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Introduction AT2R agonist C21 elicits nephroprotective effects in ischemia/reperfusion (IR)-induced acute kidney injury (AKI) by preventing tubular cell damage. IR-induced AKI associates with tubular cell deciliation. Primary cilia are sensory organelles that regulate signaling pathways central for tubular cell survival, whose stability depends on α-tubulin acetylation. Extracellular signal-regulated kinases (ERK) activates Histone-deacetylase 6 (HDAC6), which is the main α-tubulin deacetylase involved in loss of primary cilia stability in epithelial cells. The aim of this study was to get mechanistic insight into C21 nephroprotective effect during renal-IR.
Methods Male Wistar rats were pretreated 24h with C21 (Vicore Pharma, 0,3 mg/kg). Controls and one C21 treated rats were subjected to 40 min of unilateral renal ischemia followed by 24 h of reperfusion (IR and C21-IR, respectively), whereas another group of controls and C21 treated rats were subjected to simulated surgery (C and C21, respectively) (n=3-5 rats per group; Institutional Animal Care and Use Committee-FBIOyF #023-2020). C21 effect was also assessed using a serum/ATP depletion model of IR in MDCK cells (n=3-4 independent experiments). C21, HDAC6-inhibitor (Tubastatin A 1 μM, TubA) or MEK1/2 inhibitors (PD98059 5μM or U0126 10μM) were added to the media 24h before IR. Cell ciliation, relative cilia levels of acetylated-α-tubulin (c-Ac-αtub) and ERK1/2 localization were analyzed by immunofluorescence microscopy, activated ERK (pERK) by immunoblotting and cell viability by Trypan blue exclusion. Results are expressed as media±SEM. ANOVA/Holm-Sidak-test, Kruskal-Wallis/Dunn’s-test or t-test were applied. *p<0.05 vs control (C), #p<0.05 vs C-IR.
Results C21 prevented IR-induced cilia shortening (length (um): C:3.7+/-0.3; C21:3.4+/-0.3; C-IR:2.1+/-0.1*; C21-IR:2.9+/-0.3) and cell deciliation (% ciliated cells C: 94.2+/-1.7, C21: 96.5+/-0.9; IR: 63.5+/-3.8*; C21-IR: 82.5+/-2.8#) and increased basal c-Ac-αtub (C: 2.4+/-0.2; C21-B 3.3+/-0.2*; n=3*) in rat kidney tubular cells. C21 also increased c-Ac-αtub in MDCK cells (C: 8.9+/-1.1; C21: 11.5+/-1.6*; n=4*), whereas it inhibited ERK1/2 activation (% of C: pERK1: C: 1, C21: 0.70+/-0.06* pERK2: C: 1, C21: 0.65+/-0.07*). ERK1/2 inhibitors partially prevented IR-induced decrease in cell viability (% viable cells: C: 94.2+/-0.1, U0126: 93.1+/-0.3; IR: 62.1+/-2.8*; U0126-IR: 75.5+/-2.9*#; n=3; C: 96.1+/-0.2, PD98059: 95.1+/-0.6; IR: 74.9+/-2.6*; PD98059-IR: 81.8+/-1.8*#; n=3). Analysis of ERK1/2 subcellular distribution in differentiated tubular cells revealed a major ERK1/2 localization at the primary cilia. Concomitantly, ERK1/2 inhibition increased basal c-Ac-αtub (C: 9.0+/-0.3; U0126: 11.0+/-0.6*; n=4; C: 10.4+/-1.2; PD98059: 12.1+/-0.7*; n=4). Loss of viability induced by IR in tubular cells was also prevented by HDAC6 inhibition with TubA (% viable cells: C: 95+/-1, Tub A: 95+/-1, IR: 71+/-4*, Tub-IR: 82+/-3*#, n=6).
Conclusions AT2R nephroprotective effect during renal IR is associated with prevention of tubular cell deciliation in the rat. The cellular mechanism underlying this effect involves AT2R mediated inhibition of ERK1/2 kinases, and concomitant increase in primary cilia stability. Our results support a model where AT2R induced inhibition of ERK1/2 activation leads to cilia stabilization by prevention of HDAC6 activation.