Getting the “Inside” Scoop on EphrinB2 Signaling in Pericytes and the Effect on Peritubular Capillary Stability
Authors: David P. Basile, Mervin C. Yoder
Journal: Journal of the American Society of Nephrology (2013)

Progressive CKD develops from a lack of fidelity in tissue remodeling after acute injury. A lack of successful repair following even subtle injuries, regardless of their cause, suggests that complete resolution of function may be difficult to achieve, even if initiating deficits are corrected. This pathologic repair further burdens the adaptive capacity of the kidney and contributes to steady decline in function.1 Interstitial fibrosis is the commonly recognized histologic feature of this pathologic remodeling,1 which is present in all models of CKD, and is highly correlated with progression to ESRD. The last 10–15 years have witnessed the articulation of new hypotheses on the development of interstitial renal fibrosis. For example, it is now clear that all forms of CKD are associated with a reduction of peritubular capillaries, which results in exacerbation of hypoxia and the activation of molecular pathways associated with scar formation.2 The recognition that capillary rarefaction precedes fibrosis in models of AKI indicates that capillary loss is not solely attributable to encroaching fibrosis but rather may initiate or contribute to the fibrosis.2 Despite the presence of a hypoxic environment that should support angiogenesis, it is unclear why the kidney capillaries seem incapable of mounting an effective repair response. In addition to an increased interest in capillaries, several theories have been promulgated regarding the nature and origin of the myofibroblast, the primary matrix-producing kidney cell type driving scar formation. Several cell types probably contribute to the origin of these cells, such as resident fibroblasts, circulating fibrocytes, epithelial cells, and endothelial cells.3 Recent studies by Duffield's group have identified blood vessel pericytes as possible contributors to the myofibroblast population.4 Pericytes are vascular mural cells contained in the basement membrane of microvessels. Their role as a vascular stabilizing factor during developmental angiogenesis has been firmly established, and abnormal interactions between endothelial cells and pericytes are implicated in many human pathologic conditions.5 In response to injury, pericytes may lose contact with adjacent endothelial cells and escape from the vascular wall, leading to activation and differentiation into a myofibroblastic cell type.6 The focus on the pericyte in the kidney is intriguing not only because of its potential contribution to fibrosis but also because it allows for a novel perspective on capillary stability. Recent studies indicate that renal pericytes, activated in response to injury, lose their ability to stabilize capillaries in vitro, providing a possible mechanism for why renal vascular repair responses are not sustained.7 A more detailed understanding of the molecular interactions between pericytes and endothelial cells is an important goal to overcome obstacles related to renal revascularization. Several molecules mediating pericyte and endothelial cell-cell interactions have been identified, including members of the TGF-β family, angiopoietin/Tie-2 signaling, and Notch pathway activation.5 In addition, Eph receptor tyrosine kinase signaling is regulated by interaction with their cognate receptors, known as ephrins upon cell-cell contact. Mice with mutations in the ephrinB2/EphB4 pathway show developmental vascular defects.8 These molecules have also been associated with the development of the renal vasculature during embryogenesis.9 Given that repair processes after renal injury may recapitulate developmental paradigms,10 it is reasonable to hypothesize that ephrin/Eph activation may participate in renal vascular remodeling after injury. In this issue of JASN, Kida et al. investigated the role of ephrin signaling on capillary rarefaction and pericyte/endothelial interactions after AKI.11 The molecular expression of ephrin family members following ureteral obstruction (UUO) provided impetus to focus on the ephrinB2/Eph4 receptor pathway known to be important in embryonic vascular development. Of note, ephrinB2/Eph4 binding generates bidirectional signals that affect both Eph receptor–expressing and ephrin-expressing cells. The activation of signaling in ligand-producing cells is known as reverse signaling and, in the case of ephrinB2, can encompass both activation of tyrosine kinase activity and coordinating of other signaling mechanisms by a cytoplasmic PDZ moiety that has the potential to interact with multiple other signaling pathways.12 Targeted mutations of ephrinB2 that prevent tyrosine phosphorylation (referred to as 5Y mutations) or mutations that truncate the ephrinB2 cytoplasmic PDZ signaling domain (referred to as ΔV) allow the functional assessment of this reverse signaling pathway, while presumably maintaining ephrinB2 activation of Eph4 receptors. The authors used transgenic animals bearing these mutations with the UUO injury model. This model is reported to manifest an early but transient angiogenic response, which peaks at day 4 in wild-type