PART Ⅱ: Key role for EphB2 receptor in renal fibrosis
Mar 21, 2022
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Zhimin Huang, Simeng Liu, Anna Tang, Laith Al-Rabadi, Mark Henkemeyer, Patrice N. Mimche and Yufeng Huang
Erythropoietin producing hepatocellular (Eph)-Eph receptor-interacting (Ephrin)receptor-ligand signaling has been implicated in the development of tissue fibrosis, though it has not been well defined in the kidney. We detected substantial up-regulation of expression and phosphorylation of the EphB2 receptor tyrosine kinase in fibrotic kidney tissue obtained both from mice subjected to unilateral renal ischemia-reperfusion (IR)model at 14 days and in patients suffering from chronic kidney disease (CKD). Knockout (KO) mice lacking EphB2 expression exhibited a normal renal structure and function, indicating no major role for this receptor in kidney development or action. Although IR injury is well-known to cause tissue damage, fibrosis, and renal dysfunction, we found that kidneys from EphB2 KO mice showed much less renal tubular injury and retained a more preserved renal function. IR-injured kidneys from EphB2 KOs exhibited greatly reduced fibrosis and inflammation compared with injured wildtype (WT) littermates, and this correlated with a significant reduction in renal expression of profibrotic molecules, inflammatory cytokines, NADPH oxidases, and markers for cell proliferation, tubular epithelial-to-mesenchymal transition (EMT), myofibroblast activation, and apoptosis. A panel of 760 fibrosis-associated genes was further assessed, revealing that 506 genes in WT mouse kidneys following IR injury changed their expression. However, 70.9% of those genes were back to or close to normal in expression when EphB2 was deleted. These data indicate that endogenous EphB2 expression and signaling are abnormally activated after kidney injury and subsequently contribute to the development of renal fibrosis via the regulation of multiple profibrotic pathways.
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Discussion
In the present study, we used the unilateral renal IR mouse model that is well established for studying the transition of renal actual kidney disease(AKI) to CKD [31,32]. Features of an extensive inflammatory response, renal tubular epithelial cell apoptosis, and fibrosis were observed while EphB2 was up-regulated and activated at the site of IR injury in mouse kidneys. Importantly, up-regulation and activation of EphB2 are consistently found in diabetes or hypertension-induced CKDmouse models and the human biopsy tissues showing glomerulosclerosis and interstitial fibrosis. These observations are consistent with ERCB data in the publicly available kidney transcriptome database, Nephroseg. mRNA expression levels of EphB2 in kidneys procured from patients with FSGS were significantly higher than those in healthy living donors (www.nephroseg.org). Collectively, these data suggest that EphB2 up-regulation/activation may be a basic response to chronic injury-induced renal fibrosis, regardless of the initiating agent or disorder. Furthermore, EphB2 KO attenuates renal inflammation, and fibrosis induced by IR injury in mice, and this amelioration is correlated with an overall reduction in profibrotic markers and inflammatory cytokines, oxidative stress, and tubular cell death. Our results indicate that EphB2 signaling is crucial in kidney injury and potentially contributes to the development of renal fibrosis. Our observation using the NanoString gene expression demonstrating that the majority of molecules involved in renal fibrosis are regulated by or related to EphB2 further supports its role as a critical driver of kidney damage following IR injury.
The mechanistic pathways involved in injury-induced EphB2 signaling and activation have not been thoroughly investigated. We postulate that injury-induced increased ligands such as EphrinB1 and EphrinB2 as we observed in the IR-injured mouse kidneys and diabetes or hypertension-induced CKDmouse models may activate the EphB2 receptor via enhanced interaction of Eph-Ephrin.In addition, a few studies have shown that activation of the Notch signaling pathway regulates EphB2 through the glycogen synthase kinase 1(GSK)3β- mediated suppression of Wnt-/β-catenin signaling in intestinal epithelial cells both in vitro and in vivo in various gene-manipulated mouse models [33,34]. Whether this pathway also regulates EphB2 in the kidney during injury is not investigated yet. Interestingly, GSK-3β has been recently implicated in the pathogenesis of kidney injury [35]. On the other hand, it is likely that up-regulation/activation of EphB2 upon kidney injury may aggravate kidney cell injury, death, and EMT thereby causing renal fibrosis via multiple pathways, including potentiation of NF-kB-mediated inflammation response, activation of TGFβ1/Smad3-mediated EMT, and other pathways, as indicated by the NanoString gene expression and verified by the signaling assessment in the present study.
