*3.3. Measurements of Vascular Compliance*

Echocardiography and ultrasonography studies were performed upon completion of Phases I and III. At the end of Phase I, FHS diet significantly increased PWV of the ascending thoracic aorta, thereby indicating decreased aortic compliance compared to the GCS-GCS group (Figure 2). By the end of Phase III, maintenance of the FHS diet throughout all three phases of the protocol led to a significantly greater PWV in the in FHS-FHS group compared with the GCS-GCS group that had glucose and control salt diet for the same duration. Notably, PWV was significantly higher in the FHS-FHS group at the end of Phase III compared with the same animals at the end of Phase I.

**Figure 2. Pulse Wave Velocity at the end of Phases I and III.** Pulse wave velocity (PWV) of the ascending aorta assessed (**A**) at the end of Phase I and (**B**) at the end of Phase III. Group labels are as described in the legend for Table 1. Values are the mean ± SE; *n* as depicted on the graphs. \* *p* < 0.05 vs. GCS-GCS (by one-way ANOVA); † *p* < 0.05 vs. FHS-FHS in Phase I (by two-way ANOVA with repeated measures).

No differences were observed in the RRI among any of the groups at the end of Phase I (Figure 3). Similar to PWV values, FHS-GHS and FHS-FHS groups in Phase III displayed a statistically higher RRI than GCS-GCS rats. These trends were only elevated in Phase III and demonstrated no intra-group variability when compared to Phase I by two-way ANOVA. Note that due to anatomic and/or technical reasons, PWV and RRI were not able to be reliably assessed in each animal.

**Figure 3. Renal Resistive Index at the end of Phases I and III**. Renal resistive index (RRI) of the left main renal artery using doppler imaging (**A**) at the end of Phase I and (**B**) at the end of Phase III. Values are the mean ± SE; *n* as depicted on the graphs. \* *p* < 0.05 vs. GCS-GCS in Phase III.

#### *3.4. Echocardiographic Assessments*

Physical assessment of the LV was performed using a short axis view and M-mode imaging. Table 4 depicts the results of echocardiography of the LV performed at the end of Phase III. No significant differences of standard systolic function, such as ejection fraction and fractional shortening, were observed among the groups. LV mass was significantly greater in the FHS-GHS and FHS-FHS groups vs. the GCS-GCS group. Further assessment of structural morphology revealed significant increases in anterior and posterior wall thickness in the FHS-GHS group. These changes were only apparent during diastole. In the FHS-FHS group, LV thickness only reached significance in the anterior wall. When assessed as a ratio of total wall thickness (sum of anterior and posterior walls) to inner diameter of the LV during diastole, there was a significant reduction in the ratio in FHS-GHS rats when compared with that of the GCS-GCS group (Figure 4A–D). Figure 4E shows histological views typical of the LV myocardium from the four groups. Notably, collagen staining was observed in the tissues taken from the FHS-GHS and FHS-FHS rats. Taken together, these measurements are consistent with ventricular hypertrophy and concentric remodeling.

Figure 5A shows representative Doppler images of transmitral flow from GCS-GCS and FHS-FHS rats at the end of Phase III. Table 5 shows values of diastolic function conducted by way of pulsed and tissue Doppler imaging at the end of Phase III. The ratio of early to late phase filling (E/A) demonstrated a significant reduction in the FHS-FHS group compared with the GCS-GCS group. The E/A ratios for each of the groups at the end of both Phase I and Phase III indicate that this parameter of diastolic dysfunction is significantly suppressed only after 12 weeks of the diet high in both fructose and salt (Figure 5B). Reductions in the E/A ratio in the FCS-GHS and FHS-GHS test groups were also observed, although these values did not achieve significance (*p* = 0.064 vs. GCS-GCS). Likewise, decreases in mitral valve deceleration time were also observed across all groups when compared to the GCS-GCS group. However, these were also only significant in the FHS-FHS group.


