages. To eliminate these concerns, we generated inducible podocyte-specific MAGI-2 knockout (MAGI-2 IpdKO) mice by crossing MAGI-2flox/flox mice with NPHS2-CreERT2 mice41Yokoi H. Kasahara M. Mukoyama M. et al.Podocyte-specific expression of tamoxifen-inducible Cre recombinase in mice.Nephrol Dial Transplant. 2010; 25: 2120-2124Crossref PubMed Scopus (14) Google Scholar (Figure 2a). Four weeks after the MAGI-2 IpdKO mice received an injection of tamoxifen, a Western blot of glomerular lysates and immunofluorescence demonstrated MAGI-2 downregulation (Figure 2b and 2c). Twenty weeks after a tamoxifen injection, MAGI-2 IpdKO mice also exhibited significantly massive proteinuria (0.03 g/gCre vs. 2.40 g/gCre, P < 0.05; Figure 2d). Six months after the injection, periodic acid–Schiff staining of the kidney specimens in MAGI-2 IpdKO mice revealed sclerotic lesions, whereas no sclerotic lesions were observed in the control mice (Figure 2e). The glomerulosclerosis index in MAGI-2 IpdKO mice was significantly higher (0 vs. 5.8 %, P = 0.03; Supplementary Figure S2A). In fact, the immunofluorescence of WT-1, a marker for the podocyte nucleus, revealed that the number of podocyte nuclei is significantly lower in MAGI-2 IpdKO mice (Supplementary Figure S2B and S2C). This result indicates that the podocytes are more detached in the MAGI-2 IpdKO group. In addition, electron microscopy showed an abnormal morphology for the podocyte ultrastructure in MAGI-2 IpdKO mice (Figure 2f and g). These results again supported the finding that MAGI-2 deletion leads to proteinuria and glomerulosclerosis. To investigate the MAGI-2 function for SD components, we next analyzed the expression change in the SD components 3 weeks after tamoxifen injection and found that MAGI-2 expression was simply downregulated in MAGI-2 IpdKO mice, whereas little proteinuria or few sclerotic lesions were seen in the control group (Figure 2b and d). Immunofluorescence showed that the expressions of nephrin and neph1 were downregulated in MAGI-2 IpdKO mice, although there were no apparent differences in other scaffold proteins, such as CD2-associated protein, ZO-1, and podocin (Figure 2h). In addition, the staining of nephrin in MAGI-2 IpdKO mice adopted a cytosolic pattern, whereas a capillary pattern was retained in the control mice. Similarly, a Western blot of glomeruli showed that the nephrin and neph1 expressions began to decrease from 2 weeks after tamoxifen injection in a manner similar to that of MAGI-2 (Figure 2i). Meanwhile, there were no evident differences in the expressions of other scaffold proteins within these time frames. These results show that the acquired deletion of MAGI-2 results in a reduced expression of the SD main components, which supports the hypothesis that MAGI-2 may maintain localization of the SD main components similar to other scaffold proteins. In the previous sections, our results showed that MAGI-2 expression is necessary to maintain the apparent abundance of neph1 in this experimental context. Meanwhile, prior studies have demonstrated that ZO-1 also supports the neph1 structure as a scaffold protein.23Huber T.B. Schmidts M. Gerke P. et al.The carboxyl terminus of Neph family members binds to the PDZ domain protein zonula occludens-1.J Biol Chem. 2003; 278: 13417-13421Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar,42Sagar A. Arif E. Solanki A.K. et al.Targeting Neph1 and ZO-1 protein-protein interaction in podocytes prevents podocyte injury and preserves glomerular filtration function.Sci Rep. 2017; 7: 12047Crossref PubMed Scopus (16) Google Scholar This interaction between neph1 and ZO-1 has been adequately demonstrated in biochemical assays and in in vitro and in vivo studies.23Huber T.B. Schmidts M. Gerke P. et al.The carboxyl terminus of Neph family members binds to the PDZ domain protein zonula occludens-1.J Biol Chem. 2003; 278: 13417-13421Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar,42Sagar A. Arif E. Solanki A.K. et al.Targeting Neph1 and ZO-1 protein-protein interaction in podocytes prevents podocyte injury and preserves glomerular filtration function.Sci Rep. 2017; 7: 12047Crossref PubMed Scopus (16) Google Scholar,43Wagner M.C. Rhodes G. Wang E. et al.Ischemic injury to kidney induces glomerular podocyte