Ouabain Enhances ADPKD Cell Apoptosis via the Intrinsic Pathway

Abstract

Progression of autosomal dominant polycystic kidney disease (ADPKD) is highly influenced by factors circulating in blood. We have shown that the hormone ouabain enhances several characteristics of the ADPKD cystic phenotype, including the rate of cell proliferation, fluid secretion and the capacity of the cells to form cysts. In this work, we found that physiological levels of ouabain (3 nM) also promote programmed cell death of renal epithelial cells obtained from kidney cysts of patients with ADPKD (ADPKD cells). This was determined by Alexa Fluor 488 labeled-Annexin-V staining and TUNEL assay, both biochemical markers of apoptosis. Ouabain-induced apoptosis also takes place when ADPKD cell growth is blocked; suggesting that the effect is not secondary to the stimulatory actions of ouabain on cell proliferation. Ouabain alters the expression of BCL family of proteins, reducing BCL-2 and increasing BAX expression levels, anti- and pro-apoptotic mediators respectively. In addition, ouabain caused the release of cytochrome c from mitochondria. Moreover, ouabain activates caspase-3, a key "executioner" caspase in the cell apoptotic pathway, but did not affect caspase-8. This suggests that ouabain triggers ADPKD cell apoptosis by stimulating the intrinsic, but not the extrinsic pathway of programmed cell death. The apoptotic effects of ouabain are specific for ADPKD cells and do not occur in normal human kidney cells (NHK cells). Taken together with our previous observations, these results show that ouabain causes an imbalance in cell growth/death, to favor growth of the cystic cells. This event, characteristic of ADPKD, further suggests the importance of ouabain as a circulating factor that promotes ADPKD progression.

Keywords: BCL-2 proteins; K-ATPase signaling; Na; caspase; cytochrome c; polycystic kidney disease; programmed cell death.

Figure 1

Ouabain induces apoptosis in ADPKD, but not in NHK cells. (A) Alexa Fluor 488 labeled-Annexin V and PI staining. After treatment with ouabain for 24 h, cells were labeled and sorted using flow cytometry. The top panel shows representative plots for the sorted NHK and ADPKD cells, without and with of ouabain. Different cell populations undergoing necrosis, early and late apoptosis, identified by differential annexin-V/PI labeling, were quantified and expressed as percent of total cells (bottom panel). (B) DNA fragmentation assays. Cells seeded onto glass coverslips were incubated with 3 nM ouabain and the indicated experimental conditions for 24 h, fixed and analyzed for apoptosis using the Dead-End Fluormetric TUNEL System. Fragmented DNA was labeled by incorporation of fluorescein-12-dUTP(a) at 3′-OH DNA ends. The nuclei were counterstained with DAPI and visualized by immunofluorescence microscopy. At least 10 random fields were analyzed from each of three different ADPKD kidneys. In all graphs, bars represent the mean ± SEM of three different experiments. The asterisks indicate values that are statistically different compared to the corresponding untreated control, with P < 0.05.

Figure 2

Ouabain modifies the expression of BCL-2 and BAX protein levels in ADPKD, but not NHK cells. NHK (A) and ADPKD (B) cells were treated in the absence and presence of 3 nM ouabain and 24 h later, expression levels of BCL-2 and BAX were determined by immunoblot. Tubulin was used as a loading control. Top panels show the densitometric analysis of the protein bands, while bottom panels show representative blots. Bars are the mean ± SEM of three experiments. Asterisks indicate statistically different values, with P < 0.05 vs. untreated control.

Figure 3

Ouabain stimulates cytochrome C release from mitochondria in ADPKD, but not NHK cells. (A) Immunoblot analysis. After treatment with or without 3 nM ouabain for 24 h, cells were processed to obtain cell cytoplasmic and mitochondrial fractions, and samples were subjected to immunoblot analysis to determine cytochrome c levels. Tubulin was used as a loading control, while VDAC was used as a mitochondrial marker. The bottom panels show representative immunoblots. Relative densitometric levels for cytochrome c in the cytoplasm are shown in the upper panels and they represent data compiled from four different experiments. Due to the difference between mitochondrial and cytoplasmic cytochrome c levels, and to better quantify the cytoplasmic bands, different exposure times for the cytochrome c cytosolic and mitochondrial samples from the same immunoblots were used. (B) Immunocytochemical analysis. After treatment with 3 nM ouabain for 24 h, NHK and ADPKD cells were labeled for cytochrome c and MitoTracker, to visualize mitochondria. Cytochrome c release was quantified and expressed as the cytosol to mitochondrial ratio. Bars represent the compiled data from 3 different experiments. In A and B, bars represent the mean ± SEM. Asterisks indicate statistically different values, with P < 0.05 vs. untreated control.

Figure 4

Ouabain stimulates caspase-3 cleavage and activity in ADPKD, but not NHK cells. (A) Caspase-3 cleavage. After treatment with 3 nM ouabain for 24 h, the total (35 kDa) and cleaved products (17 and 12 kDa) of caspase-3 were determined in NHK and ADPKD cells by immunoblot and densitometric analysis. Upper panels show representative blots and bottom panels the densitometric analysis from three different experiments. Bars are the mean ± SEM of three experiments. (B) Relative caspase-3 and -7 activity levels. ADPKD cells were treated in the absence and presence of 3 nM ouabain and caspase activity was measured using the Caspase-3/7 Glo Assay. Data are the mean ± SEM of sextuplicate experiments. (C) PARP-1 cleavage. After ouabain treatment for 24 h, NHK and ADPKD cell lysates were subjected to immunoblot to determine fragmentation of the caspase-3 substrate, PARP-1. The top panels show the densitometric analysis of the total and cleaved (89 kDa) PARP-1 bands. Cleaved PARP-1 is expressed relative to the corresponding untreated controls. The bottom panels show representative immunoblots. Bars represent the mean ± SEM of 5 different experiments. Asterisks show statistically different values, compared to untreated controls and with P < 0.05.

Figure 5

Ouabain does not affect the extrinsic pathway for apoptosis in ADPKD cells. NHK (A) and ADPKD (B) cells were treated with 3 nM ouabain for 24 h and the total (41/43 kDa) and cleaved (37 kDa) forms of caspase-8 were determined by immunoblot analysis and quantified by densitometry. The top panels show the densitometric analysis of the bands from 4 different experiments. The bottom panels show representative blots. Values are the mean ± SEM. Asterisks indicate statistically different values, with P < 0.05 vs. untreated control.

Figure 6

Ouabain induces ADPKD cell apoptosis independent from its proliferative effects. NHK (A) and ADPKD (B) cells were treated with or without 3 nM ouabain, in the presence and absence of 2.5 M thymidine to arrest cell growth. Alexa fluor-labeled-annexin-V and PI staining and cell sorting were utilized to detect apoptosis/necrosis. Bars represent the mean ± SEM of 3 different experiments. While the values for ouabain induced apoptosis were statistically significant between NHK and ADPKD cells (P < 0.05), no statistical differences were found between cells treated with ouabain in the presence and absence of thymidine, both for NHK or ADPKD samples.