**Table 4.** Echocardiographic Parameters at the End of Phase III.

Group names as in Table 1: LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; LVIDs, left ventricular systolic internal diameter; LVIDD, left ventricular diastolic internal diameter; LVAW, left ventricular anterior wall width; LVPW, left ventricular posterior wall width; LVTW, left ventricular total wall width; LV Mass, left ventricular mass. Values are the mean ± SE. \* *p* < 0.05 vs. GCS-GCS.

**Figure 4. Assessment of Left Ventricular Parameters in Phase III.** Images were acquired using a short axis view of the left ventricle (LV) via M-Mode. (**A**) Cardiac mass was the total wet weight of the heart after harvesting. (**B**) Total wall thickness was the sum of anterior and posterior LV wall widths. (**C**) LV diastolic diameter was the diameter of the LV at end diastole. (**D**) LV diastolic wall/diameter was calculated as the ratio of total wall thickness to the diameter of the LV at end-diastole. Values are the mean ± SE, *n* as indicated per group; \* *p* < 0.05 vs. GCS-GCS. (**E**) Representative histological sections of LV tissue from each group. Group labels are as described in the legend for Table 1. Mason's trichrome; 40× magnification.

**Figure 5. Assessment of Diastolic Function.** (**A**) Representative Doppler images of transmitral flow patterns from GCS-GCS and FHS-FHS rats at the end of Phase III. (**B**) Ratio of early (E-wave) to late (A-wave) left ventricular filling (E-wave) left ventricular filling as index of diastolic function. Group labels are as described in the legend for Table 1. Values are the mean ± SE; *n* as indicated per group, \* *p* < 0.05 vs. GCS-GCS.


**Table 5.** Echocardiographic Parameters Associated with Diastolic Function at the End of Phase III.

Group names as in Table 1: E, early phase ventricular filling; A, late phase ventricular filling; DT, mitral valve deceleration time; IVRT, isovolumetric relaxation time; E', mitral annulus early phase filling; A', mitral annulus late phase filling. Values are the mean ± SE. \* *p* < 0.05 vs. GCS-GCS, \*\* *p* < 0.01 vs. GCS-GCS.

#### **4. Discussion**

The major findings of this study support the hypothesis that consumption of fructose plus high-salt diet during pre- and early adolescence results in measurable deleterious cardiovascular effects in adulthood when ingesting high dietary sodium without fructose. Specifically, ingestion of high fructose either alone or with high salt during this early critical period of life resulted in salt-sensitive hypertension in maturity, despite the rats resuming a diet that was free of fructose and had normal salt content during young adulthood. The elevation in mean and systolic blood pressures in FCS-GHS and FHS-GHS rats was comparable to rats that had ingested high-fructose and high-salt diet throughout the entire protocol. The cardiovascular parameters such as aortic and renal artery compliance, LV mass and wall thickness, and LV diastolic function were impaired only in the rats that had ingested high fructose and high salt during adolescence. Notably, the magnitude of salt-sensitive blood pressure elevation was similar in all groups fed fructose in early life. Taken together, these findings suggest that the reduced vascular compliance and LV

diastolic dysfunction are not simply due to the elevated blood pressure or fructose alone in early life but to the combination of fructose with high salt in adolescence.

An extensive body of work has accrued to show that maternal exposure to environmental and dietary conditions can profoundly influence not only fetal development but also subsequent cardiac and renal function in offspring [31]. Importantly, moderately high maternal salt with [32] or without [33,34] concurrent fructose intake leads to hypertension in male rats. In contrast, the impact of factors on cardiovascular function in the adolescent period of plasticity has received scant attention.

The comparable increases in MAP in each of the four groups during Phase I was consistent with the ~10 mmHg increase typically observed as rats grow and mature [35]. Except for the FHS-FHS group that entered Phase III with elevated blood pressure, rats in the other groups, all of which were on GCS during Phase II, entered Phase III with normal MAPs similar to the GCS-GCS control group. The hypertension that developed in response to high-salt intake in rats that ingested fructose during the critical developmental period was driven by elevation in systolic pressure to levels equivalent to the rats that had consumed fructose and high salt throughout. Slight increases in diastolic blood pressure occurred which prevented any statistically significant increases in pulse pressure which were, nonetheless, nearly two-fold higher than in the GCS-GCS group. Importantly, systolic blood pressure and pulse pressure are strongly correlated with subsequent major adverse cardiac events [36,37]. Thus, fructose alone or combined with high salt during the critical adolescent period predisposes to salt sensitivity and hypertension in maturity.