effacement and dissociation of slit diaphragm proteins Neph1 and ZO-1.J Biol Chem. 2008; 283: 35579-35589Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar Hence, we investigated the interaction among neph1, ZO-1, and MAGI-2 using conditionally immortalized podocyte cell lines. First, because neph1 and ZO-1 are expressed in cultured differentiated podocytes (Figure 3a and b), we established cultured ZO-1 KO podocytes via a clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 gene-editing system (CRISPR Therapeutics, Cambridge, MA) (Figure 3c).44Li H.L. Gee P. Ishida K. Hotta A. Efficient genomic correction methods in human iPS cells using CRISPR-Cas9 system.Methods (San Diego, Calif). 2016; 101: 27-35Crossref PubMed Scopus (42) Google Scholar Sanger sequence analysis of the KO cell line revealed 5 base pair deletions in the target sequence (Figure 3d). Immunofluorescence analysis showed that the ZO-1 expression was not observed in the cell-cell interface in ZO-1 KO podocytes, but it remained detectable in control podocytes (Figure 3e). In ZO-1 KO podocytes, the neph1 expression also vanished in the cell-cell contact area but remained unchanged in control podocytes (Figure 3f). Other ZO-1 KO cell lines also showed similar phenomena (Supplementary Figure S3A and B). Western blot analysis of these cell lysates showed that the neph1 expression in ZO-1 KO podocytes was significantly lower compared with the control and wild-type (WT) podocytes (Figure 3g and 3h). These results provide further evidence that ZO-1 is essential for maintaining the neph1 expression in podocytes. One possible reason for the neph1 downregulation in ZO-1 KO podocytes might have been the off-target mutations after the CRISPR-Cas9 system. However, no mutations were detected in these off-target candidate sites (Supplementary Figure S4A). In addition, the mRNA expression of neph1 was not significantly different among these cell lines (Supplementary Figure S4B). These results suggest little possibility that neph1 downregulation is due to off-target mutagenesis. Based on these results, we considered that ZO-1 deletion leads to neph1 degradation due to an unstable condition because ZO-1 is a scaffold protein of neph1.23Huber T.B. Schmidts M. Gerke P. et al.The carboxyl terminus of Neph family members binds to the PDZ domain protein zonula occludens-1.J Biol Chem. 2003; 278: 13417-13421Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar Then, to clarify how neph1 was degraded in ZO-1 KO podocytes, we examined the potential degradation pathway of neph1 in ZO-1 KO podocytes by administering inhibitors of either protease or proteasome. The neph1 expression in ZO-1 KO podocytes was elevated in the bortezomib administration group (Supplementary Figure S4C). Thus, these results indicated that ZO-1 deficiency induces instability in neph1 localization, which leads to neph1 degradation via the ubiquitin-proteasome pathway. As suggested by the finding in MAGI-2 IpdKO mice, MAGI-2 may regulate neph1 expression. However, MAGI-2 is not expressed in the cultured podocytes (Figure 3a). Thus, the phenomena of diminishing neph1 expression in ZO-1 KO podocytes were observed in the absence of MAGI-2. To investigate the role of MAGI-2 in stabilizing neph1, we needed to transfect MAGI-2 transiently to cultured ZO-1 KO podocytes. In reality, however, the efficacy of lipofection methods for transfecting cultured differentiated podocytes is quite low. Hence, we examined the possibility of using a super electroporator, NEPA21 (Nepa Gene, Chiba, Japan) and green fluorescence protein (GFP)–control plasmid DNA to transfect cultured differentiated podocytes (Supplementary Table S1). After optimizing the conditions, this instrument provided high transfection efficiency and viability of cultured differentiated podocytes compared with conventional lipofection methods (Supplementary Figure S5A). With an optimized process of electroporation in hand, MAGI-2 was expressed at the cellular edge of ZO-1 KO podocytes but was not detected in control podocytes (Figure 3i). Then the endogenous neph1 expression returned to the cellular edge in ZO-1 KO podocytes with the overexpression of MAGI-2 (Figure 3j). Meanwhile, the neph1 expression remained undetectable in the ZO-1 KO podocytes with an empty vec