**4. Discussion**

These results of this study demonstrate that SR rats are protected against hypertension during diet-induced obesity. We found that a HFD did not induce hypertension in SR rats, unlike in SS rats, although it increased both body weights and tPVAT weights. The HFD did not affect the mRNA expression of adipocyte markers or contractile factors in tPVAT of the SS and SR rats. There were no significant differences in the mRNA levels of RAAS genes in the kidneys of SS and SR rats before and after the HFD.

Salt sensitivity is defined as a decrease in mean arterial blood pressure of >5 mmHg during a low-sodium intervention or an increase of >5 mmHg during a high-sodium intervention, whereas salt resistance is defined as a change in mean arterial blood pressure of <5 mmHg during low-sodium or high-sodium interventions. The Genetic Epidemiology Network of Salt Sensitivity (GenSalt) study conducted in rural northern China demonstrated that ~32.4% of Chinese adults are sodium sensitive [21]. Physical activity and dietary potassium intake were associated with reduced sodium sensitivity, whereas older age, female gender, Black race, obesity, metabolic syndrome, and elevated BP were associated with increased sodium sensitivity [21]. In 65% of individuals with high blood pressure, the amount of salt intake had a greater effect on blood pressure in patients with metabolic syndromes such as hypertension, diabetes, and obesity than in those without the syndrome [22]. This model showed similar results in a previous study in that SS rats but not SR rats developed hypertension after high-fructose intake [19]. Similarly, SBP increased in spontaneous hypertensive rats (SHRs) after 12 weeks of a HFD, whereas SBP did not begin to increase in Wistar–Kyoto rats (WKY) until 12 weeks of a HFD [23]. In the present study, we characterized the development of hypertension in SS and SR rats by monitoring SBP, DBP, body weight, and food intake during a HFD (Figure 1). The HFD increased the SBP in the SS but not in the SR rats, although the HFD caused diet-induced obesity and tPVAT enlargement in both rats. Therefore, SR rats have a unique trait in that they are resistant to the development of diet-induced hypertension.

At a given renal perfusion pressure, the SR rats had higher renal blood flow with lower resistance than the SS rats did [24] because signaling via NO is more dominant than that via O2− in the thick ascending limb cells of the renal tubule [25]. However, in the SS rats, signaling via O2− is more dominant than that via NO in the thick ascending limb cells of the renal tubule, which results in higher vascular resistance with constriction of the renal blood vessels. Because the genetic traits that determine salt resistance in SR rats may also determine fructose or fat resistance, it remains to be investigated which genes of the SR rats determine salt resistance.

Obesity-induced hypertension has been linked to increased coagulability, endothelial dysfunction, and inflammation [26], as well as more conventional risk factors for cardiovascular diseases such as insulin resistance, hypertension, and atherogenic dyslipidemia consisting of hypertriglyceridemia, high low-density lipoprotein cholesterol particles, and suboptimal high-density lipoprotein cholesterol levels [27]. Hypertension mediates abnormal kidney function and vascular pathology. The mediators are physical compression of the kidneys by fat in and around the kidneys, activation of the RAAS, and increased activity of the sympathetic nervous system [28]. Obesity is associated with volume expansion of extracellular fluid, which increases blood flow in many tissues and, in turn, increases venous return and cardiac output [29]. Therefore, obesity is associated with functional vasodilation that is probably due to the increased metabolic rate and higher tissue oxygen consumption [28].

Because visceral fat and PVAT increase the risk for cardiovascular diseases [30], an alteration in body fat distribution might have a significant impact on high blood pressure. In states of obesity, as the PVAT mass increases, the contractile and the proinflammatory cytokine TNFα increase, whereas anticontractile factors significantly decrease [31]. We observed no significant differences between the SS and SR rats fed a CD or HFD in the mRNA expression of angiotensin II-related genes such as *Ace* and *angiotensinogen*, a norepinephrine-related gene such as *tyrosine hydroxylase*, chemerin-related genes such as *Rarres2* and *Cmklr1*, or a serotonin-related gene such as *Slc6a4* in tPVAT (Figure 5). Although there were no significant differences in the levels of the proinflammatory cytokines IL-6 and TNFα, the anti-inflammatory cytokine adiponectin was higher in the SR rats than in the SS rats fed a CD (Figure 4). PVAT-derived factors such as IL-6 [32] and TNFα [33] had a contractile or an anticontractile effect depending on the microenvironment. Regardless of whether the rats were fed a CD or HFD, there were no significant differences in the mRNA expression or cytokine levels in the SS and SR rats. Therefore, the factors with anticontractile or contractile effects may exert a contractile effect in obese SS rats.

