The myeloid mineralocorticoid receptor controls inflammatory and fibrotic responses after renal injury via macrophage interleukin-4 receptor signaling

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The myeloid mineralocorticoid receptor controls inflammatory and fibrotic responses after renal injury via macrophage interleukin-4 receptor signaling Jonatan Barrera-Chimal1,2, Gabriel R. Estrela1,9, Sebastian M. Lechner1,9, Se´bastien Giraud3,4,5, Soumaya El Moghrabi1, Shiem Kaaki3,6, Peter Kolkhof7, Thierry Hauet3,4,5 and Fre´de´ric Jaisser1,8 1

INSERM, UMRS 1138, Team 1, Centre de Recherche des Cordeliers, Pierre et Marie Curie University, Paris Descartes University, Paris, France; 2Molecular Physiology Unit, Instituto de Investigaciones Biomedicas, UNAM and Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico; 3INSERM U1082 IRTOMIT, Poitiers, France; 4Université de Poitiers, Faculté de Médecine et de Pharmacie, Poitiers, France; 5CHU Poitiers, Service de Biochimie, Poitiers, France; 6CHU Poitiers, Service d’Anatomopathologie, Poitiers, France; 7Bayer AG, Cardiology Research, Wuppertal, Germany; and 8INSERM, Clinical Investigation Center 1433, French-Clinical Research Infrastructure Network (F-CRIN) INI-CRCT, Nancy, France

Acute kidney injury induced by ischemia/reperfusion is an independent risk factor for chronic kidney disease. Macrophage recruitment plays an essential role during the injury and repair phases after an ischemic episode in the kidney. Here we show that the novel non-steroidal mineralocorticoid receptor antagonist finerenone or selective myeloid mineralocorticoid receptor ablation protects against subsequent chronic dysfunction and fibrosis induced by an episode of bilateral kidney ischemia/ reperfusion in mice. This protection was associated with increased expression of M2-antiinflamatory markers in macrophages from finerenone-treated or myeloid mineralocorticoid receptor-deficient mice. Moreover, the inflammatory population of CD11bD, F4/80D, Ly6Chigh macrophages was also reduced. Mineralocorticoid receptor inhibition promoted increased IL-4 receptor expression and activation in the whole kidney and in isolated macrophages, thereby facilitating macrophage polarization to an M2 phenotype. The long-term protection conferred by mineralocorticoid receptor antagonism was also translated to the Large White pig pre-clinical model. Thus, our studies support the rationale for using mineralocorticoid receptor antagonists in clinical practice to prevent transition of acute kidney injury to chronic kidney disease. Kidney International (2018) 93, 1344–1355; https://doi.org/10.1016/ j.kint.2017.12.016 KEYWORDS: inflammation; ischemic injury; macrophage polarization Copyright ª 2018, International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Correspondence: Frédéric Jaisser, INSERM U1138, Centre de Recherche de Cordeliers, Team 1, 15 rue de l’Ecole de Médecine, 75006, Paris, France. E-mail: [email protected] 9

These authors contributed equally to this work.

Received 31 May 2017; revised 6 December 2017; accepted 15 December 2017; published online 13 March 2018 1344

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hronic kidney disease (CKD) has become a global public health problem that threatens the health care system. According to a recent meta-analysis, it is estimated that the global prevalence of CKD is approximately 11% to 13%.1 In recent years, it has been recognized that patients surviving an episode of acute kidney injury (AKI) are at increased risk of adverse outcomes, such as de novo CKD, worsening of pre-existing CKD, and end-stage kidney disease.2 It has been demonstrated in experimental models that a single episode of renal ischemia-reperfusion (IR) may result in maladaptive repair and lead to chronic kidney fibrosis and dysfunction.3,4 Intensive research in the field over the past few years has shed light on mechanisms involved in the AKI-to-CKD transition, such as capillary rarefaction, tubular epithelial cell G2/M arrest, sustained oxidative stress, and chronic inflammation.5 The infiltration of inflammatory cells early after an AKI episode plays an important role in defining effective versus maladaptive repair.6 Nevertheless, there are no therapeutic approaches in current clinical practice that have proven to be useful in preventing progression to CKD after an AKI episode.7 There is thus a need to identify novel underlying mechanisms that facilitate CKD development to aid the development of more precise targeted therapy. We previously demonstrated that pharmacological mineralocorticoid receptor (MR) antagonism is an efficient strategy to prevent CKD after a single episode of AKI.4,8 However, the detailed mechanisms of MR-mediated kidney fibrosis are poorly understood. MR is expressed in myeloid cells and has been shown to play a key role in aldosterone and angiotensin II–mediated heart fibrosis.9,10 Thus, we hypothesized that myeloid MR may potentially be involved in the development of kidney fibrosis after an ischemic AKI episode. RESULTS Nonsteroidal MR antagonism is beneficial in AKI-induced CKD

