**2. Materials and Methods**

All animal procedures and protocols were approved by the Wayne State University Institutional Animal Care and Use Committee (Protocol #19-03-1001). Animal care and experimentation was conducted in accordance with the guidelines and principles articulated in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague Dawley rats (Envigo Sprague Dawley, Shelby, MI, USA) were housed under controlled conditions (21–23 ◦C; 12 h light and 12 h dark cycles, lighting period beginning at 6 a.m.).

#### *2.1. Dietary Regimen*

Upon arrival, rats were permitted to acclimate for at least 48 h and provided standard lab chow and water, ad libitum. As depicted in Figure 1, when rats reached ~4.5 weeks of age, a hemodynamic transmitter was implanted (as described in surgical procedures) and the animal was permitted to recover in individual standard polyurethane caging. One week later, rats were placed into metabolic housing units (Tecniplast USA, West Chester, PA, USA) and provided milled chow containing either 20% glucose and 0.4% Na<sup>+</sup> (glucose control salt, GCS; ModTest Diet® 5755-5WZZ; St. Louis, MO, USA) or 20% fructose and 0.4% Na<sup>+</sup> (fructose control salt, FCS; ModTest Diet® 5755-5W3Y; St. Louis, MO, USA). Rats were permitted a 3 day acclimation period followed by a 3 day baseline period where food and water were provided ad libitum and baseline hemodynamic data were recorded by telemetry. Then, the rats entered Phase I (Figure 1; study weeks 2 to 4, inclusive): GCS rats

(*n* = 9) continued on the same diet. Rats receiving FCS chow were then randomly assigned to continue FCS (*n* = 9) or placed on 20% fructose and 4.0% Na<sup>+</sup> (fructose high salt, FHS; ModTest Diet® 5755-5WZ8; *n* = 18; St. Louis, MO, USA) for three weeks. At this time, a pair feeding paradigm was initiated to achieve equal caloric intake among the groups on a day-to-day basis. Water continued to be provided ad libitum. Food and water intake and urine output were assessed daily. In Phase II (Figure 1, study weeks 5 to 7, inclusive), all rats were returned to standard individual shoebox housing units. Rats on GCS feed were maintained on this diet for the remainder of this study, including Phase III. Rats on FCS feed were then placed on GCS chow. The rats on FHS chow were then further randomly assigned to receive either GCS feed (*n* = 9) or to continue the FHS diet (*n* = 9). The rats on FHS chow during Phase II remained on FHS through to the end of this study.

**Figure 1. Schematic of the Timeline of Experimental Protocols and Study Phases.** Rat age and study week are depicted across the timeline. R, recovery period; A, acclimation to metabolic cages; B, baseline. Surgery for telemetry transmitter placement and ultrasound studies are as indicated. Groups are subsequently depicted by their sugar-salt intake in Phases I and III.

After 3 weeks, the rats were again placed into metabolic cages and permitted to acclimate to the change in caging for three days prior to initiating Phase III (Figure 1; study weeks 9 to 11, inclusive). FCS- and FHS-fed rats that had been shifted to a GCS feed in Phase II were then subjected to a high-salt challenge without fructose for the remainder of the protocol using a 20% glucose and 4.0% Na+ chow (glucose high salt, GHS; ModTest Diet® 5755-5WOW). This produced four groups characterized by their dietary regimens in the early and late phases—Phase 1 and Phase III, respectively (Figure 1). The groups are named based on their diets during Phases I and III: (a) GCS-GCS, (b) FCS-GHS, (c) FHS-GHS, and (d) FHS-FHS. Rats were maintained on these diets for an additional three weeks; thereafter, terminal studies were performed.

#### *2.2. Ultrasonography*

At the end of Phases I and III, rats were anesthetized in an induction chamber using 3% isoflurane and transferred to a pre-heated electronic ECG platform where 1–1.5% isoflurane was delivered via nosecone to maintain a sufficient plane of anesthesia. Fur from the chest

and abdominal area was removed using an electric shaver followed by application of depilatory cream (Church & Dwight Co., Inc., Erwing, NJ, USA). Electrode gel was placed on each of the ECG strips where the rat's limbs were held in place using tape. Body temperature was measured via a rectal probe and contact gel preheated to 37 ◦C was applied before performing echocardiography according to standard methods [24,25].

Image acquisition was conducted using the Vevo3100 Imaging system and MX250S transducer (Fujifilm Visualsonics, Inc., Toronto, ON, Canada). Assessment of left ventricular (LV) dimensions and systolic function was performed using a short axis view in M-mode at the level of the papillary muscle. Left ventricular (LV) diastolic filling and function were assessed using pulsed wave Doppler of transmitral blood flow velocities. These were located using color imaging superimposed over an apical four-chamber view. Further assessment of LV diastolic function was conducted using tissue Doppler imaging (TDI) near the mitral annulus measured along the apical axis. Pulse wave velocity (PWV) determination within the aortic arch was made via the determination of pulse transit time from the aortic root to a point within the aortic arch. Distance between these points was measured using a B-mode image of this anatomical segment. Aortic PWV was calculated as the difference in pulse transit time (calculated using the ECG tracing as a reference) measured at these two points divided by the distance between them.

Renal resistive index (RRI) was determined using pulsed Doppler measurements along the left main renal artery. RRI was calculated by taking the difference between systolic and diastolic velocity divided by the diastolic velocity during each respective cardiac cycle [26]. Data analysis was performed offline using VevoLab and VevoVasc software (Fujifilm Visualsonics, Inc., Toronto, ON, Cananda) in blinded fashion.
