Endothelial autophagy and Endothelial-to-Mesenchymal Transition (EndoMT) in eEPC treatment of ischemic AKI (pdf)

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Endothelial autophagy and Endothelial-to-Mesenchymal Transition (EndoMT) in eEPC treatment of ischemic AKI

J Nephrol DOI 10.1007/s40620-015-0222-0 ORIGINAL ARTICLE Endothelial autophagy and Endothelial-to-Mesenchymal Transition (EndoMT) in eEPC treatment of ischemic AKI Daniel Patschan1 • Katrin Schwarze1 • Elvira Henze1 • Susann Patschan1 • Gerhard Anton Müller1 Received: 19 May 2015 / Accepted: 24 July 2015 Ó The Author(s) 2015. This article is published with open access at Springerlink.com Abstract Background Autophagy enables cells to digest endogenous/exogenous waste products, thus potentially prolonging the cellular lifespan. Early endothelial progenitor cells (eEPCs) protect mice from ischemic acute kidney injury (AKI). The mid-term prognosis in AKI critically depends on vascular rarefication and interstitial fibrosis with the latter partly being induced by mesenchymal transdifferentiation of endothelial cells (EndoMT). This study aimed to determine the impact of eEPC preconditioning with different autophagy inducing agents [suberoylanilide hydroxamic acid (SAHA)/temsirolimus] in ischemic AKI. Methods Male C57/Bl6 N mice were subjected to bilateral renal ischemia (40 min). Animals were injected with either untreated, or SAHA- or temsirolimus-pretreated syngeneic murine eEPCs at the time of reperfusion. Mice were analyzed 48 h and 4 weeks later. In addition, cultured eEPCs were treated with transforming growth factor (TGF)-b ± SAHA, autophagy (perinuclear LC3-II), and stress-induced premature senescence (SIPS—senescenceassociated b-galactosidase, SA-b-Gal), and were evaluated 96 h later. Results Cultured eEPCs showed reduced perinuclear density of LC3-II ? vesicles and elevated levels of SA-bGal after treatment with TGF-b alone, indicating impaired autophagy and aggravated SIPS. These effects were completely abrogated by SAHA. Systemic administration of either SAHA or tems pretreated eEPCs resulted in elevated & Daniel Patschan 1 Clinic of Nephrology and Rheumatology, University Medical Center of Göttingen, Robert-Koch-Straße 40, 37075 Göttingen, Germany intrarenal endothelial p62 at 48 h and 4 weeks, indicating stimulated endothelial autophagy. This effect was most pronounced after injection of SAHA-treated eEPCs. At 4 weeks endothelial expression of mesenchymal alphasmooth muscle actin (aSMA) was reduced in animals receiving untreated and SAHA-pretreated cells. In addition, SAHA-treated cells reduced fibrosis at week 4. Tems in contrast aggravated EndoMT. Postischemic renal function declined after renal ischemia and remained unaffected in all experimental cell treatment groups. Conclusion In ischemic AKI, intrarenal endothelial autophagy may be stabilized by systemic administration of pharmacologically preconditioned eEPCs. Early EPCs can reduce postischemic EndoMT and fibrosis in the mid-term. Autophagy induction in eEPCs either increases or decreases the mesenchymal properties of intrarenal endothelial cells, depending on the substance being used. Thus, endothelial autophagy induction in ischemic AKI, mediated by eEPCs is not a renoprotective event per se. Keywords EndoMT AKI Endothelial autophagy EPCs Introduction Early endothelial progenitor (or outgrowth) cells (eEPCs/ eEOCs) have been proven as an effective therapeutic tool in murine ischemic acute kidney injury (AKI) [1, 2]. As opposed to so-called late EPCs (lEPCs), early EPCs predominantly act by indirect mechanisms (e.g., modulation of the perivascular milieu by producing proangiogenic substances and/or by releasing vasoprotective microparticles, enriched by certain micro-RNA molecules). In the current literature, lEPCs are defined as true progenitors of 123 J Nephrol endothelial cells whereas eEPCs are classified as ‘hematopoietic cells with proangiogenic activity’. Nevertheless, early EPCs have successfully been used to treat ischemic diseases under diverse experimental conditions (e.g., ischemic heart and cerebrovascular disease). In 2006, the cells were shown to promote post-AKI repair as well. Meanwhile, a significant number of pharmacological strategies have been established, helpful to increase renoprotective competence of eEPCs in ischemic AKI [3–7]. The microenvironmental alterations associated with ischemia can significantly aggravate renal damage since ischemia does not only induce tubular malfunction/damage but also interstitial inflammation and severe microvasculopathy [8]. The latter causes ongoing ischemia even if the initial cause of hypoperfusion has been eliminated. Both postischemic interstitial inflammation and microvasculopathy significantly result from alterations of the paracrinic milieu. The proximal tubule releases numerous factors including tumor necrosis factor (TNF)-a, interleukin (IL)-6, IL-1b, TGF-b, monocyte chemoattractant protein-1 [MCP-1], IL-8, and RANTES [9–11]. In such a context, mature endothelial cells undergo aggravated senescence or stress-induced premature senescence (SIPS) [12]. eEPCs, after invading the kidney via the renal artery, are most likely also altered by deleterious effects of diverse humoral factors and it can be argued that SIPS decreases the anti-ischemic competence of eEPCs in AKI. A few years ago, studies by the group of Goligorsky elegantly showed a dynamic cascade of hyperglycemia-induced inhibition of endothelial autophagy, followed by increased endothelial SIPS [13]. Autophagy is widely regarded as an endogenous defense mechanism against numerous endogenous and exogenous stressors. The role of autophagy in modulating the ‘renal injury-response’ has especially been analyzed in the tubular compartment. Periyasamy-Thandavan and colleagues identified autophagy as a cytoprotective mechanism in cisplatin-induced damage of proximal tubular epithelial cells [14]. Another study showed autophagy to have a protective role during in vitro hypoxia and in vivo ischemia–reperfusion injury (IRI) [15]. Finally, Quercetin-mediated attenuation of renal IRI was shown to critically depend on autophagy activation in an AMP-activated protein kinase-dependent manner [16]. Therefore one may conclude that stimulated (tubular) autophagy generally increases tissue resistance against toxic/ischemic damage. Nevertheless, de facto no study has so far evaluated the role of endothelial autophagy in AKI. Thus, the aim of our study was to analyze whether stabilization of autophagy in therapeutically administered eEPCs increases renoprotective cell competence in murine ischemic AKI. 123 Methods Animal models The animal study protocol was in accordance with the guidelines of the German Institute of Health Guide for the Care and Use of Laboratory Animals and ap-proved by the Institutional Animal Care and Use Committee. C57BL/6 N mice were originally obtained from Jackson Labs (Bar Harbor, ME, USA) and bred in the local animal facility of the Göttingen University Hospital. As in previous studies, male 8-12 week-old C57Bl/6 N mice were used in all experiments. All animals were separately caged with a 12:12-h light–dark cycle and had free access to water and (...truncated)