Persistent activation of autophagy in kidney tubular cells promotes renal interstitial fibrosis during unilateral ureteral obstruction - PubMed (original) (raw)
Persistent activation of autophagy in kidney tubular cells promotes renal interstitial fibrosis during unilateral ureteral obstruction
Man J Livingston et al. Autophagy. 2016.
Abstract
Renal fibrosis is the final, common pathway of end-stage renal disease. Whether and how autophagy contributes to renal fibrosis remains unclear. Here we first detected persistent autophagy in kidney proximal tubules in the renal fibrosis model of unilateral ureteral obstruction (UUO) in mice. UUO-associated fibrosis was suppressed by pharmacological inhibitors of autophagy and also by kidney proximal tubule-specific knockout of autophagy-related 7 (PT-Atg7 KO). Consistently, proliferation and activation of fibroblasts, as indicated by the expression of ACTA2/α-smooth muscle actin and VIM (vimentin), was inhibited in PT-Atg7 KO mice, so was the accumulation of extracellular matrix components including FN1 (fibronectin 1) and collagen fibrils. Tubular atrophy, apoptosis, nephron loss, and interstitial macrophage infiltration were all inhibited in these mice. Moreover, these mice showed a specific suppression of the expression of a profibrotic factor FGF2 (fibroblast growth factor 2). In vitro, TGFB1 (transforming growth factor β 1) induced autophagy, apoptosis, and FN1 accumulation in primary proximal tubular cells. Inhibition of autophagy suppressed FN1 accumulation and apoptosis, while enhancement of autophagy increased TGFB1-induced-cell death. These results suggest that persistent activation of autophagy in kidney proximal tubules promotes renal interstitial fibrosis during UUO. The profibrotic function of autophagy is related to the regulation on tubular cell death, interstitial inflammation, and the production of profibrotic factors.
Keywords: Autophagy; kidney injury; proximal tubule; renal fibrosis; unilateral ureteral obstruction.
Figures
Figure 1.
Autophagy is persistently induced in renal proximal tubules during UUO. C57Bl/6 (A, B, F, G and H) or CAG-RFP-GFP-LC3 (C, D and E) mice were subjected to either sham operation or UUO surgery. The mice were sacrificed at the indicated time points (2 days, 4 d, 1 wk and 2 wk) and left kidneys were collected for histological and immunoblot analyses. (A) Representative images of immunohistochemical staining of LC3B. Occasionally, there was LC3B-positive staining in nuclei; however, no LC3B-positive staining was observed in either sham control or UUO kidney tissues in the absence of primary antibody (data not shown), suggesting the specificity of this LC3B staining. Scale bar: 20 µm. (B) Quantitative analysis of punctate LC3B staining. Data are expressed as mean ± SD. *, P < 0.05, significantly different from the sham group. (C) Representative images of GFP-LC3 and RFP-LC3 fluorescence staining. Scale bar: 15 µm. (D) Quantitative analysis of GFP-LC3 and RFP-LC3 puncta. Data are expressed as mean ± SD. *, P < 0.05, significantly different from the sham group; #, P < 0.05, values of RFP-LC3 puncta significantly different from the relevant values of GFP-LC3 puncta. (E) Analysis of autophagic flux rate. Data are expressed as mean ± SD. *, P < 0.05, significantly different from the sham group. (F) Representative images of immunoblot analysis of LC3B, SQSTM1, ACTA2, and FN1. PPIB was used as a loading control. (G) Densitometric analysis of LC3B, SQSTM1, ACTA2, and FN1 signals. After normalization with PPIB, the protein signal of the sham was arbitrarily set as 1, and the signals of other conditions were normalized with the sham control to calculate fold changes. Data are expressed as mean ± SD. *, P < 0.05, significantly different from the sham group. (H) Representative images of Masson trichrome staining. Scale bar: 50 µm.
Figure 2.
