Autophagy in proximal tubules protects against acute kidney injury

Abstract

Autophagy is induced in renal tubular cells during acute kidney injury; however, whether this is protective or injurious remains controversial. We address this question by pharmacologic and genetic blockade of autophagy using mouse models of cisplatin- and ischemia-reperfusion-induced acute kidney injury. Chloroquine, a pharmacological inhibitor of autophagy, blocked autophagic flux and enhanced acute kidney injury in both models. Rapamycin, however, activated autophagy and protected against cisplatin-induced acute kidney injury. We also established a renal proximal tubule-specific autophagy-related gene 7-knockout mouse model shown to be defective in both basal and cisplatin-induced autophagy in kidneys. Compared with wild-type littermates, these knockout mice were markedly more sensitive to cisplatin-induced acute kidney injury as indicated by renal functional loss, tissue damage, and apoptosis. Mechanistically, these knockout mice had heightened activation of p53 and c-Jun N terminal kinase, the signaling pathways contributing to cisplatin acute kidney injury. Proximal tubular cells isolated from the knockout mice were more sensitive to cisplatin-induced apoptosis than cells from wild-type mice. In addition, the knockout mice were more sensitive to renal ischemia-reperfusion injury than their wild-type littermates. Thus, our results establish a renoprotective role of tubular cell autophagy in acute kidney injury where it may interfere with cell killing mechanisms.

Figure 1. Autophagy is induced in proximal tubules during cisplatin nephrotoxicity in C57BL/6 mice

C57BL/6 mice (male, 8–10 weeks old) were injected with 25mg/kg cisplatin or saline as control. (A) After the indicated time, kidneys were harvested to collect cortical and outer medulla tissues for immunoblot analysis of LC3 and β-actin (loading control). For densitometry, the LC3-II signals were divided by the β-actin signal of the same samples to determine the ratios. (B) Three days after treatment, kidneys were collected for immunofluorescence staining of LC3, FITC-labeled PHA, and Hoechst33342 (×630). Arrows point to LC3 puncta (autophagosomes) and insets show LC3 puncta at a higher magnification. (C) Quantification of LC3 dots in individual proximal tubule of the cisplatin-treated group. Data are expressed as mean ± SD. Control tissues did not show LC3 dots.

Figure 2. Inhibition of autophagy by chloroquine worsens cisplatin-AKI in C57BL/6 mice

C57BL/6 mice (male, 8 to 10 weeks old, littlermates or age-matched non-littermates) were divided into three groups for following treatments respectively: 1) saline control (n=3); 2) cisplatin+saline (n=4); 3) cisplatin+chloroquine (n=5). The mice were sacrificed on day 4. (A) Whole tissue lysate of kidney cortex was collected for immunoblot analysis of LC3, p62 and β-actin. (B) Densitometry of LC3-II and p62 signals. The blot in (A) was analyzed by densitometry. The LC3-II and p62 signals were divided by the β-actin signal of the same samples to determine the ratios. Data are expressed as mean ± SD. * P < 0.05, significantly different from the control group; # P < 0.05, significantly different from the cisplatin+saline group. (C) and (D) Blood samples were collected for measurements of BUN and serum creatinine. Data are expressed as mean ± SD. * P < 0.05, significantly different from the control (or day 0) group; # P < 0.05, significantly different from the cisplatin+saline group. (E) Representative histology of kidney cortex (hematoxylin-eosin (H-E) staining, ×200). (F) Pathological score of tubular damage in cisplatin+saline and cisplatin+chloroquine groups. (G) Representative images of TUNEL staining (×200). (H) Quantification of TUNEL-positive cells in cisplatin+saline and cisplatin+chloroquine groups. Data in (F) and (H) are expressed as mean ± SD. * P < 0.05, significantly different from the cisplatin+saline group. Control tissues without cisplatin treatment did not show tubular damage or apoptotic cells.

Figure 2. Inhibition of autophagy by chloroquine worsens cisplatin-AKI in C57BL/6 mice

C57BL/6 mice (male, 8 to 10 weeks old, littlermates or age-matched non-littermates) were divided into three groups for following treatments respectively: 1) saline control (n=3); 2) cisplatin+saline (n=4); 3) cisplatin+chloroquine (n=5). The mice were sacrificed on day 4. (A) Whole tissue lysate of kidney cortex was collected for immunoblot analysis of LC3, p62 and β-actin. (B) Densitometry of LC3-II and p62 signals. The blot in (A) was analyzed by densitometry. The LC3-II and p62 signals were divided by the β-actin signal of the same samples to determine the ratios. Data are expressed as mean ± SD. * P < 0.05, significantly different from the control group; # P < 0.05, significantly different from the cisplatin+saline group. (C) and (D) Blood samples were collected for measurements of BUN and serum creatinine. Data are expressed as mean ± SD. * P < 0.05, significantly different from the control (or day 0) group; # P < 0.05, significantly different from the cisplatin+saline group. (E) Representative histology of kidney cortex (hematoxylin-eosin (H-E) staining, ×200). (F) Pathological score of tubular damage in cisplatin+saline and cisplatin+chloroquine groups. (G) Representative images of TUNEL staining (×200). (H) Quantification of TUNEL-positive cells in cisplatin+saline and cisplatin+chloroquine groups. Data in (F) and (H) are expressed as mean ± SD. * P < 0.05, significantly different from the cisplatin+saline group. Control tissues without cisplatin treatment did not show tubular damage or apoptotic cells.