The mechanisms underlying later salt-sensitive hypertension in rats that consumed fructose in youth remain to be defined. Failure to suppress PRA in the FHS-GHS and FHS-FHS suggests involvement of the renin-angiotensin system. Angiotensin II serves as a pressor inducing hormone that can act systemically on the vasculature to increase vasoconstriction or on target organs such as the kidney to increase extracellular fluid volume by facilitating fluid reabsorption [38]. Notably, adult rats fed similar fructose and high-salt diets exhibit increased proximal tubular sodium-hydrogen exchange [39–42] and stimulation of thick ascending limb sodium-potassium-2-chloride cotransporter expression [43] as well as enhanced renal sympathetic nerve activity [7]. Increased extracellular volume due to positive net sodium balance together with neurohumorally mediated vasoconstriction over the course of Phase III in rats fed a high-fructose diet in Phase I likely plays a role in producing the increases in MAP [6]. However, increased extracellular volume is unlikely to be the only governing factor. Prolonged fructose feeding has been associated with hyperinsulinemia which can cause increased levels of other vasoactive factors such as endothelin-1 [28], reactive oxygen species and uric acid [4,27,44–47]. Although no differences in basal plasma glucose or insulin levels were observed among all four groups, the significantly lower glucose:insulin ratio in the groups that consumed fructose and high salt in adolescence indicates a possible role for insulin resistance, a hallmark of pre-diabetes. Whether these or other mechanisms remain "primed" by high-fructose intake during the plastic adolescent period and are then brought into play to induce hypertension upon ingestion of high-salt diet in the absence of fructose later in life will need further investigation.

Increased arterial pressure over time can induce cellular and molecular alterations that deform the vascular wall and increase afterload to the left ventricle [48]. It is noteworthy that the FCS-GHS group displayed hypertension equivalent to that of the other fructosefed groups by the end of Phase III, but vascular stiffening and LV diastolic dysfunction occurred only in the groups exposed to both fructose and high salt in early life. In fact, evidence of vascular dysfunction became evident in FHS groups by the end of Phase I as demonstrated by increased PWV despite similar arterial pressures across all groups. In Phase III, PWV is augmented in both FHS-GCS and FHS-FHS groups when compared to Phase III GCS-GCS controls. Notably, in rats fed fructose and high salt for the entire protocol, the decline in aortic compliance progressed further in Phase III when compared to initial Phase I measurements. These findings suggest that the combination of fructose and

high-salt diet has a direct effect on vascular function that is independent of the elevated arterial pressure. Failure of optimal suppression of PRA in the FHS-GHS and FHS-FHS groups supports an angiotensin-associated mechanism underlying the reduced arterial compliance in these groups, but does not exclude additional potential mechanisms such as increased sympathetic activity [7] or increased sodium reabsorption that could lead to an expanded extracellular volume [39–41]. Notably, these mechanisms are not necessarily independent of each other as increased renal sympathetic activity enhances renin secretion and Ang II increases proximal tubule sodium reabsorption by the kidney. Importantly, the present data indicate that fructose alone during the early adolescent phase does not impair the normal suppression of PRA with ingestion of high salt later in life. Rather the combined ingestion of fructose and high salt in this early plastic phase does predispose to salt-sensitive hypertension later in life.

Several studies have demonstrated the prognostic ability of the renal resistive index (RRI) to predict the decline in renal function associated with the progression of hypertension, chronic nephropathy, and diabetes mellitus in humans [49–52] and adverse cardiac and renal outcomes in hypertension [53,54]. While some controversy remains over the reliability of RRI as a measurement across all diseases [55], there is a general consensus that elevated RRI is linked closely with systemic vascular stiffness. Additional studies have found functional correlation between elevated RRI and intrarenal perfusion as well as histopathological findings such as tubulointerstitial damage and renal atherosclerosis [56–58].