We developed a model of bilateral IR-induced CKD in mice and demonstrated that pharmacological MR inhibition with Kidney International (2018) 93, 1344–1355

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the nonsteroidal MR antagonist (MRA) finerenone can prevent CKD development after IR. Finerenone was administered at 48, 24, and 1 hour before the IR procedure, while no finerenone was given thereafter until killing (D30) (Supplementary Figure S1). In this model with important bilateral IR, the protection conferred by finerenone against the acute effects of IR (24 hours) was partial as shown by a limited reduction in plasma creatinine and urea levels (Supplementary Figure S2). Four weeks after transient ischemia, plasma creatinine (Figure 1a), urea (Figure 1b), and proteinuria levels (Figure 1c) were increased in the IR untreated mice. This was associated with increased expression of transforming growth factor beta as an indicator of kidney fibrosis (Figure 1d). These alterations were efficiently prevented by prophylactic finerenone administration. Histology analysis of Sirius Red–stained slides revealed substantially greater collagen deposition in the IR group than in sham- and finerenone-treated mice (Figure 1e–h). We tested whether

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finerenone was also protecting toward CKD progression in a more severe ischemic injury episode by using the 30-minute unilateral renal ischemia model and 4 weeks of follow-up. Unilateral IR induced severe tubulo-interstitial fibrosis as evidenced in the Sirius Red staining and fibrosis scoring (Supplementary Figure S2A–D). In contrast, in mice treated with finerenone before IR, the severity of kidney fibrosis was significantly reduced. Proteinuria was elevated in the untreated IR group compared with sham, and finerenone treatment prevented the increased proteinuria (Supplementary Figure S2E). Role of myeloid MR in CKD progression and macrophage polarization

In previous studies, we have reported a role for the MR expressed in smooth muscle cells in acute kidney injury after IR.11 Transient renal ischemia was induced in smooth muscle cell MR knockout mice (MRSMCKO), and kidney injury was

Figure 1 | Finerenone protects against the transition from acute kidney injury to chronic kidney disease. Renal function was determined by quantifying the plasma levels of (a) creatinine and (b) urea. (c) The urinary protein excretion was determined. (d) We measured kidney mRNA levels of transforming growth factor (TGF)-b1 by real-time polymerase chain reaction as a marker of kidney fibrosis. Representative Sirius Red– stained images for the (e) sham, (f) ischemia-reperfusion (IR), and (g) IR þ finerenone (Fine) groups. (h) The fibrosis score was blindly quantified on 8 fields per mouse. The mRNA levels of the proinflammatory cytokines (i) interleukin (IL)-6 and (j) IL1-beta were quantified in whole kidney. Kidney macrophages (CD45þ, F4/80þ, CD19–, CD3–, and Ly6g– cells) were sorted and the RNA extracted. The mRNA levels of (k) M1 markers and (l) M2 markers were determined in the IR (black bars) and IR þ Fine (gray bars) groups. n ¼ 6 per group. Arg1, arginase 1; MCP, monocyte chemoattractant protein; Pai, plasminogen activator inhibitor; PPAR, peroxisome proliferator-activated receptor; TNF, tumor necrosis factor. One-way analysis of variance or Student t-test was performed. Bar ¼ 100 mm. *P < 0.05, **P < 0.01, ***P < 0.001. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International (2018) 93, 1344–1355

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evaluated after 30 days. MR deficiency in smooth muscle cells of MRSMCKO mice prevented renal dysfunction 24 hours after IR (plasma creatinine: 14.7  0.9 mmol/l in MRSMCKO vs. 34.5  2.7 mmol/l in MRf/f, n ¼ 6, P < 0.05), but had no effect on the prevention of chronic kidney alterations after IR (Supplementary Figure S4). Therefore, other cells that express the MR and that are targeted by MRAs must contribute to the chronic alterations induced by kidney IR. Inflammation is an important player in AKI-induced CKD. Thus, we evaluated the expression of the proinflammatory cytokines interleukin (IL) 6 and IL1-ß in the whole kidney 24 hours after reperfusion. The levels of both cytokines increased after induction of IR in untreated mice. This effect was prevented by finerenone administration (Figure 1i and j), suggesting that MR antagonism may act through the modulation of inflammation. Macrophages play a crucial role in the early phases after an ischemic episode and may determine the fate of kidney repair depending on their phenotype. We investigated whether macrophage polarization after IR was altered by MR inhibition. Finerenone treatment did not modify the number of infiltrating macrophages or the expression of CD80 or Ly6C versus that in untreated IR mice (Supplementary Figure S5A–C). However, the reduction in the expression of the M2 marker CD206, which was observed in the ischemic group, was prevented by finerenone administration (Supplementary Figure S5D). We also sorted macrophages (CD45þ, F4/80þ, CD3–, Ly6G–, CD19– cells) from kidney tissue after 24 hours of ischemia and performed gene expression analysis for several M1 and M2 markers. MR

