Suppressed autophagic response underlies augmentation of renal ischemia/reperfusion injury by type 2 diabetes

Abstract

Diabetes mellitus is a major risk factor for acute kidney injury (AKI). Here, we hypothesized that suppression of autophagic response underlies aggravation of renal ischemia/reperfusion (I/R) injury by type 2 diabetes mellitus (T2DM). In OLETF, a rat model of T2DM, and its non-diabetic control, LETO, AKI was induced by unilateral nephrectomy and 30-min occlusion and 24-h reperfusion of the renal artery in the contralateral kidney. Levels of serum creatinine and blood urea nitrogen and tubular injury score after I/R were significantly higher in OLETF than in LETO. Administration of chloroquine, a widely used autophagy inhibitor, aggravated I/R-induced renal injury in LETO, but not in OLETF. In contrast to LETO, OLETF exhibited no increase in autophagosomes in the proximal tubules after I/R. Immunoblotting showed that I/R activated the AMPK/ULK1 pathway in LETO but not in OLETF, and mTORC1 activation after I/R was enhanced in OLETF. Treatment of OLETF with rapamycin, an mTORC1 inhibitor, partially restored autophagic activation in response to I/R and significantly attenuated I/R-induced renal injury. Collectively, these findings indicate that suppressed autophagic activation in proximal tubules by impaired AMPK/ULK1 signaling and upregulated mTORC1 activation underlies T2DM-induced worsening of renal I/R injury.

Figure 1

Type 2 diabetes and chloroquine increased susceptibility of the kidney to ischemia/reperfusion injury. Serum creatinine (A) and blood urea nitrogen (BUN) (B) levels of LETO (N = 12), OLETF (N = 12), LETO treated with chloroquine (LETO+CQ, N = 8), and OLETF treated with chloroquine (OLETF+CQ, N = 10) at 24 h after ischemia and reperfusion. (C) Representative images of hematoxylin-eosin staining of renal tissues at 24 h after ischemia/reperfusion. (D) Tubular injury score of renal tissues of before ischemia (Pre) and 24 h after ischemia/reperfusion (I/R) in the four groups. *P < 0.05. Scale bar, 100 μm. NS = not significant.

Figure 2

Autophagic response in proximal tubular cells after ischemia/reperfusion was attenuated in OLETF. (A) Representative immunofluorescence images of LC3 (green) and the proximal tubular marker lotus tetragonolobus lectin conjugated with Texas Red (LTL; red) in the kidneys of LETO, OLETF, and LETO treated with chloroquine (LETO+CQ). Nuclei were stained with Hoechst33342. Pre: pre-ischemia, I/R: 24 h after ischemia/reperfusion. (B) Summary data of LC3 dot numbers. N = 4 in each group. *P < 0.05, NS = not significant. Scale bar, 50 μm.

Figure 3

Activation of renal autophagy after ischemia/reperfusion was reduced in OLETF. (A) Representative immunoblot images for p62 and LC3 in the kidney. Summary data for p62 (B) and LC3-II/LC3-I ratio, LC3-II, and LC3-I (C) in total kidney tissues before ischemia (Pre) and 24 h after ischemia/reperfusion (I/R). Ratio of LC3-II to LC3-I after I/R was significantly reduced in OLETF. Representative immunoblots for beclin-1 (D) and summary data (E) in kidney tissues. Beclin-1 protein level was increased after I/R only in LETO. N = 8 in each group. *P < 0.05, NS = not significant.

Figure 4

Analysis of AMPK/ULK1 signal pathways. (A) Representative immunoblots for phospho-Thr172 and total AMPKα in renal tissues before ischemia and 24 h after reperfusion in LETO and OLETF. Pre: pre-ischemia, I/R: 24 h after ischemia/reperfusion. (B) Summary data for phospho-Thr172 AMPKα level normalized by β-actin. Phospho-AMPKα level was significantly increased after I/R in LETO but not in OLETF. (C) Representative blots for phospho-Ser555 and total ULK1. The blot of β-actin is identical to that in Fig. 3D because blots of both beclin-1 and ULK1 were from the same membrane. (D) Summary data for phospho-ULK1 level normalized by total ULK1. Phospho-ULK1 level was also elevated after I/R only in LETO. N = 8 in each group. (E) Representative blots for SIRT1 in renal tissues. (F) Summary data for SIRT1 protein level normalized by β-actin. In contrast to LETO, I/R failed to increase SIRT1 protein level in OLETF. N = 6 in each group. *P < 0.05. NS = not significant.

Figure 5

Analysis of Akt/mTORC1 signal pathways. Representative immunoblots (A) and summary data (B) for phospho-Thr389 and total p70S6 kinase (p70S6K), phospho-Ser235/236 and total S6, and actin in renal tissues before ischemia and 24 h after reperfusion in LETO and OLETF. Pre: pre-ischemia, I/R: 24 h after ischemia/reperfusion. (B) Before ischemia, phospho-p70S6K and phospho-S6 levels were reduced in OLETF. Levels of phospho-p70S6K and phospho-S6 were increased after I/R in both groups. The change in phospho-S6 level (ΔP-S6/T-S6) from the pre-ischemic period to 24 h after I/R was greater in OLETF than in LETO. (C) Representative immunoblots for phospho-Ser473 and total Akt. (D) Phospho-Akt level before ischemia was lower in OLETF than in LETO. I/R increased phospho-Akt levels in both OLETF and LETO, and phospho-Akt levels after I/R were similar in LETO and OLETF. N = 8 in each group. (E) Representative images of immunostaining for phospho-S6 in kidney sections before and 24 h after ischemia/reperfusion in LETO and OLETF. Scale bar, 100 μm. *P < 0.05.

Figure 6

Effects of rapamycin on kidney injury after ischemia/reperfusion in OLETF. Levels of serum creatinine (A) and blood urea nitrogen (BUN) (B) 24 h after renal ischemia/reperfusion in OLETF treated with a vehicle (N = 7) or rapamycin (Rapa, 0.25 mg/kg, N = 7) 30 min prior to ischemia in OLETF. (C) Representative images of hematoxylin-eosin staining of renal tissues at 24 h after ischemia/reperfusion. (D) Tubular injury score before ischemia and 24 h after reperfusion in OLETF treated with vehicle or rapamycin. Pre: pre-ischemia, I/R: 24 h after ischemia/reperfusion. *P < 0.05. Scale bar, 100 μm.

Figure 7

Restoration of autophagic response by rapamycin in OLETF. (A) Representative images of immunoblotting for phospho-Thr389 and total p70S6 kinase (p70S6K) and for phospho-Ser235/236 and total S6 of kidney tissues before ischemia and 24 h after reperfusion in OLETF pretreated with a vehicle (N = 7) or rapamycin (N = 7). Pre: pre-ischemia, I/R: 24 h after ischemia/reperfusion, Rapa; rapamycin-treated group. (B) I/R-induced increases in phospho-p70S6K and phospho-S6 levels were completely suppressed by rapamycin. (C) Representative immunofluorescence images of LC3 (green) and the proximal tubular marker lotus tetragonolobus lectin conjugated with Texas Red (LTL; red) in the kidneys from OLETF administered a vehicle or rapamycin. Nuclei were stained with Hoechst33342. (D) Summary data for LC3 dot level. (E) Representative blots for p62 and LC3. (F) Protein level of p62 after I/R was significantly reduced by rapamycin in OLETF. (G) LC3-II/LC3-I ratio after I/R was significantly increased by rapamycin, and the change was associated with a significant increase in LC3-II protein level. Scale bar, 50 μm. *P < 0.05.