Effect of sodium (S)-2-hydroxyglutarate in male, and succinic acid in female Wistar rats against renal ischemia-reperfusion injury, suggesting a role of the HIF-1 pathway

Abstract

<jats:sec> <jats:title>Background</jats:title> <jats:p>Ischemia–reperfusion (IR) injury is the main cause of delayed graft function in solid organ transplantation. Hypoxia-inducible factors (HIFs) control the expression of genes related to preconditioning against IR injury. During normoxia, HIF-α subunits are marked for degradation by the egg-laying defective nine homolog (EGLN) family of prolyl-4-hydroxylases. The inhibition of EGLN stabilizes HIFs and protects against IR injury. The aim of this study was to determine whether the EGLN inhibitors sodium (<jats:italic>S</jats:italic>)-2-hydroxyglutarate [(<jats:italic>S</jats:italic>)-2HG] and succinic acid (SA) have a nephroprotective effect against renal IR injury in Wistar rats.</jats:p> </jats:sec> <jats:sec> <jats:title>Methods</jats:title> <jats:p>(<jats:italic>S</jats:italic>)-2HG was synthesized in a 22.96% yield from commercially available L-glutamic acid in a two-step methodology (diazotization/alkaline hydrolysis), and its structure was confirmed by nuclear magnetic resonance and polarimetry. SA was acquired commercially. (<jats:italic>S</jats:italic>)-2HG and SA were independently evaluated in male and female Wistar rats respectively after renal IR injury. Rats were divided into the following groups: sham (SH), nontoxicity [(<jats:italic>S</jats:italic>)-2HG: 12.5 or 25 mg/kg; SA: 12.5, 25, or 50 mg/kg], IR, and compound+IR [(<jats:italic>S</jats:italic>)-2HG: 12.5 or 25 mg/kg; SA: 12.5, 25, or 50 mg/kg]; independent SH and IR groups were used for each assessed compound. Markers of kidney injury (BUN, creatinine, glucose, and uric acid) and liver function (ALT, AST, ALP, LDH, serum proteins, and albumin), proinflammatory cytokines (IL-1β, IL-6, and TNF-α), oxidative stress biomarkers (malondialdehyde and superoxide dismutase), and histological parameters (tubular necrosis, acidophilic casts, and vascular congestion) were assessed. Tissue HIF-1α was measured by ELISA and Western blot, and the expression of Hmox1 was assessed by RT-qPCR.</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>(<jats:italic>S</jats:italic>)-2HG had a dose-dependent nephroprotective effect, as evidenced by a significant reduction in the changes in the BUN, creatinine, ALP, AST, and LDH levels compared with the IR group. Tissue HIF-1α was only increased in the IR group compared to SH; however, (<jats:italic>S</jats:italic>)-2HG caused a significant increase in the expression of Hmox1, suggesting an early accumulation of HIF-1α in the (<jats:italic>S</jats:italic>)-2HG-treated groups. There were no significant effects on the other biomarkers. SA did not show a nephroprotective effect; the only changes were a decrease in creatinine level at 12.5 mg/kg and increased IR injury at 50 mg/kg. There were no effects on the other biochemical, proinflammatory, or oxidative stress biomarkers.</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusion</jats:title> <jats:p>None of the compounds were hepatotoxic at the tested doses. (<jats:italic>S</jats:italic>)-2HG showed a dose-dependent nephroprotective effect at the evaluated doses, which involved an increase in the expression of Hmox1, suggesting stabilization of HIF-1α. SA did not show a nephroprotective effect but tended to increase IR injury when given at high doses.</jats:p> </jats:sec>

Peer Review #1 of "Treatment with sodium (S)-2-hydroxyglutarate prevents liver injury in an ischemia-reperfusion model in female Wistar rats (v0.1)"

“…In the 13 C-NMR spectrum, the following signals were observed: 33.91, 36.38, 75.01, 184.10, and 185.71 ppm (Figure S2). Both spectra were consistent with the reported in the literature for this compound (Bal & Gryff-Keller 2002;Cienfuegos-Pecina et al 2020). A value of [α] D 20°C = 8.4°cm 3 g -1 dm -1 was observed, and it was consistent with previously reported data (Cienfuegos-Pecina et al 2020; Ritthausen 1872).…”

“…(S)-2HG was synthesized using a two-step methodology from L-glutamic acid, by using the optimized synthetic route reported by our laboratory (Cienfuegos-Pecina et al 2020). Briefly, L- Manuscript to be reviewed glutamic acid was subjected to a diazotization reaction in the presence of NaNO 2 and H 2 SO 4 for 24 hours.…”

“…A 50 mg sample of (S)-2HG was dissolved in double-distilled water and analyzed using sodium 3-(trimethylsilyl)propionate-2,2,3,3-d 4 (TSP) in D 2 O (Sigma-Aldrich) as an internal standard. The 1 H-NMR and 13 C-NMR spectra were acquired using the noesypr1d pulse sequence for water signal suppression and compared with those reported in the literature (Bal & Gryff-Keller 2002;Cienfuegos-Pecina et al 2020).…”

“…The specific optical rotation ([α] D 20°C ) of the synthetic (S)-2HG was measured in a PerkinElmer 341 Polarimeter (PerkinElmer, Waltham, MA, USA). Data were compared with previous reports in the literature (Cienfuegos-Pecina et al 2020;Ritthausen 1872).…”

“…On the other hand, its (S) enantiomer is naturally produced in vivo as a product of non-specific reactions of several enzymes, such as the malate dehydrogenase or the lactate dehydrogenase A (Du & Hu 2021;Intlekofer et al 2017). The (S)-2-hydroxyglutarate [(S)-2HG] has a higher inhibitory effect against EGLN-1 than against EGLN-2 or 3 (Koivunen et al 2012), and a previous report of our research group showed that the oral administration of its disodium salt has a nephroprotective effect against IR injury in Wistar rats, suggesting the involvement of the HIF-1 pathway in its mechanism (Cienfuegos-Pecina et al 2020). Given the pharmacologic potential of this compound, we aimed to evaluate whether the administration of (S)-2HG has a hepatoprotective effect against IR injury in Wistar rats.…”