Fyn‐binding protein ADAP supports actin organization in podocytes

Abstract The renal podocyte is central to the filtration function of the kidney that is dependent on maintaining both highly organized, branched cell structures forming foot processes, and a unique cell‐cell junction, the slit diaphragm. Our recent studies investigating the developmental formation of the slit diaphragm identified a novel claudin family tetraspannin, TM4SF10, which is a binding partner for ADAP (also known as Fyn binding protein Fyb). To investigate the role of ADAP in podocyte function in relation to Fyn and TM4SF10, we examined ADAP knockout (KO) mice and podocytes. ADAP KO mice developed glomerular pathology that began as hyalinosis and progressed to glomerulosclerosis, with aged male animals developing low levels of albuminuria. Podocyte cell lines established from the KO mice had slower attachment kinetics compared to wild‐type cells, although this did not affect the total number of attached cells nor the ability to form focal contacts. After attachment, the ADAP KO cells did not attain typical podocyte morphology, lacking the elaborate cell protrusions typical of wild‐type podocytes, with the actin cytoskeleton forming circumferential stress fibers. The absence of ADAP did not alter Fyn levels nor were there differences between KO and wild‐type podocytes in the reduction of Fyn activating phosphorylation events with puromycin aminonucleoside treatment. In the setting of endogenous TM4SF10 overexpression, the absence of ADAP altered the formation of cell‐cell contacts containing TM4SF10. These studies suggest ADAP does not alter Fyn activity in podocytes, but appears to mediate downstream effects of Fyn controlled by TM4SF10 involving actin cytoskeleton organization.


Introduction
The slit diaphragm is a specialized cell junction that forms between foot processes of adjacent podocytes and is a major component of the filtration apparatus of the kidney. Its formation is a multi-step process during development, beginning as a simple cadherin-based junction that migrates to the basal cell surface and remodels into the mature structure containing the many prototypic proteins of the slit diaphragm including Nephrin, Podocin, CD2AP, and Neph-1 (Reeves et al. 1978;Huber and Benzing 2005;Garg et al. 2007;Patrakka and Tryggvason 2010). Defects in or loss of these key slit diaphragm proteins are known to cause many genetic and acquired kidney diseases, and underscores the critical contribution of the podocyte-produced components of the filtration barrier to kidney function (Grahammer et al. 2013).
Our recent studies investigating the early molecular events in the formation and assembly of the slit diaphragm identified a novel tetraspannin, TM4SF10 (gene name: Tmem47), as participating in the development of this unique cell junction (Bruggeman et al. 2007;Azhibekov et al. 2011;Dong and Simske 2016). TM4SF10 is a 20 kDa cell junction protein in the Claudin/PMP 22/EMP family (Bruggeman et al. 2007), and is one of the few claudin family proteins known to be expressed in podocytes (Yu 2015). TM4SF10 is highly expressed during kidney development, being transiently expressed at the basal-most region of podocyte precursors in comma and s-shaped bodies, but is absent by the capillary loop stage. Although its expression is absent in adult glomeruli, it can be re-expressed in mature podocytes during glomerular injury repair processes (Bruggeman et al. 2007;Azhibekov et al. 2011). In Caenorhabditis elegans, the TM4SF10 ortholog VAB-9 has been well characterized. VAB-9 has a transient but essential role organizing contractile F-actin filaments to insert into the developing adherens junction complex of the worm epidermis, and has a redundant role in cell adhesion (Simske et al. 2003). In cultured MDCK cells, TM4SF10 also has a role in regulating cell junction dynamics (Dong and Simske 2016).
To determine the functional role of TM4SF10 in the mammalian kidney, we previously screened a murine fetal kidney library to identify TM4SF10 interacting partners and identified the large cytoplasmic adapter protein ADAP, also known as the Fyn binding partner Fyb (gene name: Fyb) or Slap-130 (Azhibekov et al. 2011). ADAP, like the slit diaphragm protein CD2AP (Shih et al. 1999), was originally described in T cells. ADAP is part of the T-cell receptor signaling complex where it associates with numerous effector proteins involved in actin dynamics, integrin binding, and NF-jB signaling (Griffiths and Penninger 2002;Medeiros et al. 2007;Menasche et al. 2007;Burbach et al. 2008Burbach et al. , 2011Wang and Rudd 2008;Srivastava et al. 2010;Pauker et al. 2011). Upon recruitment to the T-cell receptor, ADAP becomes tyrosine phosphorylated, creating binding sites for the SH2 domains of Fyn and SLP-76, another adapter protein which binds Nck (although there is evidence Nck binds ADAP directly (Sylvester et al. 2010)). ADAP is an intriguing TM4SF10 interacting protein since it consolidates multiple processes in actin cytoskeleton remodeling during integrin binding at the immunologic synapse. Evaluating similar roles for ADAP in podocytes may shed light on the unique functions of podocyte cell-cell and cell-matrix interactions that govern slit diaphragm and podocyte foot process formation during development and foot process loss during disease pathogenesis.
Although our prior screening studies identified ADAP as a binding partner of TM4SF10 in the renal cells, the function of kidney-expressed ADAP has not been previously examined. The goal of this study is to begin to investigate the role of ADAP in podocytes, and its function in kidney physiology by examining an ADAP knockout mouse model and ADAP knockout podocyte cell lines.

