Impact of Nrf2 signaling on thiol homeostasis and renal physiology

Cells evolved robust homeostatic mechanisms to protect against oxidation or alkylation by electrophilic species. Antioxidant signaling responses under control of the Kelch ECH associating protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor were studied. Glutathione (GSH) is the most abundant intracellular thiol and acts as the chief intracellular reduction-oxidation couple through oxidation to glutathione disulfide (GSSG) and subsequent enzymatic reduction back to GSH by glutathione reductase (GR). The fundamental effects of electrophilic Nrf2 activators on the GSH/GSSG antioxidant couple were studied in RAW264.7 macrophages. Electrophiles increased intracellular GSH through Nrf2-dependent transcription of GSH biosynthetic enzymes, yet paradoxically reduced the ratio of GSH to GSSG, which is commonly used as an index of oxidative stress. GSH was found to regulate GR activity via product inhibition in an oxidative stress-independent manner, as determined by biochemistry studies, cell biology experiments, and kinetic modeling approaches. Additionally, inhibition of GR by electrophilic nitrated fatty acid (NO2-FA) by covalent modification of Cys61 at the catalytic site was observed. Together, these results demonstrate that electrophiles differentially modulate the GSH/GSSG couple through two independent mechanisms, and electrophile-mediated increases in GSSG promote S-glutathionylation of cellular proteins. Pharmacologic activators of Nrf2 are in preclinical and clinical development for treatment of a broad spectrum of renal disorders. To characterize the effects of this pathway on renal physiology a murine model of pharmacomimetic Nrf2 activation and primary human renal cortical epithelial cells in culture were studied. Nrf2 signaling was found to be constitutively high in the proximal tubule of the nephron and inducible in the distal convoluted tubule and collecting duct. Hyperactivation of Nrf2 was found to protect against polydipsia in lithium-induced nephrogenic diabetes insipidus (Li-NDI) without improving the urine concentrating defect due to down-regulation of aquaporin 2 (Aqp2). Nitric oxide-independent changes in vascular tone were observed in isolated resistance arteries as well as in conscious ambulatory animals. These effects were attributed to Nrf2-dependent downregulation of the vasodilatory autocoids prostacyclin and kynurenine. Together these findings suggest that Nrf2 protects against Li-NDI through alteration of tubuloglomerular feedback, and may offer a novel therapeutic approach for treatment of this condition