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antagonism with finerenone led to a 50% reduction of tumor necrosis factor alpha and Il-1 beta mRNA levels (Figure 1k), whereas mRNA expression of M2 markers, such as plasminogen activator inhibitor 1, mannose receptor, peroxisome proliferator-activated receptor gamma, IL-10, and arginase 1 (Arg1), was higher than in nontreated IR mice (Figure 1l). We evaluated the specific contribution of the myeloid MR in CKD progression after IR using a model of MR inactivation in myeloid cells (MRMyKO) (Supplementary Figure S6A). MR deletion in macrophages isolated from MRMyKO mice was confirmed by Western blotting (Supplementary Figure S6B). After the induction of IR, MRf/f and MRMyKO mice developed the same degree of injury 24 hours after reperfusion, shown by a similar increase in plasma creatinine (Supplementary Figure S7A) and urea levels (Supplementary Figure S7B). Moreover, the degree of tubular injury was comparable between the 2 genotypes (Supplementary Figure S7C–E). Four weeks after ischemia, MRf/f mice had higher plasma creatinine levels than did sham mice (Figure 2a). We did not observe this effect in MRMyKO mice (Figure 2a). Moreover, transforming growth factor beta and fibronectin mRNA levels were not altered in mice deficient for the MR in myeloid cells, in contrast with the increase observed in MRf/f ischemic mice (Figure 2b and c). Finally, MRMyKO mice did not develop kidney fibrosis 4 weeks after kidney ischemia, whereas the MRf/f mice did (Figure 2d–h). Macrophage polarization during the acute phase after an ischemic insult may determine effective or maladaptive repair. We assessed the macrophage phenotype in MRMyKO mice 24 hours after reperfusion.

Figure 2 | Mineralocorticoid receptor (MR) deficiency in myeloid cells protects against chronic dysfunction and fibrosis induced by ischemia-reperfusion (IR). (a) Renal function was determined by quantifying the plasma levels of creatinine. We determined the kidney mRNA levels of (b) transforming growth factor (TGF) b1 and (c) fibronectin by real-time polymerase chain reaction as markers of kidney fibrosis. (d) The fibrosis score was blindly quantified on 8 fields per mouse. Representative Sirius Red–stained images for the (e) sham MRf/f, (f) IR MRf/f, (g) sham MRMyKO, and (h) IR MRMyKO groups. Sham is indicated by the white bars and IR by the black bars. n ¼ 6 per group. Two-way analysis of variance was performed. Bar ¼ 100 mm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. MRf/f, mice with the exon 3 of the MR gene flanked by LoxP sites; MRMyKO, mineralocorticoid receptor inactivation in myeloid cells. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. 1346

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CD11bþ and F4/80þ cells were identified as the macrophage population. The percentage of infiltrating macrophages was similar between MRf/f (10.5  3.7%) and MRMyKO (11.2  2.7%) mice (Figure 3a), as well as the number of CD80þ (an M1 marker) cells (Figure 3b). There was a trend toward a higher number of CD206þ (an M2 marker) cells in the MRMyKO mice (Figure 3c), whereas the number of Ly6Chipositive cells (proinflammatory cells) decreased (Figure 3d). There were no changes in T lymphocyte, dendritic cell, or granulocyte accumulation in the kidney (Supplementary Figure S8). We further characterized the expression of M1 and M2 markers in macrophages isolated from kidneys 24 hours after reperfusion. M1 marker levels were unchanged in MRMyKO mice (Figure 3e), whereas we observed increased expression of several M2 macrophage markers, such as plasminogen activator inhibitor 1, mannose receptor, YM1, and matrix metallopeptidase 9 (Figure 3f). Of note, the levels of the macrophage markers CD80, CD206, and Ly6C did not change in the MRSMCKO mice after renal IR relative to control littermates (Supplementary Figure S9). MR modulates IL-4 receptor signaling in macrophages

We isolated peritoneal macrophages from wild-type mice and cultured them in pro-M1 (LPS) or pro-M2 (IL-4) conditions

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and evaluated the effect of MR antagonism to understand how MR inhibition could affect macrophage polarization after an inflammatory stimulus and promote an M2-like phenotype. LPS treatment induced a reduction in CD206 expression, which was significantly less pronounced in macrophages incubated with finerenone (Supplementary Figure S10A). The addition of IL-4 to macrophages subjected to MR inhibition induced an increase in the expression of the M2 marker CD206 (Supplementary Figure S10B). These data suggest that MR might act by modulating IL-4 receptor signaling, which plays an essential role in M2 macrophage polarization.12,13 We explored the in vivo relevance of IL-4 receptor by determining the mRNA levels of IL-4 receptor in the whole kidney in mice that underwent bilateral renal ischemia with or without finerenone treatment. We observed a significant increase in the mRNA levels of IL-4 receptor in the kidneys of mice with MR inhibition by finerenone (Figure 4a). Moreover, we assessed the activation state of IL-4 receptor by analyzing its phosphorylation in whole kidney. Finerenone administration in the ischemic setting induced an increase in the phosphorylation of IL-4 receptor (Figure 4b). Next, we quantified the IL-4 receptor expression by flow cytometry in macrophages (F4/80þ and CD11bþ cells) isolated from kidneys of these experimental

