Dietary fructose enhances angiotensin II-stimulated Na⁺ transport via activation of PKC-α in renal proximal tubules

Abstract

Angiotensin II (ANG II) stimulates proximal nephron transport via activation of classical protein kinase C (PKC) isoforms. Acute fructose treatment stimulates PKC and dietary fructose enhances ANG II’s ability to stimulate Na⁺ transport, but the mechanisms are unclear. We hypothesized that dietary fructose enhances ANG II’s ability to stimulate renal proximal tubule Na⁺ reabsorption by augmenting PKC-α activation and increases in intracellular Ca²⁺. We measured total and isoform-specific PKC activity, basal and ANG II-stimulated oxygen consumption, a surrogate of Na⁺ reabsorption, and intracellular Ca²⁺ in proximal tubules from rats given either 20% fructose in their drinking water (fructose group) or tap water (control group). Total PKC activity was measured by ELISA. PKC-α, PKC-β, and PKC-γ activities were assessed by measuring particulate-to-soluble ratios. Intracelluar Ca²⁺ was measured using fura 2. ANG II stimulated total PKC activity by 53 ± 15% in the fructose group but not in the control group (−15 ± 11%, P < 0.002). ANG II stimulated PKC-α by 0.134 ± 0.026 but not in the control group (−0.002 ± 0.020, P < 0.002). ANG II increased PKC-γ activity by 0.008 ± 0.003 in the fructose group but not in the control group (P < 0.046). ANG II did not stimulate PKC-β in either group. ANG II increased Na⁺ transport by 454 ± 87 nmol·min⁻¹·mg protein⁻¹ in fructose group, and the PKC-α/β inhibitor Gö6976 blocked this increase (−96 ± 205 nmol·min⁻¹·mg protein⁻¹, P < 0.045). ANG II increased intracellular Ca²⁺ by 148 ± 53 nM in the fructose group but only by 43 ± 10 nM in the control group (P < 0.035). The intracellular Ca²⁺ chelator BAPTA blocked the ANG II-induced increase in Na⁺ transport in the fructose group. We concluded that dietary fructose enhances ANG II’s ability to stimulate renal proximal tubule Na⁺ reabsorption by augmenting PKC-α activation via elevated increases in intacellular Ca²⁺.

INTRODUCTION

Renal proximal tubules reabsorb two-thirds of the water and fluid filtered by the glomerulus. Na⁺ entry into proximal tubule cells is mediated by a number of cotransporters and exchangers. Exit is mediated by basolateral Na⁺-K⁺-ATPase, which pumps 3 Na⁺ into the extracellular medium while transporting 2 K⁺ into the cell and cleaving one ATP to ADP. The ADP is rephosphorylated in the mitochondria. Because of the high rates of active Na⁺ transport dependent on basolateral Na⁺-K⁺-ATPase, Na⁺ reabsorption in this segment is stoichiometrically related to oxygen consumption at a ratio of 18 Na⁺/O₂ (7, 14, 19, 27). Several hormones and factors regulate proximal nephron Na⁺ reabsorption; chief among these is angiotensin II (ANG II) (4, 30).

ANG II stimulates proximal nephron Na⁺ reabsorption via activation of ANG II type 1 receptors (6, 35). These receptors activate both G_q and G_i α-subunits of heteromeric G proteins. Consequently, protein kinase C (PKC) and reductions in cAMP mediate the effects of ANG II on Na⁺ reabsorption in the proximal nephron (31, 34). There are at least 11 isoforms of PKC, several of which are activated by ANG II. Classical PKC isoforms, α, β, and γ, are activated by increases in intracellular Ca²⁺ and acyl glycerols (21, 36) and appear to be the primary mediators of ANG II-induced increases in Na⁺ reabsorption in proximal tubules (8, 22, 24, 25).

Dietary fructose consumption has increased since the early 1970s due to the addition of high-fructose corn syrup to many beverages and foods. In the United States, fructose consumption went from <2 to >40 lbs/yr per person (23, 28, 39) in the past 5 decades. On average, Americans consume >11% of their daily calories as fructose (32, 39), while more than 17 million people in the United States acquire >20% of their calories as fructose (39).

