Protein Kinase A/CREB Signaling Prevents Adriamycin-Induced Podocyte Apoptosis via Upregulation of Mitochondrial Respiratory Chain Complexes

Abstract

Previous work showed that the activation of protein kinase A (PKA) signaling promoted mitochondrial fusion and prevented podocyte apoptosis. The cAMP response element binding protein (CREB) is the main downstream transcription factor of PKA signaling. Here we show that the PKA agonist 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate-cyclic AMP (pCPT-cAMP) prevented the production of adriamycin (ADR)-induced reactive oxygen species and apoptosis in podocytes, which were inhibited by CREB RNA interference (RNAi). The activation of PKA enhanced mitochondrial function and prevented the ADR-induced decrease of mitochondrial respiratory chain complex I subunits, NADH-ubiquinone oxidoreductase complex (ND) 1/3/4 genes, and protein expression. Inhibition of CREB expression alleviated pCPT-cAMP-induced ND3, but not the recovery of ND1/4 protein, in ADR-treated podocytes. In addition, CREB RNAi blocked the pCPT-cAMP-induced increase in ATP and the expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1-α). The chromatin immunoprecipitation assay showed enrichment of CREB on PGC1-α and ND3 promoters, suggesting that these promoters are CREB targets. In vivo, both an endogenous cAMP activator (isoproterenol) and pCPT-cAMP decreased the albumin/creatinine ratio in mice with ADR nephropathy, reduced glomerular oxidative stress, and retained Wilm's tumor suppressor gene 1 (WT-1)-positive cells in glomeruli. We conclude that the upregulation of mitochondrial respiratory chain proteins played a partial role in the protection of PKA/CREB signaling.

Keywords: CREB; PKA signaling; apoptosis; mitochondria; podocyte.

FIG 1

PKA signaling prevented ADR-induced podocyte apoptosis. (A and B) Representative immunoblot and corresponding graph of podocytes treated with ADR (0.25 μg/ml) in the presence or absence of pCPT-cAMP (50 μmol/liter) for the indicated periods. Each bar represents data obtained from five independent experiments. (C and D) Detection of cell apoptosis by annexin V-PI staining. Data were obtained from at least three independent assays. (E) Evaluation of cell viability using the CCK-8 assay. (F) JC-1 staining of podocytes (original magnification, ×200). Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as a positive control of mitochondrial membrane potential decrease. *, P < 0.05 compared with the control group; **, P < 0.05 compared with the ADR group. pCPT, pCPT-cAMP.

FIG 2

CREB silencing weakened the protective effect of PKA signaling in podocytes. (A to C) Western blots of podocytes treated with pCPT (50 μmol/liter) for the indicated periods. Each bar represents data obtained from five independent experiments. (D) Detection of p-CREB expression in podocytes by immunofluorescence staining (original magnification, ×400). DAPI, 4,6-diamidino-2-phenylindole; COX, cytochrome c oxidase IV (used as a reference mitochondrial protein). (E) Representative immunoblots and corresponding graph of CREB in podocytes treated with CREB RNAi or a negative-control siRNA. (F) Representative immunoblots and corresponding graph of cleaved caspase 3 in podocytes. *, P < 0.05 compared with the control group; **, P < 0.05 compared with the ADR group; ***, P < 0.05 compared with the ADR-plus-pCPT-plus-ng group. ng, negative-control siRNA.

FIG 3

PKA signaling enhanced mitochondrial function. (A) Podocytes were pretreated or not with pCPT (50 μmol/liter) for 48 h and then incubated with ADR (0.25 μg/ml) for another 24 h. Fluorescence microscopy showed the generation of mitochondrial ROS using DCF fluorescence or the mitochondrial superoxide indicator MitoSOX red. (B) Oxygen consumption rate (OCR) in podocytes from three groups. Each error bar represents data from three wells. Oligomycin (arrow A), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (arrow B), and a combination of rotenone and antimycin A (arrow C) were added sequentially to the podocyte culture. (C) The baseline OCR was defined as the OCR before the application of oligomycin. (D) ATP-linked OCR was calculated as the baseline OCR minus the nadir following oligomycin. (E) Maximum respiration was defined as the peak OCR following the application of FCCP. (F) Effects of CREB RNAi on ATP; each bar represents data obtained from five independent experiments. (G and H) CREB silencing blocked the pCPT-cAMP-induced expression of peroxisome PGC1-α. (I) The regions within the PGC1-α promoter were analyzed by ChIP-qPCR. (J) Enrichment of CREB at the PGC1-α promoter. *, P < 0.05 compared with the control group; **, P < 0.05 compared with the ADR group; ***, P < 0.05 compared with the ADR-plus-pCPT-plus-ng group.

FIG 4

PKA signaling upregulated the mRNA expression of respiratory chain complex I. (A) Heat map of 13 mitochondrial DNA-expressed genes. The red color indicates a high relative expression and the green color indicates a low relative expression. (B to D) Real-time PCR analysis of the expression of mRNAs in ADR-treated podocytes in the absence or presence of pCPT. Each bar represents data obtained from five independent experiments. *, P < 0.05 compared with the control group; **, P < 0.05 compared with the ADR group.

FIG 5

CREB silencing blocked the pCPT-cAMP-induced increase in the protein expression of mitochondrial respiratory chain complexes. (A and B) Representative immunoblots and corresponding graph of mitochondrial respiratory chain complex proteins in podocytes treated with ADR in the absence or presence of pCPT for the indicated periods. (C to F) Representative immunoblots and corresponding graph of mitochondrial respiratory chain complex proteins in podocytes treated with CREB siRNA or a negative-control siRNA. Each bar represents data obtained from five independent experiments. (G) The regions within the ND3 promoter were analyzed by ChIP-qPCR. (H) Enrichment of CREB at the ND3 promoter. *, P < 0.05 compared with the control group; **, P < 0.05 compared with the ADR group; ***, P < 0.05 compared with the ADR-plus-pCPT-plus-ng group.

FIG 6

pCPT-cAMP alleviated ADR-induced proteinuria, renal oxidative stress, and loss of podocytes. (A) pCPT-cAMP increased p-CREB expression in mouse glomeruli (original magnification, ×400). (B) Albuminuria was expressed as the albumin-to-creatinine ratio (ACR) in four groups. Data are expressed as means ± SEM. (C) Urine proteins were stained with Coomassie brilliant blue and were resolved by gel electrophoresis. BSA, bovine serum albumin. (D) Immunohistochemical staining was performed to detect WT-1-positive cells in glomeruli (original magnification, ×400). WT-1-positive cells were counted by a single renal pathologist using a blinded method. At least 50 glomeruli per kidney were found. (E) Real-time PCR analysis of the expression of mitochondrial complex mRNAs in isolated glomeruli. (F) Immunofluorescence staining of 3-nitrotyrosine in mouse kidney (original magnification, ×400). Nestin is a marker protein of mature podocytes. *, P < 0.05 compared with the control group; **, P < 0.05 compared with the ADR group.