ARF6 mediates nephrin tyrosine phosphorylation-induced podocyte cellular dynamics

Abstract

ADP-ribosylation factor 6 (ARF6) is a small GTPase necessary for regulating cellular structure, motility, and vesicle trafficking. In several cellular systems, ARF6 was shown to regulate actin dynamics in coordination with Rac1, a Rho small GTPase. We examined the function of ARF6 in the kidney podocyte because Rac1 was implicated in kidney diseases involving this cell. We found that ARF6 expression was enriched in human podocytes and that it modulated podocyte cytoskeletal dynamics through a functional interaction with nephrin, an intercellular junction protein necessary for podocyte injury-induced signaling requiring activation by tyrosine phosphorylation of its cytoplasmic domain. ARF6 was necessary for nephrin activation-induced ruffling and focal adhesion turnover, possibly by altering Rac1 activity. In podocyte-specific Arf6 (ARF6_PodKO) knockout mice, ARF6 deficiency did not result in a spontaneous kidney developmental phenotype or proteinuria after aging. However, ARF6_PodKO mice exhibited distinct phenotypes in two in vivo glomerular injury models. In the protamine sulfate perfusion model, which induced acute podocyte effacement, ARF6_PodKO mice were protected from podocyte effacement. In the nephrotoxic serum nephritis model, which induced immune-complex mediated injury, ARF6_PodKO mice exhibited aggravated proteinuria. Together, these observations suggest that while ARF6 is necessary for nephrin tyrosine phosphorylation-induced cytoskeletal dynamics in cultured podocytes, ARF6 has pleotropic podocyte roles in vivo, where glomerular injury-specific mechanisms might activate distinct signaling pathways that dictate whether ARF6 activity is beneficial or deleterious for maintaining the integrity of the glomerular filtration barrier.

Angiotensin II promotes podocyte injury by activating Arf6-Erk1/2-Nox4 signaling pathway

“…Arf6 activity was determined with the Arf6-GTP Pull-Down Kit following manufacturer's instructions (Cytoskeleton, Denver, CO, USA) and the protocol described previously [11]. Briefly, 500 μg of cell lysates were incubated in 4˚C cold room on a rotator for 1 h with 15 μl of GGA3-PBD bead, then centrifuged at 3,000g at 4˚C for 2 min.…”

“…Nephrin, an essential podocyte protein, is a critical component of podocyte slit diaphragm protein complex [27]. Nephrin tyrosine phosphorylation triggers cytoskeletal dynamics associated with increased lamellipodia activity and focal adhesion turnover [11,28]. It has been demonstrated that Arf6 mediates nephrin tyrosine phosphorylation-induced podocyte focal adhesion remodeling and lamellipodia formation [11].…”

“…Nephrin tyrosine phosphorylation triggers cytoskeletal dynamics associated with increased lamellipodia activity and focal adhesion turnover [11,28]. It has been demonstrated that Arf6 mediates nephrin tyrosine phosphorylation-induced podocyte focal adhesion remodeling and lamellipodia formation [11]. In addition, it has been reported that overexpression of dominant-negative Arf6 T27N completely inhibits VEGF-induced Rac1 activation and ROS production in cultured endothelial cells [29].…”

“…Just recently, it has been reported that the small GTPase ADP-ribosylation factor 6 (Arf6) belonging to the Ras superfamily is present in glomerular podocytes, and elevation of Arf6 activity is required for focal adhesion turnover and lamellipodia formation following induction of nephrin tyrosine phosphorylation in vitro and in vivo [11]. The role of Arf protein is controlled by guanine exchange factors (GEFs) and GTPase activating proteins (GAPs), respectively.…”

TRPC Channels in Proteinuric Kidney Diseases

“…Moreover, accumulating evidence suggests that dynamic signaling between the SD and focal adhesions may promote both podocyte adherence to the GBM and maintenance of glomerular filtration barrier function [7,17,18]. In support of this hypothesis, lamellipodia formation at focal adhesions are necessary for cell migration and are induced downstream of nephrin phosphorylation in cultured podocytes [19,20]; conversely, β1 integrin activation induces tyrosine phosphorylation of nephrin [21]. This crosstalk between the SD and focal adhesions may help podocytes adapt to changes in the microenvironment including alterations in glomerular filtration rate or glomerular pressure.…”