An HFD tends to increase urinary protein excretion with marginal kidney damage and further accelerates salt-induced proteinuria and renal histological aggravation [34]. Although several studies have reported that HFD-induced hypertension increases the expression of RAAS in the kidneys of rodent models [35,36], no differences were found in the expression of the RAAS components (*Renin, Ace, Ace2, Angiotensinogen, At1ar, At1br, At2r,* and *Mas1*) in kidneys of SS and SR rats fed a CD or HFD. The increase in SBP did not correlate with the mRNA expression of the RAAS components in the kidney; these findings suggest that the mRNA expression of the RAAS components did not affect the BP in the SS and SR rats. Another study demonstrated that a HFD for 12 weeks did not induce a definite diabetic state despite hypertension in SHRs and suggested that a HFD for 24 weeks induced definite dyslipidemia and insulin resistance as well as systemic RAAS activation [12].

In particular, Nox2 represents endothelial and vascular dysfunction in metabolic disease and hypertension [37]. HFD-induced hypertension shows that endothelial Nox2 derived superoxide plays a critical role in endothelial dysfunction, whereas Nox2 deficiency or pharmacologic Nox2 inhibition protects against vascular oxidative stress. Nox1 and Nox2 primarily produce O2 ·−. Nox4 is a functional source of reactive oxygen species generation in the mitochondria of the kidney cortex, wherein mitochondrial superoxide dismutase (SOD) effectively dismutates Nox4-derived superoxide to H2O2 [38]. In our

study, *Nox2* was markedly higher in the kidney of SR rats than in SS rats, with no significant changes in *Nox1, Nox3,* or *Nox4* (Figure S1).

The constriction of the mesenteric vascular bed provides an important contribution to the total peripheral resistance [39]. Structural or functional alterations in the mesenteric vascular bed contribute to the hypertensive process [40]. The mesenteric arteries of SHR express a greater density of calcium channels than those of WKY, which exhibited exaggerated constrictor responses to a variety of stimuli in SHR [41,42]. A high-salt diet was shown to decrease SOD activity in the mesenteric arteries of SS rats [43]. An HFD induced endothelial dysfunction in the mesenteric vascular bed due to a proinflammatory and contractile state of the PVAT [44].

This study has some limitations. First, we measured SBP and DBP by the tail-cuff method. Second, PVATs at different anatomical locations present different phenotypes [1]. The brown adipose tissues in the thoracic aorta were not whitened by diet-induced obesity/inflammation and did not have the characteristics of white adipocytes [45,46]. Although the HFD increased the body weights and tPVAT weights as well as adipocyte areas of the rats, it did not affect the properties (Figures 2 and 3). Although SS rats had similar cellularity in white and brown adipocytes as compared to SR rats, SS rats had higher cellularity in beige adipocytes than SR rats, which were not affected by the HFD. It would be of interest to determine whether an HFD affects abdominal or mesenteric PVAT, as the mesenteric vascular bed plays a key role in blood pressure regulation. Finally, although the HFD groups and CD groups had a similar calorie intake, it is possible that reduced energy expenditure contributed to increases in body weight in the HFD groups [17].

In summary, the blood pressure of the SR rats fed an HFD was not affected by the fat accumulation, properties of tPVAT, or RAAS in the kidney. Therefore, our results indicate that the SR rat is protected against hypertension during diet-induced obesity. This finding implies that the genetic trait that determines salt sensitivity may also determine fructose and fat sensitivity and that it is associated with the prevention of hypertension.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/nu14183843/s1, Figure S1: The effects of a high-fat diet on the expression of genes related to nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) in kidneys from SS and SR rats; Table S1: Primers for RT-qPCR.

**Author Contributions:** I.K. conceived the study and attained the funding; S.L. and S.J. developed and designed the experiments; S.L., S.J., and J.Y.K. performed the experiments; S.L. and S.J. analyzed the data; S.L. wrote the paper which was revised critically by I.K. for important intellectual content. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology (NRF-2021R1A2B502001763, and 2021R1A4A1021617), and a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI15C0001).

**Institutional Review Board Statement:** The Institutional Review Board of Kyungpook National University approved the care and use of animals (approval No. 2021-0192). Every effort was made to minimize both the number of animals used and their suffering. SS rats (DIS/EisSlc; Dahl-Iwai S) and SR rats (DIR/EisSlc; Dahl-Iwai R) were purchased from Japan SLC, Inc. (Hamamatsu, Shizoka, Japan).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** Informed consent was obtained from all subjects involved in the study.

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