Pharmacological inhibition of autophagy reduces interstitial fibrosis during UUO. C57Bl/6 mice were divided into 4 groups: 1. sham operation; 2. UUO + saline; 3. UUO + 60 mg/kg chloroquine (CQ); 4. UUO + 30 mg/kg 3-methyladenine (3-MA). The mice were sacrificed at the indicated time points (1 wk and 2 wk) and left kidneys were collected for histological and immunoblot analyses. (A) Representative images of immunoblot analysis of LC3B, SQSTM1, ACTA2, and FN1. ACTB was used as a loading control. (B) Densitometric analysis of LC3B, SQSTM1, ACTA2, and FN1 signals. After normalization with ACTB, the protein signal of the sham was arbitrarily set as 1, and the signals of other conditions were normalized with the sham to calculate fold changes. Data are expressed as mean ± SD. *, P < 0.05, significantly different from the sham group; #, P < 0.05, significantly different from the UUO + saline group. (C) Representative images of Masson trichrome staining. Scale bar: 50 µm. (D) Quantitative analysis of Masson trichrome staining. Data are expressed as mean ± SD. *, P < 0.05, significantly different from the sham group; #, P < 0.05, significantly different from the UUO + saline group.
Figure 3.
Pharmacological inhibition of autophagy attenuates tubular cell apoptosis induced during UUO. Animals and their treatment were the same as described in Figure 2. (A) Representative images of TUNEL staining. Scale bar: 50 µm. (B) Quantitative analysis of TUNEL staining. Data are expressed as mean ± SD. *, P < 0.05, significantly different from the UUO + saline group.
Figure 4.
Autophagy in proximal tubules is impaired in PT-Atg7 knockout mice during UUO. Floxed control (PT-Atg7 FC) and PT-Atg7 KO mice (littermates or age-matched) were subjected to either sham operation or UUO surgery. The mice were sacrificed at the indicated time points (4 d, 1 wk and 2 wk) and left kidneys were collected for histological and immunoblot analyses. (A) Representative images of immunohistochemical staining of LC3B. Scale bar: 20 µm. (B) Enlarged images of LC3B staining. Scale bar: 20 µm. (C) Representative images of immunoblot analysis of LC3B, SQSTM1, and ATG7. PPIB was used as a loading control.
Figure 5.
Autophagy deficiency in PT-Atg7 knockout mice suppresses renal interstitial fibrosis, tubular atrophy and nephron loss during UUO. Animals and their treatment were the same as described in Fig. 4. (A) Representative images of immunoblot analysis of ACTA2, VIM, FN1, and FGF2. PPIB was used as a loading control. (B) Densitometric analysis of ACTA2, VIM, FN1, and FGF2 signals. After normalization with PPIB, the protein signal of PT-Atg7 FC sham was arbitrarily set as 1, and the signals of other conditions were normalized with PT-Atg7 FC sham to calculate fold changes. Data are expressed as mean ± SD. *, P < 0.05, significantly different from PT-Atg7 FC sham group; #, P < 0.05, significantly different from the relevant PT-Atg7 FC group. (C) Representative images of Masson trichrome staining. Scale bar: 50 µm. (D) Quantitative analysis of Masson trichrome staining. Data are expressed as mean ± SD. *, P < 0.05, significantly different from PT-Atg7 FC sham group; #, P < 0.05, significantly different from the relevant PT-Atg7 FC group. (E) Representative images of PAS staining. Scale bar: 50 µm. (F) Representative images of LTL staining. Scale bar: 50 µm. (G) Quantitative analysis of LTL staining. Data are expressed as mean ± SD. *, P < 0.05, significantly different from PT-Atg7 FC sham group; #, P < 0.05, significantly different from the relevant PT-Atg7 FC group.
Figure 6.
Tubular apoptosis and interstitial inflammation are also attenuated in PT-Atg7 knockout mice during UUO. Animals and their treatment were the same as described in Figure 4. (A) Representative images of TUNEL staining. Scale bar: 50 µm. (B) Quantitative analysis of TUNEL staining. Data are expressed as mean ± SD. *, P < 0.05, significantly different from the relevant PT-Atg7 FC group. (C) Representative images of macrophage staining. Scale bar: 50 µm. (D) Quantitative analysis of macrophage staining. Data are expressed as mean ± SD. *, P < 0.05, significantly different from PT-Atg7 FC sham group; #, P < 0.05, significantly different from the relevant PT-Atg7 FC group.