High fructose intake has been associated with the progression of renal damage in rats (37). Dietary fructose also causes salt-sensitive hypertension (5). This is likely a direct effect, because acute fructose treatment stimulates nephron Na⁺ transport (1). Acute fructose treatment stimulates proximal nephron Na⁺ reabsorption via activation of the PKC signaling pathway (5). Dietary fructose enhances the ability of ANG II to stimulate Na⁺ reabsorption in this segment without altering its maximum effect (10, 12). In these studies, the effect of different concentrations of ANG II on Na⁺ transport was studied in control and fructose-fed rats. Although the higher concentrations of ANG II stimulated transport in both groups, the lowest concentration (1 pM) augmented it in fructose-fed rats but not in control rats. Therefore, 1 pM was used to maximize the difference in response between the control and fructose groups. Although PKC is likely involved (10), the mechanisms by which dietary fructose augments the stimulatory action of ANG II on proximal nephron Na⁺ reabsorption are poorly understood. We hypothesized that dietary fructose enhances ANG II’s ability to increase intracellular Ca²⁺, which, in turn, augments PKC-α activation and thereby Na⁺ transport.

MATERIALS AND METHODS

Reagents.

Unless specified, all drugs and reagents were obtained from Sigma-Aldrich (St. Louis, MO). The Pierce Coomassie (Bradford) Protein Assay Kit was obtained from Thermo Scientific (Rockford, IL). HEPES-buffered physiological saline solution contained (in mM) 10 HEPES (pH 7.4 at 37°C), 130 NaCl, 4 KCl, 0.4 NaH₂PO₄, 2.1 Na₂HPO₄, 1.2 MgSO₄, 5.5 glucose, 6.0 dl-alanine, 2.0 Ca(lactate)₂, and 1.0 Na₃citrate, and the osmolality was adjusted to 310 ± 5 mosmol/L with mannitol.

Animals.

Male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA) weighing between 104 and 120 g were randomly assigned to one of two experimental groups. One group received 20% fructose solution (fructose group), whereas the other group received tap water (control group). The 20% fructose solution was prepared fresh every 2–3 days.

Animals in both experimental groups were housed under normal rat housing conditions with a 12:12-h light-dark cycle and ad libitum provision of food and fluids. After 6–8 days of dietary treatment, animals underwent terminal surgery. Rats were anesthetized with ketamine (100 mg/kg body wt ip) and xylazine (20 mg/kg body wt ip) and given 100 units heparin (ip). This study was approved by the Case Western Reserve University Institutional Animal Care and Use Committee. All experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

Proximal tubule suspensions.

Proximal tubule suspensions were generated using methods similar to those we have previously used (10) with the modifications described here. Briefly, rats were anesthetized and an abdominal U-shaped incision was made. The left kidney was retroperfused from the abdominal aorta with 80 mL of HEPES-buffered physiological saline solution at 4°C. The physiological saline solution contained 2 mg/mL collagenase type I and 1 U/mL heparin and was infused at 4 mL/min for 20 min. The digested kidney was excised before flow was stopped and then immediately immersed in physiological saline solution at 4°C. It was then transferred to a cold Lucite plate, and the cortex was gently scraped with a razor blade to collect proximal tubules. This tissue was minced and transferred to a 5-mL conical tube containing 4 mL of cold HEPES-buffered physiological saline solution. Tissue was disrupted by passing it through a pipette tip. The resulting suspension was filtered sequentially through 390- and 250-µm meshes and recovered by centrifugation at 100 g for 2 min at 4°C. The final pellet was resuspended in 3−5 mL of HEPES-buffered physiological saline solution with protease inhibitor cocktail (catalog no. P8340, Sigma-Aldrich).

PKC activity measurements.