animals. However, this angiogenic response is not sustained, and ultimately capillary rarefaction ensues. Mice bearing mutations in ephrinB2 reverse signaling (those lacking the PDZ signaling domain) manifested a less robust early angiogenic response and a more significant reduction in capillary density.11 Similarly, more severe vascular injury was also observed when these mice were subjected to ischemia/reperfusion-induced AKI, and subsequent development of fibrosis was more severe. Kidney pericytes isolated from ephrinB2 ΔV mice lacked the ability to stabilize capillary structures of cultured endothelial cells in vitro, whereas the addition of an exogenous inhibitor, soluble ephrinB2-Fc, did not. Thus, ephrin B2 reverse signaling was required for kidney pericytes to mediate capillary stabilization in these injury model systems. Several questions derive from these interesting observations. First, because the ephrinB2 ΔV mutant mice displayed the greatest deficits, how does PDZ signaling result in the stabilizing capillary effect provided by renal pericytes? The PDZ domain is important in modulating cell shape, as evidenced by increased motility and contraction of human umbilical vein endothelial cells overexpressing ephrinB2, but only if the PDZ domain is intact.13 However, little information is available regarding potential signaling partners. One potential interacting partner candidate, PDZ-RGS3, was recently shown to enhance Wnt signaling during the epithelial-mesenchymal transition process.14 A second important question is whether the endogenous ephrinB2 signaling becomes impaired after injury and contributes to the rarefaction seen in most models of CKD. Interestingly, this group previously demonstrated that injury-activated kidney pericytes lose their endothelial cell–stabilizing activity,7 but it is not known whether such activation impairs ephrinB2 signaling, leading to the capillary rarefaction, or whether ephrinB2 reverse signaling is activated but insufficient to mitigate the resultant capillary rarefaction. Should the exogenous overexpression of ephrin B2 reverse signaling restore the capillary stabilizing activity in activated pericytes, an exciting new approach toward vascular repair may ensue. Besides altered pericyte activity, other factors surely contribute to renal capillary rarefaction. For example, these investigators also suggest that proliferation of kidney endothelial cells is influenced by ephrinB2 reverse signaling because ephrin B2 ΔV mice showed less endothelial cells proliferation after injury relative to wild type, and kidney endothelial cells from these mice lacked VEGF responsiveness in vitro relative to control kidney cells. However, the degree to which kidney endothelial cells are capable of proliferating and mediating repair in response to AKI is not yet clear and is potentially controversial. Our laboratory has focused on the intrinsic capacity of renal capillary endothelial cells to participate in repair. Using repetitive BrdU administration, we determined that proliferation of rat kidney endothelial cells is extremely rare. Moreover, neither ischemia/reperfusion injury nor the exogenous administration of VEGF-121 significantly enhanced proliferation.15 In addition, sustained culture of renal-derived endothelial cells is typically difficult to attain. Compared with other endothelial cells cell types, kidney endothelial cells from rat grow slowly, have low colony-forming potential, and fail to form stable branched structures in vitro.16 In our hands, both rat kidney endothelial cells16 and mouse kidney endothelial cells (unpublished data) fail to proliferate in response to VEGF stimulation, despite a high level of VEGF-receptor expression. In the current study by Kida et al., although the proliferative and angiogenic response were reduced in the early phase of the UUO in ephrinB2 ΔV mice, the number of proliferating endothelial cells present even in wild type animals was modest. In this context, whether further reduction in the already nominal proliferation of kidney endothelial cells observed in ephrinB2 ΔV mice actually contributes to a greater degree of rarefaction is difficult to determine considering the multiple cell types being influenced by the mutations used in this study. In recent years, several strategies have been proposed to overcome the vascular defects that develop after acute injury or during progression of CKD. These include the administration of exogenous vascular growth factors15 or the use of hematopoietic derived proangiogenic cells,17 also called endothelial progenitor cells.18 In the kidney, these treatments may prevent, but have not been shown to reverse, capillary loss. The report provided by Kida et al. may help investigators shape appropriate questions in this area. The study provides a greater understanding of the intrinsic capacity of endothelial cells to undergo repair and the capacity of pericytes to stabilize nascent vascular cells. It also highlights the fact that the molecular interactions between these two cells types is an important determinant of the final structure generated in response to injury. By highlighting these points, the study is a welcome advance to an underappreciated problem. Disclosures None.