Mutations of LMX1B lead to the hereditary disease NPS, which is associated with renal symptoms (Witzgall, 2017). Within the kidney, the transcription factor LMX1B is exclusively expressed in podocytes and plays an essential role in the maturation and maintenance of the cell. Previous studies revealed not only the putative Lmx1b target genes Abra, Arl4c and Sm22, but also evidence of an impact of Lmx1b on the regulation of the podocyte actin cytoskeleton and focal adhesions (Burghardt et al., 2013; Stepanova, 2016). Therefore, the main purpose of the present work was to identify and investigate pathways linking Lmx1b and its target genes to the actin cytoskeleton and focal adhesions and to clarify the nature of actin and focal adhesion dysregulation in disease.
By the use of an inducible podocyte-specific Lmx1b knock-out mouse line with an mTmG reporter construct, knock-out of Lmx1b could be induced at a desired time point in podocytes of mature mice, and those podocytes could be separated from other glomerular cells. Analysis of the protein expression in podocytes by western blotting confirmed an increased expression of Arl4c and transgelin following Lmx1b knock-out. While Arl4c is most likely a direct target of Lmx1b, transgelin expression seems to be a general response to podocyte damage (Marshall et al., 2011). The expression of Abra in wild-type or knock-out podocytes could not be confirmed free of doubt.
Primary murine podocytes were used to clarify the impact of Lmx1b on the actin cytoskeleton. To better resemble the physiological conditions, cells were plated on laminin 521 in all of those experiments. As some effects of Lmx1b knock-out might only be visible during cell stress and not in the steady state, many experiments were performed with both spreading and also full-grown, steady state podocytes. The knock-out of Lmx1b led to an increased F actin staining in both spreading and steady state podocytes. Moreover, the circularity and roundness in the late phase of spreading and also at steady state were increased. The cell area of spreading podocytes and also the cell size measured by forward light scatter in FACS experiments was independent of Lmx1b knock-out, but the cell area of Lmx1b-deficient steady state podocytes was markedly increased. Thus, primary Lmx1b knock-out podocytes are flatter and rounder and contain more actin fibers. Untreated podocytes revealed similar random movement velocities and spreading rates independent of Lmx1b expression. Treatment of podocytes with cytochalasin D induced cell shrinkage at similar rates and accumulation of short actin fibers at spots near the nucleus and at the base of small cell extensions. Removal of cytochalasin D, in turn, led to cell spreading, which was reduced in the absence of Lmx1b. The cytoskeleton remained atypically organized two hours after wash-out of cytochalasin D in knock-out cells as well as wild-type controls. The different observations of cell spreading with or without prior cytochalasin D treatment can be explained by different initial states of the actin cytoskeleton and focal adhesions at the beginning of the experiment. Thus, only some actin-dependent dynamic processes are influenced by Lmx1b and its target genes.
As the impact of Lmx1b on the actin cytoskeleton was obvious, one of the main goals was to identify the signaling pathways involved. Therefore, two different strategies were carried out. One possibility was to examine the activities of proteins involved in signaling cascades. This was achieved either by specific binding of activated proteins to effectors or by analyzing the amount of phosphorylation utilizing specific antibodies. The other possibility was the investigation of the podocyte spreading after the removal of cytochalasin D in the presence of inhibitors blocking specific signaling proteins. In case the inhibitor would block a dysregulated protein, the spreading curves of knock-out and wild-type podocytes were expected to converge. The Rho GTPases are key actin regulators (Sadok and Marshall, 2014) and therefore, were subject of investigation. The activity of RhoA and Cdc42 was significantly reduced in glomerular lysates of podocyte-specific Lmx1b knock-out mice, while the activity of Rac1 was equal to controls. Additionally, the phosphorylation of the myosin light chain and thereby activation of myosin-2 was decreased after de novo expression of LMX1B in a human podocyte cell line (hPCL). By the use of inhibitors in spreading experiments, LIMK but not ROCK could be identified to be dysregulated in primary murine Lmx1b knock-out podocytes. Many of the actin cytoskeleton related dysregulations of podocytes can be explained by the reduced activity of Cdc42, but not RhoA, including increased circularity and roundness, decreased spreading after removal of cytochalasin D and the dysregulation of LIMK. Nevertheless, further investigation of the Cdc42 pathway is required to prove this connection.
As Lmx1b knock-out podocytes revealed increased adhesion to laminin-111 (Burghardt et al., 2013), the potential role of focal adhesions downstream of Lmx1b was investigated. Moreover, outside-in signaling at focal adhesions impacts the actin cytoskeleton. Focal adhesions are accumulations of a multitude of proteins, which anchor cells to the extracellular matrix and establish a mechanical as well as a signaling link to the actin cytoskeleton. Transmembrane integrins are the most important proteins within focal adhesions, and α3β1-integrin is the most abundant integrin in podocytes (Sachs and Sonnenberg, 2013). Staining with an antibody specific for active β1-integrin revealed no differences between Lmx1b knock-out and wild-type control podocytes by confocal microscopy. On the other hand, a slightly increased activation of β1-integrin in knock-out podocytes was detectable by flow cytometry. The amount of total β1-integrin in podocytes and also the activity of β1-integrin in other glomerular cells remained unchanged, thus indicating primary intracellular rather than secondary extracellular causes for increased β1-integrin activation in podocytes. Further investigations regarding the pathway of increased integrin activation are of interest, for instance, talin or kindlin-2 expression and localization (Askari et al., 2009) as well as measurement of the mechanical tension at focal adhesions with FRET-based biosensors (Grashoff et al., 2010).
Transgelin is an actin-binding protein previously shown to be highly expressed in Lmx1b-deficient podocytes, but not in wild-type controls. Transgelin is localized to the cell cortex in both spreading and steady state podocytes, but colocalization with actin bundles was only observed in steady state podocytes. Surprisingly, podocyte outgrowth led to transgelin expression in wild-type controls, although to a lower extent when compared to steady state Lmx1b knock-out podocytes. To address the impact of de novo transgelin expression on the podocyte and on renal health, a double Sm22 and Lmx1b (podocyte-specific) knock-out mouse line was created. Investigation of the survival and proteinuria of those mice revealed a strong dependence on the genetic background, but also an increased percentage of mice with prolonged lifetime and delayed onset of proteinuria compared to podocyte-specific Lmx1b knock-out control mice. A deeper analysis of double knock-out mice 8 days postnatally, on the other hand, demonstrated worse proteinuria of most mice, except for one animal without any pathological phenotype. No influence of additional Sm22 knock-out on kidney histology, kidney ultrastructure, filtration slit density and glomerular phalloidin staining could be revealed. The body weight of some, but not all, double knock-out mice was reduced compared to controls. Supported by a previous report (Marshall et al., 2011), this led to the assumption of beneficial aspects of transgelin expression in early podocyte damage and detrimental effects in the later disease progression