After the generation of proximal tubule suspensions, two 4-mL aliquots from each suspension were equilibrated for 5 min at 37°C while stirred. One served as a control (untreated), whereas the other was treated for 5 min with 1 pM ANG II. Each sample was centrifuged at 100 g for 2 min at 4°C. Pelleted tubules were lysed in 0.4 mL CelLytic M (catalog no. C2978, Sigma-Aldrich) and sonicated for 30 cycles at output 2, 30% duty cycle (Branson 450 Analog Sonifier, catalog no. 101-063-198R). Ten microliters of sonicate were assayed using the PKC kinase activity kit (catalog no. ADI-EKS-420A, Enzo) with a reaction time of 1 h. PKC activity measurements (relative to the provided active PKC standard) were normalized to total protein.

Western blots of PKC-α, PKC-β, and PKC-γ isoforms.

After proximal tubule suspensions were generated, two 1.5-mg aliquots of proximal tubules were taken from the suspension and warmed to 37°C with agitation for 5 min. Tubules were then treated with either 1 pM ANG II or vehicle for 5 min. After treatment, tubules were cooled to 4°C on ice and centrifuged at 300 g for 2 min at 4°C. Tubules were resuspended in 400 µL of homogenization buffer containing 50 mM Tris·HCl, 50 mM NaCl, and 2× protease inhibitor cocktail. Tubules were sonicated on ice for 30 cycles at duty cycle 30 and output 2 (Branson 450 Analog Sonifier). The solution was centrifuged at 1,150 g for 10 min at 4°C to pellet cell debris and nuclei, and the supernatant was subjected to centrifugation at 64,400 g for 100 min at 4°C. The supernatant was considered to be the soluble fraction. The pellet was resuspended in homogenization buffer containing 0.2% Triton X-100 and 2× protease inhibitor cocktail and then centrifuged at 64,400 g for 40 min at 4°C, with the resulting supernatant considered to be the particulate fraction. Protein concentrations were determined by the Pierce Coomassie (Bradford) Protein Assay Kit, and the remaining protein samples were stored at −80°C. After being thawed, samples were loaded onto 10% Criterion TGX Stain-Free polyacrylamide gels (Bio-Rad Laboratories, Hercules, CA). Soluble and particulate protein samples with vehicle or ANG II treatment from the fructose and control groups were run in pairs on the same gel. Electrophoresis was performed to separate proteins, which were transferred onto PVDF membranes via the iBlot 2 Dry Blotting System (ThermoFisher Scientific, Waltham, MA).

The antibody incubations and washes varied slightly for each PKC isoform. For PKC-α, membranes were incubated in blocking buffer composed of 5% nonfat milk in PBS-Tween 20 (PBS-T; 137 mM NaCl, 2.7 mM KCl, 10 mM NaH₂PO₄, and 0.1% Tween 20) for 60 min and then incubated with primary antibody (Table 1). Membranes were washed with PBS-T for 15 min and incubated with secondary antibody in PBS-T with 5% nonfat milk. Membranes were washed with PBS-T for 15 min and incubated with a luminol-based chemiluminescent horseradish peroxidase substrate (Pierce Biotechnology, Rockford, IL) and exposed in an Azure C600 imaging system (Azure Biosystems, Dublin, CA) for 100 s. An image was captured, and densitometry was performed using ImageJ (imagej.nih.gov). Particulate-to-soluble ratios were calculated to assess changes in PKC isoform activation.

Table 1.

Antibodies and blotting conditions

Antibody	Provider	Catalog No.	Source	Dilution
PKC-α	BD Biosciences	610108	Mouse	1:1,000
Secondary HRP anti-mouse IgG	BD Biosciences	554002	Goat	1:1,000
PKC-βI	Abcam	ab195039	Rabbit	1:1,000
Secondary HRP anti-rabbit IgG	GE Healthcare Life Sciences	NA9340	Donkey	1:1,000
PKC-γ	Santa Cruz Biotechnology	sc-166385	Mouse	1:200
Secondary HRP anti-mouse IgG	GE Healthcare Life Sciences	Na931	Sheep	1:400

HRP, horseradish peroxidase.

