Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018;14(1):53-65.
doi: 10.1080/15548627.2017.1391428.

Antioxidant role of autophagy in maintaining the integrity of glomerular capillaries

Jun Matsuda  1 , Tomoko Namba  1 , Yoshitsugu Takabatake  1 , Tomonori Kimura  1 , Atsushi Takahashi  1 , Takeshi Yamamoto  1 , Satoshi Minami  1 , Shinsuke Sakai  1 , Ryuta Fujimura  1 , Jun-Ya Kaimori  2 , Isao Matsui  1 , Takayuki Hamano  3 , Yoko Fukushima  4 , Keiko Matsui  5 , Tomoyoshi Soga  6 , Yoshitaka Isaka  1
Affiliations

Antioxidant role of autophagy in maintaining the integrity of glomerular capillaries

Jun Matsuda et al. Autophagy. 2018.

Abstract

Autophagy is a lysosomal degradation system by which cytosolic materials and damaged organelles are broken down into basic components. To explore the physiological role of autophagy in glomerular endothelial cells (GEnCs), we compared the autophagic flux among cells in the kidney under starvation. Inhibition of autophagy by chloroquine administration significantly increased the number of autophagosomes or autolysosomes in GEnCs and proximal tubular cells, but not in podocytes, suggesting that the GEnCs exhibit substantial autophagic activity. Next, we analyzed endothelial and hematopoietic cell-specific atg5-deficient mice (atg5-conditional KO [cKO] mice). Glomeruli of 4-wk-old atg5-cKO mice exhibited slightly distended capillary loops accompanied by an accumulation of reactive oxygen species (ROS). Glomeruli of 8-wk-old atg5-cKO mice showed a lobular pattern with thickening of the capillary loops and mesangial matrix expansion; however, the vasculature of other organs was preserved. The atg5-cKO mice died by 12 wk of age, presumably due to pancytopenia resulting from the defect in their hematopoietic lineages. Therefore, we subjected 4-wk atg5-cKO mice to irradiation followed by bone marrow transplantation from normal littermates. Transplanted mice recapitulated the glomerular phenotypes of the atg5-cKO mice with no obvious histological changes in other organs. Twelve-mo-old transplanted mice developed mesangiolysis and glomerulosclerosis with significant deterioration of kidney function. Administration of N-acetyl-l-cysteine, a ROS scavenger, to atg5-cKO mice rescued the glomerular phenotypes. These data suggest that endothelial autophagy protects glomeruli from oxidative stress and maintains the integrity of glomerular capillaries. Enhancing endothelial autophagy may provide a novel therapeutic approach to minimizing glomerular diseases.

