Benefit of Mineralocorticoid Receptor Antagonism In
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BASIC RESEARCH www.jasn.org Benefit of Mineralocorticoid Receptor Antagonism in AKI: Role of Vascular Smooth Muscle Rac1 Jonatan Barrera-Chimal,* Gwennan André-Grégoire,* Aurelie Nguyen dinh Cat,* †‡ | Sebastian M. Lechner,* Jérôme Cau, Sonia Prince,* Peter Kolkhof,§ Gervaise Loirand, | †‡ Vincent Sauzeau, Thierry Hauet, and Frédéric Jaisser*¶ *Unité Mixte de Recherche Scientifique 1138, Team 1, Institut National de la Santé et de la Recherche Médicale, Centre de Recherche des Cordeliers, Pierre et Marie Curie University, Paris Descartes University, Paris, France; †Unité U1082 Ischemie Reperfusion en Transplantation d’Organes Mécanismes et Innovations Thérapeutiques, Institut National de la Santé et de la Recherche Médicale, Université de Poitiers, Poitiers, France; ‡Service de Biochimie, Centre Hospitalier Universitaire de Poitiers, Pôle BIOlogie Santé publique PHARMacie, Poitiers, France; §Cardiology Research, BAYER Pharma AG, Wuppertal, Germany; |Unité Mixte de Recherche Scientifique 1087, Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique Unité Mixte de Recherche Scientifique 6291, l’Institut du Thorax, Nantes, France; and ¶Clinical Investigation Centre 1433, Institut National de la Santé et de la Recherche Médicale, Vandoeuvre-lès-Nancy, France ABSTRACT AKI is a frequent complication in hospitalized patients. Unfortunately, there is no effective pharmacologic approach for treating or preventing AKI. In rodents, mineralocorticoid receptor (MR) antagonism prevents AKI induced by ischemia-reperfusion (IR). We investigated the specific role of vascular MR in mediating AKI induced by IR. We also assessed the protective effect of MR antagonism in IR-induced AKI in the Large White pig, a model of human AKI. In mice, MR deficiency in smooth muscle cells (SMCs) protected against kidney IR injury. MR blockade by the novel nonsteroidal MR antagonist, finerenone, or genetic deletion of MR in SMCs associated with weaker oxidative stress production. Moreover, ischemic kidneys had higher levels of Rac1- GTP, required for NADPH oxidase activation, than sham control kidneys, and genetic deletion of Rac1 in SMCs protected against AKI. Furthermore, genetic deletion of MR in SMCs blunted the production of Rac1-GTP after IR. Pharmacologic inhibition of MR also prevented AKI induced by IR in the Large White pig. Altogether, we show that MR antagonism, or deletion of the MR gene in SMCs, limited the renal injury induced by IR through effects on Rac1-mediated MR signaling. The benefits of MR antagonism in the pig provide a rational basis for future clinical trials assessing the benefits of this approach in patients with IR-mediated AKI. J Am Soc Nephrol 28: ccc–ccc, 2017. doi: 10.1681/ASN.2016040477 AKI is a frequent complication that affects almost 5% Received April 25, 2016. Accepted September 22, 2016. – of hospitalized patients and 40% 70% of intensive J.B.-C. and G.A.-G. contributed equally to this work. care unit patients.1 It is now recognized as a risk fac- Published online ahead of print. Publication date available at 2 tor for the development of CKD. One of the princi- www.jasn.org. pal features of ischemic AKI is a decrease in renal Present address: Dr. Jonatan Barrera-Chimal, Unidad de Fisiología blood flow, leading to lower levels of oxygen delivery Molecular, Instituto de Investigaciones Biomedicas, National Autono- to kidney tissues, resulting in endothelial alterations, mous University of Mexico and Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico. tubular cell injury, oxidative stress, and inflamma- tion.3,4 The reduction of renal perfusion is linked to Correspondence: Dr. Frédéric Jaisser, U1138, Institut National de la Santé et de la Recherche Médicale, Centre de Recherche de changes in the balance between vasoconstrictors and Cordeliers, Team 1, 15 rue de l’Ecole de Médecine, 75006 Paris, vasodilators.5 For example, during kidney ischemia- France. Email: [email protected] reperfusion (IR), nitric oxide (NO) bioavailability is Copyright © 2017 by the American Society of Nephrology J Am Soc Nephrol 28: ccc–ccc, 2017 ISSN : 1046-6673/2804-ccc 1 BASIC RESEARCH www.jasn.org impaired by the low levels of eNOS activity and NO oxidation by RESULTS locally produced reactive oxygen species (ROS).6,7 DespiterecentadvancesinourunderstandingofAKI,there MR Antagonism with Finerenone Prevents AKI Induced is still no effective treatment. New innovative treatments tar- by IR in Mice geting common mechanisms involved in AKI of different We first investigated whether the novel nonsteroidal MR antagonist etiologies are therefore required. We have shown that miner- finerenone had a beneficial effect on AKI induced by IR in mice, as alocorticoid receptor (MR) antagonist before, oreven just after previously reported for spironolactone9 and BR-42688 in rats. As ischemic damage is beneficial, and