Stiripentol protects against calcium oxalate nephrolithiasis and ethylene glycol poisoning

Abstract

Increased urinary oxalate excretion (hyperoxaluria) promotes the formation of calcium oxalate crystals. Monogenic diseases due to hepatic enzymes deficiency result in chronic hyperoxaluria, promoting end-stage renal disease in children and young adults. Ethylene glycol poisoning also results in hyperoxaluria promoting acute renal failure and frequently death. Stiripentol is an antiepileptic drug used to treat children affected by Dravet syndrome, possibly by inhibiting neuronal lactate dehydrogenase 5 isoenzyme. As this isoenzyme is also the last step of hepatic oxalate production, we hypothesized that Stiripentol would potentially reduce hepatic oxalate production and urine oxalate excretion. In vitro, Stiripentol decreased in a dose-dependent manner the synthesis of oxalate by hepatocytes. In vivo, Stiripentol oral administration reduced significantly urine oxalate excretion in rats. Stiripentol protected kidneys against calcium oxalate crystal deposits in acute ethylene glycol intoxication and chronic calcium oxalate nephropathy models. In both models, Stiripentol improved significantly renal function. Patients affected by Dravet syndrome and treated with Stiripentol had a lower urine oxalate excretion than control patients. A young girl affected by severe type I hyperoxaluria received Stiripentol for several weeks: urine oxalate excretion decreased by two-thirds. Stiripentol is a promising potential therapy against genetic hyperoxaluria and ethylene glycol poisoning.

Keywords: Nephrology; Urology.

Figure 1. Inhibition of oxalate synthesis by stiripentol in vitro and in vivo.

(A) HepG2 cells were grown in a hydroxyproline-enriched medium to produce oxalate (red bars). Oxalate synthesis (mM) was reduced in a dose-dependent manner when stiripentol was added to the medium. *P = 0.03, n = 4 experiments. (B) siRNA targeting LDHA reduced significantly oxalate synthesis and the addition of 10 μg/ml stiripentol to SiRNA reduced mildly oxalate synthesis, suggesting that oxalate synthesis is mostly performed by LDH5. **P = 0.03 versus control, n = 4 experiments. (C) Stiripentol given orally for 2 days significantly reduced urine oxalate excretion. ^#P = 0.002, n = 6 animals. After wash-out, urine oxalate excretion was restored. Data are mean ± SEM. Mann-Whitney tests (B and C) and Kruskall-Wallis with Dunn’s multiple comparison tests (A) were used to compare the different groups.

Figure 2. Stiripentol protects against ethylene glycol intoxication.

(A) Representative crystalluria showing the presence of calcium oxalate monohydrate crystals (black arrow) and to a lesser extent calcium oxalate dihydrate crystals (white arrow) in urine. Original magnification, ×400. (B) Mean crystalline volume in urine was significantly lower in animals receiving stiripentol in addition to ethylene glycol (EG + stiripentol, red squares) than in animals receiving ethylene glycol alone (EG, black circles). *P = 0.004, n = 6 animals/group). (C) The weight of kidneys from rats exposed to ethylene glycol only was increased when compared with kidneys from rats treated by stiripentol. *P = 0.004, n = 6). (D–F) Kidney crystalline accumulation was prevented by stiripentol. **P = 0.002, n = 6. (G) Stiripentol protected rats against ethylene glycol–induced renal failure. **P = 0.002, n = 6. (H) Crystalline deposits were stained by Yasue coloration, evidencing their calcic nature. (I) Fourier transform infrared spectroscopy revealed the exclusive presence of COM among tubular deposits. Data are mean ± SEM. Mann-Whitney tests were used to compare the different groups. Original magnification E, F, H, ×200.

Figure 3. Stiripentol protects against calcium oxalate nephropathy.

(A) Urine oxalate excretion was increased by hydroxyproline-enriched diet, and the daily administration of stiripentol (red bars) protected partly against hyperoxaluria. *P = 0.0002 at days 8 and 15, **P = 0.0019 at day 11; n = 8 animals/group. (B) Daily stiripentol also reduced urine crystalline volume. ^#P = 0.015 at day 8, ^##P = 0.007 at day 15; n = 8 animals/group. (C–E) Kidney crystal deposits were reduced by stiripentol. ^†P = 0.004, n = 8 animals/group. (F) Stiripentol protected rats against hydroxyproline-induced renal failure. ^‡P = 0.028, n = 8 animals/group. Hydroxyproline + stiripentol, red squares; hydroxyproline, black circles. Data are mean ± SEM. Mann-Whitney and Kruskall-Wallis with Dunn’s multiple comparison tests were used to compare the different groups.

Figure 4. Urine glycolate excretion was increased by a hydroxyproline-enriched diet in rats.

At day 2 and day 8 glycolate excretion was significantly more increased in animals exposed to stiripentol than in controls. *P = 0.049 and **P = 0.004, respectively; n = 8 animals/group. Hydroxyproline, black circles and empty bars; hydroxyproline + stiripentol, blue squares and bars. Data are mean ± SEM. Kruskall-Wallis with Dunn’s multiple comparison tests was used to compare the different groups.

Figure 5. Stiripentol decreases urine oxalate excretion in humans.

(A) Urine oxalate excretion (indexed to creatinine, mmol oxalate/mmol creatinine) was lower in children affected by Dravet syndrome and treated with stiripentol than in controls (i.e., children affected by cystinuria). *P = 0.002; Dravet + stiripentol, red squares, n = 8; controls, black circles, n = 40). Mann-Whitney test was used to compare the 2 groups. (B) Urine oxalate excretion (indexed to creatinine, mmol oxalate/mmol creatinine) was measured before and after stiripentol therapy (25 mg/kg/day and 50 mg/kg/day) in a young girl affected by type I primary hyperoxaluria.