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

View Article: PubMed Central - PubMed

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)-β ± SAHA, autophagy (perinuclear LC3-II), and stress-induced premature senescence (SIPS—senescence-associated β-galactosidase, SA-β-Gal), and were evaluated 96 h later.

Results: Cultured eEPCs showed reduced perinuclear density of LC3-II + vesicles and elevated levels of SA-β-Gal after treatment with TGF-β 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 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 alpha-smooth muscle actin (αSMA) 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.

No MeSH data available.


Related in: MedlinePlus

Intrarenal endothelial autophagy after IRI with versuswithout systemic administration of unconditioned or preconditioned eEPCs. a Percentages of endothelial p62 in the short-term (48 h). Endothelial presence of p62 increased after IRI in all experimental groups. Administration of unconditioned and preconditioned eEPCs further elevated p62 with a peak in the ‘eEPC + SAHA’-group. b Endothelial p62 at week 4 after IRI. c–j p62 (green) in the endothelium of a small artery (CD31—red; c–e control, f–h post-IRI at 48 h). Images i and j magnify the endothelial layer. The white ovals in i and j surround the endothelium in areas of increased p62 presence (Tems: temsirolimus; magnification in C–H ×20, in I and J ≈ ×40; Data in A and B are mean ± SEM, ‘*’ indicates significant differences (p < 0.05) as compared to untreated controls, ‘#’ indicates significant differences as compared to the ‘IRI’-group, ‘+’ indicates the difference between ‘+eEOCs’ and ‘+ eEOCs + SAHA’—for exact p-values see text). IRI, ischemia–reperfusion injury; eEOCs, early endothelial outgrowth cells. For other abbreviations, see previous figure (color figure online)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5016542&req=5

Fig2: Intrarenal endothelial autophagy after IRI with versuswithout systemic administration of unconditioned or preconditioned eEPCs. a Percentages of endothelial p62 in the short-term (48 h). Endothelial presence of p62 increased after IRI in all experimental groups. Administration of unconditioned and preconditioned eEPCs further elevated p62 with a peak in the ‘eEPC + SAHA’-group. b Endothelial p62 at week 4 after IRI. c–j p62 (green) in the endothelium of a small artery (CD31—red; c–e control, f–h post-IRI at 48 h). Images i and j magnify the endothelial layer. The white ovals in i and j surround the endothelium in areas of increased p62 presence (Tems: temsirolimus; magnification in C–H ×20, in I and J ≈ ×40; Data in A and B are mean ± SEM, ‘*’ indicates significant differences (p < 0.05) as compared to untreated controls, ‘#’ indicates significant differences as compared to the ‘IRI’-group, ‘+’ indicates the difference between ‘+eEOCs’ and ‘+ eEOCs + SAHA’—for exact p-values see text). IRI, ischemia–reperfusion injury; eEOCs, early endothelial outgrowth cells. For other abbreviations, see previous figure (color figure online)

Mentions: Next, we aimed to analyze whether systemic administration of preconditioned eEPCs to postischemic mice would modulate autophagy in mature endothelial cells of smaller arteries/arterioles. Cell preconditioning was performed using two different autophagy inducing agents, the substance SAHA and the mammalian target of rapamycin (mTOR) inhibitor temsirolimus. Endothelial autophagy was measured by staining of p62 in CD31+ cells, and analyzed at 48 h and 4 weeks postischemia. The results shall be given separately for 48 h and 4 weeks. At 48 h (p62 in CD31+ cells in  %): Control 4.5 ± 1.1 %, IRI 13.1 ± 2.5 %, IRI+eEPCs 18.6 ± 2.7 %, IRI+eEPCs+SAHA 34.5 ± 7.6 %, IRI+eEPCs+temsirolimus 18 ± 2.8 %. The following differences were significant: Control vs. IRI (p = 0.0051) vs. IRI+eEPCs (p = 0.001) vs. IRI+eEPCs+SAHA (p < 0.0001) vs. IRI+eEPCs+temsirolimus (p < 0.0001); IRI vs. IRI+eEPCs (p = 0.028) vs. IRI+eEPCs+SAHA (p = 0.0032) vs. IRI+eEPCs+temsirolimus (p = 0.047); IRI+eEPCs vs. IRI+eEPCs+SAHA (p = 0.033). At 4 weeks: IRI 13.4 ± 1.7 %, IRI+eEPCs 21 ± 2.3 %, IRI+eEPCs+SAHA 27.3 ± 2.4 %, IRI+eEPCs+temsirolimus 18.5 ± 3.2 %. The following differences were significant: Control vs. IRI (p = 0.0017) vs. IRI+eEPCs (p = 0.0003) vs. IRI+eEPCs+SAHA (p < 0.0001) vs. IRI+eEPCs+temsirolimus (p = 0.00017); IRI vs. IRI+eEPCs (p = 0.023) vs. IRI+eEPCs+SAHA (p = 0.0029) (Fig. 2).Fig. 2