can considerably reduce IR we planned to address the cell-specificroleofMRinthevascula- injury in rats.8 However, the mechanisms underlying the ben- ture, we investigated the protective effects of finerenone against IR efits of MR antagonists (MRAs) remain unclear. The role of in mice with a genetic background similar to that of the ECs-MR MR activation in modulating renal perfusion is highlighted by and SMCs-MR knockout mice used in subsequent experiments. the normalization of renal blood flow and limitation of renal Finerenone prevented the renal dysfunction induced by bilateral injury by pharmacologic MR antagonism.9,10 It remains un- ischemia (Figure 1, A and B). Nontreated mice with AKI had high clear whether the MR expressed in the vasculature is involved renal mRNA levels of NGAL and Kim-1, two markers of tubular in the beneficial effects of MRAs. There is strong evidence to injury. Finerenone blunted the increase in the levels of these two implicate the MR expressed in either endothelial cells (ECs) or markers (Figure 1, C and D). It also prevented IR-induced tubular smooth muscle cells (SMCs) in the modulation of vascular injury, as assessed by measuring cast formation and tubular cell tone, and thus, BP.11–13 We recently demonstrated that dele- detachment, as shown by comparison with the sham treatment tion of the MR gene in SMCs prevented the increase in renal (Figure 1, E–H). Plasma potassium concentration was similar and vascular resistance in a mouse model of cyclosporin A (CsA) unchanged in the two groups (Supplemental Figure 1). nephrotoxicity, because of increased L-type calcium channel activity.14 This finding suggests that MR activation in renal MR Deficiency in SMCs, but Not in ECs, Protects Mice ECs or SMCs may contribute to IR injury. The goal of this Against AKI Induced by IR study was to investigate the specificroleoftheMRinECsand We investigated the role of the MR expressed in ECs or SMCs SMCs during the kidney injury induced by IR, and to under- in kidney IR injury, by generating two knockout mouse stand the mechanisms underlying MR-mediated IR injury. models with cell-specificandinducibleMRinactivationinECs We also assessed the protective effect of MRAs in the Large (MRendoKO)orSMCs(MRSMCKO). Control MRf/f mice with bilat- White pig, an animal model of human AKI, to provide a ra- eral renal ischemia developed AKI, as shown by an increase in tional basis for future clinical trials assessing the benefits of plasma creatinine (Figure 2A), an increase in mRNA levels for MRA in patients with IR-mediated AKI. NGAL and Kim-1 (Figure 2, B and C), and the presence of Figure 1. Benefits of finerenone (10 mg/kg) for preventing kidney injury induced by IR. Renal function was evaluated by quantifying the plasma levels of (A) creatinine and (B) urea. As markers of tubular injury, (C) levels of mRNA in the kidney for neutrophil gelatinase- associated lipocalin and (D) kidney injury molecule-1 were assessed by RT-PCR. Representative hematoxylin and eosin staining images are shown for the (E) sham, (F) IR, and (G) IR and finerenone (IR+Fine) groups. (H) The percentage of injured tubules was quantified blind, on ten fields per mouse. Scale bar, 20 mm. n=8 per group. One-way ANOVA was performed. ***P,0.001; ****P,0.001. 2 Journal of the American Society of Nephrology J Am Soc Nephrol 28: ccc–ccc,2017 www.jasn.org BASIC RESEARCH Figure 2. MR deficiency in ECs does not protect against kidney IR injury. Plasma levels of (A) creatinine were measured as an index of renal function. As markers of tubular injury, we used RT-PCR to determine mRNA levels for (B) neutrophil gelatinase-associated lipocalin and (C) kidney injury molecule-1. (D) The percentage of injured tubules was quantified blind on ten fields per mouse. Representative hematoxylin and eosin staining images are shown for (E) MRf/f sham, (F) MRf/f IR, (G) MRendoKO sham, and (H) MRendoKO IR. Scale bar, 20 mm. White bars represent the sham groups and black bars represent the IR groups. n=8 per group. Two-way ANOVA was per- formed. *P,0.01; **P,0.001; ***P,0.001. injured tubules (Figure 2, D and F). Similar changes were in- concentration (Figure 3A) and the significantly lower levels duced by IR in the MRendoKO mice: plasma creatinine levels of mRNA for NGAL and Kim-1 (Figure 3, B and C). MR increased (Figure 2A), as did NGAL and Kim-1 mRNA levels knockout in SMCs also prevented the development of tubular (Figure 2, B–C), and tubular injury similar to that in control lesions (Figure 3, D and H). This suggests that the MR ex- mice was observed (Figure 2, D and H). In contrast, MR de- pressed in SMCs, but not in the endothelium, is crucial for ficiency in SMCs protects against IR-induced renal injury, as the development of IR injury and is a mandatory target for demonstrated by the lack of increase in plasma creatinine the beneficial effects of the MRA finerenone. Figure 3. MR deficiency in SMCs protects against kidney IR injury.