For PKC-β, membranes were incubated in blocking buffer composed of 5% nonfat milk in Tris-buffered saline-Tween 20 (TBS-T; 140 mM NaCl, 3 mM KCl, 25 mM Tris base, and 0.1% Tween 20) for 60 min and then incubated with primary monoclonal antibody (Table 1). Membranes were washed with TBS-T for 15 min and incubated with secondary antibody in TBS-T with 5% nonfat milk. Membranes were washed with TBS-T for 15 min and developed and analyzed using methods similar to those used for PKC-α. For PKC-γ, membranes were incubated in blocking buffer composed of 2.5% nonfat milk in TBS-T and then incubated with primary antibody (Table 1). Membranes were washed with TBS-T for 15 min and incubated with secondary antibody in 2.5% nonfat milk in TBS-T. Membranes were washed with TBS-T for 15 min and developed and analyzed using methods similar to those used for PKC-α.

Oxygen consumption.

After proximal tubule suspensions had been generated, they were resuspended in 3 mL of HEPES-buffered saline solution with protease inhibitor to prevent ANG II degradation. One-milliliter aliquots were added to each of two chambers containing 5 mL of HEPES-buffered physiological saline solution equilibrated with 100% O₂ in a Yellow Springs Instruments model 5301B bath assembly (Yellow Springs, OH) at 37°C for 5 min. Gö6976 (100 nM), a commonly used (16, 38, 40, 41) classical PKC-α/β inhibitor (29), was added to one chamber, whereas vehicle was added to the other. The chambers were then sealed, and oxygen tension was monitored with a Yellow Springs Instruments 5300 Biological Oxygen Monitor using methods similar to those we have previously described (10). Once a stable baseline was achieved, 1 pM ANG II was added to each chamber while oxygen tension was continuously recorded. At the end of the experiment, total protein in each chamber was measured. Oxygen consumption was calculated from the chamber volume, the rate of decrease in oxygen tension, and the amount of protein. All Na⁺ transport in proximal tubules ultimately depends on Na⁺-K⁺-ATPase. Furthermore, essentially all ATP generation in this segment is a result of aerobic metabolism and oxidative phosphorylation. Six ATP are generated per molecule of O₂ consumed in the proximal nephron (14), and 3 Na⁺ are transported per ATP used by the pump (19). Thus, 18 Na⁺ are transported per O₂ molecule (7, 19, 27). About 60–70% of total oxygen consumption is due to Na⁺ transport in the proximal nephron (2). Oxygen consumption has been used by many investigators as a surrogate measure of Na⁺ transport in the renal epithelium. Therefore, based on this stoichiometry, oxygen consumption was converted to Na⁺ transported using the factor of 18 Na⁺/O₂ (27). ANG II-stimulated Na⁺ transport in the absence and presence of Gö6976 was compared.

To measure the effect of chelating intracellular Ca²⁺ on ANG II-stimulated Na⁺ transport, the above protocol was repeated except that BAPTA (25 μM) was used instead of Gö6976.

Intracellular Ca²⁺ in isolated proximal tubules.

Proximal tubules were isolated and perfused as previously described (5). Briefly, control and fructose-fed rats rats weighing 100–150 g were anesthetized with ketamine (100 mg/kg body wt ip) and xylazine (20 mg/kg body wt ip). The abdominal cavity was opened, and the left kidney superfused with ice-cold 150 mM NaCl, removed, and then placed in physiological saline solution (4°C). Slices were cut, and proximal tubules were dissected from medullary rays under a stereomicroscope at 4–10°C. Tubules were transferred to a temperature-regulated chamber and perfused using concentric glass pipettes at 37 ± 1°C. They were loaded with 1 μM fura 2-AM (Life Technolgies, Carlsbad, CA) for 30 min and washed for 15 min using dye-free physiological saline solution at 37 ± 1°C. A xenon arc lamp and a lambda 10-2 filter wheel (Sutter Instrument, Novato, CA) were used to excite the dye alternately at 340 and 380 nm. Emitted fluorescence was collected with a ×40 oil-immersion objective mounted on an inverted microscope (Nikon Eclipse TE300) equipped with a 400-nm dichroic mirror and a 470- to 550-nm barrier filter and digitally imaged with a CoolSnap HQ digital camera (Photometrics, Tucson, AZ). Ratiometric data (340-to-380-nm fluorescence), which is proportional to intracellular Ca²⁺, were recorded with MetaFluor version 6.2r6 imaging software (Molecular Devices, Downingtown, PA). Intracellular Ca²⁺ was measured every 10 s for 2 min (basal) and then for 3 min in the presence of 1 nM ANG II added to the bath and then an additional 3 min with 100 nM ANG II, a concentration that should give maximal stimulation. Intracellular Ca²⁺ was calibrated at the end of each experiment with 20 μM 4-bromo-A23187 (Life Technologies) and 10 mM MnCl₂. Intracellular Ca²⁺ was calculated using the following equation as previously described.

