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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

Renal fibrosis and EndoMT in the respective groups. Significant fibrosis exclusively occurred after 4 weeks but not at 48 h postischemia (a). Administration of SAHA treated cells mediated anti-mesenchymal effects at week 4. b EndoMT at 48 h postischemia. Endothelial cells within small arteries/arterioles displayed increased expression of alpha-smooth muscle actin in all experimental groups, the strongest pro-mesenchymal transformation was observed in animals receiving temsirolimus-treated eEPCs. c EndoMT at week 4 postischemia. While temsirolimus-treated cells further aggravated EndoMT, administration of native and SAHA preconditioned eEPCs stabilized endothelial properties of the cells [data as mean ± SEM, ‘*’ indicates significant differences (p < 0.05) as compared to untreated controls, ‘#’ in a difference between ‘IRI’ and ‘+eEPCs+SAHA 4 w’; ‘#’ in b difference between ‘IRI’ and ‘+eEPCs+Tems’; ‘#’ in c differences as compared to ‘IRI’; ‘+’ in b difference between ‘+eEPCs’ and ‘+eEPCs+Tems’; ‘+’ in c difference between ‘IRI’ and ‘+eEPCs+Tems’; ‘##’ in b difference between ‘+eEPCs+SAHA’ and ‘+eEPCs+Tems’–for exact p-values see text]. EndoMT, mesenchymal transdifferentiation of endothelial cells. For other abbreviations, see previous figures
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Fig4: Renal fibrosis and EndoMT in the respective groups. Significant fibrosis exclusively occurred after 4 weeks but not at 48 h postischemia (a). Administration of SAHA treated cells mediated anti-mesenchymal effects at week 4. b EndoMT at 48 h postischemia. Endothelial cells within small arteries/arterioles displayed increased expression of alpha-smooth muscle actin in all experimental groups, the strongest pro-mesenchymal transformation was observed in animals receiving temsirolimus-treated eEPCs. c EndoMT at week 4 postischemia. While temsirolimus-treated cells further aggravated EndoMT, administration of native and SAHA preconditioned eEPCs stabilized endothelial properties of the cells [data as mean ± SEM, ‘*’ indicates significant differences (p < 0.05) as compared to untreated controls, ‘#’ in a difference between ‘IRI’ and ‘+eEPCs+SAHA 4 w’; ‘#’ in b difference between ‘IRI’ and ‘+eEPCs+Tems’; ‘#’ in c differences as compared to ‘IRI’; ‘+’ in b difference between ‘+eEPCs’ and ‘+eEPCs+Tems’; ‘+’ in c difference between ‘IRI’ and ‘+eEPCs+Tems’; ‘##’ in b difference between ‘+eEPCs+SAHA’ and ‘+eEPCs+Tems’–for exact p-values see text]. EndoMT, mesenchymal transdifferentiation of endothelial cells. For other abbreviations, see previous figures

Mentions: Endothelial-to-mesenchymal transition significantly contributes to tissue fibrosis in the kidney and in other organs such as the heart [19, 21, 22]. Fibrosis was analyzed by Masson trichrome staining, the results being expressed in relative units. At 48 h: Control 0.27 ± 0.06, IRI 0.33 ± 0.09, IRI+eEPCs 0.53 ± 0.18, IRI+eEPCs+SAHA 0.38 ± 0.13, IRI+eEPCs+temsirolimus 0.45 ± 0.17. The differences were not statistically significant at 48 h. Four weeks: IRI 2.9 ± 0.8, IRI+eEPCs 2.6 ± 0.23, IRI+eEPCs+SAHA 0.36 ± 0.05, IRI+eEPCs+temsirolimus 3.86 ± 0.73. The following differences were significant: Control vs. IRI (p < 0.0001) vs. IRI+eEPCs (p < 0.0001) vs. IRI+eEPCs+temsirolimus (p < 0.0001). Endothelial-to-mesenchymal transition was evaluated by tissue staining of αSMA and Cat D31. The results are given separately for 48 h and 4 weeks. At 48 h (αSMA in CD31+ cells in  %): Control 0.8 ± 0.28 %, IRI 2.14 ± 0.42 %, IRI+eEPCs 3.07 ± 0.49 %, IRI+eEPCs+SAHA 2.18 ± 0.51 %, IRI+eEPCs+temsirolimus 7.3 ± 0.86 %. The following differences were significant: Control vs. IRI (p = 0.017) vs. IRI+eEPCs (p = 0.0025) vs. IRI+eEPCs+SAHA (p = 0.0075) vs. IRI+eEPCs+temsirolimus (p < 0.0001); IRI+eEPCs+temsirolimus vs. IRI (p < 0.0001) vs. IRI+eEPCs (p = 0.003) vs. IRI+eEPCs+SAHA (p < 0.0001). At 4 weeks: IRI 4.7 ± 0.4 %, IRI+eEPCs 1.8 ± 0.6 %, IRI+eEPCs+SAHA 1.65 ± 0.4 %, IRI+eEPCs+temsirolimus 15.8 ± 2.5 %. The following differences were significant: Control vs. IRI (p < 0.0001) vs. IRI+eEPCs+temsirolimus (p < 0.0001); IRI vs. IRI+eEPCs (p = 0.0037) vs. IRI+eEPCs+SAHA (p = 0.005); IRI+eEPCs+temsirolimus vs. IRI (p < 0.0001) vs. IRI+eEPCs (p = 0.0014) vs. IRI+eEPCs+SAHA (p < 0.0001) (Fig. 4).Fig. 4


