Volume 73, Issue 4, Pages 430-445 (February 2008) The phosphoinositide-3 kinase γ–Akt pathway mediates renal tubular injury in cisplatin nephrotoxicity  H. Kuwana, Y. Terada, T. Kobayashi, T. Okado, J.M. Penninger, J. Irie-Sasaki, T. Sasaki, S. Sasaki  Kidney International  Volume 73, Issue 4, Pages 430-445 (February 2008) DOI: 10.1038/sj.ki.5002702 Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 1 Survival curves of PI3Kγ+/+ and PI3Kγ−/− mice after the cisplatin administration. (a) A low dose of cisplatin (15 mg kg−1) was injected to PI3Kγ+/+ mice (●) and PI3Kγ−/− mice (■) intraperitoneally (N=25, each group). (b) A conventional cisplatin dose (20 mg kg−1) was injected to PI3Kγ+/+ mice (●) and PI3Kγ−/− mice (■) intraperitoneally (N=25, each group). (c) A higher dose of cisplatin (30 mg kg−1) was injected to PI3Kγ+/+ mice (●) and PI3Kγ−/− mice (■) intraperitoneally (N=25, each group). Statistically significant differences (*P<0.05) are indicated at 5 days. Kidney International 2008 73, 430-445DOI: (10.1038/sj.ki.5002702) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 2 Blood urea nitrogen (BUN) and serum creatinine (Cre) levels in PI3Kγ+/+ and PI3Kγ−/− mice after the cisplatin injection. A low dose of cisplatin (15 mg kg−1) was injected to PI3Kγ+/+ mice (white bar) and PI3Kγ−/− mice (black bar) intraperitoneally (N=28, each group). Blood was collected at baseline (control) and at 2, 3, and 5 days after the cisplatin injection. Blood urea nitrogen (a) and serum creatinine (b) were measured at the indicated times. Data are mean±s.e.m. of seven mice per group. Statistically significant differences (*P<0.05) are indicated. Kidney International 2008 73, 430-445DOI: (10.1038/sj.ki.5002702) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 3 Renal histopathology and scores for characteristic histological signs of renal injury and leukocyte infiltration in PI3Kγ+/+ and PI3Kγ−/− mice with cisplatin-induced acute kidney injury. Kidneys were removed 72 h after injection with cisplatin (15 mg kg−1). (a) The kidneys of PI3Kγ+/+ mice treated with a low dose (15 mg kg−1) of cisplatin show a very slight loss of tubular epithelial cells (TECs) and very low levels of intratubular debris and cast formation at low-power magnification. Cort, cortex; OM, outer medulla (original magnification × 50). (b) The kidneys of PI3Kγ−/− mice treated with a low dose (15 mg kg−1) of cisplatin show severe loss of TECs and severe tubular dilation, intratubular debris, and cast formation at low-power magnification (original magnification × 50). (c) The kidneys of cisplatin-treated PI3Kγ+/+ mice show a very slight loss of TECs and very little intratubular debris and cast formation at high-power magnification (original magnification × 400). (d) The kidneys of cisplatin-treated PI3Kγ−/− mice show severe loss of TECs and severe tubular dilation, intratubular debris, and cast formation at high-power magnification (original magnification × 400). (e) The kidneys of PI3Kγ+/+ and PI3Kγ−/− mice manifest the characteristic histological signs of renal injury. Tubular damage was significantly higher in the kidneys of PI3Kγ−/− mice (black bars) than in the kidneys of PI3Kγ+/+ mice (white bars). Data are mean±s.e.m. of seven mice per group. Statistically significant differences (*P<0.05) are indicated. (f) Leukocyte infiltration was measured by naphthol AS-D chloroacetate esterase staining. The cisplatin injection (after 2 and 3 days) brought about an increase in leukocytes within the kidney cortex in both PI3Kγ+/+ mice (white bars) and PI3Kγ−/− mice (black bars). The increments were larger in PI3Kγ−/− mice than in PI3Kγ+/+ mice (*P<0.05). Kidney International 2008 73, 430-445DOI: (10.1038/sj.ki.5002702) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 4 Tubular cell death in the renal tissues of PI3Kγ+/+ and PI3Kγ−/− mice with cisplatin-induced acute kidney injury. Kidneys were removed 72 h after an injection with the low dose of cisplatin (15 mg