Endothelial autophagy and Endothelial-to-Mesenchymal Transition (EndoMT) in eEPC treatment of ischemic AKI
Intrarenal endothelial autophagy after IRI with versuswithout systemic administration of unconditioned or preconditioned eEPCs. a Percentages of endothelial p62 in the short-term (48 h). Endothelial presence of p62 increased after IRI in all experimental groups. Administration of unconditioned and preconditioned eEPCs further elevated p62 with a peak in the ‘eEPC + SAHA’-group. b Endothelial p62 at week 4 after IRI. c–j p62 (green) in the endothelium of a small artery (CD31—red; c–e control, f–h post-IRI at 48 h). Images i and j magnify the endothelial layer. The white ovals in i and j surround the endothelium in areas of increased p62 presence (Tems: temsirolimus; magnification in C–H ×20, in I and J ≈ ×40; Data in A and B are mean ± SEM, ‘*’ indicates significant differences (p < 0.05) as compared to untreated controls, ‘#’ indicates significant differences as compared to the ‘IRI’-group, ‘+’ indicates the difference between ‘+eEOCs’ and ‘+ eEOCs + SAHA’—for exact p-values see text). IRI, ischemia–reperfusion injury; eEOCs, early endothelial outgrowth cells. For other abbreviations, see previous figure (color figure online)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016542&req=5

Fig2: Intrarenal endothelial autophagy after IRI with versuswithout systemic administration of unconditioned or preconditioned eEPCs. a Percentages of endothelial p62 in the short-term (48 h). Endothelial presence of p62 increased after IRI in all experimental groups. Administration of unconditioned and preconditioned eEPCs further elevated p62 with a peak in the ‘eEPC + SAHA’-group. b Endothelial p62 at week 4 after IRI. c–j p62 (green) in the endothelium of a small artery (CD31—red; c–e control, f–h post-IRI at 48 h). Images i and j magnify the endothelial layer. The white ovals in i and j surround the endothelium in areas of increased p62 presence (Tems: temsirolimus; magnification in C–H ×20, in I and J ≈ ×40; Data in A and B are mean ± SEM, ‘*’ indicates significant differences (p < 0.05) as compared to untreated controls, ‘#’ indicates significant differences as compared to the ‘IRI’-group, ‘+’ indicates the difference between ‘+eEOCs’ and ‘+ eEOCs + SAHA’—for exact p-values see text). IRI, ischemia–reperfusion injury; eEOCs, early endothelial outgrowth cells. For other abbreviations, see previous figure (color figure online)
Mentions: Next, we aimed to analyze whether systemic administration of preconditioned eEPCs to postischemic mice would modulate autophagy in mature endothelial cells of smaller arteries/arterioles. Cell preconditioning was performed using two different autophagy inducing agents, the substance SAHA and the mammalian target of rapamycin (mTOR) inhibitor temsirolimus. Endothelial autophagy was measured by staining of p62 in CD31+ cells, and analyzed at 48 h and 4 weeks postischemia. The results shall be given separately for 48 h and 4 weeks. At 48 h (p62 in CD31+ cells in  %): Control 4.5 ± 1.1 %, IRI 13.1 ± 2.5 %, IRI+eEPCs 18.6 ± 2.7 %, IRI+eEPCs+SAHA 34.5 ± 7.6 %, IRI+eEPCs+temsirolimus 18 ± 2.8 %. The following differences were significant: Control vs. IRI (p = 0.0051) vs. IRI+eEPCs (p = 0.001) vs. IRI+eEPCs+SAHA (p < 0.0001) vs. IRI+eEPCs+temsirolimus (p < 0.0001); IRI vs. IRI+eEPCs (p = 0.028) vs. IRI+eEPCs+SAHA (p = 0.0032) vs. IRI+eEPCs+temsirolimus (p = 0.047); IRI+eEPCs vs. IRI+eEPCs+SAHA (p = 0.033). At 4 weeks: IRI 13.4 ± 1.7 %, IRI+eEPCs 21 ± 2.3 %, IRI+eEPCs+SAHA 27.3 ± 2.4 %, IRI+eEPCs+temsirolimus 18.5 ± 3.2 %. The following differences were significant: Control vs. IRI (p = 0.0017) vs. IRI+eEPCs (p = 0.0003) vs. IRI+eEPCs+SAHA (p < 0.0001) vs. IRI+eEPCs+temsirolimus (p = 0.00017); IRI vs. IRI+eEPCs (p = 0.023) vs. IRI+eEPCs+SAHA (p = 0.0029) (Fig. 2).Fig. 2

View Article: PubMed Central - PubMed

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&nbsp;N mice were subjected to bilateral renal ischemia (40&nbsp;min). Animals were injected with either untreated, or SAHA- or temsirolimus-pretreated syngeneic murine eEPCs at the time of reperfusion. Mice were analyzed 48&nbsp;h and 4&nbsp;weeks later. In addition, cultured eEPCs were treated with transforming growth factor (TGF)-&beta;&nbsp;&plusmn;&nbsp;SAHA, autophagy (perinuclear LC3-II), and stress-induced premature senescence (SIPS&mdash;senescence-associated &beta;-galactosidase, SA-&beta;-Gal), and were evaluated 96&nbsp;h later.

Results: Cultured eEPCs showed reduced perinuclear density of LC3-II&nbsp;+&nbsp;vesicles and elevated levels of SA-&beta;-Gal after treatment with TGF-&beta; 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 intrarenal endothelial p62 at 48&nbsp;h and 4&nbsp;weeks, indicating stimulated endothelial autophagy. This effect was most pronounced after injection of SAHA-treated eEPCs. At 4&nbsp;weeks endothelial expression of mesenchymal alpha-smooth muscle actin (&alpha;SMA) 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.

No MeSH data available.


Related in: MedlinePlus