Intracellular Ca²⁺ concentration = K_d(F_min/F_max)(R − R_min)/(R_max − R)where K_d is the Ca²⁺ dissociation constant of fura 2 (224 nM), F_min is the fluorescence intensity of 380 nm during MnCl₂, F_max is the fluorescence intensity of 380 nm during 4-bromo-A23187, R is the ratio between 340 and 380 nm of each sample, R_min is the minimum ratio between 340 and 380 nm during MnCl₂, and R_max is the maximum ratio between 340 and 380 nm during 4-bromo-A23187. To assure that data were obtained from viable tubules, experiments in which tubules exhibited maximal stimulation <50 nM intracellular Ca²⁺ were excluded.

Statistical analysis.

Results are expressed as means ± SE. P values were calculated using paired or unpaired t tests as noted in the figures. Corrections for multiple testing were made using Hochberg’s method (15). P < 0.05 was considered significant. Corrected P values are reported.

RESULTS

We have shown that ANG II stimulates proximal nephron PKC activity (3) and that acute fructose treatment also activates PKC (5). Thus, to begin to test our hypothesis, we first measured the effect of dietary fructose on ANG II-stimulated total PKC activity in proximal tubule suspensions from fructose-fed and control rats (Fig. 1). We found that ANG II increased total PKC activity in tubules from the fructose group by 53 ± 15% but not in the control group (−15 ± 11%, n = 9 for each group, P < 0.002, fructose vs. control group).

Fig. 1.

Angiotensin II (ANG II)-induced changes in total protein kinase C (PKC) activity in proximal tubules from rats treated with 20% fructose in drinking water (fructose group) or tap water (control group) for 6−8 days. n = 9 for each group. An unpaired t test was used.

The classical isoforms of PKC, α, β and γ, are considered key regulators of ANG II-stimulated Na⁺ reabsorption. As a result, we next tested activation of these isoforms by acute ANG II treatment in proximal tubules from fructose-fed and control rats (Fig. 2). PKC translocates from the cytosol to the membrane when activated. Therefore, we assessed PKC activity by measuring the change in the particulate-to-soluble ratio of each isoform. ANG II treatment increased the particulate-to-soluble ratio of PKC-α by 0.134 ± 0.026 in tubules from the fructose group and −0.002 ± 0.020 in tubules from the control group (n = 9 for each group, P < 0.002, fructose vs. control group).

Fig. 2.

Angiotensin II (ANG II)-induced changes in protein kinase C (PKC)-α activity in proximal tubules from rats treated with 20% fructose in drinking water (fructose group) or tap water (control group) for 6−8 days. A: representative blot; B: mean data. n = 9 for each group. An unpaired t test was used. Sol., soluble; Par., particulate.

PKC-β may also contribute to increased total PKC activity. Consequently, we next measured ANG II-stimulated PKC-β activation in tubules from fructose-fed and control rats (Fig. 3). Acute ANG II treatment did not alter the particulate-to-soluble ratio in tubules from the fructose group (−0.005 ± 0.007, n = 5) or the control group (−0.002 ± 0.006, n = 5).

Fig. 3.