kg−1). (a) The kidneys of cisplatin-treated PI3Kγ+/+ mice show very few TUNEL-positive cells (original magnification × 200). (b) The kidneys of cisplatin-treated PI3Kγ−/− mice show an elevated number of TUNEL-positive renal tubular cells (original magnification × 200). (c) The number of TUNEL-positive tubular cells was significantly higher in the kidneys of PI3Kγ−/− mice (black bars) than in the kidneys of PI3Kγ+/+ mice (white bars). Data are mean±s.e.m. of seven mice per group. Statistically significant differences (*P<0.05) are indicated. Kidney International 2008 73, 430-445DOI: (10.1038/sj.ki.5002702) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 5 Quantitative analysis of TNF-α and ICAM-1 mRNA expression and protein content in the renal tissues of PI3Kγ−/− and PI3Kγ+/+ mice with cisplatin-induced acute kidney injury. (a) PI3Kγ+/+ and PI3Kγ−/− mice were administered 15 mg kg−1 cisplatin or vehicle, and then killed 12 h after the injection. Extracted total RNAs were subjected to quantitative PCR using the LightCycler Real-Time PCR to estimate the TNF-α mRNA and ICAM-1 mRNA levels relative to the levels of GAPDH mRNA, as described in the Materials and Methods section. Each column with a bar shows mean±s.e.m. (n=6). (b) The representative agarose gels of PCR products for TNF-α, ICAM-1, and GAPDH are shown. (c) PI3Kγ+/+ and PI3Kγ−/− mice were administered 15 mg kg−1 of cisplatin or vehicle, and then killed at 24 or 48 h after the injection. TNF-α was quantitated in extracted renal proteins by ELISA, as described in the Materials and Methods section. Data are mean±s.e.m. of six mice per group. (d) PI3Kγ+/+ and PI3Kγ−/− mice were administered 15 mg kg−1 cisplatin or vehicle, and then killed at 24 or 48 h after the injection. ICAM-1 was quantitated in extracted renal proteins by ELISA, as described in the Materials and Methods section. Data are mean±s.e.m. of six mice per group. NS, no significant difference between WT vs KO. Kidney International 2008 73, 430-445DOI: (10.1038/sj.ki.5002702) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 6 Phosphorylation of Akt and Bad in the renal tissues of PI3Kγ+/+ and PI3Kγ−/− mice with cisplatin-induced acute kidney injury. PI3Kγ+/+ and PI3Kγ−/− mice were administered 15 mg kg−1 cisplatin or vehicle, and then killed at 24, 48, or 72 h after the injection. Extracted renal proteins were examined by immunoblot analyses for phosphorylated (Ser473) Akt, total Akt, phosphorylated (Ser136) Bad, total Bad, and actin. Mice injected with vehicle at 0 h served as controls. Extracted proteins (20 μg) from the renal tissue were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Phosphorylated Akt, total Akt, phosphorylated (Ser136) Bad, total Bad, and actin protein levels of PI3Kγ+/+ mice kidneys (a) and PI3Kγ−/− mice kidneys (b) were detected by western blot analysis. (c) Quantitative analyses of the levels of the phosphorylated form of Akt vs the total Akt levels after the cisplatin administration are shown for both PI3Kγ+/+ mice (white bar) and PI3Kγ−/− mice (black bar). (d) Quantitative analyses of the levels of the phosphorylated form of Bad vs the total Bad levels after the cisplatin administration are shown for both PI3Kγ+/+ mice (white bar) and PI3Kγ−/− mice (black bar). Data are mean±s.e.m. of six mice per group. *P<0.05. Kidney International 2008 73, 430-445DOI: (10.1038/sj.ki.5002702) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 7 Immunohistological examination of phosphorylated Akt (Ser473) in the renal tissues of PI3Kγ+/+ mice after the cisplatin administration. (a) The renal cortex from PI3Kγ+/+ mice was immunostained with phosphorylated Akt (Ser473)- and AQP1-specific antibodies. Positive staining of phosphorylated Akt was detected after the cisplatin injection (original magnification × 100). (b) The renal medulla from PI3Kγ+/+ mice was immunostained with phosphorylated Akt (Ser473)- and AQP1-specific antibodies. Positive staining of phosphorylated Akt was