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

Background 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 (S)-2-hydroxyglutarate [(S)-2HG] and succinic acid (SA) have a nephroprotective effect against renal IR injury in Wistar rats. Methods (S)-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. (S)-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 [(S)-2HG: 12.5 or 25 mg/kg; SA: 12.5, 25, or 50 mg/kg], IR, and compound+IR [(S)-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. Results (S)-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, (S)-2HG caused a significant increase in the expression of Hmox1, suggesting an early accumulation of HIF-1α in the (S)-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. Conclusion None of the compounds were hepatotoxic at the tested doses. (S)-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.

196 Evaluation of treatment with sodium (S)-2HG 197 Rats were randomized and divided into the following groups. 198 1. Sham group (SH), n = 6: Rats were treated with double-distilled water administered p.o. 199 twice per day for 2 days. They then underwent a laparotomy without induction of kidney 200 IR injury and were allowed to recover for 15 h, after which they were sacrificed, and blood 201 and kidney tissue samples were obtained.   213 Evaluation of treatment with SA 214 Rats were randomized and divided into the following groups.  2. Nontoxicity groups, n = 4 each: Rats were treated with SA at a dose of 12.5, 25, or 50 220 mg/kg (12.5Tox, 25Tox, and 50Tox, respectively) in double-distilled water under the same 221 conditions as the SH group. Eight hours after treatment, the rats received the same 222 procedure as the SH group. 3. IR group (IR), n = 6: Rats were treated with double-distilled water as for the SH group.

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After treatment, they received a laparotomy with induction of kidney IR injury comprising 225 45 min of ischemia and 15 h of reperfusion. After reperfusion, the rats were sacrificed, and 226 blood and kidney tissue samples were obtained.  ). Rats were anesthetized by intraperitoneal injection with 100 mg/kg of 235 ketamine (Anesket, PiSA Agropecuaria, S.A. de C.V. Reg. SAGARPA Q7833-028, Guadalajara, 236 Jal., Mexico) and 10 mg/kg of xylazine (Sedaject, Vedilab S.A. de C.V. Reg. SAGARPA Q-237 0088-122, Querétaro, Qro., Mexico). After anesthesia, rats were shaved, and asepsis of the 238 abdominal region was performed using Microdacyn antiseptic solution (Oculus Technologies of 239 Mexico, S.A. de C.V., Guadalajara, Jal., Mexico) followed by a 20% solution of chlorhexidine 240 gluconate (Farmacéuticos Altamirano de México, S.A. de C.V., Mexico City, Mexico). A 241 midline incision was then performed, both kidneys were exposed, the kidneys were dissected at 242 both renal hila, and these structures were occluded using atraumatic vascular clamps for 45 min. 243 After ischemia, the clamps were withdrawn, and the incision was sutured. 244 245 Rats were transferred to cages containing UV-sterilized sawdust, and tramadol dissolved in water 246 was administered as an analgesic (50 mg/L, ad libitum) (Grünenthal GmbH, Stolberg, Germany). 247 The reperfusion follow-up occurred over the next 15 h. The rats were then anesthetized by 248 intraperitoneal injection of 50 mg/kg of ketamine and 5 mg/kg of xylazine. The incision was 249 reopened, and 5-7 mL of blood was withdrawn by cava vein phlebotomy, which caused death by 250 exsanguination. When the heart had stopped, both kidneys were removed. Half of each kidney 251 was conserved in a phosphate-buffered 10% formalin solution (pH 7.4), and the other half was 252 frozen at -80°C. Serum was separated from blood samples by centrifugation at 2000 g for 12 253 min and stored at -80°C until use.

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255 Analysis of biochemical markers, oxidative stress markers, and proinflammatory cytokines 256 To evaluate renal function, the serum concentrations of blood urea nitrogen (BUN) and 257 creatinine were measured. To assess liver function, the serum activities of alanine 258 aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and 259 alkaline phosphatase (ALP) were measured. The serum concentrations of glucose (GLU), uric 260 acid (UA), total proteins (TP), and albumin (ALB) were also measured. The biochemical 261 analysis was performed using kinetic or end-point UV-visible spectrophotometric methods in an 262 ILab Aries instrument (Instrumentation Laboratory SpA, Milan, Italy).