Figure 3 | Myeloid mineralocorticoid receptor (MR) knockout mice show fewer inflammatory macrophages and more M2 macrophages in the kidney after ischemia-reperfusion (IR). A cell suspension was prepared from the whole kidney 24 hours after ischemia and the mononuclear cells enriched and the percentage of various cell populations determined. (a) Percentages of macrophages: F4/80þ, CD11bþ cells. (b) Percentage of macrophages positive for the M1 marker CD80. (c) Percentage of macrophages positive for the M2 marker CD206. (d) Percentage of macrophages expressing the inflammatory macrophage marker Ly6C at low, medium, or high levels. The mRNA levels of (e) M1 markers and (f) M2 markers. Arg1, arginase 1; Igf1, insulin-like growth factor 1; IL, interleukin; MCP, monocyte chemoattractant protein; MMP, matrix metalloproteinase; MRf/f, mice with the exon 3 of the MR gene flanked by LoxP sites; MRMyKO, mineralocorticoid receptor inactivation in myeloid cells; Pai, plasminogen activator inhibitor; TNF, tumor necrosis factor. White bars represent the MRf/f IR mice and black bars the MRMyKO IR mice. n ¼ 6 per group. The Student t-test was performed. *P < 0.05. Kidney International (2018) 93, 1344–1355

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Figure 4 | The mineralocorticoid receptor (MR) modulates interleukin-4 receptor (IL4R) expression and activation. (a) The mRNA levels of IL4R expression were quantified by real-time polymerase chain reaction in RNA extracted from whole kidney of sham, ischemia-reperfusion (IR), and IR þ finerenone (Fine) groups. (b) Western Blot analysis showing the total and phosphorylated IL4R (p-IL4R) as well as the densitometric analysis. (c) The percentage of macrophages positive for IL4R expression was determined in the gated F4/80þ - CD11bþ cell population. (d) Peritoneal macrophages were cultured and the expression levels of IL4R were determined in nontreated macrophages, and macrophages treated with Fine (10 mM), lipopolysaccharide (LPS; 100 ng/ml), or LPS þ Fine by flow cytometry. (e) The mRNA levels of IL4R expression were quantified by real-time polymerase chain reaction in RNA extracted from whole kidney of sham, MRf/f IR, and MRMyKO IR groups. (f) Phosphorylation status of STAT6 in macrophages isolated from MRf/f or MRMyKO mice in the presence of IL-4 (5 ng/ml) during 30 minutes. n ¼ 5 per group. One-way analysis of variance or Student t-test was performed. *P < 0.05, **P < 0.01, ***P < 0.001. MRf/f, mice with the exon 3 of the MR gene flanked by LoxP sites; MRMyKO, mineralocorticoid receptor inactivation in myeloid cells.

groups. We found that IL-4 receptor expression was significantly higher in kidney macrophages isolated from mice that underwent bilateral renal ischemia and received finerenone compared with sham-treated and untreated ischemic mice (Figure 4c), thus confirming that MR inhibition increases IL-4 receptor expression specifically in macrophages. 1348

Moreover, the expression of the IL-4 receptor alpha-subunit (CD124) increased in isolated macrophages subjected to LPS challenge and MR inhibition (Figure 4d). IL-4 receptor mRNA levels also increased in the kidneys of mice deficient of MR in myeloid cells, and MRMyKO mice presented an increase in IL4R mRNA kidney levels compared with sham-treated Kidney International (2018) 93, 1344–1355

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mice (Figure 4e). Finally, we analyzed the downstream signaling of the IL-4 receptor by measuring the phosphorylation of STAT6 in macrophages from MRf/f and MRMyKO mice stimulated with IL-4. IL-4 addition induced a significant increase in STAT6 phosphorylation in MRMyKO mice relative to controls (Figure 4f). Benefit of MRA in CKD progression after AKI in the Large White pig

We have previously shown that inflammation is critical for the AKI-to-CKD transition in the Large White pig preclinical model.14 We tested whether MR antagonism given at the time of AKI only can prevent the progression to CKD after an episode of renal ischemia (Figure 5a). In kidney biopsies at

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day 14 after the ischemic insult, we observed a reduced inflammatory infiltrate in the soludactone-treated pigs (inflammation score: 0.35  0.04) compared with the vehicle group (inflammation score: 1.66  0.02). Ninety days after the induction of ischemia, the pigs that underwent kidney IR and received the MR antagonist soludactone (potassium canreonate) at the early time points displayed lower levels of plasma creatinine than did the vehicle group (Figure 5b). Histological quantification of the Sirius Red–positive area (Figure 5c) showed severe interstitial fibrosis in the cortex and medulla in the IR plus vehicle group (Figure 5d). In contrast, the pigs that received soludactone at the time of AKI were protected against the later development of chronic fibrosis (Figure 5e).