detected in the outer medulla after the cisplatin injection (original magnification × 100). IM, inner medulla; OM, outer medulla; p-Akt, phosphorylated Akt; AQP1, aquaporin-1; Merge, merged condition. Kidney International 2008 73, 430-445DOI: (10.1038/sj.ki.5002702) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 8 Immunohistological examination of phosphorylated Akt (Ser473) in the renal tissues of PI3Kγ−/− mice after the cisplatin administration. (a) The renal cortex from PI3Kγ−/− mice was immunostained with phosphorylated Akt (Ser473)- and AQP1-specific antibodies. Staining of phosphorylated Akt was not detected after the cisplatin injection or vehicle injection (original magnification × 100). (b) The renal medulla from PI3Kγ−/− mice was immunostained with phosphorylated Akt (Ser473)- and AQP1-specific antibodies (original magnification × 100). IM, inner medulla; OM, outer medulla; p-Akt, phosphorylated Akt; AQP1, aquaporin-1; Merge, merged condition. Kidney International 2008 73, 430-445DOI: (10.1038/sj.ki.5002702) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 9 Immunohistological examination of phosphorylated Akt (Ser473) in the renal tissues of PI3Kγ+/+ mice after the cisplatin administration, at high-power magnification. (a) The renal outer medulla taken from PI3Kγ+/+ mice after the cisplatin injection was immunostained with phosphorylated Akt (Ser473)- and AQP1-specific antibodies. Positive staining of phosphorylated Akt was detected after the cisplatin injection (original magnification × 600). The renal outer medulla taken from PI3Kγ+/+ mice after injection of vehicle was immunostained with phosphorylated Akt (Ser473)- and AQP1-specific antibodies (original magnification × 600). p-Akt, phosphorylated Akt; AQP1, aquaporin-1; Merge, merged condition. (b) The renal cortex taken from PI3Kγ+/+ mice after the cisplatin injection was immunostained with phosphorylated Akt (Ser473)- and AQP2-specific antibodies (original magnification × 600). The renal cortex taken from PI3Kγ+/+ mice after injection of vehicle was immunostained with phosphorylated Akt (Ser473)- and AQP2-specific antibodies (original magnification × 600). p-Akt, phosphorylated Akt; AQP2, aquaporin-2; Merge, merged condition. Kidney International 2008 73, 430-445DOI: (10.1038/sj.ki.5002702) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 10 Caspase-3 activity assay and cell death ELISA assay of primary cultured TECs of PI3Kγ+/+ and PI3Kγ−/− mice with or without the cisplatin administration. (a) Caspase-3 enzymatic activity was measured colorimetrically by the procedure described in the Materials and Methods section. Caspase-3 activity was measured in the TECs from PI3Kγ+/+ mice (●) and PI3Kγ−/− mice (■) at indicated times of cisplatin (50 μmol l−1) treatment. Data are mean±s.e.m. of six experiments per group. *P<0.05, WT vs KO. (b) The TECs from PI3Kγ+/+ mice (white bar) and PI3Kγ−/− mice (black bar) were treated with the indicated dose of cisplatin for 25 h. Cisplatin increased caspase-3 activity dose dependently. The TECs from PI3Kγ−/− mice (black bar) show higher caspase-3 activity than the TECs from PI3Kγ+/+ mice (white bar). Data are mean±s.e.m. of six experiments per group. *P<0.05, WT vs KO. (c) Cell death detection ELISA assays were measured in the TECs from PI3Kγ+/+ mice (●) and PI3Kγ−/− mice (■) at indicated times of cisplatin (50 μmol l−1) treatment. Data are mean±s.e.m. of six experiments per group. *P<0.05, WT vs KO. (d) The TECs from PI3Kγ−/− mice (black bar) and PI3Kγ+/+ mice (white bar) were treated with the indicated dose of cisplatin for 25 h. Cisplatin increased cell death ELISA absorbance dose dependently. The TECs from PI3Kγ−/− mice (black bar) show higher absorbance than the TECs from PI3Kγ+/+ mice (white bar). Data are mean±s.e.m. of six experiments per group. *P<0.05, WT vs KO. Kidney International 2008 73, 430-445DOI: (10.1038/sj.ki.5002702) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 11 Transfection of