Hani Suleiman

Arhgap24 inactivates Rac1 in mouse podocytes, and a mutant form is associated with familial focal segmental glomerulosclerosis

The specialized epithelial cell of the kidney, the podocyte, has a complex actin-based cytoskeleton. Dynamic regulation of this cytoskeleton is required for efficient barrier function of the kidney. Podocytes are a useful cell type to study the control of the actin cytoskeleton in vivo, because disruption of components of the cytoskeleton results in podocyte damage, cell loss, and a prototypic injury response called focal segmental glomerulosclerosis (FSGS). Searching for actin regulatory proteins that are expressed in podocytes, we identified a RhoA-activated Rac1 GTPase-activating protein (Rac1-GAP), Arhgap24, that was upregulated in podocytes as they differentiated, both in vitro and in vivo. Increased levels of active Rac1 and Cdc42 were measured in Arhgap24 knockdown experiments, which influenced podocyte cell shape and membrane dynamics. Consistent with a role for Arhgap24 in normal podocyte functioning in vivo, sequencing of the ARHGAP24 gene in patients with FSGS identified a mutation that impaired its Rac1-GAP activity and was associated with disease in a family with FSGS. Thus, Arhgap24 contributes to the careful balancing of RhoA and Rac1 signaling in podocytes, the disruption of which may lead to kidney disease.

Nanoscale protein architecture of the kidney glomerular basement membrane

In multicellular organisms, proteins of the extracellular matrix (ECM) play structural and functional roles in essentially all organs, so understanding ECM protein organization in health and disease remains an important goal. Here, we used sub-diffraction resolution stochastic optical reconstruction microscopy (STORM) to resolve the in situ molecular organization of proteins within the kidney glomerular basement membrane (GBM), an essential mediator of glomerular ultrafiltration. Using multichannel STORM and STORM-electron microscopy correlation, we constructed a molecular reference frame that revealed a laminar organization of ECM proteins within the GBM. Separate analyses of domains near the N- and C-termini of agrin, laminin, and collagen IV in mouse and human GBM revealed a highly oriented macromolecular organization. Our analysis also revealed disruptions in this GBM architecture in a mouse model of Alport syndrome. These results provide the first nanoscopic glimpse into the organization of a complex ECM. DOI: http://dx.doi.org/10.7554/eLife.01149.001

Rac1 activation in podocytes induces rapid foot process effacement and proteinuria.

ABSTRACT The kidneys vital filtration function depends on the structural integrity of the glomerulus, the proximal portion of the nephron. Within the glomerulus, the architecturally complex podocyte forms the final cellular barrier to filtration. Injury to the podocyte results in a morphological change called foot process effacement, which is a ubiquitous feature of proteinuric diseases. The exact mechanism underlying foot process effacement is not known, but recently it has been proposed that this change might reflect activation of the Rac1 GTPase. To test this hypothesis, we generated a podocyte-specific, inducible transgenic mouse line that expressed constitutively active Rac1. When the Rac1 transgene was induced, we observed a rapid onset of proteinuria with focal foot process effacement. Using superresolution imaging, we verified that the induced transgene was expressed in damaged podocytes with altered foot process morphology. This work sheds new light on the complex balance of Rho GTPase signaling that is required for proper regulation of the podocyte cytoskeleton.

LMX1B is Essential for the Maintenance of Differentiated Podocytes in Adult Kidneys

Mutations of the LMX1B gene cause nail-patella syndrome, a rare autosomal-dominant disorder affecting the development of the limbs, eyes, brain, and kidneys. The characterization of conventional Lmx1b knockout mice has shown that LMX1B regulates the development of podocyte foot processes and slit diaphragms, but studies using podocyte-specific Lmx1b knockout mice have yielded conflicting results regarding the importance of LMX1B for maintaining podocyte structures. In order to address this question, we generated inducible podocyte-specific Lmx1b knockout mice. One week of Lmx1b inactivation in adult mice resulted in proteinuria with only minimal foot process effacement. Notably, expression levels of slit diaphragm and basement membrane proteins remained stable at this time point, and basement membrane charge properties also did not change, suggesting that alternative mechanisms mediate the development of proteinuria in these mice. Cell biological and biophysical experiments with primary podocytes isolated after 1 week of Lmx1b inactivation indicated dysregulation of actin cytoskeleton organization, and time-resolved DNA microarray analysis identified the genes encoding actin cytoskeleton-associated proteins, including Abra and Arl4c, as putative LMX1B targets. Chromatin immunoprecipitation experiments in conditionally immortalized human podocytes and gel shift assays showed that LMX1B recognizes AT-rich binding sites (FLAT elements) in the promoter regions of ABRA and ARL4C, and knockdown experiments in zebrafish support a model in which LMX1B and ABRA act in a common pathway during pronephros development. Our report establishes the importance of LMX1B in fully differentiated podocytes and argues that LMX1B is essential for the maintenance of an appropriately structured actin cytoskeleton in podocytes.

