Low dose ouabain stimulates NaK ATPase α1 subunit association with angiotensin II type 1 receptor in renal proximal tubule cells

Abstract

Our laboratory has recently demonstrated that low concentrations of ouabain increase blood pressure in rats associated with stimulation of NaK ATPase activity and activation of the Src signaling cascade in NHE1-dependent manner. Proteomic analysis of human kidney proximal tubule cells (HKC11) suggested that the Angiotensin II type 1 receptor (AT1R) as an ouabain-associating protein. We hypothesize that ouabain-induced stimulation of NaK ATPase activity is mediated through AT1R. To test this hypothesis, we examined the effect of ouabain on renal cell angiotensin II production, the effect of AT1R inhibition on ouabain-stimulated NKA activity, and the effect of ouabain on NKA-AT1R association. Ouabain increased plasma angiotensin II levels in rats treated with ouabain (1μg/kg body wt./day) for 9days and increased angiotensin II levels in cell culture media after 24h treatment with ouabain in human (HKC11), mouse (MRPT), and human adrenal cells. Ouabain 10pM stimulated NKA-mediated ⁸⁶Rb uptake and phosphorylation of EGFR, Src, and ERK1/2. These effects were prevented by the AT1R receptor blocker candesartan. FRET and TIRF microscopy using Bodipy-labeled ouabain and mCherry-NKA or mCherry-AT1R demonstrated association of ouabain with AT1R and NKA. Further our FRET and TIRF studies demonstrated increased association between AT1R and NKA upon treatment with low dose ouabain. We conclude that ouabain stimulates NKA in renal proximal tubule cells through an angiotensin/AT1R-dependent mechanism and that this pathway contributes to cardiac glycoside associated hypertension.

Keywords: AT1R; FRET-TIRF microscopy; Human kidney cells; NaK ATPase; Ouabain; Proteomics.

Figure 1. Identification of ouabain-binding proteins

HKC11 cells were treated with 10 pM or 100 nM ouabain for 15 min at 37°C in humidified incubator. Cells were washed with ice-cold PBS, homogenized, and crude membranes were prepared as described in Methods. Cell membranes were solubilized in immunoprecipitation buffer and ouabain-binding proteins were immunoprecipitated with 1 μg Digibind (an antibody against cardioglycosides) per 100 μg protein as described in Methods. Proteins were eluted with 100 mM glycine pH 2 and collected in 1/10 volume 1 mM Tris-base pH 10 to neutralize the pH. Half of the proteins were subjected to LC/MS for identification of the proteins (Table 1–3 and Supplementary Tables 1–4) and the rest were used for western blot. A, Representative western blot from two individual experiments for NKA, NHE1, Src, EGFR, and caveolin-1 is shown. B, Cells transiently transfected with mCherry-labeled AT1R were treated with 10 nM Bodipy-ouabain for 15 min. Cells were washed with PBS to remove excess Bodipy-ouabain and live cells were imaged by confocal microscopy using Olympus FV1000 confocal microscope. A representative image from three independent experiments is shown.

Figure 2. Association between ouabain and NKA or AT1R in kidney proximal tubule cells

Immortalized human proximal tubule cells (HKC11) were transfected with either mCherry-tagged rat NKA or AT1R (red fluorescence). Cells were treated with bodipy-ouabain (10 pM (AT1R transfected cells) or 100 nM (rat NKA transfected cells), green fluorescence) for 15 min. Excess ouabain was washed with PBS and the cells were imaged for epifluorescence (A), sensitized FRET (B), or TIRF (D) microscopy as described in Methods. Representative images from 8 individual experiments are shown (n=8). C, Sensitized FRET after photo bleaching was calculated using SlideBook4 software as described in Methods. Each bar represents corrected FRET (FRETc) as mean±se from 8 individual experiments (n=8). In each experiment 20–30 cells were imaged and data from all the cells were pooled and considered as one data point.

Figure 2. Association between ouabain and NKA or AT1R in kidney proximal tubule cells

Immortalized human proximal tubule cells (HKC11) were transfected with either mCherry-tagged rat NKA or AT1R (red fluorescence). Cells were treated with bodipy-ouabain (10 pM (AT1R transfected cells) or 100 nM (rat NKA transfected cells), green fluorescence) for 15 min. Excess ouabain was washed with PBS and the cells were imaged for epifluorescence (A), sensitized FRET (B), or TIRF (D) microscopy as described in Methods. Representative images from 8 individual experiments are shown (n=8). C, Sensitized FRET after photo bleaching was calculated using SlideBook4 software as described in Methods. Each bar represents corrected FRET (FRETc) as mean±se from 8 individual experiments (n=8). In each experiment 20–30 cells were imaged and data from all the cells were pooled and considered as one data point.