Angiotensin II (ANG II)-induced changes in protein kinase C (PKC)-β activity in proximal tubules from rats treated with 20% fructose in drinking water (fructose group) or tap water (control group) for 6−8 days. A: representative blot; B: mean data. n = 5 for each group. An unpaired t test was used. Sol., soluble; Par., particulate.

Finally, we measured PKC-γ activation (Fig. 4). ANG II treatment increased the particulate-to-soluble ratio of PKC-γ by 0.008 ± 0.003 in tubules from the fructose group but not from the control group (−0.002 ± 0.003, n = 5 for each group, P < 0.046, fructose vs. control group).

Fig. 4.

Angiotensin II (ANG II)-induced changes in protein kinase C (PKC)-γ activity in proximal tubules from rats treated with 20% fructose in drinking water (fructose group) or tap water (control group) for 6−8 days. A: representative blot; B: mean data. n = 5 for each group. An unpaired t test was used. Sol., soluble; Par., particulate.

To show that the increase in PKC activity has a physiological effect and to investigate the roles of PKC-α and PKC-γ, we used the PKC-α/β inhibitor Gö6976 and measured its effect on ANG II-stimulated Na⁺ transport in proximal tubule suspensions (Fig. 5). We found that ANG II stimulated Na⁺ transport by 454 ± 87 nmol Na⁺·min⁻¹·mg protein⁻¹ in tubules from the fructose group in the absence of Gö6976. However, in its presence, ANG II was unable to stimulate transport in tubules from the fructose group (change of −96 ± 205 nmol Na⁺·min⁻¹·mg protein⁻¹, n = 5, P < 0.045, ANG II vs. ANG II + Gö6976).

Fig. 5.

Effect of the selective protein kinase C-α/β inhibitor Gö6976 (100 nM) on angiotensin II (ANG II)-induced stimulation of Na⁺ transport by proximal tubules in fructose-fed rats. Fructose-fed rats were given 20% fructose in drinking water for 6−8 days. n = 5 for each group. A paired t test was used.

Since PKC-α and PKC-γ are Ca²⁺ dependent, their enhanced activation may be caused by elevated increases in intracellular Ca²⁺ in proximal tubule cells. Figure 6 shows the effect of ANG II on intracellular Ca²⁺ in tubules from fructose-fed and control rats. In tubules from the fructose group, ANG II increased intracellular Ca²⁺ by 148 ± 53 nmol (n = 7), while in the control group (n = 10), the increase was significantly less (43 ± 10 nmol, P < 0.035, fructose vs. control group).

Fig. 6.

Angiotensin II (ANG II)-induced changes in intracellular Ca²⁺ (Ca_i) in proximal tubules from rats treated with 20% fructose in drinking water (fructose group) or tap water (control group) for 6−8 days. n = 7 rats in the fructose group; n = 10 rats in the control group. An unpaired t test was used.

To further examine the role of increases in intracellular Ca²⁺ in enhanced ANG II-stimulated Na⁺ transport, we used the intracellular Ca²⁺ chelator BAPTA and measured its effect in proximal tubule suspensions from fructose-fed rats (Fig. 7). In the absence of BAPTA, ANG II stimulated Na⁺ transport in tubules from the fructose group by 432 ± 47 nmol Na⁺·min⁻¹·mg protein⁻¹. However, in the presence of the Ca²⁺ chelator, ANG II did not stimulate transport in tubules from the fructose group (change of 47 ± 62 nmol Na⁺·min⁻¹·mg protein⁻¹, n = 5 P < 0.004, ANG II vs. ANG II + BAPTA).

Fig. 7.

Effect of the Ca²⁺ chelator BAPTA (25 μM) on angiotensin II (ANG II)-induced stimulation of Na⁺ transport by proximal tubules in fructose-fed rats. Fructose-fed rats were given 20% fructose in drinking water for 6−8 days. n = 5 for each group. A paired t test was used.

