The role of malonic acid derivate in the modulation of metabolic pathways in the kidneys during chronic heart failure

Introduction. Chronic heart failure (CHF) often leads to progressive renal dysfunction; however, pharmacological protection strategies aimed at correcting metabolic and oxidative disturbances in renal tissue remain underdeveloped. In this regard, it seems relevant to study the effect of new compounds, such as malonic acid derivatives, including 4-[(3-ethoxy-3-oxopropanoyl) amino]benzoic acid (etmaben), on the expression of genes encoding key enzymes of metabolic and antioxidant pathways in the kidneys in CHF. Previously, a more pronounced nephroprotective effect was detected for etmaben than for another malonate, maloben.

Aim. To evaluate the effect of 4-[(3-ethoxy-3-oxopropanoyl)amino]benzoic acid on the expression of genes regulating enzymatic pathways in the kidneys of rats with experimental CHF.

Materials and methods. The study was conducted on 30 outbred white male rats (300–350 g) housed under standard conditions (12-hour light/dark cycle, temperature 22 ± 2 °C, humidity 50–60 %, with ad libitum access to food and water). The animals were divided into three groups. Group 1 (Control–) consisted of healthy animals (n = 10) that received purified water (1 mL/kg/day, intragastrically). Group 2 (CHF–) included animals with chronic heart failure (CHF, n = 10) that were administered purified water (1 mL/kg/day, intragastrically) starting from day 30 after the surgery. Group 3 (CHF+) was composed of animals with CHF (n = 10) that received etmaben (60 mg/kg/day, intragastrically) starting from day 30 after the surgery. CHF was modeled by ligation of the left coronary artery. Successful model induction was confirmed echocardiographically (ejection fraction <40 %). Operated animals were randomized using a random selection method. Etmaben or water was administered daily for 30 days, starting from day 30 after surgery. On day 61, the animals were euthanized, and kidney tissues were homogenized in Lira reagent (LLC "Biolabmix", Russia). RNA was extracted using chloroform, isopropanol, and NaOAc, and purified with LiCl. RNA concentration was measured on a Nanophotometer N60, and quality was assessed by gel electrophoresis. Reverse transcription was performed from 1 µg of RNA (MMLV-RT-Kit, Eurogen). Real-time PCR was run on a QuantStudio 5 with SYBR Green (Eurogen), analyzing the genes Gpx1, Nrf2, Nox1, Glud1, Hes1, mTOR, Txnrd1, Hif1a, Cpt1b, and B2M (reference). Statistical analysis was performed in R-Studio (version 4.3.3) using the RQdeltaCT package. One-way ANOVA, the Kruskal – Wallis test, post-hoc Tukey and Dunn tests, as well as odds ratio analysis and logistic regression were applied. Gene expression analysis was performed using the 2^–ddCt method with normalization to the reference gene B2M.

Results and discussion. It was found that CHF led to significant activation of the transcription factor Nrf2; however, the expression level of its target gene, GPx1, remained unchanged both in CHF and after etmaben treatment, indicating impaired functioning of this signaling pathway. Administration of 4-[(3-ethoxy-3-oxopropanoyl)amino]benzoic acid caused a statistically significant increase in the expression of the Cpt1b gene (p < 0.05), suggesting a shift in cellular metabolism towards fatty acid β-oxidation. Furthermore, significant suppression of the pro-oxidant enzyme Nox1 and activation of the thioredoxin reductase 1 (Txnrd1) gene (p < 0.05) were recorded in the treatment groups. The Notch signaling pathway was activated by etmaben, as evidenced by increased expression of the Hes1 gene (p < 0.05), while no significant effect on the expression of the mTOR gene was detected.

Conclusion. Etmaben functions as a metabolic modulator and redox regulator. Its action is not associated with direct antioxidant activity but is due to the activation of adaptive mechanisms: reducing the demand for antioxidant defense by suppressing sources of reactive oxygen species (Nox1), activating a key regulator of the antioxidant response (Nrf2), and altering energy metabolism through the induction of Cpt1b and Glud1.

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