Figure 5 | Mineralocorticoid receptor antagonism in the Large White pig protects against chronic kidney disease and chronic fibrosis induced by ischemia-reperfusion. (a) Experimental protocol. (b) Renal function was determined by quantifying the plasma levels of creatinine. (c) The percentage of Sirius Red–positive area was quantified. Representative Sirius Red–stained images for the (d) vehicle and (e) soludactone groups. n ¼ 6 per group. Statistical analyses were performed using the Mann-Whitney test. Bar ¼ 50 mm. **P < 0.01, ***P < 0.001. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org. Kidney International (2018) 93, 1344–1355

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DISCUSSION

In the present study, we show that MR antagonism in the mouse or Large White pig preclinical model effectively protects against progression to CKD after an episode of ischemic AKI. Moreover, the AKI-to-CKD transition was prevented in a selective myeloid MR knockout model. The M1 and M2 macrophage marker expression pattern in the acute phase, after the ischemic insult, suggests that pharmacological MR inhibition and/or genetic MR inactivation in myeloid cells reduces infiltration of proinflammatory macrophages after kidney IR and increase that of wound-healing M2 macrophages. Finerenone administration prior to the ischemic insult resulted in partial protection from the acute effects of renal IR; therefore, it is possible that CKD prevention is in part mediated by a reduced AKI severity after finerenone administration. However, in the MRMyKO mouse model, there is no evidence of protection against the acute injury induced by the ischemia insult compared with MRf/f control mice; despite similar AKI severity in MRMyKO and MRf/f mice, there was a complete protection from the chronic consequences of the ischemic insult in MRMyKO mice, thus suggesting a critical role for myeloid MR in mediating an efficient repair after AKI and protection against CKD and excluding the possibility of reduced AKI severity as mediator of CKD protection. Our data highlight the important role of inflammatory cells in the development of kidney fibrosis after IR and are in accord with studies showing macrophage involvement in effective repair after an ischemic insult, resulting in protection against chronic dysfunction and fibrosis. Infiltrating inflammatory cells play an essential role in the injury and repair phases after ischemic processes. In particular, macrophages are abundantly recruited to the kidney after IR and play dual roles in ischemic AKI.15 We observed a reduction in the number of Ly6Chi inflammatory cells after renal ischemia in MRf/f mice. This finding is in accordance with a previous report showing that CD11bþ Ly6Chi cells are associated with increased and nonresolved injury and inflammation in the ischemic kidney.16 In the early phase following IR, macrophages promote inflammation and amplify injury through the release of proinflammatory cytokines, causing vascular congestion.6 On the contrary, in the late phase after IR, macrophages play an important role in the repair process, acting as scavengers of cell debris and promoting regeneration.17 This dual effect can be explained by the different phenotypes that a macrophage can adopt, either pro- or antiinflammatory. Macrophages that express M1 markers are essentially proinflammatory and produce cytokines, such as IL-6, tumor necrosis factor alpha, and IL-1 beta, whereas macrophages that express M2 markers, such as mannose receptors, IL-10, IL-4, peroxisome proliferator-activated receptor gamma, Arg1, and matrix metallopeptidase 9, are essentially antiinflammatory.18–20 Many intermediate and dynamic populations have also been characterized. Macrophages display great plasticity and can switch between these phenotypes and subpopulations.21 Macrophages that infiltrate the kidney during the early phase of AKI promote IRI in mice 1350