Figure 3. Creation and characterization of the PT-Atg7-KO) mouse model

(A) Breeding protocol for generating PT-Atg7-KO mice. Male littermate mice of 8–9 weeks old were used for experiments after genotypes confirmed. (B) Representative images of PCR-based genotyping. Genomic DNA was extracted from tail biopsy and amplified to detect wild-type (WT) and floxed alleles of Atg7 and PEPCK-Cre allele as indicated. (C) Whole tissue lysate of kidney cortex was collected from PT-Atg7-KO and wild-type (PT-Atg7-WT) littermate mice for immunoblot analysis of Atg7, LC3, Atg5 (Atg12 conjugated), p62, and β-actin. (D) Immunohistochemical staining of p62 (×200) in kidney cortical tissues of wild-type and PT-Atg7-KO mice. The selected areas were shown at high magnification in the middle panels.

Figure 4. Cisplatin-induced autophagy is inhibited in renal proximal tubules in PTAtg7-KO mice

Wild-type and PT-Atg7-KO mice were injected with 25mg/kg cisplatin or saline as control. (A) After the indicated time, kidneys were harvested to collect cortical tissues for immunoblot analysis of Atg7, LC3, Atg5 (Atg12 conjugated), p62, and β-actin. (B) Three days after injection, kidneys were collected for immunofluorescence staining of LC3, FITC-labeled PHA, and Hoechst33342 (×630). Representative images of cisplatin-treated wild-type and PT-Atg7-KO groups were shown. Arrows point to LC3 dots (autophagosomes) and inset shows LC3 dots at higher magnifications. (C) Quantification of LC3 dots in individual proximal tubule of cisplatin-treated wild-type and PT-Atg7-KO groups. Data are expressed as mean ± SD. * P < 0.05, significantly different from the wild-type group. (D) After 4 days of treatment, kidneys were collected for immunohistochemical staining of p62 (×200). The selected areas were shown at high magnifications.

Figure 4. Cisplatin-induced autophagy is inhibited in renal proximal tubules in PTAtg7-KO mice

Wild-type and PT-Atg7-KO mice were injected with 25mg/kg cisplatin or saline as control. (A) After the indicated time, kidneys were harvested to collect cortical tissues for immunoblot analysis of Atg7, LC3, Atg5 (Atg12 conjugated), p62, and β-actin. (B) Three days after injection, kidneys were collected for immunofluorescence staining of LC3, FITC-labeled PHA, and Hoechst33342 (×630). Representative images of cisplatin-treated wild-type and PT-Atg7-KO groups were shown. Arrows point to LC3 dots (autophagosomes) and inset shows LC3 dots at higher magnifications. (C) Quantification of LC3 dots in individual proximal tubule of cisplatin-treated wild-type and PT-Atg7-KO groups. Data are expressed as mean ± SD. * P < 0.05, significantly different from the wild-type group. (D) After 4 days of treatment, kidneys were collected for immunohistochemical staining of p62 (×200). The selected areas were shown at high magnifications.

Figure 5. Cisplatin-AKI is aggravated in PT-Atg7-KO mice

Wild-type (n=15) and PT-Atg7-KO littermate mice (n=21) were injected with 25mg/kg cisplatin or saline as control. (A) and (B) Blood samples were collected for measurements of BUN and serum creatinine. Data are expressed as mean ± SD. * P < 0.05, significantly different from the control (or day 0) groups; # P < 0.05, significantly different from the relevant wild-type group. (C) Representative histology of kidney cortex (H-E staining, ×200). (D) Pathological score of tubular damage in cisplatin-treated wild-type and PT-Atg7-KO mice. (E) Representative images of TUNEL staining (×200). (F) Quantification of TUNEL-positive cells in cisplatin-treated wild-type and PT-Atg7-KO groups. Data in (D) and (F) are expressed as mean ± SD. * P < 0.05, significantly different from the wild-type group.

