*3.4. Effects of Empagliflozin on Renal Expression of Selected Pro-Inflammatory Genes*

The changes in renal expression of selected genes (*Ccl2*, *Il6*, *Tgfb*, *Tnf*) indicated that ageing was related to the substantial aggravation of inflammation in SHR-CRP rats (Figure 3). In addition, empagliflozin treatment reduced the expression of *Ccl2* (to 63% in young and to 66% in adult rats) and *Il6* (to 47% in young and to 61% in adult rats) genes in both age groups, while the expression of *Tgfb* and *Tnf* genes was not affected.

#### *3.5. Effects of Empagliflozin on Renal Function Markers and Histological Analysis*

In both age groups, urinary glucose excretion was more than twenty times higher, and sodium excretion two times higher, in empagliflozin-treated SHR-CRP rats (data not shown), suggesting an effective blockade of the SGLT-2 transporter. Microalbuminuria was substantially aggravated in adult SHR-CRP rats as compared with young animals, which is in accordance with the established phase of the incipient nephropathy associated with transgenic CRP expression at the age of one year. This was also confirmed by slightly increased histological markers found in old rats (Figures 2 and 4) and also supported by increased gene expression levels of pro-inflammatory markers MCP-1 and IL-6 (Figure 3). Unexpectedly, empagliflozin treatment decreased microalbuminuria only in young, but not in adult animals. However, this was not paralleled by a decrease of glomerulosclerosis index or tubulointerstitial injury index. Both control and empagliflozin-treated adult SHR-CRP rats exhibited moderate changes with focal segmental glomerular sclerosis accompanied by smaller areas of tubular atrophy when compared to empagliflozin-treated rats. Contrary to adult rats, young SHR-CRP rats exhibited substantially reduced histopathological changes in the kidney and showed no effects of empagliflozin treatment (Figure 4).

**Figure 3.** Relative mRNA expression of selected pro-inflammatory genes in the kidneys—monocyte chemoattractant protein-1 (MCP-1; (**A**)), Interleukin-6 (IL-6; (**B**)), Transforming growth factor-β (TGF-β; (**C**)), and Tumor necrosis factor-α (TNF-α; (**D**)). # *p* < 0.05 vs. respective young group. \* denotes *p* < 0.05; \*\* denotes *p* < 0.01; ## denotes *p* < 0.01; ### denotes *p* < 0.001. Data are means ± SEM; n = 7–8 for each group.

**Figure 4.** Representative histological images of renal cortex (PAS, 200×) (PAS, original objective 20×) of young (**A**,**B**) and adult (**C**,**D**) untreated (**A**,**C**) and empagliflozin-treated SHR-CRP rats (**B**,**D**). A scale bar is shown in Figure 4D.

#### **4. Discussion**

*4.1. Renoprotective Effects of Empagliflozin Are Associated with Reduced Ectopic Fat Accumulation and Lower Inflammation and Oxidative Stress*

The results of the current study demonstrated the significant renoprotective effects of empagliflozin in SHR-CRP rats. Specifically, reduced ectopic fat accumulation in the kidney was associated with decreased inflammation and oxidative stress and reduced microalbuminuria in young rats. Moreover, we confirmed that in the spontaneously hypertensive rats expressing human C-reactive protein (a model of metabolic syndrome, inflammation and organ damage), there is an age-dependent increase of insulin resistance, inflammatory markers in kidneys (MCP-1, TGF-β, TNF-α and IL-6), and deterioration of kidney function [19].

The reduced weight of renal fat following empagliflozin treatment was also associated with a reduced tissue expression of genes coding for enzymes that regulate inflammation (MCP-1 and IL-6) in both young and adult rats, which is consistent with the reduced serum levels of these pro-inflammatory markers. Conversely, the effects of empagliflozin treatment on gene expression of other pro-inflammatory cytokines TGF-β, TNF-α were not demonstrated. However, we and others found ambiguous effects of gliflozins therapy on distinct pro-inflammatory markers in non-diabetic rat strains—sometimes affecting more TNF-α [17], MCP-1 [18], or IL-1β and NF-κB [18]. Moreover, it could not be excluded that only those pro-inflammatory parameters which were upregulated by the insertion of the CRP transgene (IL-6, but not TNF-α) [19], could be influenced by empagliflozin therapy. The higher albuminuria found in adult control and treated animals is compatible with the long-term effect of high blood pressure, as well as the pro-hypertensive effect of CRP transgene in these animals. Thus, the absence of an empagliflozin effect on the reduction of albuminuria in adult rats could be explained either by the existing long-term changes in the kidneys of ageing animals or by the higher susceptibility of young animals to pharmacological interventions [26]. Similarly, our previous study performed in another non-diabetic hypertensive model—adult Ren-2 transgenic rats—did not show any effect of empagliflozin on proteinuria or albuminuria [17]; while a reduction of albuminuria was disclosed in a pre-diabetic rat model—hereditary hypertriglyceridemic rats [18]. Conversely, a reduction of albuminuria in young rats was associated with substantial attenuation of several parameters of oxidative stress (reduced levels of lipoperoxidation products TBARS and conjugated dienes, increased activity of glutathione peroxidase, increased glutathione levels, etc.) suggesting that intervention applied at an early age (critical developmental window) could be more effective [26]. Altogether, these results support the theory that empagliflozin treatment protects against the incipient nephropathy associated with the overexpression of the CRP transgene by reducing oxidative stress and inflammation either directly or as a consequence of reduced ectopic fat accumulation in the kidney.