DISCUSSION

Dietary fructose augments the ability of low concentrations of ANG II to stimulate proximal nephron Na⁺ reabsorption, but the signaling mechanisms by which this occurs remain unknown. We hypothesized that dietary fructose enhances ANG II’s ability to stimulate renal proximal tubule Na⁺ reabsorption by augmenting PKC-α activation and increases in intracellular Ca²⁺. We found that 20% fructose in the drinking water enhanced the ability of ANG II to stimulate total PKC, PKC-α, and PKC-γ activity in rat proximal tubules. Additionally, a PKC-α/β inhibitor blocked ANG II-stimulated active Na⁺ transport in proximal tubules from fructose-fed rats. We found that dietary fructose elevated ANG II-induced increases in intracellular Ca²⁺. Finally, we found that chelating intracellular Ca²⁺ blocked ANG II-stimulated Na⁺ transport, thereby supporting the link between ANG II, increases in intracellular Ca²⁺, PKC activation, and enhanced transport.

Our data showing that 20% fructose in drinking water enhanced total PKC activity stimulated by ANG II in proximal tubules is novel. These results are supported by data showing that a PKC inhibitor can prevent acute fructose treatment from augmenting Na⁺/H⁺ exchange activity (5) and the ability of dietary fructose to elevate ANG II-stimulated Na⁺ reabsorption in proximal tubules (10). However, neither of these reports measured PKC activity or investigated the isoform(s) involved. Furthermore, they did not study the mechanism by which PKC was activated.

Given that many investigators have reported that ANG II stimulates Na⁺ reabsorption in the proximal nephron via PKC, one might well ask why we did not observe an increase in activity in control suspensions. The concentration of ANG II that we used for most experiments was 1 pM. This concentration is the threshold at which ANG II begins to stimulate transport. Thus, it has no effect on Na⁺ reabsorption or PKC activity in control tubules. We chose it because this is the most sensitive portion of the concentration-response curve to demonstrate a difference in the sensitivity of frucose-treated and control tubules. We have used this strategy several times and have also reported that while the concentration-response relationship is shifted to lower values, the maximum response is not changed by dietary fructose (10, 12).

Here, for the first time, we show that dietary fructose augments the ability of ANG II to stimulate the activity of the classical PKC isoforms α and γ, suggesting that one or both of these isoforms mediate the stimulatory effects of ANG II on Na⁺ reabsorption in fructose-fed rats. Given that the magnitude of the increases in particulate-to-soluble ratios for PKC-α were much greater than that for PKC-γ, PKC-α is most likely the primary isoform involved in the effect of fructose. These data are similar to those showing that the ability of ANG II to stimulate proximal nephron Na⁺ reabsorption changes with aging (9), as does its effects on the classical isoforms (3). Although there are at least 11 different PKC isoforms, we focused on the Ca²⁺- and lipid-dependent isoforms because they have been reported to mediate most of the effects of ANG II on Na⁺ reabsorption in the proximal nephron (10, 22, 24–26).

Because both PKC-α and PKC-γ activities were stimulated by ANG II in the fructose group, we investigated the ability of the selective PKC-α/β inhibitor Gö6976 to prevent ANG II-induced increases in Na⁺ reabsorption. Gö6976 completely prevented the augmentation of ANG II-induced increases in Na⁺ transport by proximal tubules in fructose-fed rats. These data, in addition to the greater magnitude of increases in PKC-α versus PKC-γ activities discussed above, indicate that PKC-α is the primary isoform responsible for this effect. Our data showing that PKC-α mediates the augmented response to ANG II in fructose-fed rats have not been previously reported. However, it is in agreement with much of the literature, which shows that this isoform mediates the stimulatory actions of ANG II on proximal nephron Na⁺ reabsorption mediated by Na⁺/H⁺ exchange (10, 22, 24, 25).

PKC-α activity is stimulated by acyl glycerols and increases in intracellular Ca²⁺ (21, 36). As a result, we investigated whether ANG II-stimulated intracellular Ca²⁺ was augmented in tubules from fructose-fed rats. We found that although intracellular Ca²⁺ was increased by ANG II in perfused tubules from both groups, the increase in intracellular Ca²⁺ was greater in the fructose group. Thus, it is likely that the elevated increases in intracellular Ca²⁺ explain at least part of the ability of dietary fructose to enhance ANG II-induced activation of PKC-α. To our knowledge, there are no similar reports in the literature regarding this matter.