ADAP KO mouse model and KO podocyte cell lines
All animal studies were approved and conducted under the oversight of the Animal Care and Use Committee of Case Western Reserve University. The Fyb À/À global null mouse has been previously described and is on the C57Bl/6 background (Peterson et al. 2001). Standard breeding practices were used to maintain this line, and for all experiments including the aging study, mice were housed in a specific pathogen free (modified barrier) facility with microisolator caging in ventilated racks, handled with aseptic technique in laminar flow hoods, and given sterile ad libitum food and water.
To create ADAP knockout (KO) podocyte cell lines, Fyb À/À mice were intercrossed with the Immortomouse (Charles River Laboratories) which ubiquitously expresses a temperature sensitive SV40 large T antigen (tsA58). This temperature sensitive T antigen permits cell proliferation at reduced temperature (33°C, permissive conditions), but at physiologic temperature (37°C, nonpermissive conditions) the proliferative effect is eliminated and the cells resume a state of terminal differentiation. A homozygous male for the tsA58 transgene was intercrossed with a female Fyb À/À mouse, and the heterozygous F1 offspring were crossed to create F2 offspring, regenerating some homozygotes for the knockout allele. The F2 offspring were genotyped for presence of the tsA58 transgene using a PCR method previously described (Kern and Flucher 2005). Homozygosity for the Fyb À/À allele was verified using PCR primers as previously described (Peterson et al. 2001). Offspring that were positive for the tsA58 transgene that were Fyb +/+ were also used for cell line development to create wild-type (WT) cells for comparison. Glomeruli were isolated using the standard sieving method and primary podocyte outgrowths were cultured as previously described (Mundel et al. 1997). Cells were cloned by limiting dilution and podocyte phenotype was confirmed by expression of typical podocyte markers including Nephrin (Nphs1), Synaptopodin (Synpo), WT-1 (Wt1), and Collagen IVa3 (Col4a3) by rt/PCR. Three clones each for both WT and KO podocytes were analyzed for experiments. For experiments, cells were used after culture for 7-10 at 37°C as previously described (Shankland et al. 2007).
Podocyte cultures were treated with puromycin aminonucleoside ("PAN", Sigma) at doses as noted on figures as previously described (Rico et al. 2005). Cells were lysed for Western blotting or fixed with neutral formalin for immunostaining as previously described (Azhibekov et al. 2011). For cell attachment kinetics, podocytes were cultured on type I collagen-coated plates for specified times, fixed in neutral formalin, and stained with 0.1% crystal violet. Final graphical data are a composite of all clones tested (performed three times) and data are reported as mean AE SD with statistical significance determined by unpaired t test; P > 0.05 were considered significant. 2017