Keywords: Atg5; autophagic flux; autophagy; glomerular endothelial cells; oxidative stress.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
GEnCs exhibit high autophagic activity. In vivo evaluation of the autophagic flux among the different cell lineages in the kidney during the fasted state. (A to C) Representative electron micrographs of GEnCs (A), PTECs (B), and podocytes (C) of 8-wk-old WT mice that were subjected to 24 h of starvation with or without chloroquine administration 6 h before euthanasia (n = 3 in each group). Arrows indicate autophagosomes or autolysosomes. The lower panels show a magnification of the indicated areas (white squares) in the upper panels. Bars: 1 μm. (D) Quantification of the number of autophagosomes or autolysosomes per cell among GEnCs, PTECs, or podocytes. For each condition, at least 10 cells were examined in each mouse. Data are provided as the mean ± SD. NS, not significant. Statistically significant differences (**P < 0.01 versus the conditions without chloroquine administration) are indicated.
Figure 2.
Figure 2.
Evidence of autophagy deficiency in the ECs of atg5-cKO mice. (A to C) SQSTM1 accumulation in kidney ECs of atg5-cKO mice. Representative images of the immunofluorescence analysis using 8-wk-old Atg5F/+;Tek-Cre+;EGFP-ChAT (upper) and atg5-cKO;EGFP-ChAT (lower) mice. Sections containing glomeruli (A), peritubular capillaries (B), and renal small arteries (C) were immunostained for SQSTM1 (red) and for 4’, 6-diamidino-2-phenylindole (DAPI; blue) as counterstaining. (A) The right panels show a magnification of the indicated areas (white squares) in the left panels. Arrowheads indicate SQSTM1-positive aggregates localized in GFP-positive ECs of atg5-cKO;EGFP-ChAT mice. Bars: 20 μm. (D) Western blot analysis using isolated glomeruli from 4-wk-old atg5-cKO mice and their Atg5-CTRL littermates. (E and F) Densitometric quantification of the levels of ATG5 protein (E) and the conversion of MAP1LC3B-I to MAP1LC3B-II (F). n = 3 in each group. Data are provided as the mean ± SD. Statistically significant differences (**P < 0.01 versus the Atg5-CTRL mice) are indicated.
Figure 3.
Figure 3.
Glomerular abnormalities of atg5-cKO mice. Representative images of PAS staining (A), electron micrographs (B), and immunohistochemical analysis for PECAM1 (C) of glomeruli from 4- and 8-wk-old atg5-cKO and Atg5-CTRL mice (n = 4 to 6 in each group). (A) Distended capillary loops (arrows). (B) Segmental loss of glomerular endothelial fenestra (arrowhead) accompanied by foot process effacement of podocytes (arrow) adjacent to the transformed ECs. The right panels show a magnification of the indicated areas (white squares) in the left panels. Bars: 20 μm (A and C) and 1 μm (B). Plasma UN and creatinine, urinary albumin/creatinine ratio (D), and blood pressure (E) of 4- (D) and 6- (E) wk-old atg5-cKO and Atg5-CTRL mice are shown. n = 6 for plasma UN and creatinine; n = 9 to 14 for urinary albumin/creatinine ratio; and n = 3 or 4 (E) in each group. Data are provided as the mean ± SD. NS, not significant. SBP, systolic blood pressure; DBP, diastolic blood pressure.
Figure 4.
Figure 4.
Accumulation of ROS in the glomeruli of atg5-cKO mice. (A) Representative images of the immunohistochemical analysis for dityrosine in kidney sections from 4- or 8-wk-old Atg5-CTRL (upper) and atg5-cKO (lower) mice. Small arteries are indicated with arrowheads. The lower panels show a magnification of the indicated areas (black squares) in the upper panels. Bars: 20 μm. (B) Western blot analysis for dityrosine, nitrotyrosine, and 4-HNE using isolated glomeruli homogenates from 8-wk-old atg5-cKO mice and their Atg5-CTRL littermates (n = 3 in each group).
Figure 5.
Figure 5.
