Cytoprotective properties of novel nonpeptide calpain inhibitors in renal cells

Calpains are cytosolic, Ca²⁺, -activated, neutral cysteine proteases. Rabbit renal proximal tubule (RPT) cells express both μ- and m-calpain. Although multiple calpain inhibitors protect against RPT cell death, most calpain inhibitors lack specificity, membrane permeability, and/or potency. A group of novel catalytic site-directed calpain inhibitors, including chloroacetic acid N'-[6,7-dichloro-4-(4-methoxy-phenyl)-3-oxo-3,4-dihydroquinoxalin-2-yl] hydrazide (SJA7019) and chloroacetic acid N'-(6,7-dichloro-4-phenyl-3-oxo-3,4-dihydroquinoxalin-2-yl) hydrazide (SJA7029), were identified to be potent calpain inhibitors in vitro. The goals of this study were to determine the action of these two compounds on 1) RPT calpain activity using fluorescein isothiocyanate-casein zymography, 2) antimycin A-induced RPT extracellular ⁴⁵Ca²⁺ influx and cell death, and 3) hypoxia/reoxygenation-induced RPT cellular dysfunction and death. The results showed that the SJA compounds inhibited RPT μ- and m-calpain with equal potency (approximate IC₅₀, 30 μM) and efficacy, and blocked antimycin A-induced extracellular Ca²⁺ influx and cell death. In addition, SJA7029 blocked cell death and allowed the recovery of mitochondrial function and active Na⁺ transport in RPTs subjected to hypoxia/reoxygenation. In summary, the SJA compounds 1) were more potent inhibitors of calpains than catalytic site-directed peptide inhibitors in this model, 2) prevented extracellular Ca²⁺ influx during the late phase of cell death, and 3) are true cytoprotectants and allow recovery of RPT cellular functions after injury.