Renal pathology and immunohistochemistry
Kidney pathology was assessed using standard periodicacid Schiff (PAS) staining on formalin-fixed, paraffinembedded 4 lm sections. Transmission electron micrographs were obtained using standard methods on Karnovsky-fixed, epon-embedded sections. On light microscopy, glomeruli were scored for pathological changes using a scale of 0-4 where 0 = normal, and abnormalities were scored as: 1 = hyalinosis only, changes restricted to glomerular hilum, 2 ≤ 50% segmental progression of sclerosis/mesangial matrix expansion, 3 ≥ 50% segmental changes, 4 = obsolete glomeruli. Examples of glomeruli with these scoring differences are shown in Figure 1. A minimum of 100 glomeruli in full sagittal sections were counted per animal. Percentage of glomeruli scoring abnormal (categories 1-4 above) were multiplied by their disease score and summed to generate a composite score, such that mice with 100% normal glomeruli would score zero and mice with 100% obsolete glomeruli would score four. Group means AE SD are reported and statistical differences were determined by unpaired t test.

ADAP expression in normal mouse kidney
ADAP expression was evident in developing mouse kidney glomeruli at the capillary loop stage and co-labeled cells that were positive for the podocyte marker Synaptopodin (Fig. 1A). Glomerular ADAP expression levels detected in adult normal mouse kidney were considerably lower in glomeruli, except for immune cells in vascular spaces (data not shown). This expression pattern of higher levels during glomerular development that wanes in the adult kidney was similar to the expression pattern of TM4SF10, which has highest during glomerular development, but absent in normal, adult glomeruli (Bruggeman et al. 2007).

ADAP KO kidney phenotype
A mouse model of a global knockout of ADAP (ADAP KO or Fyb À/À ) has been previously described. These mice are viable with normal fecundity and have minor abnormalities in immune responses due to a partial disruption of integrin interactions between T cells and antigen presenting cells resulting in reduced signaling through the immunologic synapse (Peterson et al. 2001). A renal phenotype, however, was not been previously reported. Weaning age animals had no obvious histopathological changes, but a progressive glomerular pathology developed with age. These changes began as hyalinosis and progressed to focal and segmental sclerosis (Fig. 1B). On electron microscopy, there was evidence of foot process widening or effacement and basement membrane thickness irregularities in ADAP KO mice (Fig. 1C). Although it has been reported C57Bl/6 mice from some commercial suppliers can develop an age-related kidney phenotype (Yabuki et al. 2006;Schmitt et al. 2009;Yang et al. 2010), the observed glomerular changes in the ADAP KO mice were histopathologically different, occurred in younger animals, and were significantly greater compared to agematched normal mice (Fig. 1D). Quantitative scoring of glomerular lesions indicated the pattern of pathology was clearly both focal (~50% of glomeruli were affected), and segmental (few fully obsolete glomeruli, see methods). Some mice >300 days of age also developed proteinuria, which was more common in male mice, but was not observed in age-matched normal mice (Fig. 1E). This sex difference was evident in both the degree of histopathological changes and in proteinuria. Although male mice consistently exhibited low levels of albuminuria, female mice rarely developed albuminuria at 1 year of age.