Figure 2. Association between ouabain and NKA or AT1R in kidney proximal tubule cells

Immortalized human proximal tubule cells (HKC11) were transfected with either mCherry-tagged rat NKA or AT1R (red fluorescence). Cells were treated with bodipy-ouabain (10 pM (AT1R transfected cells) or 100 nM (rat NKA transfected cells), green fluorescence) for 15 min. Excess ouabain was washed with PBS and the cells were imaged for epifluorescence (A), sensitized FRET (B), or TIRF (D) microscopy as described in Methods. Representative images from 8 individual experiments are shown (n=8). C, Sensitized FRET after photo bleaching was calculated using SlideBook4 software as described in Methods. Each bar represents corrected FRET (FRETc) as mean±se from 8 individual experiments (n=8). In each experiment 20–30 cells were imaged and data from all the cells were pooled and considered as one data point.

Figure 3. Effect of ouabain on Angiotensin II release

Cells (A: adrenal cells, B: MDCK, C: HKC11, and D: MRPT) were treated with indicated ouabain concentrations for 24 h. Cell media (A–D) or plasma (E) from ouabain treated rats was collected and proteins were purified using C18 HPLC columns followed by determination of angiotensin concentration EIA as described in Methods. Each bar represents data as pg/mL (mean±se) from 4 independent experiments (n=4 for cell culture) or plasma from 6 animals (n=6 for plasma). * indicates P < 0.05 as calculated by two-way ANOVA followed by Bonferroni analysis.

Figure 4. Effect of ouabain on ACE and AGT expression

Human kidney proximal tubule cells (HKC11) were treated with 100 nM or 10 pM ouabain for 24 h at 37°C in humidified incubator. Cells were washed with ice-cold PBS and expression of ACE and AGT was determined by western blot in nuclear-free homogenates. A representative blot from 4 individual experiments is shown. Each bar represents data as ratio of arbitrary densitometry units between ACE or AGT and actin (mean±se) from 4 independent experiments (n=4). * indicates P < 0.05 as calculated by two-way ANOVA followed by Bonferroni analysis.

Figure 5. Effect of candesartan on ouabain-stimulated NKA activity in renal proximal tubule cells

Human (HKC11) or mouse (MRPT) kidney proximal tubule cell lines were treated for 15 min with ouabain (10 pM, HKC11; 100 nM, MRPT) in the presence or absence of AT1R blocker, candesartan (100 nM). Ouabain (1 mM)-sensitive ⁸⁶Rb uptake was determined as a measure of NKA-mediated ion transport. Each bar represents data as nmoles ⁸⁶Rb/mg protein/10 min (mean±se) from 6 individual experiments (n=6) performed in triplicate. * indicates P < 0.05 as calculated by one-way ANOVA followed by Bonferroni analysis.

Figure 6. Effect of candesartan on ouabain-stimulated NKA phosphorylation and expression in human kidney proximal tubule cells

HKC11 (A and C) or LLCPK1 cells stably expressing AT1R (B) were treated for 15 min with 10 pM or 1 nM ouabain in the presence or absence of AT1R blocker, candesartan (100 nM). A and B, cell membranes were immunoprecipitated with NKA antibodies (α6F) followed by western blot using phospho-tyrosine antibodies. Nitrocellulose membranes were stripped and reprobed with NKA antibodies to show equal loading (bottom panel). A representative blot from 4 (HKC11, n=4) or 2 (LLCPK1, n=2) independent experiments. Each bar represents data as ratio of band intensity of phospho to total NKA (mean±se) from 4 independent experiments (n=4) as Arbitrary Units (AU). C, cell surface was biotinylated as described in Methods. Crude membrane proteins were separated by 10% SDS-PAGE and analyzed by immunoblot using NKA (upper panel) or caveolin-1 (lower panel) antibodies. Each bar represents data (mean±se) densitometry data (Arbitrary Units (AU)) from three independent experiments as fold difference from the vehicle-treated group as. * indicates P < 0.05 as calculated by two-way ANOVA followed by Bonferroni analysis.