Consistent with PWV, we observed significant increases in RRI in each of the groups fed fructose in early adolescence only in Phase III. Importantly, as a measure of pulsatility, RRI reflects intrinsic renal artery compliance but is also influenced substantially by changes in upstream systemic and downstream intrarenal vascular properties [26]. The elevated RRI is thus consistent with the increased aortic PWV observed in this present study but suggests that the decline in renal artery compliance is delayed compared with changes in the ascending aorta and aortic arch where hydrostatic and shear forces are greater [59]. Oxidative stress [27,47], impaired nitric oxide generation [6], and inflammatory mechanisms [60] have been implicated in vascular changes during fructose and high-salt exposure. Again, whether these same factors contribute to the impaired compliance of the aorta and renal artery observed after exposure to fructose and high salt in youth is likely but remains to be proven.

Total peripheral resistance is a function of MAP and heart rate—an increase in either factor without a corresponding decrease in the other elevates total peripheral resistance [61]. In the present study, this physiologic dysfunction was observed as both an increase in systemic resistance and lack of vascular compliance. The net effect of these factors was an increase in ventricular afterload leading to left ventricular remodeling and subsequent hypertrophy evidenced by increased LV mass and total wall thickness. Together with the augmented ratio of ventricular wall thickness to end-diastolic cavity radius, these findings are consistent with concentric remodeling with preserved ejection fraction [62,63]. The decrease in the ratio of early to late diastolic filling was accompanied by an increase in isovolumetric relaxation time and decrease in mitral valve deceleration time. Shortening of the mitral valve deceleration time implies restrictive filling and has been positively correlated to severe adverse cardiac events [64]. Each of these measurements are indicative of diastolic dysfunction and are phenotypes associated with either the development of cardiomyopathy in rats that had consumed fructose and high salt in the critical adolescent period [65–68]. Despite the lack of rigorous morphometric studies, collagen deposition was apparent only in the two groups of rats fed FHS in early life. The present finding is consistent with the findings of Abdelhaffez et al. [69] who reported increased cardiac interstitial fibrosis after rats ingested 12 weeks of 20% fructose in their drinking water. Unfortunately, that study did not provide functional data. Intriguingly, long-chain non-coding mRNAs that are co-expressed with mRNAs involved the fructose metabolic pathways have been implicated in myocardial fibrosis after myocardial infarction in humans [70]. Whether

similar cellular and biochemical pathways are implicated in fructose-high salt-induced cardiovascular dysfunction will be crucial avenues of investigation.

#### *4.1. Limitations*

The present study used 20% glucose with 0.4% NaCl as the reference group to control for caloric intake rather than a standard rat chow which is typically ~7% simple sugars. Previous studies have found no significant changes in arterial pressure or bodily sodium balance in rats fed 20% glucose with either low- or high-salt diet for short (1 week) or more prolonged periods of time (3 weeks) [6,7]. It should be noted that in these other studies, the sugars (glucose and fructose) were in the drinking water rather than in the chow. Incorporating the carbohydrate in the chow permitted more accurate assessment of intake and equalization across groups. Nonetheless, it is important to note that, while the timeline of the study at present exceeds that of these prior studies, the values for hemodynamic, vascular, and cardiac parameters are comparable in our GCS-GCS rats that we used as reference group to parameters observed after three weeks of 20% glucose in drinking water with either 0.4% or 4% NaCl [27]. The 9- to 10-week exposure to dietary fructose in the FHS-FHS group was certainly longer than that in previous studies that evaluated these cardiovascular parameters. PRA was not suppressed in the FCS-FHS and the FHS-FHS groups. Ideally, concurrent plasma Ang II measurements would have been confirmatory as in our previous studies [7,27]. The volume of plasma required for plasma Ang II measurements by validated assay in our laboratory is 0.8–1.0 mL. Obtaining blood from conscious rats via indwelling catheter while avoiding hypotension that could potentially induce an increase in PRA and Ang II independent of the dietary condition was a primary goal. Due to the need to assess other plasma factors, we were only able to obtain sufficient plasma to reliably assess PRA, which only required 50 μL of plasma. Sex hormones play an important role in the development of hypertension following a high-fructose diet [71,72]. We only studied male rats in the present cohort in part due to restrictions in the number of animals permitted during the pandemic restrictions. We acknowledge that female rats have been shown to be particularly resistant to the development of insulin resistance and, therefore, may prove to be less prone to the consequences of fructose and high-salt diet [73–75]. Studies in female rats will be needed in the future. Finally, the nature of the ultrasonographic imaging precluded obtaining all parameters in each of the rats due to anatomical variations or issues with technique. Blood samples obtained at the end of this study via the indwelling arterial catheter were performed in conscious animals to avoid confounders such as hypotension and anesthesia; however, in some cases, this limited the volume of plasma that could be obtained due clotting or kinking of the catheter. Although statistical analyses for missing data were performed by imputation using the mean, the limitation still exists.

