*3.4. HFD Did Not Affect the Expression of the Inflammatory or Contractile Factors in tPVAT of SS and SR Rats*

To determine whether the HFD affected the inflammatory factors (IL-6, TNFα, and adiponectin) in tPVAT, we conducted ELISA analysis (Figure 4). The HFD did not affect the proinflammatory factors (IL-6 and TNFα) in tPVAT of SS and SR rats (Figure 4A,B). The tissue level of the anti-inflammatory factor (adiponectin) in tPVAT was higher in SR rats than in SS rats fed a CD (Figure 4C). We measured the expression of genes related to contractile factors (angiotensin II, norepinephrine, chemerin, and serotonin) to investigate whether the HFD affected the contractile factors in tPVAT (Figure 5). The HFD did not affect the gene expressions of *Ace*, *Angiotensinogen*, *Tyrosine hydroxylase*, *Rarres2*, *Cmklr1*, or *Slc6a4* in tPVAT of SS or SR rats.

**Figure 2.** The effects of a high-fat diet on fat deposits, weight, histological characteristics, and adipocyte area of the thoracic perivascular adipose tissues (tPVAT) in SS and SR rats. (**A**) Representative pictures visualizing tPVAT from the SS and SR rats fed a CD or HFD for 12 weeks. The HFD increased fat deposits (**A**) and PVAT weight (**B**) in both the SS and SR rats. (**C**,**D**) Representative microscopic images of the thoracic aortas and tPVAT from the SS and SR rats fed a CD or HFD for 12 weeks. The thoracic aortas and tPVAT sections were stained with hematoxylin and eosin (H&E, upper) and trichrome (lower) stains. The scale bars for 100×, 200×, and 400× magnification were 200, 100, and 50 μm, respectively. The HFD increased the PVAT adipocyte area (black arrow) in SS rats but not SR rats. Graph, mean ± SEM of 6 independent experiments. Two-way analysis of variance followed by Tukey's post hoc multiple comparisons test. \* *p* < 0.05 and \*\* *p* < 0.01 vs. the SS CD group. ## *p* < 0.01 vs. the SR CD group.

## *3.5. HFD Did Not Affect the Expression of Genes Related to RAAS in the Kidney of SS and SR Rats*

Based on our findings that the HFD increased blood pressure only in SS rats without affecting the expression of contractile factors in tPVAT, we investigated whether the HFD increased the mRNA expression of RAAS genes in the kidney. The HFD did not increase the mRNA expression of *Renin*, *Ace*, *Ace2*, or *Angiotensinogen* (Figure 6), nor that of angiotensin receptors (*At1ar*, *At1br*, *At2r*, *Mas1*; Figure 7). We also investigated the effect of HFD on the mRNA expression of NADPH oxidase-related factors in the kidneys of SS and SR rats. These data showed that there were no differences in gene expression between the CD and HFD groups (Figure S1).

**Figure 3.** The effects of a high-fat diet on the expression of adipocyte marker genes in PVAT from SS and SR rats. The mRNA expression of brown adipocyte-related genes such as *uncoupling protein 1* (*Ucp1*, **A**), *peroxisome proliferator-activated receptor γ coactivator 1-α* (*Pgc-1α*, **B**), and *peroxisome proliferator-activated receptor γ* (*Pparγ*, **C**), a beige adipocyte-related gene such as *transmembrane protein 26* (*Tmem26*, **D**), and a white adipocyte-related gene such as *Leptin* (**E**) were measured by RT-qPCR in PVAT from SS and SR rats fed a CD or HFD for 12 weeks. Graph, mean ± SEM of 6 independent experiments. Two-way analysis of variance followed by Tukey's post hoc multiple comparisons test. \*\* *p* < 0.01 vs. the SS CD group.

**Figure 4.** The effects of a high-fat diet on adipose tissue derived inflammatory factors in PVAT from SS and SR rats. Tissue levels of proinflammatory cytokines (interleukin-6 [IL-6, **A**] and tumor necrosis factor α [TNFα, **B**]) and an anti-inflammatory cytokine (adiponectin, **C**) were detected via enzyme-linked immunosorbent assay (ELISA) in SS and SR rats fed a CD or HFD for 12 weeks. Graph, mean ± SEM of 6 independent experiments. Two-way analysis of variance followed by Tukey's post hoc multiple comparisons test. \* *p* < 0.05 vs. the SS CD group.

**Figure 5.** The effects of a high-fat diet on the expression of genes related to contractile factors in PVAT from SS and SR rats. The mRNA expression of angiotensin II-related genes such as *angiotensinconverting enzyme* (*Ace*, **A**) and *angiotensinogen* (*Agt*, **B**), a norepinephrine-synthesizing gene such as *tyrosine hydroxylase* (**C**), chemerin-related genes such as *rarres2* (**D**) and chemerin receptor 23 (*Cmklr1*, **E**), and a serotonin-related gene such as serotonin transporter (*Slc6a4*, **F**) were measured by RT-qPCR in PVAT from SS and SR rats fed a CD or HFD for 12 weeks. Graph, mean ± SEM of 6 independent experiments. Two-way analysis of variance followed by Tukey's post hoc multiple comparisons test.

**Figure 6.** The effects of a high-fat diet on the expression of genes related to angiotensin II in kidneys from SS and SR rats. The mRNA expressions of angiotensin II-related genes such as *Renin* (**A**), *Ace* (**B**), *Ace2* (**C**), *Agt* (**D**) were measured by RT-qPCR in the kidneys of SS and SR rats fed a CD or HFD for 12 weeks. Graph, mean ± SEM of 6 independent experiments. Two-way analysis of variance followed by Tukey's post hoc multiple comparisons test.

**Figure 7.** The effects of a high-fat diet on the expression of genes related to angiotensin II receptor in kidney from SS and SR rats. The mRNA expression of angiotensin receptor related genes such as *angiotensin II type 1a receptor* (*At1ar*, **A**), *angiotensin II type 1b receptor* (*At1br*, **B**), *angiotensin II type 2 receptor* (*At2r*, **C**), and *mas1 proto-oncogene* (*Mas1*, **D**) were measured by RT-qPCR in kidney from SS and SR rats fed a CD or HFD for 12 weeks. Graph, mean ± SEM of 6 independent experiments. Two-way analysis of variance followed by Tukey's post hoc multiple comparisons test.