Intravital and Kidney Slice Imaging of Podocyte Membrane Dynamics

In glomerular disease, podocyte injury results in a dramatic change in cell morphology known as foot process effacement. Remodeling of the actin cytoskeleton through the activity of small GTPases was identified as a key mechanism in effacement, with increased membrane activity and motility in vitro However, whether podocytes are stationary or actively moving cells in vivo remains debated. Using intravital and kidney slice two-photon imaging of the three-dimensional structure of mouse podocytes, we found that uninjured podocytes remained nonmotile and maintained a canopy-shaped structure over time. On expression of constitutively active Rac1, however, podocytes changed shape by retracting processes and clearly exhibited domains of increased membrane activity. Constitutive activation of Rac1 also led to podocyte detachment from the glomerular basement membrane, and we detected detached podocytes crawling on the surface of the tubular epithelium and occasionally, in contact with peritubular capillaries. Podocyte membrane activity also increased in the inflammatory environment of immune complex-mediated GN. Our results provide evidence that podocytes transition from a static to a dynamic state in vivo, shedding new light on mechanisms in foot process effacement.

Role of Transcription Factors in Podocytes

Despite a wealth of information on structural proteins, comparatively little is known on the transcriptional regulation of podocyte structure and function. In this review we will highlight those transcription factors which, by gene inactivation or classical transgenic experiments, have been shown to be essential for podocytes or probably will turn out to be so. The tumor suppressor protein WT1 is not only indispensable for the initial stages of kidney development, but also very likely maintains the integrity of the fully differentiated podocyte. In the kidney, the LIM homeodomain transcription factor LMX1B is specifically synthesized in podocytes, and mutations in LMX1B lead to nail-patella syndrome and the associated nephropathy. Other transcription factors such as hypoxia-inducible factors and PAX2 are likely to play a role in podocytes, whereas the significance of others, e.g. of POD1 and CITED2, is more speculative at this point.

Albuminuria associated with CD2AP knockout mice is primarily due to dysfunction of the renal degradation pathway processing of filtered albumin.

Here we address the assumption that the massive intact albuminuria accompanying mutations of structural components of the slit diaphragm is due to changes in glomerular permeability. The increase in intact albumin excretion rate in Cd2ap knockout mice by >100‐fold was not accompanied by equivalent changes in urine flow rate, glomerular filtration rate or increases in dextran plasma clearance rate, which demonstrates that changes in glomerular permeability alone could not account for the increase in intact albumin excretion. The albuminuria could be accounted for by inhibition of the tubule degradation pathway associated with degrading filtered albumin. There are remarkable similarities between these results and all types of podocytopathies in acquired and toxin‐induced renal disease, and nephrotic states seen in mice with podocyte mutations.

