The synthetic triterpenoid RTA dh404 (CDDO-dhTFEA) restores Nrf2 activity and attenuates oxidative stress, inflammation, and fibrosis in rats with chronic kidney disease

Abstract

1. Chronic oxidative stress and inflammation are major mediators of chronic kidney disease (CKD) and result in impaired activation of the cytoprotective transcription factor Nrf2. Given the role of oxidative stress and inflammation in CKD pathogenesis, strategies aimed at restoring Nrf2 activity may attenuate CKD progression. 2. The present study investigated whether the synthetic triterpenoid RTA dh404 (2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-9,11-dihydro-trifluoroethyl amide or CDDO-dhTFEA) would afford renal protection in a 5/6 nephrectomized rat model of CKD. RTA dh404 (2 mg/kg/day) was orally administered once daily for 12 weeks after 5/6 nephrectomy surgery. 3. The remnant kidneys from the vehicle-treated CKD rats showed activation of nuclear factor kappaB (NF-κB), upregulation of NAD(P)H oxidase, glomerulosclerosis, interstitial fibrosis and inflammation, as well as marked reductions in Nrf2 and its target gene products (i.e. catalase, heme oxygenase-1, thioredoxin 1, thioredoxin reductase 1 and peroxiredoxin 1). The functional and structural deficits in the kidney were associated with increased (∼30%) mean arterial pressure (MAP). Treatment with RTA dh404 restored MAP, increased Nrf2 and expression of its target genes, attenuated activation of NF-κB and transforming growth factor-β pathways, and reduced glomerulosclerosis, interstitial fibrosis and inflammation in the CKD rats. 4. Thus, chronic treatment with RTA dh404 was effective in restoring Nrf2 activity and slowing CKD progression in rats following 5/6 nephrectomy.

Figure 1.

Representative western blots and group data depicting protein abundance of phospho-IκB and nuclear contents of p65 active subunit of NF-κB, iNOS and COX-2 in the renal tissues of sham-operated control (CTL; n = 6) and a 5/6 nephrectomized rat CKD treated with vehicle (CKD; n = 9) or RTA dh404 (CKD + RTA dh404; n = 9); *p < 0.05 versus CTL, #p < 0.05 versus CKD.

Figure 2.

Representative western blots and group data depicting protein abundance of NAD(P)H oxidase subunits (p22^phox, gp91^phox and rac1) and NT in the renal tissues of sham-operated control (CTL; n = 6) and a 5/6 nephrectomized rat CKD treated with vehicle (CKD; n = 9) or RTA dh404 (CKD + RTA dh404; n = 9); *p < 0.05 versus CTL, **p < 0.01 versus CTL, #p < 0.05 versus CKD.

Figure 3.

Representative western blots and group data depicting protein abundance of Nrf2, Keap1 and Nrf2 downstream gene products: catalase, HO-1 and catalytic (GCLC) and modulatory (GCLM) subunits of glutamate-cysteine ligase in the renal tissues of sham-operated control (CTL; n = 6) and a 5/6 nephrectomized rat CKD treated with vehicle (CKD; n = 9) or RTA dh404 (CKD + RTA dh404; n = 9); *p < 0.05 versus CTL, **p < 0.01 versus CTL, #p < 0.05 versus CKD.

Figure 4.

Messenger RNA expression of peroxiredoxin 1 (Prdx1), thioredoxin 1 (Txn1) and thioredoxin reductase 1 (Txnrd1) in the renal tissues of sham-operated control (CTL; n = 6) and a 5/6 nephrectomized rat CKD treated with vehicle (CKD; n = 9) or RTA dh404 (CKD + RTA dh404; n = 9); **p < 0.01 versus CTL, #p < 0.05 versus CKD.

Figure 5.

Representative western blots and group data depicting protein abundance of TGF-β, α-SMA, extracellular signal-regulated kinase 1/2 (ERK 1/2) and SMAD7 in the renal tissues of sham-operated control (CTL; n = 6) and a 5/6 nephrectomized rat CKD treated with vehicle (CKD; n = 9) or RTA dh404 (CKD + RTA dh404; n = 9); *p < 0.05 versus CTL, **p < 0.01 versus CTL, #p < 0.05 versus CKD, ##p < 0.01 versus CKD.

Figure 6.

Representative photomicrographs of the H&E stained renal tissue (20X) in a sham-operated control (A) and a 5/6 nephrectomized rat (CKD)] treated with vehicle (B) or RTA dh404 (C). The remnant kidney in the CKD animals (B) exhibited significant glomerulosclerosis, tubulo-interstitial injury and heavy inflammatory cell infiltration. RTA dh404 treatment-reduced inflammatory cell infiltration and glomerular and tubulo-interstitial injury (C).

Figure 7.

Representative photomicrographs of the PAS-stained renal tissue in a sham-operated control (A) and a 5/6 nephrectomized rat (CKD) treated with vehicle (B) or RTA dh404 (C). The remnant kidney in the CKD animals exhibited significant fibrosis (B) which was reduced with RTA dh404 treatment (C). 20 × magnification.