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To assess the oxidative-stress-induced injury, the tissue concentration of malondialdehyde 265 (MDA), and the tissue activity of total superoxide dismutase (SOD) was measured. Briefly, 200 266 mg of kidney tissue was homogenized, and the tissue homogenates were centrifuged three times 267 at 10,000 g for 10 min at 4°C. MDA and SOD quantification was performed using the 268 supernatant. 269 270 MDA is one of the final products of lipid peroxidation, mainly arachidonic acid, and 271 polyunsaturated fatty acids, and its activity was measured using the thiobarbituric acid 272 colorimetric method with a thiobarbituric acid-reactive substances (TBARS) assay kit (Cayman 273 Chemical Company, Ann Arbor, MI, USA). The product of this reaction was measured 274 spectrophotometrically at 535 nm and normalized to the amount of homogenized tissue. SOD 275 represents several metalloenzymes that form a crucial part of the cell enzymatic antioxidant 276 defenses. SOD catalyzes the dismutation of the superoxide anion to O 2 and H 2 O 2 . Total SOD 277 activity was measured using a method of inhibition of the reduction of Dojindo's water-soluble 278 tetrazolium salt [WST-1; 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H 279 tetrazolium, monosodium salt] in the presence of xanthine oxidase, xanthine, and oxygen 280 (Sigma-Aldrich). SOD activity ameliorates the reduction in tetrazolium and was measured 281 spectrophotometrically at 450 nm.

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The serum concentrations of the proinflammatory cytokines interleukin 1 (IL-1), IL-6, and 284 tumor necrosis factor- (TNF-) were measured using a commercial sandwich enzyme-linked 285 immunosorbent assay (ELISA) (Pepro-Tech, Mexico City, Mexico). Briefly, 96-well microplates 286 were covered with rabbit antibodies specific to the measured cytokine. Plates were washed with 287 phosphate-buffered saline containing 0.05% Tween 20 (pH 7.2) and then blocked with 1% serum 288 bovine albumin. Samples and standards were added and detected using specific biotinylated 289 detection antibodies and avidin-conjugated horseradish peroxidase (HRP). 2,2-Azino-bis(3-290 ethylbenzothiazoline-6-sulfonic) acid was used as the substrate for HRP; the reaction produced a 291 green chromogen whose concentration was proportional to the concentration of the evaluated 292 cytokine. The concentration of the end-product was measured spectrophotometrically at 405 nm, 293 with an additional 650 nm wavelength measurement for correction. 294 295 Renal histopathology evaluation 296 Fixed kidney tissue was paraffin embedded and processed using standard histological techniques. 297 Paraffin blocks were cut using a microtome at a thickness of 4 m. Sections were deparaffinized, 298 hydrated, stained with hematoxylin-eosin, and evaluated microscopically with the assessor 299 blinded to the identity of the groups. 306 307 Tissue HIF-1 concentration measurement 308 ELISA 309 HIF-1 concentration was assessed in tissue homogenates as described for oxidative stress 310 biomarkers. The analysis was performed using a sandwich ELISA method (Sigma-Aldrich) with 311 specific capture and detection antibodies, the latter conjugated to HRP. 3,3,5,5-312 Tetramethylbenzidine was used as the substrate, and the reaction was stopped by the addition of 313 a 0.2 M solution of H 2 SO 4 . Absorbance was measured at 450 nm. HIF-1 concentration is 314 reported relative to the amount of homogenate tissue. 315 316 Western blot 397 Because one of our aims was to identify any evidence of hepato-or nephrotoxic effects caused 496 497 Several animal models have been used to study renal acute kidney injury caused by IR. Initial 498 studies were performed in animals of relatively large size, such as dogs, pigs, and rabbits, and 499 since the 1960s rat models have been one of the most reported in the literature (Wei & Dong 500 2012). However, in recent years, mouse models have become popular mainly due to the 501 availability of standardized genetically engineered strains. Rat models of IR injury have been 502 well characterized (Heyman et al. 2002;Owji et al. 2018), and rats have the advantage of larger 503 anatomical structures and a higher blood volume than mice, yielding larger amounts of serum 504 after exsanguination, making easier to perform a diversity of biochemical assays. Hence, we 505 decided to use a model of renal IR in rats to assess whether (S)-2HG and SA exert a 506 nephroprotective effect. 517 It has been widely demonstrated that susceptibility to IR injury is gender-dependent. Studies both 518 in mice and rats have shown that the estradiol-androgen ratio has a key role in the modulation of 519 IR injury. High concentrations of testosterone are associated with a more aggressive injury and 520 higher mortality (Robert et al. 2011), and orchiectomy has been shown to attenuate the post-521 ischemic oxidative stress and the IR injury in mice (Kim et al. 2006). We decided to use male 522 and female rats because of the availability of animals in our laboratory. For that reason, the use 523 of independent SH and IR control groups was imperative. The fact that we did not assess each 524 compound both in male and female rats is a limitation of this study. Additional experiments are 525 needed to demonstrate the effect of these compounds on both genders. 526 527 Metabolism of (S)-2HG has not been well characterized in animal models. Biochemical studies 528 have reported an (S)-2HG dehydrogenase activity of 4.5 ± 5.5 nmol/min/mg of protein in rat 529 liver, significantly higher than the activity in the kidney (less than 1 nmol/min/mg of protein) and 530 other organs (Jansen & Wanders 1993), but pharmacokinetic experiments are still needed. 531 Studies assessing the chronic exposure to (S)-2HG have not been performed, however, the 532 chronic exposure to the (R)-2HG enantiomer has already been reported. A study showed that the 533 daily injection of a dose of 250 mg/kg for 32 days caused significant skeletal atrophy and a 534 decrease of the body weight in mice (Karlstaedt et al. 2016). Nevertheless, these results cannot 535 be extrapolated to our model, because of the different effects produced by each of the 536 enantiomers of 2-HG in vitro (Koivunen et al. 2012). Besides, the application of EGLN 537 inhibitors in the field of solid organ transplantation does not require the use of a chronic 538 exposition, but just an acute administration of the drug. In our study, (S)-2HG caused no toxic 539 effects at the hepatic or renal level at the evaluated doses in our experimental conditions, as 540 shown by the normal values of the biochemical markers. Comparison between the nontoxicity 541 groups and the SH group showed no effects of (S)-2HG on the biomarkers of oxidative stress, 542 proinflammatory cytokines, and histological parameters. By contrast, (S)-2HG exerted a dose-543 dependent nephroprotective effect in the groups that underwent IR injury, as manifested by the 544 significant amelioration of the changes in the serum concentrations of BUN and creatinine after 545 IR injury. IR injury caused an increase of the serum ALP, AST, and LDH activities, whereas (S)-546 2HG caused a significant amelioration of the activity of these enzymes, which suggests a 547 protective effect of this compound. The TP concentrations differed significantly between the IR 548 and 25+IR groups, although these values were within the reported reference interval for this rat 549 strain (Boehm et al. 2007). 550 551 Oxidative stress is one of the main mechanisms involved in IR injury. However, we observed no 552 changes in the biomarkers of oxidative stress after IR injury in our rat model. The magnitude of 553 MDA production and the decrease in tissue SOD activity after IR injury depends on the duration 554 of ischemia and reperfusion. Our results are consistent with those reported previously showing 555 that these changes are nonsignificant after 30 min of ischemia and 24 h of reperfusion (Dobashi 556 et al. 2000).