Figure 7.
MTOR activation in proximal tubules during UUO is inhibited by autophagy deficiency in PT-Atg7 knockout mice. Animals and their treatment were the same as described in Fig. 4. (A) Representative images of immunoblot analysis of phospho-RPS6KB1 and RPS6KB1. PPIB was used as a loading control. (B) Densitometric analysis of phospho-RPS6KB1 signals. After normalization with RPS6KB1 and PPIB, the protein signal of PT-Atg7 FC sham was arbitrarily set as 1, and the signals of other conditions were normalized with PT-Atg7 FC sham to calculate fold changes. Data are expressed as mean ± SD. *, P < 0.05, significantly different from PT-Atg7 FC sham group; #, P < 0.05, significantly different from the relevant PT-Atg7 FC group.
Figure 8.
Autophagy is induced by TGFB1 in BUMPT mouse proximal tubular cell line along with profibrotic changes. BUMPT cells were untreated (d 0) or treated with 5 ng/ml TGFB1 for 1 to 3 d. In some experiments BUMPT cells were transiently transfected with mRFP-GFP-LC3 and then untreated (control) or treated with 5 ng/ml TGFB1 in the absence or presence of 20 μM chloroquine (CQ). After treatment the cells were collected for morphological and immunoblot analyses. (A) Representative images of phase contrast showing cell morphology. Scale bar: 50 µm. (B) Representative images of fluorescence microscopy showing GFP-LC3 and mRFP-LC3 puncta in cells. Scale bar: 15 µm. (C) Quantitative analysis of GFP-LC3 and mRFP-LC3 puncta. Data are expressed as mean ± SD. *, P < 0.05, significantly different from the relevant control group; ˆ, P < 0.05, significantly different from the TGFB1 group. #, P < 0.05, significantly different from GFP-LC3 puncta counting. (D) Analysis of autophagic flux rate. Data are expressed as mean ± SD. *, P < 0.05, significantly different from the control group; #, P < 0.05, significantly different from the TGFB1 group. (E) Representative images of immunoblot analysis of LC3B, SQSTM1, FN1, VIM, and CDH1. ACTB was used as a loading control. (F) Densitometric analysis of LC3B, SQSTM1, FN1, VIM, and CDH1 signals. After normalization with ACTB, the protein signal of control (day 0) was arbitrarily set as 1, and the signals of other conditions were normalized with control to calculate fold changes. Data are expressed as mean ± SD. *, P < 0.05, significantly different from control group.
Figure 9.
Inhibition of autophagy suppresses FN1 accumulation in TGFB1-treated proximal tubular cells without affecting phenotypic transition. (A) BUMPT cell were untreated (day 0) or treated with 5 ng/ml TGFB1 for 1 to 3 d in the absence (−) or presence (+) of 20 μM chloroquine (CQ). After treatment the cells were collected for immunoblot analysis of LC3B, SQSTM1, FN1, VIM, and CDH1. ACTB was used as a loading control. (B) Densitometric analysis of FN1 signals. After being normalized with ACTB, the protein signal of control (day 0, CQ -) was arbitrarily set as 1, and the signals of other conditions were normalized with control to calculate fold changes. Data are expressed as mean ± SD. *, P < 0.05, significantly different from control (day 0, CQ -) group; #, P < 0.05, significantly different from TGFB1 (CQ -) group. (C) Atg7 floxed control (FC) and KO proximal tubular cells were untreated (day 0) or treated with 5 ng/ml TGFB1 for 1 to 3 d. After treatment the cells were collected for immunoblot analysis of LC3B, SQSTM1, FN1, VIM, and CDH1. ACTB was used as a loading control. (D) Densitometric analysis of FN1 signals. After normalization with ACTB, the protein signal of Atg7 FC control (d 0) was arbitrarily set as 1, and the signals of other conditions were normalized with Atg7 FC control to calculate fold changes. Data are expressed as mean ± SD. *, P < 0.05, significantly different from Atg7 FC control group; #, P < 0.05, significantly different from the relevant Atg7 FC group.
