**1. Introduction**

The inhibition of the sodium-glucose transporter-2 (SGLT-2) by gliflozins promotes glucose and sodium excretion. Positive renoprotective effects were demonstrated not only in diabetic patients who were treated with gliflozins in addition to a standard antidiabetic therapy [1,2] but also in non-diabetic patients [3]. The CREDENCE trial [4] demonstrated the renoprotective effects of canagliflozin in diabetic patients, while the DAPA-CKD trial [5] also showed the efficacy of dapagliflozin in non-diabetic patients with chronic kidney disease. Moreover, the DAPA-HF and EMPEROR-reduced trials also showed improved cardiovascular outcomes in heart failure patients [6]. Several mechanisms were suggested for their beneficial effects, from hemodynamic and natriuretic effects (the reduction in BP; to the modulation of tubulo-glomerular feedback, and the reduction in sympathetic hyperactivity); to the metabolic factors due to their glycosuric effects (the improvement of insulin sensitivity and the lowering of plasma triglycerides and uric acid); and to the

**Citation:** Hojná, S.; Kotsaridou, Z.; Va ˇnourková, Z.; Rauchová, H.; Behuliak, M.; Kujal, P.; Kadlecová, M.; Zicha, J.; Vanˇeˇcková, I. Empagliflozin Is Not Renoprotective in Non-Diabetic Rat Models of Chronic Kidney Disease. *Biomedicines* **2022**, *10*, 2509. https:// doi.org/10.3390/biomedicines10102509

Academic Editor: Ramón C. Hermida

Received: 9 September 2022 Accepted: 5 October 2022 Published: 7 October 2022

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attenuation of oxidative stress, inflammation, and fibrosis. The beneficial renoprotective effects of different gliflozins were demonstrated in various diabetic and prediabetic rat and mice models [7–11]. However, only limited and sometimes contradictory information is available in non-diabetic animals with chronic kidney disease. While Zhang et al. [12], Rajasekaran et al. [13], and Li et al. [14] found no renoprotective effects of gliflozins in rats after 5/6 nephrectomy, Kim et al. [15] and Wan et al. [16] reported on partial benefits in uninephrectomized rats, and Zeng et al. [17] demonstrated renoprotective effects due to reduced fibrosis in the same model. The anti-fibrotic effects of gliflozins in kidneys were also found in the ureteral obstruction model [18] and the angiotensin II-induced model [19]. In the latter model, the additive renoprotective effects of empagliflozin treatment combined with losartan were shown by Reyes-Pardo [20]. Moreover, Ali et al. [18] reported on the amelioration of adenine-induced CKD with canagliflozin due to a reduction in renal inflammation and oxidative stress.

Our previous studies in three non-diabetic hypertensive models (hypertensive Ren-2 transgenic rats, hereditary hypertriglyceridemic rats, and spontaneously hypertensive rats expressing the human CRP gene) [21–23] demonstrated the beneficial effects of SGLT-2 inhibition without a glucose-lowering effect. In all these models, empagliflozin treatment led to a reduction in body weight and visceral adiposity, while the additional effects depended on the model used—an improvement of metabolic parameters and hepatic metabolism (hHTG), a reduction in blood pressure (TGR), and the attenuation of oxidative stress and inflammation in kidneys (SHR-CRP).

Three different models of chronic kidney disease with different pathophysiological backgrounds were selected for the present study—a genetic model of hypertensionassociated renal failure, namely, a fawn-hooded hypertensive (FHH) rat, in which hypertension, proteinuria, and focal glomerulosclerosis already develops at young age [24]. Moreover, two models of kidney deterioration induced by experimental procedures were used in this study: either a uninephrectomy combined with increased sodium intake (UNX + HS) or a stenosis of renal artery–two-kidney, one-clip (2K1C) Goldblatt hypertension [25].

We hypothesized that empagliflozin would have renoprotective effects in these three models of chronic kidney disease with different pathological backgrounds, i.e., in rats genetically predisposed to renal impairment (FHH) and in two experimentally induced CKD models (UNX + HS or 2K1C).

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

#### *2.1. Animals*

Animals were housed at 23o C under a 12 h light/dark cycle, fed maintenance diet for rats and mice (Altromin-1320, Lage, Germany, 11% fat, 24% protein, 65% carbohydrates, 0.45 % NaCl), and given tap water ad libitum. Empagliflozin (Jardiance, Boehringer, Ingelheim, Germany), at a dose of 10 mg/kg/day, was mixed into the Altromin diet (so that the composition of both diets was the same) and given for 8 weeks. The amount of empagliflozin added to the diet was calculated according to our previous studies [21–23]. All procedures and experimental protocols were approved by the Ethical Committee of the Institute of Physiology, Czech Academy of Sciences (Protocol Nr. 47/2019), and conformed to the European Convention on Animal Protection and Guidelines on Research Animal Use (Directive 2010/63/EU). Three different models of chronic kidney disease were used within the study.

#### 2.1.1. Fawn-Hooded Hypertensive Rats

Male fawn-hooded hypertensive rats were treated with empagliflozin from the age of 12 weeks, for eight weeks.

## 2.1.2. Two-Kidney One-Clip (2K1C) Goldblatt Hypertension

Male Wistar rats underwent a sham operation or clipping of the right renal artery (2K1C) at the age of 10 weeks [25]. Eight weeks later, empagliflozin treatment was started and administered for eight weeks.

#### 2.1.3. Uninephrectomized Rats on High-Salt Intake

Male normotensive Hannover Sprague Dawley rats underwent uninephrectomy (UNX) at the age of 7 weeks [25], and control rats were sham operated. One week later, high-salt diet feeding (2% NaCl) and empagliflozin treatment were started. This regimen was applied for eight weeks (Figure 1).

**Figure 1.** Scheme of the experiment in Fawn-hooded hypertensive rats (FHH), 2-kidney-one-clip (2K1C), and uninephrectomized high salt-fed (UNX-HS) rats.

Body weight was monitored throughout the whole experiment and measured once a week. Systolic blood pressure was measured weekly during the experiment using tail plethysmography (HatterasInstruments, Cary, NC, USA). Food consumption was monitored throughout the experiment.

#### *2.2. Biochemical Analysis*

Three times during the experiment (at weeks 0, 4, and 7), the animals were placed in individual metabolic cages for the evaluation of kidney function using 24-h urine collection. Urinary proteinuria, sodium, and glucose were determined. Urinary protein was measured using the Folin method, with bovine serum albumin as a standard [26]. Plasma creatinine was measured by the FUJI DRI-CHEM analyzer using appropriate slides for creatinine CRE-P III (Fujifilm Corp, Tokyo, Japan). Plasma urea were determined using kit (Erba Lachema, Brno, Czech Republic). Lipoperoxidation products were assessed based on levels of thiobarbituric acid-reactive substances (TBARS) by assaying the reaction with thiobarbituric acid. MCP-1 levels were determined in urine using ELISA kit (Invitrogen, Carlsbad, CA, USA). At the end of the study, direct BP measurement was performed under light isoflurane anesthesia [27]. Then, the animals were sacrificed and plasma and tissue samples were collected and stored at −80 ◦C for further biochemical analysis.

#### *2.3. Statistical Analysis*

All data are expressed as means ± SEM. Statistical analysis was done by one-way analysis of variance (ANOVA) with Bonferoni test, using the Graph-Pad Prism software (Graph Pad Software, San Diego, CA, USA). The differences were considered significant at the *p* < 0.05.
