*2.8. Quantitative PCR (qPCR)*

1 μg total RNA was used to synthesize cDNA using SuperScript IV reverse transcriptase (ThermoFisher, Waltham, MA, USA) according to the manufacturer's protocol. The resulting cDNAs were then used as templates in quantitative real-time PCR (qPCR) reactions. Primers for qPCR reactions were designed using PrimerBLAST [23] to span at least one exon-exon junction: amplicon size was set to 70–150 bp. Each qPCR reaction contained equivalent of 8 ng of input RNA, 300 nM of each forward and reverse primer and 1× Power-up SYBRGreen master mix (ThermoFisher) and was amplified in 7900HT (Applied Biosystems, San Francisco, CA, USA). For the qPCR experiment, cycle threshold (Ct) values of selected genes were normalized relative to the expression of the peptidylprolyl isomerase A (*Ppia*, cyclophilin) gene (for kidney cortex), which served as the internal control, with results being determined in triplicates. Relative quantification was performed using the ΔΔCt method.

#### *2.9. Histology*

Kidneys (n = 5) from each group were cut along the longitudinal axis and processed for paraffin embedding. Multiple 4 μm thick sections were cut and stained with Hematoxylin-Eosin, PAS and Azan-Mallory trichrome stain for observation under light microscopy. Slides were observed and pictures acquired with a digitalized camera by an experienced pathologist in a blinded manner. Renal damage, as determined using the glomerulosclerosis index and tubulointerstitial injury, was examined, as described previously [24,25], using the Nikon NIS-Elements AR 3.1 morphometric program (Nikon, Tokyo, Japan).

#### *2.10. Statistical Analysis*

All data are expressed as means ± S.E.M. Differences between experimental groups were analyzed by two-way ANOVA with adjustments for multiple comparisons by Holm– Sidak testing. Statistical significance was defined as *p* < 0.05.

### **3. Results**

*3.1. Effects of Empagliflozin on Body Weight, Weights of Fat Depots, Cardiac Function and Blood Pressure*

As can be seen in Table 1, in both age groups of transgenic SHR-CRP rats, empagliflozin administration reduced body weight as well as adiposity, as evidenced by the significantly decreased relative weights of epididymal and perirenal fat depots. Left ventricular function expressed as fractional shortening (FS) tended to be reduced in adult SHR-CRP during the study, suggesting the deterioration of heart function in these ageing rats (Table 2). Moreover, anterior and posterior diastolic left ventricle diameter (AWTd and PWTd) substantially increased in untreated SHR-CRP rats while it remained stable in empagliflozin-treated animals throughout the study. Developing concentric hypertrophy is seen also in relative wall thickness, which tended to be higher in untreated rats as compared with treated animals, which corresponds to a decrease of relative heart weight at the end of the study. Nevertheless, no effect on blood pressure or heart rate was demonstrated in either age group of empagliflozin-treated rats (Table 1).


Kidney weight (g/100 g BW) 0.66 ± 0.03 0.70 ± 0.01 0.74 ± 0.01 0.75 ± 0.01 <0.01 n.s. n.s. Mean arterial pressure (mm Hg) 192 ± 11 197 ± 7 190 ± 4 197 ± 6 n.s. n.s. n.s. Heart rate (bpm) 373 ± 8 380 ± 5 377 ± <sup>8</sup> <sup>355</sup> ± <sup>8</sup> # n.s. n.s. n.s.

**Table 1.** Basal parameters in experimental groups during the study.

**#** PAGE denotes the significance of young vs. adult SHR-CRP rats (age effect); PTREATMENT denotes the significance of empagliflozin treatment (treatment effects); PINTERACTION denotes the significance of different empagliflozin effects in various strains (treatment vs. strain comparison). For multiple comparisons (empagliflozin treatment vs. non-treated controls) Fisher's LSD post-hoc test was used; \* denotes *p* < 0.05; \*\* denotes *p* < 0.01; \*\*\* denotes *p* < 0.001; n.s. denotes not significant, Data are means ± SEM; n = 7–8 for each group.

**Table 2.** Echocardiography parameters in adult animals at the end of the study.


SHR-CRP—spontaneously hypertensive rat (SHR) expressing human C-reactive protein (CRP), AWTd—anterior wall thickness diastolic, LVDd—left ventricular diameter diastolic, PWTd—posterior wall thickness diastolic, AWTs—anterior wall thickness systolic, LVDs—left ventricular diameter systolic, PWTs—posterior wall thickness systolic, FS—fractional shortening, HR—heart rate, \* *p* < 0.05 empagliflozin vs. control SHR-CRP, **@** *p* < 0.05 vs. baseline.