Long-term effects of autophagy deficiency in GEnCs. The long-term effects of autophagy deficiency in GEnCs were investigated in the Atg5-CTRL and atg5-cKO mice rescued by BMT at 4 wk of age (Atg5-CTRL recipients and atg5-cKO recipients, respectively). The blood UN (A), urinary albumin:creatinine ratio (B), body weight (C), peripheral blood cell counts (D), Kaplan-Meier survival curves (E), representative images of PAS staining (F), quantification of the ratio of sclerotic glomeruli per total glomeruli (G), and representative images of Masson-trichrome staining (H), immunostaining for ADGRE1 (I), electron micrographs of glomeruli (J), immunostaining for dityrosine (K), blood pressure (L) of mice at the indicated ages are shown. (A and B) n = 3 for 6-mo-old Atg5-CTRL recipients; n = 22 to 25 for 12-mo-old Atg5-CTRL recipients; n = 4 or 5 for 6-mo-old atg5-cKO recipients; and n = 11 or 12 for 12-mo-old atg5-cKO recipients. (C and D) n = 14 to 16 for Atg5-CTRL recipients; and n = 7 or 8 for atg5-cKO recipients. (G) n = 14 for Atg5-CTRL recipients; and n = 7 for atg5-cKO recipients. (L) n = 5 to 7 for Atg5-CTRL and atg5-cKO recipients, respectively. Data are provided as the mean ± SD. NS, not significant. Statistically significant differences (*P < 0.05, **P < 0.01 versus the Atg5-CTRL recipients) are indicated. (H to K) The right panels show a magnification of the indicated areas (black or white squares) in the left panels. Bars: 20 μm (F, H, I, and K) and 1 μm (J). SBP, systolic blood pressure; DBP, diastolic blood pressure.
Figure 6.
Figure 6.
Endothelial-specific autophagy-deficient mice exhibit GEnC injury. Representative images of staining for ICAM1 (A) and colloidal iron (B) in the Atg5-CTRL and atg5-cKO mice at the indicated ages after rescue by BMT at 4 wk of age (Atg5-CTRL recipients and atg5-cKO recipients, respectively). Bars: 20 μm.
Figure 7.
Figure 7.
Administration of NAC alleviates the abnormalities of endothelial-specific autophagy-deficient mice. (A) Western blot analysis for nitrotyrosine, dityrosine, 4-HNE, and SQSTM1 using kidney cortex lysates from 8-wk-old atg5-cKO mice, atg5-cKO mice with NAC administration (atg5-cKO+NAC), and their Atg5-CTRL littermates. (B, D to H) Representative images of PAS staining (B), immunohistochemical analysis for PECAM1 (D), colloidal iron staining (E), immunohistochemical analysis for ICAM1 (F), electron micrographs (G), and peripheral blood cell counts (H) in the glomeruli of 8-wk-old atg5-cKO, atg5-cKO+NAC, and Atg5-CTRL mice. In (B), (E), and (G), the lower panels show a magnification of the indicated areas (black or white squares) in the upper panels. (C) For each condition, the diameter of the glomeruli was calculated from at least 20 glomeruli in each mouse. n = 3 (A, B, D to G); 4 (C); and 4 to 7 (H) in each group. Data are provided as the mean ± SD. NS, not significant. Statistically significant differences (*P < 0.05, **P < 0.01 versus the Atg5-CTRL or atg5-cKO+NAC mice) are indicated. Bars: 20 μm (B, D to F) and 1 μm (G).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Mizushima N, Levine B, Cuervo AM, Klionsky DJ. Autophagy fights disease through cellular self-digestion. Nature. 2008;451:1069–75. doi:10.1038/nature06639. PMID:18305538. - DOI - PMC - PubMed
    1. Kroemer G, Mariño G, Levine B. Autophagy and the integrated stress response. Mol Cell. 2010;40:280–93. doi:10.1016/j.molcel.2010.09.023. PMID:20965422. - DOI - PMC - PubMed
    1. Mizushima N, Komatsu M. Autophagy: renovation of cells and tissues. Cell. 2011;147:728–41. doi:10.1016/j.cell.2011.10.026. PMID:22078875. - DOI - PubMed
    1. Takabatake Y, Kimura T, Takahashi A, Isaka Y. Autophagy and the kidney: health and disease. Nephrol Dial Transplant. 2014;29:1639–47. doi:10.1093/ndt/gft535. PMID:24520117. - DOI - PubMed
    1. Guo F, Li X, Peng J, Tang Y, Yang Q, Liu L, Wang Z, Jiang Z, Xiao M, Ni C, et al. . Autophagy regulates vascular endothelial cell eNOS and ET-1 expression induced by laminar shear stress in an ex vivo perfused system. Ann Biomed Eng. 2014;42:1978–88. doi:10.1007/s10439-014-1033-5. PMID:24838486. - DOI - PubMed

Publication types

MeSH terms

Grants and funding

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (JP26893137 [to T.N.], JP24591196 and JP15K09260 [to Y.T.], and JP24659416 [to Y.I.]), the Takeda Medical Research Foundation (to Y.T.), and the Osaka Kidney Foundation (OKF13-0002) (to Y.T.).

LinkOut - more resources