The Death Domain of Kidney Ankyrin Interacts with Fas and Promotes Fas-Mediated Cell Death in Renal Epithelia

Abstract

Abstract. Ankyrins are a ubiquitously expressed family of conserved proteins that mediate the linkage of integral membrane proteins such as transporters and channels with the underlying cytoskeleton. Ankyrins possess a conserved death domain, the functional significance of which has remained puzzling. In this study, the death domain of AnkG190, the isoform of ankyrin expressed in kidney tubules, was used as bait in a yeast two-hybrid screen to identify interacting partners. One of these interactions was with the proapoptotic molecule Fas. This was confirmed by coimmunoprecipitation, colocalization, and glutathione S-transferase pull-down assays in cultured renal epithelial (MDCK) cells. Site-directed mutagenesis of a conserved arginine (R1496 in AnkG190), previously shown to be critical for the binding of Fas (R234 in Fas) to FADD, abolished the interaction of ankyrin's death domain with Fas. Overexpression of constructs containing ankyrin's death domain promoted Fas-mediated apoptosis in MDCK cells. The linkage between ankyrin and Fas was confirmed in vivo in mouse kidney tubule cells by coimmunoprecipitation and colocalization. In an established mouse model of renal ischemia-reperfusion injury characterized by apoptotic tubule cell death, the expression of both ankyrin and Fas was markedly induced, and the interaction between these molecules remained intact. The results identify a novel tethering interaction between ankyrin and Fas in kidney epithelia and suggest that AnkG190 may play a role as an adapter molecule in renal tubule cell death.Tethering interactions between membrane proteins and the underlying spectrin-based cytoskeleton play key roles in several cellular activities, including organization of plasma membrane domains (1

Induction of Zf9 in the kidney following early ischemia/reperfusion

“…These included an elevation in serum creatinine (2.5 ± 0.6 mg/dL in the 24-hour reflow animals versus 0.6 ± 0.3 mg/dL in the control group) (P < 0.05), loss of brush border membranes, tubular dilatation, flattened tubular epithelium, luminal debris, and an interstitial infiltrate [11,12,18]. Also evident were tubule epithelial cells that displayed the characteristic morphology of apoptotic nuclei as previously described [11,12,18], consisting of condensed, fragmented, and intensely staining nuclei. We have previously published the results of microarray analysis of RNA samples obtained from ischemic mouse kidneys after various periods of reflow [11].…”

“…We utilized a well-established mouse model of early renal I/R injury characterized by the presence of tubule cell apoptosis and necrosis [11,12,18]. The characteristic functional derangements and histopathologic features of ischemic injury were readily evident in the 24-hour reperfusion samples.…”

“…We utilized well-established murine models in which the structural and functional consequences of brief periods of renal ischemia have been previously documented [11,12,18]. Briefly, male Swiss-Webster mice (Taconic Farms, Germantown, NY, USA) weighing 25 to 30 g were housed with 12:12 hour light:dark cycle and were allowed free access to food and water.…”

Biomarkers of renal recovery after acute kidney injury

“…(4) Despite significant improvements in our understanding of the pathophysiology of acute kidney injury (AKI) (5-8) and management with multiple clinical treatments, including volume resuscitation, vasoconstrictor and vasodilator therapies, (9) and renal replacement therapy (RRT), (10) the incidence, severity and outcome of AKI have remained similar over recent years.…”

Ischaemia Reperfusion Injury in Kidney Transplantation

“…In kidney IRI receptors that are known to induce extrinsic apoptosis (termed 'death-receptors'), such as the Fas receptor, are up-regulated (Del Rio, Imam et al 2004). This signalling results in the activation of pro-caspase 8 via the formation of the death-inducing signalling complex (DISC).…”