Endothelial autophagy and Endothelial-to-Mesenchymal Transition (EndoMT) in eEPC treatment of ischemic AKI
Renal fibrosis and EndoMT in the respective groups. Significant fibrosis exclusively occurred after 4 weeks but not at 48 h postischemia (a). Administration of SAHA treated cells mediated anti-mesenchymal effects at week 4. b EndoMT at 48 h postischemia. Endothelial cells within small arteries/arterioles displayed increased expression of alpha-smooth muscle actin in all experimental groups, the strongest pro-mesenchymal transformation was observed in animals receiving temsirolimus-treated eEPCs. c EndoMT at week 4 postischemia. While temsirolimus-treated cells further aggravated EndoMT, administration of native and SAHA preconditioned eEPCs stabilized endothelial properties of the cells [data as mean ± SEM, ‘*’ indicates significant differences (p < 0.05) as compared to untreated controls, ‘#’ in a difference between ‘IRI’ and ‘+eEPCs+SAHA 4 w’; ‘#’ in b difference between ‘IRI’ and ‘+eEPCs+Tems’; ‘#’ in c differences as compared to ‘IRI’; ‘+’ in b difference between ‘+eEPCs’ and ‘+eEPCs+Tems’; ‘+’ in c difference between ‘IRI’ and ‘+eEPCs+Tems’; ‘##’ in b difference between ‘+eEPCs+SAHA’ and ‘+eEPCs+Tems’–for exact p-values see text]. EndoMT, mesenchymal transdifferentiation of endothelial cells. For other abbreviations, see previous figures
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Fig4: Renal fibrosis and EndoMT in the respective groups. Significant fibrosis exclusively occurred after 4 weeks but not at 48 h postischemia (a). Administration of SAHA treated cells mediated anti-mesenchymal effects at week 4. b EndoMT at 48 h postischemia. Endothelial cells within small arteries/arterioles displayed increased expression of alpha-smooth muscle actin in all experimental groups, the strongest pro-mesenchymal transformation was observed in animals receiving temsirolimus-treated eEPCs. c EndoMT at week 4 postischemia. While temsirolimus-treated cells further aggravated EndoMT, administration of native and SAHA preconditioned eEPCs stabilized endothelial properties of the cells [data as mean ± SEM, ‘*’ indicates significant differences (p < 0.05) as compared to untreated controls, ‘#’ in a difference between ‘IRI’ and ‘+eEPCs+SAHA 4 w’; ‘#’ in b difference between ‘IRI’ and ‘+eEPCs+Tems’; ‘#’ in c differences as compared to ‘IRI’; ‘+’ in b difference between ‘+eEPCs’ and ‘+eEPCs+Tems’; ‘+’ in c difference between ‘IRI’ and ‘+eEPCs+Tems’; ‘##’ in b difference between ‘+eEPCs+SAHA’ and ‘+eEPCs+Tems’–for exact p-values see text]. EndoMT, mesenchymal transdifferentiation of endothelial cells. For other abbreviations, see previous figures
Mentions: Endothelial-to-mesenchymal transition significantly contributes to tissue fibrosis in the kidney and in other organs such as the heart [19, 21, 22]. Fibrosis was analyzed by Masson trichrome staining, the results being expressed in relative units. At 48 h: Control 0.27 ± 0.06, IRI 0.33 ± 0.09, IRI+eEPCs 0.53 ± 0.18, IRI+eEPCs+SAHA 0.38 ± 0.13, IRI+eEPCs+temsirolimus 0.45 ± 0.17. The differences were not statistically significant at 48 h. Four weeks: IRI 2.9 ± 0.8, IRI+eEPCs 2.6 ± 0.23, IRI+eEPCs+SAHA 0.36 ± 0.05, IRI+eEPCs+temsirolimus 3.86 ± 0.73. The following differences were significant: Control vs. IRI (p < 0.0001) vs. IRI+eEPCs (p < 0.0001) vs. IRI+eEPCs+temsirolimus (p < 0.0001). Endothelial-to-mesenchymal transition was evaluated by tissue staining of αSMA and Cat D31. The results are given separately for 48 h and 4 weeks. At 48 h (αSMA in CD31+ cells in  %): Control 0.8 ± 0.28 %, IRI 2.14 ± 0.42 %, IRI+eEPCs 3.07 ± 0.49 %, IRI+eEPCs+SAHA 2.18 ± 0.51 %, IRI+eEPCs+temsirolimus 7.3 ± 0.86 %. The following differences were significant: Control vs. IRI (p = 0.017) vs. IRI+eEPCs (p = 0.0025) vs. IRI+eEPCs+SAHA (p = 0.0075) vs. IRI+eEPCs+temsirolimus (p < 0.0001); IRI+eEPCs+temsirolimus vs. IRI (p < 0.0001) vs. IRI+eEPCs (p = 0.003) vs. IRI+eEPCs+SAHA (p < 0.0001). At 4 weeks: IRI 4.7 ± 0.4 %, IRI+eEPCs 1.8 ± 0.6 %, IRI+eEPCs+SAHA 1.65 ± 0.4 %, IRI+eEPCs+temsirolimus 15.8 ± 2.5 %. The following differences were significant: Control vs. IRI (p < 0.0001) vs. IRI+eEPCs+temsirolimus (p < 0.0001); IRI vs. IRI+eEPCs (p = 0.0037) vs. IRI+eEPCs+SAHA (p = 0.005); IRI+eEPCs+temsirolimus vs. IRI (p < 0.0001) vs. IRI+eEPCs (p = 0.0014) vs. IRI+eEPCs+SAHA (p < 0.0001) (Fig. 4).Fig. 4

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