Nephron progenitor cell organization and nephrogenesis is dependent on Shroom3

Abstract

The functional unit of the kidney, termed the nephron, develops from specialized mesenchyme cells known as the nephron progenitors. Nephron progenitors must undergo significant cell shape changes to regulate cell migration, cell movements, and cell projection formation during nephrogenesis. These dynamic cellular changes are essential for orientating and organizing the cells for progression through various stages of nephrogenesis to form a proper functioning nephron. The actin cytoskeleton regulates these dynamic morphological changes by interacting with intracellular proteins and their extracellular environment. Shroom3 is an actin-binding protein that regulates cell shape changes by modulating the actin cytoskeleton. In mice and humans, mutations in Shroom3 are strongly associated with poor nephron function and chronic kidney disease. Our previous work demonstrated unique Shroom3 expression patterns in nephron progenitor cells, yet the function of Shroom3 during nephrogenesis is not known. Here, we investigated functional roles for Shroom3 in nephron progenitor organization during nephrogenesis. Analysis of E13.5 and E18.5 kidney sections from Wildtype and Shroom3-/- null mutants demonstrated clustering abnormalities of nephron progenitor cells characterized by large abnormal gaps between cells in Shroom3 mutants. Analysis of Wildtype and Shroom3-/- nephron progenitor cell orientation by GM130 and Integrin-α8 immunofluorescence, markers of cell polarization, displayed altered cell orientation in some but not all cells. Transmission electron microscopy (TEM) confirmed that nephron progenitors of Shroom3-/- were irregularly shaped, abnormally clustered, and found at large distances from the neighboring epithelium. TEM analysis also revealed several filopodia-like long and thin actin-based membrane protrusions from both Wildtype and Shroom3-/- nephron progenitors. However, the broad, thick lamellipodia-like protrusions, found at the leading edge of the cells, were virtually absent from Shroom3-/- nephron progenitors. As nephrogenesis proceeds, Wildtype nephron progenitors develop into renal vesicles that consist of a central lumen surrounded by polarized wedge-shaped epithelial cells, which was not observed in Shroom3-/- renal vesicles. These alterations in the early stages of nephrogenesis in Shroom3-/- kidneys were also associated with a disruption in the epithelial polarization of mature nephrons demonstrated by irregular Sglt2 and Slc12a3 expression, key ion channels localized to the apical aspect in Wildtype epithelia. Our results demonstrate that Shroom3 is an important regulator of nephron progenitor organization and cell morphology in the earliest stages of nephron formation. It also suggests these abnormalities translate to a poorly polarized nephron epithelium and likely poor nephron function. These studies could explain why mutations in Shroom3 are so highly associated with poor nephron function and chronic kidney disease.