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and subsequent development of fibrosis through maladaptive repair.22 During the repair process, macrophage polarization to an M2-like phenotype is relevant for an efficient repair process and the prevention of fibrosis and CKD development.23 Indeed, ablation of macrophages early after IR protects against ischemic injury. However, if macrophage ablation is carried out after day 2 of IR, the kidneys fail to repair properly.23 Our data highlight a central role for the myeloid MR in macrophage polarization after an ischemic insult in the kidney. Of note, finerenone has a protective effect during the acute phase of injury after IR,11 whereas MRMyKO mice did not avoid AKI. However, both finerenone and genetic MR inactivation in myeloid cells affect macrophage polarization early after AKI and consequently during the later development of kidney dysfunction and fibrosis. In vitro evidence has shown that macrophage MRs play a central role in macrophage polarization toward an M2 phenotype.9 Here, we showed that the mechanism implicated in increased M2 macrophage polarization due to MR inhibition is linked to increased signaling through the IL-4 receptor, a key signaling pathway for M2 macrophage induction. This observation is in accord with a recent report showing that the JAK3/STAT6 signaling pathway plays a crucial role in renal recovery from AKI by modulating macrophage polarization.24 Excessive myeloid MR activation in pathological situations may therefore lead to reduced IL-4 receptor activation and reduced macrophage polarization to an M2 phenotype, thus increasing the pool of proinflammatory macrophages, and leading to sustained injury and maladaptive repair in the kidney. It has also been suggested that activation of the c-Jun NH2-terminal kinase pathway in macrophages after tissue injury is dependent on MR activation.10 This pathway may also play a role in reducing M1 macrophage polarization after kidney ischemia. Myeloid MR may be activated by glucocorticoid rather than aldosterone because the selectivity enzyme 11b-hydroxysteroid dehydrogenase-2 is not expressed in macrophages.25 These findings, reported in the context of AKI-induced CKD, may extend to other pathological situations. Indeed, the myeloid MR appears to be involved in end-organ damage. Frieler et al. showed that MRMyKO mice displayed fewer M1 macrophage markers in cerebral ischemia and were protected against the injury.26 In addition, the MR in myeloid cells has also been implicated in heart remodeling processes induced by aldosterone and angiotensin II.9,10 MR knockout in myeloid cells in a mouse model of glomerulonephritis lessened disease severity, due to the reduced recruitment of macrophages and neutrophils into the kidney.27 The translation of effective therapeutic approaches in rodent models to humans has been difficult. Here, we also tested the efficacy of MR antagonism to prevent CKD development after an AKI episode in the Large White pig as a preclinical animal model. This proof of concept is relevant due to the anatomical, physiological, and immunological similarities between porcine and human kidneys.28 The chronic effects of IR in the pig have been previously Kidney International (2018) 93, 1344–1355

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reported: after 60 minutes of warm ischemia, AKI resulted in the development of fibrosis by 3 months.14 The severity of IR correlates with the development of fibrosis and the level of adaptive immunity recorded at day 7.14 As observed in rodents, the administration of the injectable MR antagonist soludactone (canrenoate) in pigs undergoing bilateral renal ischemia efficiently prevented the deleterious chronic consequences of renal IR, including early and late macrophage infiltration. These translational data strongly support the testing of MR antagonism in AKI patients in clinical trials. Recently, the ADQI XVIII work group has acknowledged that there are no therapeutic interventions in current clinical practice to avoid AKI-to-CKD progression5; MR antagonism may be a potential therapeutic approach to address this devastating disease. A recent study by Barba-Navarro et al.29 failed to show a beneficial effect of MR antagonism in reducing the incidence of AKI in patients undergoing cardiovascular surgery. However, as noted by Bar-Nur and Jaber,30 the study presents some limitations, including that the placebo- and spironolactone-treated groups were not balanced, with more diabetic patients assigned to the spironolactone group and failure to reach the planned sample size, under-powering the study. Whether MR antagonism is effective in reducing AKI in humans remains unproven and requires further multicenter studies. Whether this would reduce long-term consequences of AKI also must be studied specifically in a dedicated trial. Indeed, a multicenter randomized clinical trial (EPURE, or Eplerenone in Patients Undergoing Renal Transplant, ClinicalTrials.gov identifier: NCT02490904) was recently initiated to assess the 3-month impact of acute eplerenone administration on renal function in transplant patients receiving grafts with extended criteria, which are

more prone to ischemia injury and delayed graft function. One of the possible drawbacks of MR antagonism in AKI patients is hyperkalemia due to the well-known effects of first- and second-generation MR antagonists in the distal nephron and, in particular, in patients with compromised renal function.31 This issue might be resolved with the use of third-generation nonsteroidal antagonists, such as finerenone, that have been proposed to have a better therapeutic index and lower risk of inducing hyperkaliemia in CKD patients. Potassium-chelating agents, such as patiromer, may also be useful for limiting the risk of hyperkalemia.32 Moreover, a few days of MRA treatment in a wellcontrolled nephrology environment may limit the risk of life-threatening hyperkalemia and still be sufficient to prevent the long-term consequences of an AKI episode. In summary, we found that MR antagonism or myeloid MR deficiency promotes macrophage polarization to a wound-healing phenotype after kidney IR, preventing the development of chronic kidney fibrosis and dysfunction. MR inhibition acts through the modulation of IL-4 receptor signaling to facilitate phenotype switching (Figure 6). We also observed the protective effect of MR antagonism in a preclinical model of AKI-to-CKD transition. Altogether, these data support the use of MR antagonists in the clinical setting for the prevention and/or treatment of the chronic consequences of ischemic AKI and positions myeloid MR as a novel therapeutic target. Experimental protocols

All animal experiments were performed following the INSERM guidelines and European Community directives for the care and use of laboratory animals. The animals were housed in controlled-climate conditions with a 12-hour

