ARHGEF7 (β-PIX) Is Required for the Maintenance of Podocyte Architecture and Glomerular Function

Abstract

BackgroundPrevious studies showed that Cdc42, a member of the prototypical Rho family of small GTPases and a regulator of the actin cytoskeleton, is critical for the normal development and health of podocytes. However, upstream regulatory mechanisms for Cdc42 activity in podocytes are largely unknown.MethodsWe used a proximity-based ligation assay, BioID, to identify guanine nucleotide exchange factors that activate Cdc42 in immortalized human podocytes. We generated podocyte-specific ARHGEF7 (commonly known as β-PIX) knockout mice by crossing β-PIX floxed mice with Podocin-Cre mice. Using shRNA, we established cultured mouse podocytes with β-PIX knockdown and their controls.ResultsWe identified β-PIX as a predominant guanine nucleotide exchange factor that interacts with Cdc42 in human podocytes. Podocyte-specific β-PIX knockout mice developed progressive proteinuria and kidney failure with global or segmental glomerulosclerosis in adulthood. Glomerular podocyte density gradually decreased in podocyte-specific β-PIX knockout mice, indicating podocyte loss. Compared with controls, glomeruli from podocyte-specific β-PIX knockout mice and cultured mouse podocytes with β-PIX knockdown exhibited significant reduction in Cdc42 activity. Loss of β-PIX promoted podocyte apoptosis, which was mediated by the reduced activity of the prosurvival transcriptional regulator Yes-associated protein.ConclusionsThese findings indicate that β-PIX is required for the maintenance of podocyte architecture and glomerular function via Cdc42 and its downstream Yes-associated protein activities. This appears to be the first evidence that a Rho–guanine nucleotide exchange factor plays a critical role in podocytes.

Rho GTPase regulatory proteins in podocytes

“…Loss of β-PIX promoted podocyte apoptosis, which was mediated by the reduced activity of the pro-survival transcriptional regulator, YAP (Yes-associated protein). 41 The link between Cdc42 and YAP has been reported previously in the cap mesenchyme (kidney mesenchymal progenitor cells surrounding the ureteric bud that give rise to the proximal parts of a nephron). 43 The results indicate that β-PIX is required for the maintenance of podocyte architecture and glomerular function via Cdc42 and its downstream YAP activity.…”

“…75 Recently, we used the analogous approach to identify β-PIX as a main GEF for Cdc42 in cultured human podocytes. 41 We are currently expanding this BioID study using RhoA, Rac1, and Cdc42 and their mutants (nucleotide-free mutants that bind to GEFs and constitutively active mutants that bind to GAPs) as baits with the goal to map the Rho GTPase interactome in podocytes. Second is to systematically screen large cohorts of patients with proteinuria and chronic kidney disease (CKD) for variants in Rho GTPase pathway genes.…”

“…5,6 To identify GEFs that are responsible for Cdc42 activation in podocytes, we used proximity ligation and proteomics (BioID) with Cdc42 as bait, and found ARHGEF7 (commonly known as β-PIX) to be the top Cdc42-interacting GEF in cultured human podocytes. 41 Podocyte-specific β-PIX deficient (β-PIX Pod-/-) mice developed progressive proteinuria, starting at around 8 weeks old, leading to kidney failure with global/segmental J o u r n a l P r e -p r o o f glomerulosclerosis. 41 In spite of the known dual activity of β-PIX towards Rac1 and Cdc42, 42 β-PIX deficiency in podocytes reduced Cdc42, but not Rac1 activity.…”

“…41 Podocyte-specific β-PIX deficient (β-PIX Pod-/-) mice developed progressive proteinuria, starting at around 8 weeks old, leading to kidney failure with global/segmental J o u r n a l P r e -p r o o f glomerulosclerosis. 41 In spite of the known dual activity of β-PIX towards Rac1 and Cdc42, 42 β-PIX deficiency in podocytes reduced Cdc42, but not Rac1 activity. Loss of β-PIX promoted podocyte apoptosis, which was mediated by the reduced activity of the pro-survival transcriptional regulator, YAP (Yes-associated protein).…”

Role of Rho GTPase Interacting Proteins in Subcellular Compartments of Podocytes

“…Although Rho GTPases in general are known to play important roles in cell survival, proliferation, and transcriptional regulations, to date only a few reports are available studying their roles in the nucleus of podocytes ( Figure 2 e). The best described is in association with the transcriptional regulator, Yes associated protein (YAP) [ 45 , 46 , 86 ]. YAP is a transcriptional co-activator of the Hippo signaling pathway.…”