**1. Introduction**

Diabetes is among the leading causes of mortality worldwide. As a chronic disease, it is associated with both micro- and macrovascular complications including coronary artery disease, stroke, diabetic nephropathy, neuropathy, and retinopathy. Traditional antidiabetic drugs, such as sulphonylureas or insulin, often have undesirable side effects, including hypoglycemia and weight gain; some of them potentially increase cardiovascular risk [1]. In contrast, new antidiabetics, in particular gliflozins, inhibitors of sodium-glucose cotransporter 2 (SGLT-2), show many beneficial actions beyond their antidiabetic effects. Their effects are mediated through the selective SGLT-2 inhibition at the renal proximal tubule, promoting glucose and sodium excretion, thus leading to significant improvement of glucose control together with the lowering of blood pressure and body weight [2]. The mechanisms underlying the reduction of cardiovascular events and renal protective effects are still poorly understood. Chronic low-grade inflammation is being increasingly recognized as a key feature associated with type 2 diabetes mellitus and its complications [3]. Experimental findings suggest that part of the renoprotective effects of SGLT-2 inhibition may be related to anti-inflammatory actions at the kidney level. The underlying mechanisms to explain this anti-inflammatory effect are multiple, and may involve body

**Citation:** Malínská, H.; Hüttl, M.; Marková, I.; Miklánková, D.; Hojná, S.; Papoušek, F.; Šilhavý, J.; Mlejnek, P.; Zicha, J.; Hrdliˇcka, J.; et al. Beneficial Effects of Empagliflozin Are Mediated by Reduced Renal Inflammation and Oxidative Stress in Spontaneously Hypertensive Rats Expressing Human C-Reactive Protein. *Biomedicines* **2022**, *10*, 2066. https://doi.org/10.3390/ biomedicines10092066

Academic Editor: Albrecht Piiper

Received: 1 July 2022 Accepted: 19 August 2022 Published: 24 August 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

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weight loss, reduction in adipose tissue inflammation, as well as decreased ectopic fat accumulation in the kidney or attenuation of oxidative stress.

Major large-scale clinical trials such as EMPA-REG, CANVAS, DECLAIRE-TIMI 58, and CREDENCE [4–7] demonstrated positive cardiovascular effects (decrease of the composite endpoints consisting of cardiovascular death, non-fatal myocardial infarction, and non-fatal stroke) of empagliflozin, canagliflozin and dapagliflozin in several thousand diabetic patients. Furthermore, in the DAPA-CKD trial, SGLT-2 inhibitors were also shown to offer renal protection in non-diabetic patients with proteinuria [8]. A recent systematic review and meta-analysis of the DAPA-HF and EMPEROR trials showed improvements in the composite renal endpoint regardless of the presence of diabetes or baseline estimated glomerular filtration rate [9]. Renal benefit has been attributed to metabolic and hemodynamic effects, including lowering of blood pressure, attenuation of glomerular hyperfiltration, body weight loss, and decreased plasma volume, as well as reduced renal hypoxia.

Systemic and renal inflammation is involved in the initiation and progression of diabetic and non-diabetic kidney disease. It has been previously demonstrated in diabetic animal models that the anti-inflammatory potential of SGLT-2 inhibitors is associated with decreased glomerular and tubulo-interstitial damage [10–12]. However, the underlying molecular mechanisms are not completely understood, and only limited information is available on SGLT-2 iinhibitor-mediated anti-inflammatory effects under experimental non-diabetic conditions. In fact, several studies demonstrated cardioprotective effects in experimental non-diabetic models [13,14]. The beneficial effects of gliflozins on renal function were observed in spontaneously hypertensive rats with heart failure [15] and also partly in rats after 5/6 nephrectomy [16]. Our studies in non-diabetic models, namely in hypertensive Ren-2 transgenic rats (TGR) [17], a model of angiotensin II-dependent hypertension, as well as in a model of metabolic syndrome and prediabetes—hereditary hypertriglyceridemic rats (HHTG) [18], showed the beneficial effects of empagliflozin treatment without a glucose-lowering effect. In these models, SGLT-2 inhibition led to a reduction in body weight and visceral adiposity, a decrease of insulin and leptin levels, and an improvement of hepatic metabolism, as well as the attenuation of oxidative stress and inflammation. There were also distinct effects depending on the model used, i.e., blood pressure decrease of TGR, and attenuation of oxidative stress and cell senescence in HHTG rats.