Figure 6 | Mineralocorticoid receptor antagonism (MRA) increased interleukin-4 (IL-4) receptor (IL-4R) signaling in macrophages and promotes the transcription of M2 antiinflammatory genes, leading to reduced inflammation and efficient repair. Kidney International (2018) 93, 1344–1355

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light-dark cycle and were provided free access to food and water. Male C57Bl/6 mice (Janvier Laboratories, France) were used for pharmacological experiments with finerenone (BAY 94-8862). Finerenone (10 mg/kg) was administered by oral gavage (vehicle: 40% kolliphor, 10% ethanol, and 50% water), only at 48, 24, and 1 hour before the IR procedure. Next, during the 4-week follow-up, the mice did not receive any finerenone and were allowed ad libitum food and water access (Supplementary Figure S1). Body weight was not altered during the 3 days of finerenone administration. The 10 mg/kg dose was previously identified to be effective in mice with AKI.11 Mice with MR inactivation in myeloid cells (MRMyKO) were generated by crossing MRf/f floxed mice33 (kindly provided by Dr. Berger, Heidelberg, Germany) with transgenic mice expressing the Cre recombinase under the control of the LysM promoter34 (The Jackson Laboratory, Bar Harbor, ME). Mice with MR knocked out in smooth muscle cells (MRSMCKO) were generated by crossing MRf/f floxed mice with transgenic mice expressing the inducible CreERT2 recombinase under the control of the SMA promoter (kindly provided by Dr. Metzger, Strasbourg, France).35 MRf/f littermates lacking the Cre transgene were used as controls. All mice were generated in the C57Bl/6 genetic background (The Jackson Laboratory). The sequences of the primers used for genotyping are listed in Supplementary Table S1. Mouse kidney IRI model

Eight-week-old male mice were anesthetized by i.p. injection of sodium pentobarbital (60 mg/kg) and placed on a heating pad with a rectal probe to keep the body temperature constant, at approximately 37  C. Bilateral flank incisions were made to expose the kidneys, and the renal pedicle was dissected. We induced renal ischemia by placing nontraumatic vascular clamps over the pedicles for 22.5 minutes. The clamps were then released and the mice received 0.5 ml 0.9% NaCl (37  C). Both incisions were closed in 2 layers, with 5-0 sutures, and reperfusion was allowed to occur for 24 hours or 4 weeks, depending on the experiment. Shamtreated mice were subjected to the same procedure, but without renal pedicle clamping. For the unilateral IR model, the incision was made only in the left flank and the clamp was placed on the dissected left renal pedicle for 30 minutes. After the reperfusion period, a blood sample was taken and plasma creatinine and urea concentrations were determined with an automatic analyzer (Konelab 20i, Thermo Fisher Scientific, Waltham, MA). At the end of the experiment, the right kidney was removed and fixed in Bouin fixative solution for histological studies and the left kidney was rapidly frozen for molecular studies. For fluorescence-activated cell sorting analysis, both kidneys were taken and placed in phosphatebuffered saline at 4  C for further processing. Histological analysis

The fixed kidneys were then dehydrated and embedded in paraffin. Sections (4 mm) were cut and stained with Sirius Red. For each mouse, at least 6 subcortical fields were 1352

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visualized and analyzed under a Leica DM4000 microscope at a magnification of 200. The fibrosis score was analyzed blindly and was assigned a value of 0 when fibrosis was < 5%, 1 for 5% to 25%, 2 for 26% to 50%, 3 for 51% to 75%, and 4 for 76% to 100% fibrosis. RNA extraction and real-time PCR

Total RNA was extracted from the kidney or isolated macrophages with TRIZOL reagent (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. Reverse transcription was performed with 1 mg of RNA and the Superscript II Reverse Transcriptase kit (Life Technologies). Transcript levels were analyzed by real-time polymerase chain reaction (RT-PCR) (fluorescence detection of SYBR green) in an iCycler iQ apparatus (Bio-Rad, Hercules, CA), with normalization against 18S as an endogenous control. The primer sequences for the genes analyzed are listed in Supplementary Table S1. Cell sorting and staining analysis

