Proteomic analysis reveals vital role of the transcription factor Nrf2 in the regulation of cell defence processes in the mouse kidney

Luke Shelton, Joanne Walsh, Rosalind Jenkins, Neil Kitteringham, B. Kevin Park, Ian Copple. University of Liverpool, Liverpool, UK

Drug-induced acute kidney injury is a significant clinical problem, and accounts for the cessation of development of many promising drug candidates. Increasing evidence supports a vital role for the transcription factor Nrf2 in protection of the kidney against a number of diseases and toxicities (1; Fig. 1), and the pharmacological induction of Nrf2 by CDDO-Me (methyl-2-cyano 3,12-dioxooleano-1,9-dien-28-oate, bardoxolone methyl) has shown promise for the clinical management of such pathologies. To provide a comprehensive analysis of the biological processes that are regulated by Nrf2 in the kidney, and to explain the means by which Nrf2 affords protection against a number of nephrotoxins, we have undertaken an iTRAQ-based proteomic analysis of whole kidney homogenates from wild type (Nrf2+/+) and transgenic Nrf2 knockout (Nrf2-/-) mice, both basally and following treatment with 3 mg/kg CDDO-Me (I.P. in DMSO) for 24 h (n=6 animals per group).

Fig. 1 > Overview of the Nrf2 pathway

A total of 3684 unique proteins were quantified over all samples and searched against a reverse decoy database. Only proteins identified within a 1 % global false detection rate were included in subsequent analyses. Of these, 136 proteins were altered between the Nrf2+/+ and Nrf2-/- vehicle control groups (P<0.05, unpaired t-test), including substantial reductions in the expression of the cell defence proteins glutathione S-transferase Mu 1 (relative expression versus pooled control sample 0.18 ± 0.09 in Nrf2-/-, 2.08 ± 0.40 in Nrf2+/+, 11.5-fold change), catalase (0.63 ± 0.16 in Nrf2-/-, 2.70 ± 0.71 in Nrf2+/+, 4.3-fold change) and NAD(P)H dehydrogenase [quinone] 1 (0.35 ± 0.09 in Nrf2-/-, 1.25 ± 0.25 in Nrf2+/+, 3.6-fold change). Whilst a single dose of CDDO-Me was sufficient to induce the expression of NAD(P)H dehydrogenase [quinone] 1 in the kidneys of Nrf2+/+ mice (1.70 ± 0.31 in CDDO-Me, 1.25 ± 0.25 in vehicle, 1.4-fold change), none of the 17 other proteins that were significantly altered (P<0.05, unpaired t-test) by CDDO-Me in Nrf2+/+ animals were found to be regulated by Nrf2 at the basal level.

These data, supported by Metacore pathway analysis and quantitation of mRNA, highlight the role of Nrf2 as a key regulator of cellular defence processes in the kidney, and provide a rationale for our ongoing investigations into the capacity of Nrf2 to protect against drug-induced nephrotoxicity. Moreover, these data justify further evaluation of the biological effects of CDDO-Me and other Nrf2 inducers in the kidney, in order to determine the risk:benefit of this compound class as potential modulators of kidney disease.

1. Shelton LM, Park BK, Copple IM (2013) Kidney Int. [Epub ahead of print].