As the kidney is a highly metabolic organ and uses high levels of adenosine triphosphate (ATP) to maintain electrolyte and acid-base homeostasis and reabsorb nutrients, energy depletion is a critical factor in the transition of renal AKI to CKD, even CKD to ESRD [36]. Although we did not measure renal ATP levels after IR injury in WT and EphB2 KO mice, our brief observation using the NanoString gene expression in this model provides the first evidence that the EphB2 signaling may be involved in/or mediate renal tubular cell mitochondrial energy metabolism. Deletion of EphB2 signaling appears to largely prevent cellular energy damage thereby ameliorating cellular apoptosis and kidney fibrosis. These brief observations may suggest a new possible paradigm of EphB2 signaling in the pathogenesis of kidney fibrosis via induction of cellular energy metabolic dysfunction, especially tubular cell energy metabolic dysfunction, in addition to the injurious effects of EphB2 signaling on the inflammation and EMT. The metabolic pathway is also important and needs to be determined.
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In the kidney, EphB2 is normally expressed in renal tubules [19]. Upon injuries, increased EphB2 was observed not only in proximal renal tubules but also in glomerular cells as we observed in kidney biopsy tissues from CKD patients. In IR-injured mouse kidneys, it is understandable that increased EphB2 is mainly observed in renal tubules since this injury mainly causes renal tubulointerstitial fibrosis. Of note, the process of kidney fibrosis involves multiple kidney cells, including renal tubular epithelial cells, interstitial fibroblasts, vascular ECs, and inflammation cells. In fact, besides being expressed on renal tubular cells and fibroblasts, EphB2 is also expressed in most cellular players of the immune system including monocytes/macrophages, dendritic cells, and B cells [37]. More research is underway to clearly delineate and understand the cell-specific function of the Eph-Ephrin signaling axis during renal inflammation and fibrosis.
Different from other members of receptor tyrosine kinases, the interaction of Eph-Ephrin activates bidirectional signaling cascades that are propagated into the receptor-expressing cell (forward signaling) and the ligand-expressing cell(reverse signaling)[3-5]. Recently, Ephrin-B2 reverse-signaling in blood vessels/capillaries was shown to be antifibrotic in the kidney after injury induced by UUO or IR since mice lacking the PDZ intracellular signaling domain of Ephrin-B2exhibit increased kidney fibrosis[21]. However, this study cannot dissect whether the increased fibrosis is due to the PDZ-dead Ephrin-B2 protein acting as a stronger ligand to stimulate EphB receptors forward signaling. Similarly, the EphB2 deficiency data in the present study will not be able to dissect whether amelioration of renal fibrosis is related to abrogation of reverse signaling through Ephrin ligands. Despite the importance of EphB signal-ing in renal fibrosis found in our work, a further thorough understanding of how the EphB2/Ephrin signaling axis promotes renal fibrosis will be clearly warranted in the future.
In summary, the present study shows a direct involvement of EphB2 signaling in the pathogenesis of renal fibrosis using the IR-induced renal fibrosis model in rodents. We also include our preliminary observations that EphB2 is markedly elevated in kidney biopsy tissue in patients with CKD, indicating its potential clinical importance. These findings suggest a novel and important link between up-regulation of EphB2 signaling and renal fibrosis and may open new avenues for targeting this receptor tyrosine kinase for the treatment of renal fibrosis.
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