Glyoxylic acid

Trichloroethene (≥ 99.5%), tetrachloroethene (≥ 99.5%), formic acid (98%), chloroacetic acid (99%), dichloroacetic acid (99.2%), formaldehyde, trichloroacetic acid (≥ 99%), oxalic acid (≥ 99%), glyoxylic acid (98%), chloroacetaldehyde, acetonitrile of HPLC grade (≥ 99.9%), perchloric acid (70%) all prepared from Sigma Aldrich, sulphuric acid (VWR, 95%) and chloroform (Aldrich, 99.8%) were used. All solutions were prepared using ultrapure water (resistivity 18.2 MΩ cm).

Bisphenol (BPA, 97%), graphite powder (G, <20 micron), Paraffin oil, copper chloride (99.0%), palladium (II) chloride (99%), potassium hydroxide, phosphoric acid (85%), sodium carbonate, glyoxylic acid, and sodium borohydride were all obtained from Sigma-Aldrich Company (Oakville, ON, Canada). Water purification systems, such as the Pascada LS water purification system, manufactured by Pall Co., Mississauga, ON, Canada, were employed in the preparation of all solutions. phosphoric acid electrolyte and buffer solution (PBS) with pH in the range of 2.0 to 8.0 was prepared using 0.2 M phosphoric acid and adding 5.0 M NaOH. The 0.005 M BPA was prepared by dissolving it in 100 µL of 1 M KOH and diluting it with Milli-Q water. The PdCl₂ was dissolved in hydrochloric acid to make H₂PdCl₄ precursor solution.

For peptide synthesis, amino acid derivatives were obtained from Iris Biotech (Germany). The reagents N,N’-diisopropylcarbodiimide (DIC), triisopropylsilane (TIS), 1-hydroxybenzotriazole (HOBt), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), isoniazid (INH), glyoxylic acid and 5(6)-carboxyfluorescein (Cf) were purchased from Sigma-Aldrich (Budapest, Hungary). Fmoc-Rink Amide MBHA resin was from Merck (Budapest, Hungary). Trifluoroacetic acid (TFA), N,N-dimethylformamide (DMF), dichloromethane (DCM), diethyl ether and acetonitrile (AcN) were from VWR (Budapest, Hungary).
For the in vitro assays, RPMI-1640 medium, fetal calf serum (FCS) and trypan blue were obtained from Sigma-Aldrich. Trypsin was from Gibco. HPMI buffer (9 mM glucose, 10 mM NaHCO₃, 119 mM NaCl, 9 mM HEPES, 5 mM KCl, 0.85 mM MgCl₂, 0.053 mM CaCl₂, 5 mM Na₂HPO₄×2H₂O, pH = 7.4) was prepared in-house, using components obtained from Sigma-Aldrich (Budapest, Hungary).

Copper sulfate pentahydrate (CuSO₄·5H₂O), tin(II) chloride (SnCl₂), potassium sodium tartrate (KNaC₄H₄O₆ × 4H₂O), palladium chloride (PdCl₂), formaldehyde (CHOH), ascorbic acid (Asc), glyoxylic acid (Gly), sodium lauryl sulfate, ethylenediaminetetraacetic acid (EDTA), and dimethylamine borane (DMAB) were all purchased from Sigma-Aldrich (Schnelldorf, Germany) and used without further purification. The certified reference solution of 0.1 g/L Pb(II) was purchased from Ecroskhim (Sankt-Petersburg, Russia). The water used in all the experiments was purified using a D-301 water purification system (Akvilon, Russia) with a resistivity of 18.2 MΩ/cm.

All animal experimental protocols were approved by Zhonghong Boyuan Biotechnology Co., Ltd (IACUC Issue No. 2 021 110 201). A total of 12 6‐week‐old male C57BL/6J mice (24–28 g) were purchased from Hubei Provincial Centers for Disease Control and Prevention. All mice were housed in specific pathogen‐free conditions with a steady humidity (40–70%) and temperature (22 ± 2 °C) barrier system under a 12‐h light‐dark cycle and provided with food and water ad libitum. The 12 mice were randomly divided into two groups (n = 6 per group): the vector stone model group and the sET‐GPX4 stone model group. The dRfxCas13d‐eIF4GI‐GPX4 plasmid was packaged into Adeno‐associated virus (AAV) vectors. The mice were anesthetized to expose the kidney, the ureter was blocked using a hemostatic clip, and then the established sET‐GPX4 AVV (1 × 10¹¹ µg per mouse) was injected into the renal pelvis. The expression of GPX4 was measured after 4 weeks. Thereafter, the kidney stone mouse model was constructed by injecting 80 mg kg⁻¹ glyoxylic acid (Sigma–Aldrich; Merck KGaA) i.p. every day for 14 days. Subsequently, the experimental animals were anesthetized by injecting pentobarbital (50 mg kg⁻¹) i.p. 1 day after the last treatment and the kidney tissues and blood samples were collected. Then all animals were euthanatized using pentobarbital (100 mg kg⁻¹) after surgery.