In the current study, we tested the metabolic and renoprotective effects of empagliflozin in spontaneously hypertensive rats expressing human C-reactive protein (SHR-CRP rats), a non-diabetic model of metabolic syndrome with severe hypertension, systemic inflammation, metabolic and hemodynamic disturbances, and target organ injury [19]. The expression of transgenic human CRP in these rats is associated with increased hepatic and renal oxidative tissue damage, increased plasma levels of interleukin 6, and a marked increase of microalbuminuria, which is accompanied by renal histopathologic changes such as fibrosis and inflammatory cellular infiltrates in the interstitium of the kidney [19]. To determine the mechanisms of empagliflozin beyond its antidiabetic effect, we focused our attention on its metabolic and renal effects in this non-diabetic model, both in young rats and in adult animals with the established systemic inflammation. In addition, we analyzed several genes involved in the inflammatory processes in the kidneys.

#### **2. Materials and Methods**

### *2.1. Animals*

The SHR-CRP/OlaIpcv transgenic rats (referred to as SHR-CRP) were derived by microinjections of zygotes with a construct containing the cDNA for human CRP under control of the apolipoprotein E promoter with the objective of driving expression of the CRP transgene in the liver, where CRP is normally produced [19]. To investigate the effects of empagliflozin on the metabolic parameters and on kidney injury associated with human CRP, we randomized male SHR-CRP transgenic rats at the age of 3 months (young) and 12 months (adult) into two groups, with or without empagliflozin treatment. In each group, we studied 8 males. The rats were fed ad libitum a standard laboratory diet or the same diet containing empagliflozin in a daily dose of 10 mg/kg body weight for 8 weeks. The rats were housed in an air-conditioned animal facility. All experiments were performed in compliance with the Animal Protection Law of the Czech Republic and were approved by the Ethics Committee of the Institute of Physiology, Czech Academy of Sciences, Prague (Protocol Nr. 47/2019), and conformed to the European Convention on Animal Protection and Guidelines on Research Animal Use (Directive 2010/63/EU).

#### *2.2. Metabolic Parameters in Epididymal Adipose Tissue and Myocardium*

As a marker of visceral adipose tissue insulin sensitivity, basal and insulin-stimulated lipid syntheses were determined ex vivo in isolated epididymal fat pad by measuring the incorporation of 14C-U glucose into lipids, as described previously [20]. Basal and adrenaline-stimulated lipolysis in the epididymal adipose tissue were measured ex vivo and evaluated as the release of non-esterified fatty acids (NEFA). Glucose and palmitate oxidation in the myocardium were measured ex vivo in heart tissue sections determined as the incorporation of 14C-U glucose and 14C-palmitate into CO2, respectively.

#### *2.3. Tissue Triglycerides and Cholesterol Measurements*

To determine triglycerides and cholesterol in the liver, kidneys and heart, tissues were powdered under liquid N2 and extracted in a chloroform:methanol mixture, after which, 2% KH2PO4 was added and the solution was centrifuged. The organic phase was collected and evaporated under N2. The resulting pellet was dissolved in isopropyl alcohol, and lipid concentrations were determined by an enzymatic assay (Erba-Lachema, Brno, Czech Republic).

#### *2.4. Biochemical Analyses*

Serum levels of glucose, triglycerides, total and HDL-cholesterol were measured by commercially available kits (Erba-Lachema, Brno, Czech Republic). NEFA levels were determined using an acyl-CoA oxidase-based colorimetric kit (Roche Diagnostics GmbH, Mannheim, Germany). Serum insulin, glucagon, leptin, adiponectin, MCP-1, TNFα, and IL-6 concentrations were determined using a rat insulin ELISA kit (Mercodia, Uppsala, Sweden; MyBioSource, San Diego, CA, USA; eBioscience-Beder, Wien, Austria; BioVendor, Brno, Czech Republic). Rat serum CRP and human serum CRP were also analyzed by ELISA kits (Alpha Diagnostics International, San Antonio, CA, USA). Serum and renal β-hydroxybutyrate (BHB) concentrations were determined by a colorimetric assay kit (Sigma-Aldrich, Saint Louis, MO, USA).