PI3Kγ and the dominant-active form of Akt prevents the cisplatin-induced caspase-3 activity and cell death ELISA activity of the TECs derived from PI3Kγ−/− mice. (a) The phosphorylation of Akt was significantly increased by transfection with PI3Kγ (AdPI3K), the dominant-active Akt (AdAkt). The phosphorylation of Akt was not observed in control adenovirus. (b) Caspase-3 enzymatic activity was measured colorimetrically by the procedure described in the Materials and Methods. The TECs from PI3Kγ−/− mice were transfected with AdPI3K, AdAkt, or Adnull for 48 h, and then treated with 50 μmol l−1 cisplatin for 24 h. Transfection of either AdPI3K or AdAkt significantly reduced the cisplatin induced-caspase-3 activity of the TECs from PI3Kγ−/− mice. These values were almost equal to the level of TECs derived from PI3Kγ+/+ mice (white bar). Data are mean±s.e.m. of six experiments per group. *P<0.05. (c) We also performed western blotting of cleaved caspase-3 in the presence of cisplatin with or without the transfection of PI3Kγ or Akt in the TECs of PI3Kγ−/− and PI3Kγ−/− mice. (d) Cell death detection ELISA assays were performed by the procedure described in the Materials and Methods section. The TECs from PI3Kγ−/− mice were transfected with AdPI3K, AdAkt, or Adnull for 48 h, and then treated with 50 μmol l−1 cisplatin for 25 h. Transfection of either AdPI3K or AdAkt significantly reduced the cisplatin induced-cell death ELISA absorbance of the TECs from PI3Kγ−/− mice. These values were almost equal to the levels of TECs derived from PI3Kγ+/+ mice (white bar). Data are mean±s.e.m. of six experiments per group. *P<0.05. Kidney International 2008 73, 430-445DOI: (10.1038/sj.ki.5002702) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 12 Apoptosis in PI3Kγ−/− mice-derived TECs stained with annexin-V (green fluorescence) and propidium iodide (red fluorescence) transfected with PI3Kγ and the dominant-active form of Akt. (a) Control TECs from PI3Kγ−/− mice. (b, c) TECs treated with 50 μmol l−1 cisplatin for 24 h (b, original magnification × 200; c, original magnification × 600). (d) TECs were transfected with Adnull (control virus) for 48 h and then treated with 50 μmol l−1 cisplatin for 24 h. (e) TECs were transfected with AdPI3K for 48 h and then treated with 50 μmol l−1 cisplatin for 24 h. (f) TECs were transfected with AdAkt for 48 h and then treated with 50 μmol l−1 cisplatin for 24 h. (g) The numbers of apoptotic cells were counted. Data are mean±s.e.m. of six experiments per group. *P<0.05. Kidney International 2008 73, 430-445DOI: (10.1038/sj.ki.5002702) Copyright © 2008 International Society of Nephrology Terms and Conditions

Figure 13 Blood urea nitrogen (BUN) and serum creatinine (Cre) levels after ischemia/reperfusion renal injury or injection of tunicamycin in PI3Kγ+/+ and PI3Kγ−/− mice. Blood was collected at 0 (control), 1, and 2 days after the ischemia/reperfusion renal injury in PI3Kγ+/+ mice (white bar) and PI3Kγ−/− mice (black bar) (N=6, each group). BUN (a) and serum creatinine (b) were measured. Blood was collected at 0 (control), 2, and 3 days after tunicamycin injection in PI3Kγ+/+ mice (white bar) and PI3Kγ−/− mice (black bar) (N=6, each group). BUN (c) and serum creatinine (d) were measured. Data are mean±s.e.m. of six mice per group. Caspase-3 enzymatic activity and cell death ELISA were measured by the procedure described in the Materials and Methods section (e). The TECs from PI3Kγ−/− mice (black bar) and the TECs derived from PI3Kγ+/+ mice (white bar) were incubated in a hypoxic condition for 10 h. The TECs from PI3Kγ−/− mice (black bar) and the TECs from PI3Kγ+/+ mice (white bar) incubated with tunicamycin (1 μM ml−1) for 36 h were subject to an assay for caspase-3 enzymatic activity and cell death ELISA (f). Data are mean±s.e.m. of six experiments per group. Kidney International 2008 73, 430-445DOI: (10.1038/sj.ki.5002702) Copyright © 2008 International Society of Nephrology Terms and Conditions