To further investigate the role of increases in intracellular Ca²⁺ in the enhanced response to ANG II in fructose-fed rats, we measured the effect of preventing the increase on ANG II-stimulated Na⁺ transport. We found that chelating intracellular Ca²⁺ prevented the ANG II-stimulated increase in Na⁺ transport in tubules from fructose-fed rats. These data support the involvement of intracellular Ca²⁺ increases in activating PKC to mediate ANG II-stimulated Na⁺ transport.

We used higher concentrations of ANG II in the Ca²⁺ experiments so that we could demonstrate that control tubules were viable, responding to ANG II with an increase in intracellular Ca²⁺ as expected. While it would have been possible to use lower concentrations of ANG II in these experiments so that the control tubules showed no significant increase, the response of the tubules from fructose-fed rats would have been reduced, and more experiments would have been required to demonstrate a significant increase due to the variability inherent in the measurement. This is likely due to the fact that fura 2 acts as a buffer of changes in intracellular Ca²⁺, and loading the dye varies from tubule to tubule even though the loading parameters are the same. Given that we had to use 1 μM fura 2 to obtain reproducible results and basal intracellular Ca²⁺ is around 100 nM, it is easy to understand why a higher concentration of ANG II was needed to obtain reproducible increases in intracellular Ca²⁺. It is important to recognize that 1 nM ANG II still stimulated transport in our hands (10, 12).

Our results for activation of PKC-α and intracellular Ca²⁺ are internally consistent as long as one assumes that the effect of 1 pM ANG II on PKC-α activity is not a maximum effect, which is a reasonable assumption. Our previous publications showed that 100 and 1000 pM ANG II stimulate Na⁺ transport more than 1 pM (10, 12). Although we used 1 nM ANG II to determine whether fructose enhanced ANG II-stimulated increases in intracellular Ca²⁺, the BAPTA experiments demonstrated that the stimulatory effect of 1 pM ANG II on oxygen consumption depends on an increase in intracellular Ca²⁺ in tubules from fructose-fed rats.

Our proposed mechanism linking fructose, ANG II, intracellular Ca²⁺, and transport is likely due to the fact that dietary fructose changes proximal nephron metabolism of lipids (11) and activation of classical PKCs requires lipids. We have previously reported that dietary fructose does not change the response to atrial natriuretic factor or dopamine (10, 12). Consequently, it seems unlikely that dopamine is involved in the effect of fructose.

Demonstrating that augmented PKC-α activation is responsible for the enhanced sensitivity of proximal nephron Na⁺ reabsorption in fructose-fed rats in vivo is not technically feasible for various reasons. For example, in the time required to isolate proximal tubules in which ANG II might have been manipulated in vivo, the effects of ANG II on PKC degrade back to basal levels. However, there are in vivo studies that support our findings. ANG II type 1 receptor blockers have been shown to prevent fructose-induced hypertension (17, 18, 20, 33), and the effects of ANG II on the proximal nephron have been shown to be critical to the development of ANG II-induced hypertension (13).

In summary, 20% fructose in drinking water for 6–8 days increased the ability of ANG II to stimulate PKC-α activity in proximal tubules leading to increased Na⁺ reabsorption by proximal tubules. This effect is likely initiated by augmented ANG II-induced increases in intracellular Ca²⁺.

GRANTS

This work was supported in part by National Heart, Lung and Blood Institute Grant HL-128053 (to J. L. Garvin).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

J.L.G. conceived and designed research; N.Y. and N.J.H. performed experiments; N.Y., N.J.H., and J.L.G. analyzed data; N.Y., N.J.H., and J.L.G. interpreted results of experiments; N.Y., N.J.H., and J.L.G. prepared figures; N.Y. drafted manuscript; N.Y., N.J.H., and J.L.G. edited and revised manuscript; N.Y., N.J.H., and J.L.G. approved final version of manuscript.