IQGAP1 Interacts with Components of the Slit Diaphragm Complex in Podocytes and Is Involved in Podocyte Migration and Permeability In Vitro

IQGAP1 is a scaffold protein that interacts with proteins of the cytoskeleton and the intercellular adhesion complex. In podocytes, IQGAP1 is associated with nephrin in the glomerular slit diaphragm (SD) complex, but its role remains ill-defined. In this work, we investigated the interaction of IQGAP1 with the cytoskeleton and SD proteins in podocytes in culture, and its role in podocyte migration and permeability. Expression, localization, and interactions between IQGAP1 and SD or cytoskeletal proteins were determined in cultured human podocytes by Western blot (WB), immunocytolocalization (IC), immunoprecipitation (IP), and In situ Proximity Ligation assay (IsPL). Involvement of IQGAP1 in migration and permeability was also assessed. IQGAP1 expression in normal kidney biopsies was studied by immunohistochemistry. IQGAP1 expression by podocytes increased during their in vitro differentiation. IC, IP, and IsPL experiments showed colocalizations and/or interactions between IQGAP1 and SD proteins (nephrin, MAGI-1, CD2AP, NCK 1/2, podocin), podocalyxin, and cytoskeletal proteins (α-actinin-4). IQGAP1 silencing decreased podocyte migration and increased the permeability of a podocyte layer. Immunohistochemistry on normal human kidney confirmed IQGAP1 expression in podocytes and distal tubular epithelial cells and also showed an expression in glomerular parietal epithelial cells. In summary, our results suggest that IQGAP1, through its interaction with components of SD and cytoskeletal proteins, is involved in podocyte barrier properties

Idiopathic Nephrotic Syndrome: Characteristics and Identification of Prognostic Factors

There are various histopathological forms of idiopathic nephrotic syndrome, including minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS). Whereas some relapse predictor factors have been identified in renal transplantation, the clinical future of idiopathic nephrotic syndrome in the native kidney remains uncertain. We designed a multicentric retrospective descriptive cohort study including all patients aged 15 years and over whose renal biopsy confirmed MCD or FSGS between January 2007 and December 2014. We studied 165 patients with idiopathic nephrotic syndrome; 97 with MCD and 68 with FSGS. In the MCD cohort, 91.7% of patients were treated with corticosteroids for a median total duration of 13 months. During 45 months of follow-up, 92.8% of patients achieved remission and 45.5% experienced relapse. In this cohort, 5% of patients experienced terminal kidney disease. With respect to FSGS patients, 51.5% were treated with corticosteroids for a median total duration of 15 months. During 66 months of follow-up, 73.5% of patients achieved remission and 20% experienced relapse. In this cohort, 26.5% of patients experienced terminal kidney disease. No statistical association was observed between clinical and biological initial presentation and relapse occurrence. This study describes the characteristics of a cohort of patients with the nephrotic idiopathic syndromes of MCD and FSGS from the time of renal biopsy and throughout follow-up

Characteristics of the secondary antibodies used in the study.

<p>(Abbreviations: IH = immunohistochemistry, IF = Immunofluorescence, WB = Western blot, IP = Immunoprecipitation).</p

Cellular localization of interactions between IQGAP1 and different proteins of the slit diaphragm: In situ Proximity Ligation assay.

<p>In situ Proximity Ligation assays confirmed in cells the interactions between IQGAP1 and nephrin, MAGI-1, CD2AP, podocin, NCK1/2 and podocalyxin. Cells were transfected with siRNA Luc (Luc) or siRNA IQGAP1 (siRNA).</p

siRNA used in the study.

<p>siRNA used in the study.</p

Involvement of IQGAP1 in podocyte proliferation, viability and adhesion.

<p>Influence of down regulation of IQGAP1 on cell proliferation and viability (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037695#pone-0037695-g007" target="_blank">Figure 7A and 7B</a>, n = 5, Two-way repeated measurements ANOVA). Cell survival was not altered by transfection. Increase of cytotoxicity was not the course of the migration defect. Podocyte adhesion after down regulation of IQGAP1 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037695#pone-0037695-g007" target="_blank">Figure 7C</a>, n = 3, Wilcoxon test). Adhesive properties of podocytes were conserved after siRNA IQGAP1 transfection. Control: control cells; siRNA: siRNA IQGAP1 transfected podocytes.</p

IQGAP1 interactions with proteins of the slit diaphragm and podocalyxin.

<p>IQGAP1 immunoprecipitation experiments were performed with undifferentiated podocytes (D0, 33°C) and differentiated podocytes (D16, 37°C), using equal amounts of total protein. The irrelevant antibody against tristetraprolin was used in control immunoprecipitations (Ctl). (A) Interaction between IQGAP1 and nephrin (data are representative of 6 independent experiments). (B to H) Interactions between IQGAP1 and MAGI-1, CD2AP, podocin, NCK1/2, α-actinin-4, β-catenin and podocalyxin (Data are representative of 5 independent experiments, 6 for podocalyxin).</p

Characteristics of primary antibodies used in the study.

<p>(Abbreviations: mAb = monoclonal Antibody, pAb = polyclonal Antibody, IH = immunohistochemistry, IF = Immunofluorescence, WB = Western blot, IP = Immunoprecipitation).</p

Schematic representation of the podocyte foot processes with putative IQGAP1 localization and interactions.

<p>Bottom: schematic representation of two pedicels, joined by the slit diaphragm. Top: two enlargements are depicted representing possible IQGAP1 interactions at the slit diaphragm (right) and the podocyte apical (left) regions. Proteins highlighted in grey and italics correspond to proteins not explored in this study. With the exception of nephrin, direct or indirect interactions between IQGAP1 and its partners remain to be demonstrated.</p

Effect of the down regulation of IQGAP1 expression on F- actin network.

<p>(A) IQGAP1 Western blot analysis showing IQGAP1 expression in podocytes transfected with siRNA IQGAP1 as compared with untransfected podocytes (UT) or podocytes transfected with siRNA directed at luciferase (Luc). (B) F-actin labelling after siRNA IQGAP1 transfection in comparison to control cells (untransfected cells). Scale bars, 50 µm.</p