Neonatal Fc Receptor Promotes Immune Complex–Mediated Glomerular Disease

The neonatal Fc receptor (FcRn) is a major regulator of IgG and albumin homeostasis systemically and in the kidneys. We investigated the role of FcRn in the development of immune complex-mediated glomerular disease in mice. C57Bl/6 mice immunized with the noncollagenous domain of the α3 chain of type IV collagen (α3NC1) developed albuminuria associated with granular capillary loop deposition of exogenous antigen, mouse IgG, C3 and C5b-9, and podocyte injury. High-resolution imaging showed abundant IgG deposition in the expanded glomerular basement membrane, especially in regions corresponding to subepithelial electron dense deposits. FcRn-null and -humanized mice immunized with α3NC1 developed no albuminuria and had lower levels of serum IgG anti-α3NC1 antibodies and reduced glomerular deposition of IgG, antigen, and complement. Our results show that FcRn promotes the formation of subepithelial immune complexes and subsequent glomerular pathology leading to proteinuria, potentially by maintaining higher serum levels of pathogenic IgG antibodies. Therefore, reducing pathogenic IgG levels by pharmacologic inhibition of FcRn may provide a novel approach for the treatment of immune complex-mediated glomerular diseases. As proof of concept, we showed that a peptide inhibiting the interaction between human FcRn and human IgG accelerated the degradation of human IgG anti-α3NC1 autoantibodies injected into FCRN-humanized mice as effectively as genetic ablation of FcRn, thus preventing the glomerular deposition of immune complexes containing human IgG.

The FERM protein EPB41L5 regulates actomyosin contractility and focal adhesion formation to maintain the kidney filtration barrier

Significance Loss of podocyte adhesion is a hallmark of glomerular disease progression. Here we unravel the in vivo composition of the podocyte adhesion machinery by the use of quantitative proteomics and identify the FERM domain protein EPB41L5 as a selectively enriched novel podocyte focal adhesion protein. EPB41L5 is essential to maintaining podocyte adhesion in vivo by recruiting the Rho GEF ARHGEF18, initiating a signaling cascade and ultimately resulting in increased actomyosin activity and focal adhesion stabilization. As EPB41L5 is down-regulated in various glomerular pathologies, these findings offer a perspective for interventions aiming to prevent loss of podocytes in glomerular disease. Podocytes form the outer part of the glomerular filter, where they have to withstand enormous transcapillary filtration forces driving glomerular filtration. Detachment of podocytes from the glomerular basement membrane precedes most glomerular diseases. However, little is known about the regulation of podocyte adhesion in vivo. Thus, we systematically screened for podocyte-specific focal adhesome (FA) components, using genetic reporter models in combination with iTRAQ-based mass spectrometry. This approach led to the identification of FERM domain protein EPB41L5 as a highly enriched podocyte-specific FA component in vivo. Genetic deletion of Epb41l5 resulted in severe proteinuria, detachment of podocytes, and development of focal segmental glomerulosclerosis. Remarkably, by binding and recruiting the RhoGEF ARGHEF18 to the leading edge, EPB41L5 directly controls actomyosin contractility and subsequent maturation of focal adhesions, cell spreading, and migration. Furthermore, EPB41L5 controls matrix-dependent outside-in signaling by regulating the focal adhesome composition. Thus, by linking extracellular matrix sensing and signaling, focal adhesion maturation, and actomyosin activation EPB41L5 ensures the mechanical stability required for podocytes at the kidney filtration barrier. Finally, a diminution of EPB41L5-dependent signaling programs appears to be a common theme of podocyte disease, and therefore offers unexpected interventional therapeutic strategies to prevent podocyte loss and kidney disease progression.

Injury-induced actin cytoskeleton reorganization in podocytes revealed by super-resolution microscopy

The architectural integrity of tissues requires complex interactions, both between cells and between cells and the extracellular matrix. Fundamental to cell and tissue homeostasis are the specific mechanical forces conveyed by the actomyosin cytoskeleton. Here we used super-resolution imaging methods to visualize the actin cytoskeleton in the kidney glomerulus, an organized collection of capillaries that filters the blood to make the primary urine. Our analysis of both mouse and human glomeruli reveals a network of myosin IIA-containing contractile actin cables within podocyte cell bodies and major processes at the outer aspects of the glomerular tuft. These likely exert force on an underlying network of myosin IIA-negative, noncontractile actin fibers present within podocyte foot processes that function to both anchor the cells to the glomerular basement membrane and stabilize the slit diaphragm against the pressure of fluid flow. After injuries that disrupt the kidney filtration barrier and cause foot process effacement, the podocytes contractile actomyosin network relocates to the basolateral surface of the cell, manifesting as sarcomere-like structures juxtaposed to the basement membrane. Our findings suggest a new model of the podocyte actin cytoskeleton in health and disease and suggest the existence of novel mechanisms that regulate podocyte architecture.