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558 The role of the inflammatory response in the renal IR mechanism is well known and involves the 559 promotion of IL-1 production, which stimulates tubular cells to produce IL-6 and TNF- (Daha 560 & van Kooten 2000). However, reports are inconsistent. One study noted that IR does not 561 increase the concentrations of these cytokines (Zhu et Zhang et al. 2015). In this work, we did not observe 564 significant increases in the concentrations of IL-1, IL-6, or TNF-. We also found that (S)-2HG 565 did not decrease the concentrations of these proinflammatory cytokines compared with their 566 baseline levels, which suggests that the compound has no immunomodulatory effect. HIF-1 567 promoted the expression of proinflammatory cytokines such as IL-1 (Rider et al. 2012) and IL-568 1 (Ogryzko et al. 2019;Zhang et al. 2006) in several experimental models. Because these 569 cytokines stimulate the production of IL-6 and TNF-, it is possible that this effect antagonizes 570 the potential amelioration of the inflammatory response induced by the nephroprotective effect 571 of (S)-2HG. Additional studies are required to confirm this idea. 572 573 Renal IR caused significant injury to tissue architecture, which appears as acute tubular necrosis. 574 We observed a tendency for (S)-2HG toward amelioration of tissue injury in the three 575 histological parameters evaluated, but these changes were not statistically significant. These 576 results agree with those of other studies of compounds with nephroprotective activity, in which 577 the magnitude of tissue injury was assessed using a semiquantitative approach (Kobuchi et al.  In the present study, IR injury was induced 8 h after the final dose of (S)-2HG and tissue 587 HIF-1 concentration was measured 15 h after IR injury. HIF-1α is a highly regulated protein, 588 extremely sensitive to the concentration of O 2 in the microenvironment and easily degraded (in 589 this study we observed a semi-degraded 55 kDa isoform of HIF-1α in the Western blot, as 590 referred by several manufacturers of anti-HIF-1α antibodies). For that reason, it is 591 understandable that the expression of this protein would not be stable after the long reperfusion 592 period; however, the IR group showed a significant accumulation of HIF-1α after the 15-hours 593 reperfusion period, but the 12.5+IR and 25+IR groups did not follow this behavior. The effect 594 observed in the IR group is consistent with that of a study in which HIF-1 accumulation caused 595 by IR occurred in two phases, namely an acute phase during ischemia and a late phase during 596 reperfusion (Conde et al. 2012). The findings of the present study suggest that the activation of a  Values are expressed as mean ± SD. The comparisons were between the 12.5Tox, 25Tox, and SH groups, and between the SH, 12.5+IR, 25+IR, and IR groups. One-way ANOVA test,   Biomarkers of kidney and liver injury, oxidative stress biomarkers, and proinflammatory cytokines after administration of succinic acid.
One-way ANOVA test, Tukey post hoc test. The comparisons were between the 12.5Tox, 25Tox, 50Tox, and SH groups, and between the SH, 12.5+IR, 25+IR, 50+IR, and IR groups.