Figure 6. Effect of candesartan on ouabain-stimulated NKA phosphorylation and expression in human kidney proximal tubule cells

HKC11 (A and C) or LLCPK1 cells stably expressing AT1R (B) were treated for 15 min with 10 pM or 1 nM ouabain in the presence or absence of AT1R blocker, candesartan (100 nM). A and B, cell membranes were immunoprecipitated with NKA antibodies (α6F) followed by western blot using phospho-tyrosine antibodies. Nitrocellulose membranes were stripped and reprobed with NKA antibodies to show equal loading (bottom panel). A representative blot from 4 (HKC11, n=4) or 2 (LLCPK1, n=2) independent experiments. Each bar represents data as ratio of band intensity of phospho to total NKA (mean±se) from 4 independent experiments (n=4) as Arbitrary Units (AU). C, cell surface was biotinylated as described in Methods. Crude membrane proteins were separated by 10% SDS-PAGE and analyzed by immunoblot using NKA (upper panel) or caveolin-1 (lower panel) antibodies. Each bar represents data (mean±se) densitometry data (Arbitrary Units (AU)) from three independent experiments as fold difference from the vehicle-treated group as. * indicates P < 0.05 as calculated by two-way ANOVA followed by Bonferroni analysis.

Figure 7. Effect of ouabain on association between NKA and AT1R in kidney proximal tubule cells

Immortalized human proximal tubule cells (HKC11) were transfected with mCherry-tagged AT1R (red fluorescence) and/or GFP-tagged human NKA (green fluorescence). Cells were treated with ouabain (1 nM) for 15 min. Excess ouabain was washed with PBS and the cells were imaged for epifluorescence (A), sensitized FRET (B), or TIRF (D) microscopy before and after treatment with ouabain (1 nM) as described in Methods. Representative images are shown from 8 individual experiments are shown (n=8). C, Sensitized FRET after photo bleaching was calculated using SlideBook4 software as described in Methods. Each bar represents corrected FRET (FRETc) as mean±se from 8 individual experiments (n=8). In each experiment 20–30 cells were imaged and data from all the cells were pooled and considered as one data point.

Figure 7. Effect of ouabain on association between NKA and AT1R in kidney proximal tubule cells

Immortalized human proximal tubule cells (HKC11) were transfected with mCherry-tagged AT1R (red fluorescence) and/or GFP-tagged human NKA (green fluorescence). Cells were treated with ouabain (1 nM) for 15 min. Excess ouabain was washed with PBS and the cells were imaged for epifluorescence (A), sensitized FRET (B), or TIRF (D) microscopy before and after treatment with ouabain (1 nM) as described in Methods. Representative images are shown from 8 individual experiments are shown (n=8). C, Sensitized FRET after photo bleaching was calculated using SlideBook4 software as described in Methods. Each bar represents corrected FRET (FRETc) as mean±se from 8 individual experiments (n=8). In each experiment 20–30 cells were imaged and data from all the cells were pooled and considered as one data point.

Figure 7. Effect of ouabain on association between NKA and AT1R in kidney proximal tubule cells

Immortalized human proximal tubule cells (HKC11) were transfected with mCherry-tagged AT1R (red fluorescence) and/or GFP-tagged human NKA (green fluorescence). Cells were treated with ouabain (1 nM) for 15 min. Excess ouabain was washed with PBS and the cells were imaged for epifluorescence (A), sensitized FRET (B), or TIRF (D) microscopy before and after treatment with ouabain (1 nM) as described in Methods. Representative images are shown from 8 individual experiments are shown (n=8). C, Sensitized FRET after photo bleaching was calculated using SlideBook4 software as described in Methods. Each bar represents corrected FRET (FRETc) as mean±se from 8 individual experiments (n=8). In each experiment 20–30 cells were imaged and data from all the cells were pooled and considered as one data point.

Figure 8. Proposed model of ouabain action in the proximal tubule

(1) Endogenous ouabain binds to and inhibits high-affinity NKA in the basolateral membrane of the proximal tubule, (2) resulting in an increase in intracellular sodium, ACE, and Ang II. (3) Ang II binds AT1 receptors in the basolateral membrane in an autocrine manner. (4) AT1R activation stimulates its association with NKA and (5) the activation of EGFR. (6) EGFR signaling stimulates the translocation of intracellular NKA to the plasma membrane, ultimately resulting in increased sodium reabsorption in the kidney and higher systemic blood pressure.