#### *3.2. Effects of Empagliflozin on Metabolic Parameters and Insulin Sensitivity*

Compared to untreated animals, empagliflozin-treated SHR-CRP rats of both age groups exerted no significant differences in serum lipids—triglycerides, non-esterified fatty acids (NEFA), total and HDL cholesterol (Table 3). However, these parameters were substantially affected by the ageing of the animals; adult rats showed increased levels of serum triglycerides and cholesterol compared to young rats. Both age groups of empagliflozintreated SHR-CRP rats exhibited markedly reduced serum insulin (Figure 1), while the serum levels of fasting and non-fasting glucose were not significantly changed (Table 3). Moreover, empagliflozin treatment reduced ectopic hepatic triglyceride accumulation and hepatic cholesterol concentration (Figure 1), as well as ectopic triglyceride accumulation

in kidneys (Figure 2). β-hydroxybutyrate levels were decreased following empagliflozin treatment, this effect being more prominent in young rats than in old ones (Figure 1). By contrast, there were no significant differences in the sensitivity of adipose tissue to insulin action, measured ex vivo as the incorporation of glucose into adipose tissue lipids (lipogenesis). Basal and adrenaline-stimulated lipolysis were also unchanged after empagliflozin treatment in both age groups (Table 3).


**Table 3.** Biochemical parameters in the experimental groups at the end of the study.

**#** PAGE denotes the significance of young vs. adult SHR-CRP rats (age effect); PTREATMENT denotes the significance of empagliflozin treatment (treatment effects); Interaction denotes the significance of empagliflozin in various strains (treatment vs. strain comparison). For multiple comparisons (empagliflozin treatment vs. non-treated controls) Fisher´s LSD post-hoc test was used; \* denotes *p* < 0.05; n.s. denotes not significant, Data are means ± SEM; n = 7–8 for each group. AUC—area under curve from the oral glucose tolerance (OGT) test.

#### *3.3. Effects of Empagliflozin on Inflammatory and Oxidative Stress Parameters*

In adult SHR-CRP rats, empagliflozin treatment reduced markers of systemic inflammation. The levels of rat (but not human) CRP, leptin, and MCP-1 were significantly decreased in the serum of SHR-CRP rats, while other inflammatory parameters (TNFα and IL-6) were not affected (Table 4). However, when compared with adult animals, young empagliflozin-treated SHR-CRP rats had decreased serum levels of leptin and human CRP. As shown in Table 5, there was a substantial augmentation of oxidative stress in the kidney cortex of aged SHR-CRP rats. The empagliflozin treatment attenuated oxidative stress parameters, most of these effects being more pronounced in young than in adult rats. The lipoperoxidation products TBARS and conjugated dienes (CD) were significantly decreased and the activities of antioxidant enzymes (glutathione peroxidase, catalase) and the concentration of reduced glutathione were increased.

**Figure 1.** The effect of empagliflozin treatment on plasma insulin (**A**), plasma β-hydroxybutyrate (**B**), liver cholesterol (**C**) and liver triglycerides (**D**). *p* < 0.05 empagliflozin vs. untreated group, *p* < 0.05 vs. respective young group. ## denotes *p* < 0.01, \*\* denotes *p* < 0.01; \*\*\* denotes *p* < 0.001; Data are means ± SEM; n = 7–8 for each group.

**Figure 2.** The effect of empagliflozin treatment on microalbuminuria (**A**), triglycerides in kidney (**B**), glomerulosclerosis index (**C**) and tubulointerstitial injury (**D**). \* *p* < 0.05 empagliflozin vs. untreated group, # *p* < 0.05 vs. respective young group. Data are means ± SEM; n = 7–8 for each group.


**Table 4.** Inflammatory parameters in serum in experimental groups at the end of the study.

**#** PAGE denotes the significance of young vs. adult SHR-CRP rats (age effect); PTREATMENT denotes the significance of empagliflozin treatment (treatment effects); Interaction denotes the significance of empagliflozin in various strains (treatment vs. strain comparison). For multiple comparisons (empagliflozin treatment vs. non-treated controls) Fisher´s LSD post-hoc test was used; \* denotes *p* < 0.05; \*\* denotes *p* < 0.01; \*\*\* denotes *p* < 0.001; n.s. denotes not significant, Data are means ± SEM; n = 7–8 for each group. CRP—C-reactive protein, MCP-1— Monocyte chemoattractant protein-1, IL-6—Interleukin-6.

**Table 5.** Oxidative stress parameters in kidney cortex in experimental groups at the end of the study.


**#** PAGE denotes the significance of young vs. adult SHR-CRP rats (age effect); PTREATMENT denotes the significance of empagliflozin treatment (treatment effects); PINTERACTION denotes the significance of empagliflozin in various strains (treatment vs. strain comparison). For multiple comparisons (empagliflozin treatment vs. non-treated controls) Fisher´s LSD post-hoc test was used; \* denotes *p* < 0.05; \*\* denotes *p* < 0.01; \*\*\* denotes *p* < 0.001; n.s. denotes not significant, Data are means ± SEM; n = 7–8 for each group. SOD—superoxide dismutase (U/mg), GSH-Px–glutathione peroxidase (μmol/NADPH/min/mg), GR–glutathione reductase (μmol/NADPH/min/mg), CAT—catalase (μmol/H2O2/min/mg), CD—conjugated dienes (nmol/mg), TBARS—thiobarbituric acid reactive substances (μmol/g), GSH—reduced glutathione (μmol/g), GSSG—oxidized glutathione (μmol/g).