In fact, in several animal models, including streptozotocin-induced diabetic mice and rats [27,28], Akita mice [29], OVE26 mice [30], and db/db mice [12,30], it was reported that kidney disease was associated with lipid accumulation and increased activity of pro-inflammatory cytokines, resulting in albuminuria, glomerular mesangial expansion, and tubulointerstitial fibrosis. Ectopic fat accumulation was observed in kidney biopsies of humans with type 2 diabetes mellitus [31,32]. Recently, Wang et al. [12] reported that SGLT-2 inhibition by JNJ-39933673 in db/db mice was associated with decreased renal lipid accumulation and prevention of the development of nephropathy. In addition, Hosokawa et al. [33] showed that ipragliflozin decreased ectopic lipid accumulation in tubular cells in diabetic mice. Although further studies are needed, it is plausible that, in addition to the well-known anti-inflammatory, anti-proliferative, and anti-fibrotic effects of SGLT-2 inhibitors, the reduction of tubular lipid deposition could contribute to the renoprotective mechanism of these molecules [34].

The attenuation of renal inflammation following empagliflozin treatment can lead to a decreased permeability of endothelial cells and subsequent alterations in the hemodynamics of the kidney [35], contributing to the improvement of renal function. The evidence for the anti-inflammatory potential of SGLT-2 inhibitors has been previously demonstrated in diabetic animal models [36]. Moreover, in human proximal tubular cells, SGLT-2 inhibitors (tofogliflozin) attenuated the expression of pro-inflammatory markers [37]. In diabetic patients, SGLT-2 inhibitors were found to reduce systemic levels of pro-inflammatory markers IL-6 and TNFα [8]. The SGLT-2 inhibitor-mediated anti-inflammatory effects, however, have not been shown under normoglycemic conditions.

Consistent with its anti-inflammatory properties, empagliflozin also attenuated oxidative stress—another key pathway known to cause kidney impairment. In the present study, empagliflozin markedly improved renal oxidative stress with the effect also being more pronounced in young rats. Empagliflozin regulates oxidative stress in the kidney cortex by stimulating antioxidant enzyme activity GSH-Px and catalase via the upregulation of Nrf2 rather than the direct inactivation of free radicals. In addition, increased GSH-Px activity can play a role in decreasing lipid peroxidation by participating in the removal of lipoperoxidation products. In the kidney cortex, increased GSH-Px activity following empagliflozin treatment was also linked to increased glutathione levels, a sensitive marker of oxidative damage. A markedly alleviated renal oxidative stress can be one of the pleiotropic metabolic mechanisms of empagliflozin that can also contribute to the improvement of renal function.

#### *4.2. Effects of Empagliflozin on Insulin, β-Hydroxybutyrate, NEFA and Leptin Concentrations*

In our study, empagliflozin markedly reduced hyperinsulinemia, although no significant changes in insulin sensitivity were observed in muscle and adipose tissue, as well as in fasting or non-fasting glucose levels. Thus, other factors contributing to the improved insulin sensitivity following empagliflozin treatment such as decreased NEFA and leptin levels can play a role. Although Nishimura et al. [38] have shown that SGLT-2 inhibitors promote systemic NEFA mobilization associated with the induction of ketone bodies as alternative substrate for energy metabolism, we observed no increase of NEFA

or β-hydroxybutyrate (BHB) after empagliflozin administration. On the contrary, empagliflozin even reduced circulating BHB concentration. A number of possible mechanisms of SGLT-2 inhibitors are implicated in their cardioprotective effects, which go beyond their diuretic and antihyperglycemic effects. The increase of ketone bodies is only one of the proposed mechanisms, which may not always apply. In addition, increased ketone bodies after SGLT-2 inhibitors are rather related to systemic alterations in substrate utilization and reduced glucose oxidation in preference for fatty acid oxidation. Thus, empagliflozin in a non-diabetic model with genetic hypertension and chronic inflammation affects heart function by other mechanisms than ketone bodies utilization. In our previous study with prediabetic animal model with vascular complications, we observed increased BHB in serum and the heart, but not increased BHB utilization in the heart [20]. In a study with diabetic obese rats, Abdurrachim et al. [39] reported that empagliflozin decreased ketone bodies utilization in the heart despite increasing circulating levels of BHB.