#### *4.2. Perspectives*

Pre-clinical and clinical studies have clearly shown the relationship between frank diabetes mellitus and cardiovascular complications [65,76–80]. Insulin resistance in the prediabetic state even without frank hyperglycemia may play an important role in developing cardiovascular abnormalities [81]. Alternatively, the exposure to both fructose *and* high salt early in life in Phase I is an important factor for later development of the vasculopathy and cardiomyopathy.

On the other hand, fructose feeding alone, without the addition of high dietary sodium during the critical developmental period is sufficient to induce as state of salt sensitivity later in life which renders the body susceptible to hypertension. Chronically this can lead to various cardiac and renal co-morbidities such as heart failure and chronic kidney disease [82,83]. Even FCS-GHS groups that became hypertensive only later in life had a reduction in diastolic function, indicated by the E/A ratio, but this was not significant. Contrastingly, the addition of high salt to a moderate fructose diet during pubescent, developmental years had lasting effects on cardiac and renal function evidenced

by diastolic dysfunction, ventricular hypertrophy, and failed renin suppression. This detriment occurred in rats that were fed this fructose and high-salt diet chronically and those that were allowed a period of reprise from poor dietary conditions (FHS-GHS) groups. Indeed, diets with fructose fed early in life-with or without the presence of elevated sodium—promote adaptations that render the body increasingly vulnerable to complications caused by even modest dietary insults; these insults are long lasting and with severe consequence.

#### **5. Conclusions**

In summary, consumption of 20% fructose but not glucose by male rats during preand early adolescence, a proportion of caloric intake comparable to the upper quintile of humans, results in salt-sensitive hypertension in mature animals. When ingested together with a high-salt diet during this critical plastic phase, dietary fructose also predisposes to vascular stiffening and left ventricular diastolic dysfunction in later life.

**Author Contributions:** Conceptualization, N.F.R. and P.E.L.; methodology, N.F.R., P.E.L. and C.S.C.; software, C.S.C., P.E.L. and N.F.R.; validation, N.F.R.; formal analysis, P.E.L., C.S.C. and N.F.R.; investigation, P.E.L., D.K. and N.F.R.; resources, N.F.R.; data curation, N.F.R.; writing—original draft preparation, P.E.L.; writing—review and editing, N.F.R., C.S.C. and D.K.; visualization, P.E.L. and N.F.R.; supervision, N.F.R.; project administration, N.F.R.; funding acquisition, N.F.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by a Merit Grant from the Dept. of Veterans Affairs #RX000851 to NFR, R01HL151738 to CSC, and graduate fellowship by NIH 2T32HL120822 for PEL.

**Institutional Review Board Statement:** All animal procedures and protocols were conducted according to the guidelines of the Declaration of Helsinki approved by the Wayne State University Institutional Animal Care and Use Committee (Protocol #19-03-1001). Animal care and experimentation were further conducted in accordance with the guidelines and principles articulated in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author by formal request to the Research and Development Office of the John D. Dingell VA Medical Center, Detroit, Michigan.

**Acknowledgments:** The authors thank Min Wu for her technical assistance.

**Conflicts of Interest:** The authors declare no conflict of interest.

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