ADAP KO podocyte phenotype and effects on cell adhesion
Multiple podocyte cell lines were established from ADAP KO and C57Bl/6 wild-type (WT) mice by intercrossing with the Immortomouse and backcrossing F1s to recreate the Fyb À/À and Fyb +/+ genotypes. Primary podocyte outgrowths from isolated glomeruli were cloned by limiting dilution and characterized for typical podocyte differentiation markers (Nephrin, WT-1, Synaptopodin) by RT/ PCR. Morphologically, ADAP KO podocyte had fewer of the long, branched extensions typical of normal podocytes ( Fig. 2A). These protrusions contained actin, and F-actin structures detected with phalloidin staining showed ADAP KO podocytes had a marked concentration of circumferential stress fibers compared to WT (Fig. 2A). The kinetics of cell adhesion of KO and WT podocyte lines were examined with an attachment time course. There was an initial delay in integrin-dependent attachment (type I collagen coated surfaces) of KO podocytes compared to WT, however, by 24 h there was no significant difference in the final number of adherent cells (Fig. 2B). In attached cells, vinculin-positive focal contacts similarly aligned at the tips of actin fibers in both KO and WT podocytes (Fig. 3A-D). However, since the KO cells lack the cellular protrusions typical of cultured podocytes, these focal contacts appeared blunted and aggregated at cell margins ( Fig. 3C and D). Although focal contacts were distributed differently in the cells, there was no difference in focal adhesion kinase (FAK) levels between WT and KO podocytes by Western blotting (Fig. 3E). Phosphorylation of FAK at residue Y 329 was similar between WT and KO cells and paralleled total FAK levels (quantification WT 0.71 AE 0.11 vs. KO 0.62 AE 0.08) as determined by Western blotting (Fig. 3E) and with a similar difference in distribution reflecting cell morphology differences (Fig. 3F). No differences were detected by both methods for downstream FAK phosphorylation events on Y 575/577 or Y 925 (data not shown). This suggested a similar ability to form focal adhesions although with a different distribution reflective of the cell morphology.
Puromycin aminonucleoside (PAN) is an accepted in vivo and in vitro method to study podocyte injury/repair events associated with proteinuria (D'Agati 2008). Short term treatment of WT and KO podocytes with PAN resulted in the typical retraction of cellular protrusions in WT cells, but this morphological change was less evident in KO cells since they had fewer cell protrusions initially (Fig. 4A). PAN treatment did not alter ADAP protein levels in either WT or KO cells (Fig. 4B). FAK protein levels were not different between untreated WT and KO cells, but with PAN treatment, FAK was reduced (33% reduction) in WT cells ( Fig. 4C and D). The KO cells similarly responded to PAN treatment with reduced FAK levels (22% reduction), but this was not statistically significant (Fig. 4C and D), probably reflecting the fewer initial cell protrusions in the baseline KO cell morphology.

ADAP effects on Fyn
The Src family kinase Fyn has been shown to be critical for podocyte slit diaphragm and foot process formation  Since ADAP is a known Fyn binding protein, the role of ADAP in altering Fyn activity was examined in WT and KO podocytes. For these studies, Fyn phosphorylation of at tyrosine (Y) 421 , associated with kinase activation, and Y 532 associated with kinase inhibition were examined by Western blotting ( Fig. 5A and B). We have previously shown in MDCK cells, that overexpression of TM4SF10 reduced Fyn activity commensurate with reduced Fyn Y 421 phosphorylation but with no change in Y 532 phosphorylation, and that ADAP over expression did not appear to alter the suppressive effect of TM4SF10 on Fyn activity (Azhibekov et al. 2011). In addition, we also have previously shown in podocytes that PAN treatment reactivates TM4SF10 expression in podocytes (Azhibekov et al. 2011), such that PAN treatments cause endogenous TM4SF10 overexpression (example of TM4SF10 induction with PAN shown in Figure 6). In Figure 4, in the setting of TM4SF10 overexpression (i.e., PAN treatment) the Fyn activating pY 421 levels decreased in both WT and KO podocytes. The Fyn inhibitory pY 532 levels were unchanged in WT and KO podocytes, with or without PAN treatment. This indicates the absence of ADAP does not appear to influence Fyn activation, and is consistent with our prior studies in which ADAP overexpression also does not alter Fyn activation. Together, these studies indicate ADAP, either with or without of TM4SF10, does not alter the degree of Fyn activation in renal cells.