The markedly decreased circulating leptin levels observed in this study may not only contribute to improved insulin resistance, but may also alleviate inflammation and cardiovascular damage. In addition to the effect on insulin sensitivity, leptin may have a pathophysiological role in sodium regulation, as well as in cardiac and renal inflammation and fibrosis [40]. Circulating leptin levels presage the development of heart failure in elderly people and a decline in the glomerular filtration rate in longitudinal studies [41,42]. Although the mechanism/s of leptin action in heart failure are not fully understood, one potential mechanism might be its effect on epicardial adipose expansion and on calcium handling in cardiomyocytes leading to impaired myocardial relaxation. SGLT-2 inhibitors lead to decreases in serum aldosterone, reduction in the activity of NHE1 (sodium-hydrogen exchanger isoform 1), and can probably directly suppress leptin secretion and its paracrine actions on the heart and kidneys to promote fibrosis [40]. All these effects may underlie the action of SGLT-2 inhibitors to ameliorate cardiac and renal injury. Although a positive effect on plasma leptin levels has been observed in other studies with empagliflozin [43,44], it is unclear whether empagliflozin action directly impacts on adipose tissue function or whether it is associated with visceral fat loss. Leptin is secreted by epicardial and perirenal adipose tissue, thus the reduction of these fat depots can also contribute to decreased leptin levels. Although adipose tissue insulin sensitivity was not affected, empagliflozin treatment reduced the weight of visceral adipose tissue, and it is possible that reduced adiposity positively influenced secretion of other adipocytokines, in addition to leptin.

## *4.3. Effects of Empagliflozin on Liver Triglycerides and Cholesterol*

Although circulating lipids were not affected in the present study, empagliflozin treatment significantly decreased hepatic triglycerides, as well as cholesterol content. According to our previous studies, empagliflozin modulates genes related to lipid synthesis and fatty acid metabolism, while it has no effect on genes involved in lipid oxidation and transport [20]. Thus, we speculate that reduced lipid accumulation in the liver is probably associated with the inhibited lipogenesis. The suppression of SCD1—the main lipogenic enzyme—was observed in our [20], as well as in another animal study with obese mice [45]. The reduced ectopic hepatic lipid deposition can also ameliorate insulin resistance. The intrahepatic accumulation of fatty acids and lipotoxic intermediates interferes with intracellular signaling pathways like insulin signaling, and can induce endoplasmic reticulum stress; both can contribute to the development of insulin resistance [46]. The hepatic lipid accumulation and impaired lipid metabolism in the liver are independent risk factors for cardiovascular events, thus, the reduced hepatic lipid accumulation can contribute to the cardioprotective effect of empagliflozin.

#### **5. Conclusions**

It can be concluded that treatment of SHR-CRP rats with empagliflozin is associated with reduced renal lipid accumulation, inflammation, and oxidative stress, resulting in the attenuation of renal damage; these beneficial effects being more pronounced in young rats. By contrast, the metabolic effects of empagliflozin prevailed in adult rats.

**Author Contributions:** I.V. conceived and designed the project, S.H., H.M., M.H., I.M., P.M. and J.Š. performed the experiments, F.P. and J.H. performed the echocardiography, I.V., I.M., H.M. and J.Z. analyzed data and interpreted the results, D.M. did the statistical analysis, I.V., H.M and M.P. wrote the manuscript, J.Z. edited the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by grant of Czech Science Foundation, project number 19-06199S and the Ministry of Health of the Czech Republic—conceptual development of research organisations (Institute for Clinical and Experimental Medicine—IKEM, IN 00023001) and by the project National Institute for Research of Metabolic and Cardiovascular Diseases (Programme EXCELES, ID Project No. LX22NPO5104)—Funded by the European Union—Next Generation EU. This study was also supported by institutional support of the Institute of Physiology, Czech Academy of Sciences, grant Nr. RVO 67985823.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institute of Physiology, Czech Academy of Sciences (Protocol Nr. 47/2019).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data arising from this study are contained within the article.

**Acknowledgments:** The technical assistance of Zde ˇnka Kopecká and Alena Charvátová is highly appreciated. The authors are grateful to Petr Kujal fo the histological pictures and to Bob Kotanchik for his help in editing the manuscript.

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