Autophagy protects kidney proximal tubule epithelial cells from mitochondrial metabolic stress

Figures & data

Figure 1. Cyclosporin A (CsA)-induced autophagy protects kidney proximal tubular cells. (A) Wild-type proximal tubular cells were treated with 25 μM CsA for the indicated hours in the presence or absence of bafilomycin A₁ (Baf A1). (B) Wild-type proximal tubular cells were treated with either 25 μM CsA or vehicle for 24 h in the presence or absence of bafilomycin A₁. (C) Cell survival of autophagy-competent [Atg5 (+)] and autophagy-deficient [atg5 (−)] kidney proximal tubular cells treated with indicated concentration of CsA for 24 h. (D) Autophagy-competent [Atg5 (+)] and autophagy-deficient [atg5 (−)] kidney proximal tubular cells treated with either 25 μM CsA or vehicle for 24 h. Data are means ± SE from three to five experiments. Values were normalized to the leftmost column. *P < 0.05 vs. vehicle-treated cells of the corresponding hour (A) or cells of corresponding treatment (B–D); ^#P < 0.05 vs. untreated (A) or vehicle-treated cells (B–D).

Figure 2. Autophagy protects proximal tubules from cyclosporin A (CsA)-induced mitochondrial damage. (A) Autophagy-competent [Atg5 (+)] and autophagy-deficient [atg5 (−)] kidney proximal tubular cells after treatment with either 25 μM CsA or vehicle for the indicated hours and stained with MitoTracker Red. (B) MitoSox Red staining of autophagy-competent [Atg5 (+)] and autophagy-deficient [atg5 (−)] kidney proximal tubular cells treated with either 25 μM CsA or vehicle for 24 h. Scale bars: 10 μm. The images are representative of multiple experiments (n = 3 to 8). Values are normalized to the signal intensity of untreated (A) or vehicle-treated (B) atg5 (−) cells. Data are means ± SE *P < 0.05 vs. atg5 (−) cells of the corresponding hour (A) or treatment (B); ^#P < 0.05 vs. untreated (A) or vehicle-treated cells (B).

Figure 3. Autophagy deficiency affects amino acid metabolism. Levels of amino acids in autophagy-competent [Atg5 (+)] and autophagy-deficient [atg5 (−)] kidney proximal tubular cells treated with either 25 μM cyclosporin A (CsA) or vehicle for 24 h were superimposed on a metabolic pathway map. Columns, average concentration (nmoL/mg protein); scale bars: SE. N.D., the metabolite concentration was below the detection limit of the analysis (n = 8). *P < 0.05 vs. Atg5 (+) cells of corresponding treatment; ^#P < 0.05 vs. vehicle-treated controls.

Figure 4. Autophagy deficiency affects tricarboxylic acid (TCA) cycle metabolism. (A) Metabolite concentrations of autophagy-competent [Atg5 (+)] and autophagy-deficient [atg5 (−)] kidney proximal tubular cells treated with either 25 μM cyclosporin A (CsA) or vehicle for 24 h were superimposed on a metabolic pathway map that included glycolysis and the pentose phosphate and TCA pathways (n = 8). Columns, average concentration (nmoL/mg protein); scale bars: SE. N.D., the metabolite concentration was below the detection limit of the analysis. (B) Scatter plot of the TCA ratio and essential amino acid (EAA)/total amino acid (TAA) ratio. *P < 0.05 vs. Atg5 (+) cells of corresponding treatment; ^#P < 0.05 vs. vehicle-treated controls.

Figure 5. Autophagy deficiency affects energy status and NADPH metabolism. (A) Quantified levels of metabolites involved in metabolism of nucleotides and NADH in autophagy-competent [Atg5 (+)] and autophagy-deficient [atg5 (−)] kidney proximal tubular cells treated with either 25 μM cyclosporin A (CsA) or vehicle for 24 h were superimposed on a mitochondrial metabolic pathway map. (B) Levels of metabolites involved in NADPH in Atg5 (+) and atg5 (−) kidney proximal tubular cells treated with either 25 μM CsA or vehicle for 24 h. Columns, average concentration (nmoL/mg protein); scale bars: SE (n = 8). *P < 0.05 vs. Atg5 (+) cells of corresponding treatment; ^#P < 0.05 vs. vehicle-treated controls.

Figure 6. Autophagy deficiency worsens energy status in response to cyclosporin A (CsA)-treatment. Autophagy-competent [Atg5 (+)] and autophagy-deficient [atg5 (−)] kidney proximal tubular cells were treated with CsA. (A) Glucose uptake. (B) mRNA expression level of Slc2a1. (C) Level of endoplasmic reticulum stress markers. (D and E) Protein (D) and mRNA (E) levels of Atpif1. (F) mRNA expression levels of metabolism-related genes. (G) Schematic representation of metabolic stress and autophagy. Magenta lines indicate the metabolic adaptation pathway through autophagy. Data are means ± SE of three to six experiments. Fold expression normalized to that of vehicle-treated Atg5 (+) cells. *P < 0.05 vs. Atg5 (+) cells of corresponding treatment; ^#P < 0.05 vs. vehicle-treated controls.