Figure 10.
TGFB1-induced apoptosis in primary culture of proximal tubular cells is suppressed by inhibition of autophagy. Primary cells were untreated (control) or treated with 5 ng/ml TGFB1 for 24 h in the absence or presence of 20 μM chloroquine (CQ) or 10 mM 3-methyladenine (3-MA). After treatment the cells were collected for morphological, biochemical and immunoblot analyses. (A, D) Representative images of immunoblot analysis of LC3B and SQSTM1. ACTB was used as a loading control. (B, E) Representative images of phase contrast and fluorescence microscopy showing cellular and nuclear morphology of apoptosis. Scale bar: 200 µm. (C, F) Caspase activity. Data are expressed as mean ± SD. *, P < 0.05, significantly different from control group; #, P < 0.05, significantly different from TGFB1 or Atg7 FC TGFB1 group.
Figure 11.
Inhibition of autophagy attenuates fibrotic changes in TGFB1-treated primary proximal tubular cells. Primary cells were treated the same as described in Figure 10. (A, C, E) Representative images of immunoblot analysis of FN1 and VIM. ACTB was used as a loading control. (B, D, F) Densitometric analysis of FN1 and VIM signals. After being normalized with ACTB, the protein signal of control or Atg7 FC control was arbitrarily set as 1, and the signals of other conditions were normalized with controls to calculate fold changes. Data are expressed as mean ± SD. *, P < 0.05, significantly different from control or Atg7 FC control group; #, P < 0.05, significantly different from TGFB1 or Atg7 FC TGFB1 group.
Figure 12.
Inhibition of apoptosis by Z-VAD does not affect autophagy induction and fibrotic changes in TGFB1-treated primary proximal tubular cells. Primary cells were untreated (control) or treated with 5 ng/ml TGFB1 for 24 h in the absence or presence of 100 μM Z-VAD. After treatment the cells were collected for morphological and immunoblot analyses. (A) Representative images of phase contrast and fluorescence microscopy showing cellular and nuclear morphology of apoptosis. Scale bar: 200 µm. (B) Representative images of immunoblot analysis of LC3B, SQSTM1, FN1, and VIM. ACTB was used as a loading control. (C) Densitometric analysis of LC3B, SQSTM1, FN1, and VIM signals. After normalization with ACTB, the protein signal of control was arbitrarily set as 1, and the signals of other conditions were normalized with control to calculate fold changes. Data are expressed as mean ± SD. *, P < 0.05, significantly different from control group.
References
- Yang L, Humphreys BD, Bonventre JV. Pathophysiology of acute kidney injury to chronic kidney disease: maladaptive repair. Contrib Nephrol 2011; 174:149-55; PMID:21921619; http://dx.doi.org/ 10.1159/000329385 - DOI - PubMed
- Venkatachalam MA, Griffin KA, Lan R, Geng H, Saikumar P, Bidani AK. Acute kidney injury: a springboard for progression in chronic kidney disease. Am J Physiol Renal Physiol 2010; 298:F1078-94; PMID:20200097; http://dx.doi.org/ 10.1152/ajprenal.00017.2010 - DOI - PMC - PubMed
- Venkatachalam MA, Weinberg JM, Kriz W, Bidani AK. Failed Tubule Recovery, AKI-CKD Transition, and Kidney Disease Progression. J Am Soc Nephrol 2015; 26:1765-76; PMID:25810494; http://dx.doi.org/ 10.1681/ASN.2015010006 - DOI - PMC - PubMed
- Fujigaki Y, Muranaka Y, Sun D, Goto T, Zhou H, Sakakima M, Fukasawa H, Yonemura K, Yamamoto T, Hishida A. Transient myofibroblast differentiation of interstitial fibroblastic cells relevant to tubular dilatation in uranyl acetate-induced acute renal failure in rats. Virchows Arch 2005; 446:164-76; PMID:15609048; http://dx.doi.org/ 10.1007/s00428-004-1155-5 - DOI - PubMed
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