ADAP effects on TM4SF10
We originally identified ADAP as a TM4SF10 binding partner, and we next examined the effect of ADAP on TM4SF10 in podocytes. Using PAN treatment to induce endogenous TM4SF10 expression, both WT and KO podocytes exhibited similar levels of TM4SF10 at cell-cell contacts (Fig. 6). The TM4SF10 localized at cell-cell junctions in both WT and KO podocytes, however, in ADAP KO podocytes there was a subtle alteration in the distribution of TM4SF10 at the cell contact. In ADAP KO podocytes, the typical "seam" of TM4SF10 at the point of cell contact was disrupted. Although TM4SF10 was present at cell-cell contacts in KO cells, those contacts were not adjacent. This study indicated ADAP loss did not impact the induction of TM4SF10 expression during the injury-repair response, but altered its positioning in the regenerating cell-cell contact. Overall, these studies indicated ADAP likely does not have a critical role in cell adhesion, but may have a function in cytoskeleton events that may include the organization of cell-cell contacts during injury-repair processes.

Discussion
The connection between ADAP and TM4SF10 in podocytes has been clarified in these studies. In our prior work, we established TM4SF10 is transiently expressed during early glomerular development but not in mature podocytes. Although normally absent in adult glomeruli, TM4SF10 is re-expressed in podocytes during PANinduced injury/repair processes, resulting in suppressed Fyn kinase activity and the subsequent loss of cell protrusions (Bruggeman et al. 2007;Azhibekov et al. 2011). We have speculated the function of TM4SF10 re-expression during injury/repair is to stabilize the damaged slit diaphragm that occurs with foot processes effacement (i.e., the loss of cell protrusions) by recreating a cadherin-containing cell-cell contact seen in podocyte precursors during development. In studies presented here, ADAP KO did not affect TM4SF10 re-expression or suppression of   (Jones et al. 2006), a key adapter protein in podocytes that connects the slit diaphragm protein Nephrin to the actin nucleation complex, the Wiskott-Aldrich syndrome family protein N-WASP and actinrelated proteins ARP2/ARP3 (Grahammer et al. 2013). Thus, ADAP may be mediating the cytoskeletal events that control cells protrusions as a downstream consequence of TM4SF10 expression. Although ADAP may have a role in actin cytoskeleton dynamics, we did not identify a clear role for ADAP in integrin function (i.e., cell adhesion) in podocytes. ADAP function is best described in T cells, where ADAP mediates integrin dependent events in the formation of the immunologic synapse (da Silva et al. 1997;Hunter et al. 2000;Peterson et al. 2001;Griffiths and Penninger 2002). We did not observe any alterations in integrin-dependent cell attachment or formation of focal adhesions in the cultured ADAP KO podocytes. Although the attachment kinetics of ADAP KO podocytes was slower than WT podocytes, this may be a consequence of inefficient substrate tethering associated with the actin cytoskeleton remodeling needed for filopodia/lamellipodia formation. In podocytes, and similar to VAB-9 in the worm epidermis (Simske et al. 2003), integrin-dependent cell adhesion processes mediated by TM4SF10 and ADAP may have redundant or compensatory pathways.
Both the in vivo and in vitro effects of ADAP KO were subtle. We did not observe any obvious kidney development defects associated with ADAP absence. The long term in vivo studies found ADAP absence eventually had an impact on the maintenance of kidney structure and function. Similarly, the modest changes observed in vitro for the ADAP KO podocytes may be expected considering the renal phenotype in the KO mouse model required a year to develop sufficient pathological changes to impact function. However, ADAP function was either not critical for podocytes or else redundant mechanisms to counteract its absence were present. Considering the longevity of podocytes and their importance to kidney filtration, maintaining redundant systems for key cell functions would be logical.
In summary, our studies to understand TM4SF10-ADAP function in the development, maintenance, and repair of podocytes has identified a key function to cytoskeleton rearrangements that may be related to filopodia and lamellipodia formation in foot process formation. The results of these studies are shedding new light on the molecular mechanisms controlling the formation of the podocyte foot process and slit diaphragm both The Physiological Society and the American Physiological Society during development but also in proteinuric kidney diseases characterized by foot process effacement and loss of the filtration barrier. The TM4SF10-ADAP function in podocytes may have future therapeutic use in facilitating injury repair, or possibly blockade of injury, to protect or recover podocytes from irreparable loss or detachment. Because TM4SF10 is typically a silent gene in adulthood that is re-expressed in acute injury processes, this molecule could be a target that could be exploited in novel therapy development.