Figure 5. Cisplatin-AKI is aggravated in PT-Atg7-KO mice

Wild-type (n=15) and PT-Atg7-KO littermate mice (n=21) were injected with 25mg/kg cisplatin or saline as control. (A) and (B) Blood samples were collected for measurements of BUN and serum creatinine. Data are expressed as mean ± SD. * P < 0.05, significantly different from the control (or day 0) groups; # P < 0.05, significantly different from the relevant wild-type group. (C) Representative histology of kidney cortex (H-E staining, ×200). (D) Pathological score of tubular damage in cisplatin-treated wild-type and PT-Atg7-KO mice. (E) Representative images of TUNEL staining (×200). (F) Quantification of TUNEL-positive cells in cisplatin-treated wild-type and PT-Atg7-KO groups. Data in (D) and (F) are expressed as mean ± SD. * P < 0.05, significantly different from the wild-type group.

Figure 6. Heightened p53 and JNK activation during cisplatin-AKI in PT-Atg7-KO mice

Wild-type and PT-Atg7-KO littermate mice were injected with 25mg/kg cisplatin (n=8 for wild-type and PT-Atg7-KO, respectively) or saline as control (n=5 for wild-type and PT-Atg7-KO, respectively). After 4 days of treatment, whole tissue lysate of renal cortex and outer medulla was collected for immunoblot analysis. (A) Representative blots of p53, p-p53 (ser15), and p21. Cyclophilin B was used as a loading control. (B) Densitometry of p53, p-p53 (ser15), and p21 signals. (C) Representative blots of p-ERK, ERK, p-JNK, JNK, p-p38, and p38. (D) Densitometry of p-ERK, p JNK, p-p38 and p38 signals. For densitometric analysis in (B) and (D), the protein signal of the wild-type control group (average value of 5 mice) was arbitrarily set as 1, and the signals of other conditions (average value for each condition) were normalized.

Figure 7. Proximal tubular cells isolated from PT-Atg7-KO mice are sensitized to cisplatin-induced apoptosis

Primary proximal tubular cells isolated from wild-type and PT-Atg7-KO mice were treated with 30μM cisplatin for 24 hours. Apoptosis was assessed by cell morphology and caspase activation. (A) Representative cell and nuclear morphology (×200). After treatment, cells were stained with Hoechst33342 to record cell and nuclear morphology. (B) Apoptosis percentage. Apoptotic cells were counted to determine the percentage of apoptosis. (C) Caspase activity measured by enzymatic assays using carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin as substrates. Data in (B) and (C) are expressed as mean ± SD. * P < 0.05, significantly different from the wild-type group.

Figure 8. PT-Atg7 KO mice are more sensitive to renal ischemia-reperfusion injury

Wild-type (n=14) and PT-Atg7-KO littermate mice (n=14) were subjected to sham operation or 25 minutes of bilateral renal ischemia followed by 0 to 72 hours of reperfusion. Blood samples were collected for measurements of BUN (A) and serum creatinine (B). Data are expressed as mean ± SD. * P < 0.05, significantly different from the sham control groups; # P < 0.05, significantly different from the wild-type group.

Figure 9. Induction of autophagy by rapamycin reduces cisplatin-induced AKI in C57BL/6 mice

C57BL/6 mice (male, 8 to 10 weeks) were divided into three groups for following treatments respectively: 1) saline control (n=4); 2) cisplatin+saline (n=12); 3) cisplatin+rapamycin (n=13). (A) Two days after treatment, whole tissue lysate of kidney cortex was collected for immunoblot analysis of LC3 and β-actin. (B) and (C) Blood samples were collected for measurements of BUN and serum creatinine. Data are expressed as mean ± SD. * P < 0.05, significantly different from the control (or day 0) group; # P < 0.05, significantly different from the cisplatin+saline group. (D) Representative histology of kidney cortex (H-E staining, ×200). (E) Pathological score of tubular damage in cisplatin+saline and cisplatin+rapamycin groups. Data are expressed as mean ± SD. * P < 0.05, significantly different from the cisplatin+saline group.

Figure 9. Induction of autophagy by rapamycin reduces cisplatin-induced AKI in C57BL/6 mice

C57BL/6 mice (male, 8 to 10 weeks) were divided into three groups for following treatments respectively: 1) saline control (n=4); 2) cisplatin+saline (n=12); 3) cisplatin+rapamycin (n=13). (A) Two days after treatment, whole tissue lysate of kidney cortex was collected for immunoblot analysis of LC3 and β-actin. (B) and (C) Blood samples were collected for measurements of BUN and serum creatinine. Data are expressed as mean ± SD. * P < 0.05, significantly different from the control (or day 0) group; # P < 0.05, significantly different from the cisplatin+saline group. (D) Representative histology of kidney cortex (H-E staining, ×200). (E) Pathological score of tubular damage in cisplatin+saline and cisplatin+rapamycin groups. Data are expressed as mean ± SD. * P < 0.05, significantly different from the cisplatin+saline group.