*2.3. Surgical Procedures*

All surgical procedures were conducted under intraperitoneal ketamine (80 mg/kg; Mylan Institutional, LLC Rockford, IL, USA) and xylazine (10 mg/kg; Akorn Animal Health, Inc., Lake Forest, IL, USA) anesthesia and subcutaneous administration of buprenorphine SR (0.3 mg/kg) for analgesia.

Hemodynamic Transmitter Placement: Following right femoral artery isolation, a small arterial incision was made and the gel-filled catheter of the hemodynamic transmitter (HDS-10, Data Sciences International, New Brighton, MN, USA) was inserted into the vessel and advanced into the abdominal aorta. The catheter was then anchored in place to the femoral artery using 3-0 silk suture (Ethicon, Johnson & Johnson, New Brunswick, NJ, USA) and the transmitter body was subcutaneously tunneled to the right flank. Subcutaneous adipose tissue was reapproximated around the surgical sight and the incision was closed using surgical staples.

Vascular Catheter Placement: At the end of Phase III and after the second ultrasonography study, catheters were placed into the left carotid artery and external jugular vein using ketamine and xylazine as above, and previously performed in our laboratory [7,27]. Catheters were secured with 3-0 silk suture and tunneled subcutaneously to the base of the neck and exteriorized. All incisions were closed using 4-0 prolene suture (Ethicon, Johnson & Johnson, New Brunswick, NJ, USA). The catheters were then filled with heparinized saline (1000 units/mL). The rats were then permitted to recover in individually housed polyurethane cages.

#### *2.4. Analytical Measurements and Calculations*

Hemodynamic Telemetry: Acquisition of hemodynamic data was conducted using Ponemah software (Data Sciences International, New Brighton, MN, USA). Systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), and heart rate (HR) were sampled for 10 s every 4 min at a sampling rate of 500 samples/second. Pulse pressure (PP) was calculated separately using these values. Baseline measurements were averaged over three days after cage acclimation. Sampling was performed at this rate continuously throughout Phases I and III within the metabolic cages.

Metabolic and Hormonal Assessment: During Phases I and III, daily chow consumption was measured gravimetrically and caloric and sodium (Na+) intake values were determined based on dietary profiles for each feed.

At the end of Phase III and prior to terminal harvest, food was removed from caging at the beginning of the morning light cycles and terminal procedures were conducted at a minimum of 6 h later to promote a semi-fasting state (as permitted by our Animal Use Committee). Glucose levels were determined using a One-Touch Ultra glucose monitor (LifeScan, Inc., Malpitas, CA, USA) on 50 μL blood from the arterial catheter. Arterial blood was collected into pre-chilled tubes containing sodium ethylenediaminetetraacetic acid (EDTA) for plasma renin activity (PRA) and into separate pre-chilled tubes containing 120 μL of 500 mM sodium EDTA, 125 mM phenanthroline, 1 mM phenylmethanesulfonyl fluoride, 20 mM pepstatin, 1 mM enalapril and 10× phosphatase inhibitor cocktail for insulin determinations. Once collected, blood was immediately centrifuged at 3000 rpm for 4 min at 4 ◦C. Plasma was stored at −70 ◦C until assay. PRA and insulin levels were measured using a standard Elisa kits (IBL International, Hamburg, Germany and Bertin Pharma SAS, Montingny-le-Bretonneux, France, respectively) Insulin sensitivity was calculated using the ratio of plasma glucose to insulin levels.

Rats were euthanized using sodium pentobarbital (120 mg/kg, IV) and hearts were excised and preserved in formalin solution for 24–48 h before embedding for histological assessment. Samples were stained with Mason's Trichrome dye and images were acquired at 40× magnification (Leica CTR5000, Leica Microsystems Inc., Buffalo Grove, IL, USA).

Statistics: All values are presented as mean ± standard error (SE). One-way analysis of variance (ANOVA) was used to determine differences among groups with a Sidak's multiple comparisons test for post hoc analysis. Two-way ANOVAs with repeated measures were performed to compare differences over time using a Sidak's multiple comparisons test for post hoc analysis. A *p*-value less than 0.05 was considered statistically significant. Due to the technical nature of many of these experimental techniques and acquisition of data over an extended period, some data were not able to be acquired at each time point for each animal. When *n*-values deviate from the original assignment, the reason for missingness is provided and values imputed as the mean. Consistent with the use of a repeated measures design, animals with missing data in any phase were omitted from two-way ANOVA tests for the analysis of change over time.

#### **3. Results**

#### *3.1. Metabolic and Humoral Profiles*

Metabolic parameters are shown in Table 1. Initial and final rat weights did not differ among the groups on different dietary regimens. Plasma glucose and insulin levels did not vary among the groups and were comparable to either vendor specifications for standard Sprague Dawley rats on standard diets and rats fed control diets with no added sugar in similar studies [28–30]. The glucose:insulin ratio was significantly lower in the FHS-GHS and FHS-FHS groups.

