Kidney Slice Culture for ER Stress

A modified version of a previously published protocol was used⁴⁹ . Human kidney pieces were decapsulated and embedded in 3% agarose prewarmed at 40 °C. Slices were cut in ice-cold DPBS using a vibrating microtome Compresstome® VF310-0Z (Precisionary Instruments). Immediately after, the 300-μm-thick slices were pre-incubated containing Williams’ Medium E + GlutaMAX™-I (Gibco™, ThermoFisher Scientific) supplemented with 25 mM D-glucose and 1% Penn-Strep (10,000 U/mL, Gibco™, ThermoFisher Scientific) for 1 h at 37 °C under 80% O₂ and 5% CO₂ atmosphere to allow the slices to restore their ATP levels^{71 (link)}. Slices were then cultured for an additional 24 h with 1 μM tunicamycin^{72 (link)} (#3516, Tocris Bioscience) and 0.05 μg/mL recombinant human Klotho (#5334-KL, R&D Systems) at 37 °C, 80% O₂ and 5% CO₂ on an orbital shaker at 80 RPM. Slices were harvested in 10% formalin solution (HT501128, Sigma-Aldrich) for histology analysis and in RNAlater for RNA isolation. All conditions were carried out as three technical replicates.