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The specialized epithelial cell of the kidney, the podocyte, has a complex actinbased 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., Introduction The kidneys filter plasma and reabsorb solutes and nutrients to maintain the appropriate extracellular environment. The proximal component of the nephron, the glomerulus, is the primary filtration barrier that [...]

The kidney's 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. [ABSTRACT FROM AUTHOR]

The article discusses research on how kidney podocytes regenerate foot processes of the filtration apparatus following injury. It mentions that sarcomere-like structures (SLSs) with periodically spaced myosin IIA and synaptopodin appeared in injured podocytes in vivo. Results show that SLSs in podocytes that migrated from isolated kidney glomeruli presented periodic synaptopodin-positive clusters, suggesting SLSs as mechanobiological mediators of podocyte recovery.

Ultrastructure expansion microscopy (U-ExM) involves the physical magnification of specimens embedded in hydrogels, which allows for super-resolution imaging of subcellular structures using a conventional diffraction-limited microscope. Methods for expansion microscopy exist for several organisms, organs, and cell types, and used to analyze cellular organelles and substructures in nanoscale resolution. Here, we describe a simple step-by-step U-ExM protocol for the expansion, immunostaining, imaging, and analysis of cytoskeletal and organellar structures in kidney tissue. We detail the critical modified steps to optimize isotropic kidney tissue expansion, and preservation of the renal cell structures of interest. We demonstrate the utility of the approach using several markers of renal cell types, centrioles, cilia, the extracellular matrix, and other cytoskeletal elements. Finally, we show that the approach works well on mouse and human kidney samples that were preserved using different fixation and storage conditions. Overall, this protocol provides a simple and cost-effective approach to analyze both pre-clinical and clinical renal samples in high detail, using conventional lab supplies and standard widefield or confocal microscopy.

Synaptopodin (Synpo) is an actin-associated protein in podocyte foot processes. By generating mice that completely lack Synpo, we previously showed that Synpo is dispensable for normal kidney function. However, lack of Synpo worsened adriamycin-induced nephropathy, indicating a protective role for Synpo in injured podocytes. Here, we investigated whether lack of Synpo directly impacts a genetic disease, Alport syndrome (AS), because Synpo is reduced in podocytes of affected humans and mice; whether this is merely an association or pathogenic is unknown. We used collagen type IV-α 5 ( Col4a5 ) mutant mice, which model X-linked AS, showing glomerular basement membrane (GBM) abnormalities, eventual foot process effacement, and progression to end-stage kidney disease. We intercrossed mice carrying mutations in Synpo and Col4a5 to produce double-mutant mice. Urine and tissue were taken at select time points to evaluate albuminuria, histopathology, and glomerular capillary wall composition and ultrastructure. Lack of Synpo in Col4a5 -/Y , Col4a5 -/- , or Col4a5 +/- Alport mice led to the acceleration of disease progression, including more severe proteinuria and glomerulosclerosis. Absence of Synpo attenuated the shift of myosin IIA from the podocyte cell body and major processes to actin cables near the GBM in the areas of effacement. We speculate that this is mechanistically associated with enhanced loss of podocytes due to easier detachment from the GBM. We conclude that Synpo deletion exacerbates the disease phenotype in Alport mice, revealing the podocyte actin cytoskeleton as a target for therapy in patients with AS. NEW & NOTEWORTHY Alport syndrome (AS) is a hereditary disease of the glomerular basement with hematuria and proteinuria. Podocytes eventually exhibit foot process effacement, indicating actin cytoskeletal changes. To investigate how cytoskeletal changes impact podocytes, we generated Alport mice lacking synaptopodin, an actin-binding protein in foot processes. Analysis showed a more rapid disease progression, demonstrating that synaptopodin is protective. This suggests that the actin cytoskeleton is a target for therapy in AS and perhaps other glomerular diseases.

Dendritic cells (DCs) are thought to form a dendritic network across barrier surfaces and throughout organs, including the kidney, to perform an important sentinel function. However, previous studies of DC function used markers, such as CD11c or CX3CR1, that are not unique to DCs. Here, we evaluated the role of DCs in renal inflammation using a CD11c reporter mouse line and two mouse lines with DC-specific reporters, Zbtb46 -GFP and Snx22 -GFP. Multiphoton microscopy of kidney sections confirmed that most of the dendritically shaped CD11c + cells forming a network throughout the renal interstitium expressed macrophage-specific markers. In contrast, DCs marked by Zbtb46 -GFP or Snx22 -GFP were less abundant, concentrated around blood vessels, and round in shape. We confirmed this pattern of localization using imaging mass cytometry. Motility measurements showed that resident macrophages were sessile, whereas DCs were motile before and after inflammation. Although uninflamed glomeruli rarely contained DCs, injury with nephrotoxic antibodies resulted in accumulation of ZBTB46 + cells in the periglomerular region. ZBTB46 identifies all classic DCs, which can be categorized into two functional subsets that express either CD103 or CD11b. Depletion of ZBTB46 + cells attenuated the antibody-induced kidney injury, whereas deficiency of the CD103 + subset accelerated injury through a mechanism that involved increased neutrophil infiltration. RNA sequencing 7 days after nephrotoxic antibody injection showed that CD11b + DCs expressed the neutrophil-attracting cytokine CXCL2, whereas CD103 + DCs expressed high levels of several anti-inflammatory genes. These results provide new insights into the distinct functions of the two major DC subsets in glomerular inflammation., (Copyright © 2018 by the American Society of Nephrology.)