Compared with the FCS-GHS group. Although the glucose:insulin ratio was nearly two-fold higher in the GCS-GCS rats compared with FHS-GHS and FHS-FHS rats, statistical significance was achieved only for the FHS-GHS (*p* < 0.01) but not for FHS-FHS groups (*p* = 0.0512). PRA was significantly reduced in FCS-GHS groups following high-salt challenge at the end of Phase III. FHS-GHS and FHS-FHS rats displayed a blunted inhibition of renin secretion.

Statistical differences among caloric intakes were observed following dietary changes from control salt to high-salt chow in Phase I, week 1 and in Phase III weeks 1 and 2 Caloric intakes were no different among the groups by of week 2 of Phase I and III except of the FCS-GHS group in Phase III that continued to ingest fewer calories (Table 2). Despite rigorous efforts to match intake among the groups, rats in the FHS-GHS and FHS-FHS diets had lower caloric intake only in week 1 of Phase I (and consequently lower calorie and sodium intakes compared with weeks 2 and 3 of Phase I, *p <* 0.05 vs. either calorie or sodium, respectively) after which their caloric intake was no different than that of the GCS-GCS group. High salt-fed rats consumed approximately 10-fold greater amount of sodium than that consumed by the control salt-fed rats, consistent with their respective dietary sodium diets.

**Table 1.** Initial and Final Body Weights, Heart Weights, Glycemic Parameters and Plasma Renin Activity among the Four Groups of Rats.


GCS-GCS, 20% glucose + 0.4% NaCl in Phases I–III; FCS-GHS, 20% fructose + 0.4% NaCl in Phase 1 and 20% glucose +4% NaCl in Phase III; FHS-GHS, 20% fructose + 4% NaCl in Phase 1 and 20% glucose + 4% NaCl in Phase III; FHS-FHS, 20% fructose + 4% NaCl in Phases I–III. PRA, plasma renin activity; G:I Ratio, glucose:insulin ratio. All groups except FHS-FHS were on 20% glucose + 0.4% NaCl in Phase II. Due to lack of sufficient plasma for insulin, *n* values for insulin and G:I Ratio are as follows: 5, 6, 7 and 7. Values are the mean ± SE; *n* as indicated per group. \* *p* < 0.05 vs. GCS-GCS. † *p* < 0.05 vs. FCS-GHS.



Values are the mean ± SE, *n* as indicated per group. Group names as in Table 1. Caloric intake calculated using caloric profiles of 3.98 kcal/g and 3.61 kcal/g for 0.4% and 4% NaCl chow, respectively. \* *p* < 0.05 vs. GCS-GCS; † *p* < 0.05 vs. FCS-GHS; § *p* < 0.05 vs. FHS-GHS.

#### *3.2. Hemodynamic Effects*

In Phase I, the addition of high salt in the diet of fructose-fed rats led to a progressive increase in mean arterial pressure that was significantly elevated after four days (data not shown). This increase was sustained for the subsequent two weeks. By the end of Phase I, the MAP increased in all groups consistent with expected changes in blood pressure with maturation; however, MAP was significantly higher in the rats receiving FHS diet during this phase (Table 3A). The FCS-GHS and FHS-GHS groups received GCS chow during Phase II and MAP at the beginning of Phase III did not differ from the MAP of the GCS-GCS group (data not shown). When FCS-GHS and FHS-GHS groups were placed on the GHS diet in Phase III, significant increases in MAP occurred within 1 week and these were sustained throughout the remainder of the phase. Final MAP pressure levels were comparable to those of FHS-FHS rats that had been on high-salt diet throughout all three phases (Table 3B). Systolic blood pressures at increased significantly in all groups on high-salt diet during Phase III compared with the GCS-GCS group that had ingested glucose and 0.4% salt chow throughout this study (*p* < 0.05). Diastolic blood pressures at the end of Phase I did not differ across any of the groups; therefore, the increases in MAP were driven predominately by systolic mechanisms. Pulse pressure, however, did not increase significantly. Additionally, no differences existed among groups when heart rate was assessed.



Group names as in Table 1. Values are the mean ± SE, n as indicated per group. (**A**) Mean arterial pressure (MAP) and heart rate (HR) at baseline and at the end of Phase I. (**B**) Study-wide changes in hemodynamics calculated as the difference between measurements taken at the end of Phase III and baseline values at the beginning of Phase I. Values are the mean ± SE, n as indicated per group. \* *p* < 0.05 vs. GCS-GCS; \*\* *p* < 0.01 vs. GCS-GCS.
