H ₂ O ₂ activates G protein, α 12 to disrupt the junctional complex and enhance ischemia reperfusion injury

Abstract

The epithelial cell tight junction separates apical and basolateral domains and is essential for barrier function. Disruption of the tight junction is a hallmark of epithelial cell damage and can lead to end organ damage including renal failure. Herein, we identify Gα12 activation by H 2 O 2 leading to tight junction disruption and demonstrate a critical role for Gα12 activation during bilateral renal ischemia/reperfusion injury. Madin–Darby canine kidney (MDCK) cells with inducible Gα12 (Gα12-MDCK) and silenced Gα12 (shGα12-MDCK) were subjected to ATP depletion/repletion and H 2 O 2 /catalase as models of tight junction disruption and recovery by monitoring transepithelial resistance. In ATP depleted cells, barrier disruption and recovery was not affected by Gα12, but reassembly was accelerated by Gα12 depletion. In contrast, silencing of Gα12 completely protected cells from H 2 O 2 -stimulated barrier disruption, a response that rapidly occurred in control cells. H 2 O 2 activated Src and Rho, and Src inhibition (by PP2), but not Rho (by Y27632), protected cells from H 2 O 2 -mediated barrier disruption. Immunofluorescent and biochemical analysis showed that H 2 O 2 led to increased tyrosine phosphorylation of numerous proteins and altered membrane localization of tight junction proteins through Gα12/Src signaling pathway. Gα12 and Src were activated in vivo during ischemia/reperfusion injury, and transgenic mice with renal tubular QLα12 (activated mutant) expression were delayed in recovery and showed more extensive injury. Conversely, Gα12 knockout mice were nearly completely protected from ischemia/reperfusion injury. Taken together, these studies reveal that ROS stimulates Gα12 to activate injury pathways and identifies a therapeutic target for ameliorating ROS mediated injury.

Accelerated receptor shedding inhibits kidney injury molecule-1 (KIM-1)-mediated efferocytosis

“…4E). Although both H 2 O 2 and PV inhibited phagocytosis, it remained possible that this effect was independent of its effect on KIM-1 shedding (27,74,83,84). We demonstrate that the inhibitory effect of both H 2 O 2 and PV on efferocytosis was rescued by blocking KIM-1 shedding with TAPI-0 pretreatment of cells (Fig.…”

G-ProteinβγSubunit Dimers Modulate Kidney Repair after Ischemia-Reperfusion Injury in Rats

“…More recently, the importance of Ga subunits in the recovery of the kidney after ischemic injury was documented in a study by Yu et al (2012). In their report, constitutively active QLa12 mutant transgenic mice were observed to have attenuated recovery after renal IRI with more severe tubular injury compared with the wild-type Ga12 mice.…”

“…Prior studies have demonstrated activation of Ga in models of AKI. For example, pharmacologic agonists of the adenosine A2A GPCR (Yap and Lee, 2012) or genetic modulation of Ga12 subunit function (Yu et al, 2012) have been shown to alter the severity of AKI in animal models. However, these studies primarily focused on signaling via Ga subunits through biased signaling cascades activated by upstream GPCRs.…”

Rho GTPases, oxidation, and cell redox control

“…They found a ~1.75-fold increase in RhoA-GTP levels that peaked approximately 5 min post stimulation. 137 Likewise, in the human lung adenocarcinoma epithelial cell line A549, H 2 O 2 was shown to activate RhoA by ~2.5-fold after 15 min, which was concomitant with stress fiber formation. 138 As the mechanism of RhoA activation was not investigated in any of these studies, it is unclear whether RhoA activity is regulated directly by oxidants or indirectly through the redox-sensitive LMW-PTP, another GAP, GEF, or GDI.…”