For cell sorting experiments, both kidneys from MRf/f or MRMyKO mice were harvested 24 hours after ischemia and placed on phosphate-buffered saline at 4 C, and thereafter digestion was performed in serum-free RPMI supplemented with DNAse I (1 mg/ml) and collagenase I (10 mg/ml) at 37 C for 45 minutes. Next, the kidney was filtered through a 70-mm nylon mesh (BD Biosciences, Franklin Lakes, NJ). Cells were recovered and the mononuclear cell fraction was enriched by a Percoll gradient. Erythrocytes were lysed with ammonium-chloridepotassium lysis buffer. Cells were collected by centrifugation (1800 rpm for 6 minutes at room temperature), washed in phosphate-buffered saline, 2% fetal bovine serum, and stained for the specific sorting of macrophages, gating for CD45þ, F4/80þ, CD19–, CD3– and Ly6g– cells. Sorting was performed in a BD FACSAria III (BD Biosciences) flow cytometer. After separation, the purity of macrophages was verified to be >98% in all cases. For cell population characterization, the same protocol for mononuclear cell enrichment was carried out as described above. Macrophages, T-cells, dendritic cells and granulocytes were detected using the following antibodies: CD11b-APCCy7, F4/80-PECy5, CD3-PE, CD4-PerCP, CD8-APC, CD11c-PE-Cy7, and Gr-1-PE. Fluorescence data from at least 200,000 events were collected from the live gate using a BD LSR II (BD Biosciences) cytometer. For the CD124 staining in macrophages we used the following antibodies: F4/80-perCP/ cy5.5, CD11b-APC-Cy7, and CD124-PE/Cy7. All data were processed and analyzed using FlowJo (Ashland, OR) software. Peritoneal macrophage collection and cell culture

Four days before killing, mice were injected i.p. with 2 ml of 3% Brewer thyoglycolate (Sigma-Aldrich, St. Louis, MO). Wild-type mice also received 10 mg/kg of finerenone once a day by gavage. Peritoneal macrophages were collected by injection and recollection of 7 ml cold phosphate-buffered Kidney International (2018) 93, 1344–1355

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saline in the peritoneal cavity of mice previously killed by cervical dislocation. Macrophages were then directly stained for flow cytometry analysis, lysed for MR Western blotting, or cultured in RPMI 1640 (Gibco, Waltham, MA) containing 10% fetal bovine serum (Gibco) completed if necessary by 10 mM of finerenone. After 3 hours, nonadherent cells were removed and macrophages were stimulated by either 5 ng/ml of mouse IL-4 (Biolegend, San Diego, CA) or 100 ng/ml of lipopolysaccharide. After 3 hours (Western blot) or 24 hours (flow cytometry), cells were either lysed or stained for flow cytometry using antibodies as described earlier. Western blotting

Protein electrophoresis was performed with 4% to 15% Mini-PROTEAN TGX precast polyacrylamide gels (BioRad). The proteins were transferred on nitrocellulose membranes with a trans-Blot Turbo (Bio-Rad). Membranes were blocked 2 hours in TBS-Tween 0.05% (Thermo Fisher Scientific), milk 5%, and incubated with either rabbit antiphospho-STAT6 (1/400), rabbit anti-STAT6 (1/1000) (Abcam, Cambridge, UK), mouse anti-MR (1/250) (kindly provided by Dr. Gomez-Sanchez), rabbit anti-IL-4R (1/200) (Abcam), rabbit anti-phospho-IL-4R (1/200) (Abcam), or mouse anti-b-actin (1/5000) (Sigma-Aldrich) antibody. After incubation with the primary antibody, membranes were washed and incubated with the appropriate goat anti-rabbit or goat anti-mouse secondary antibody (1/7500) (Dako, Santa Clara, CA). Finally, the proteins were detected with an enhanced chemiluminescence kit (Amersham, Little Chalfont, UK).

Statistics

The results are expressed as the mean  SEM. In the finerenone studies, the significance of between-group differences was assessed by 1-way analysis of variance (ANOVA) with the Bonferroni correction for multiple comparisons. For the knockout mouse studies, the significance of differences was determined by 2-way ANOVA with the Bonferroni correction for multiple comparisons. A Student t-test was performed to compare 2 groups. For the pig experiments, the results were compared by Mann-Whitney test. GraphPad (La Jolla, CA) Prism 6 software was used for statistical analyses and graphical representations. We considered P values < 0.05 to be statistically significant. DISCLOSURE

PK is an employee of Bayer AG. All the other authors declared no competing interests. ACKNOWLEDGMENTS

This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale, the Centre de Recherche Industrielle et Technique, the Agence Nationale de la Recherche (ANR-16-CE14-0021-01), the Fight-HF Avenir investment program (ANR-15-RHUS-0004), the French Medical Research Foundation (DEQ20160334885), and a research grant from Bayer AG (12127a10). JB-C was partially supported by a postdoctoral fellowship from the French Foundation for Medical Research (SPF20130526725). We would like to thank Dr. Berger (Heidelberg, Germany) for sharing the MRf/f line and Dr. Metzger (Strasbourg, France) for providing the SMA-CreERT2 line. We are most grateful to Sonia Prince for excellent technical assistance and the CEF crew for their support with the mice. Flow cytometry analysis was performed at the Centre d’Imagerie Cellulaire et de Cytométrie (CICC), Centre de Recherche des Cordeliers, UMRS 1138, Paris, France.

Large White pig AKI-to-CKD model

All animal experiments were performed according to the guidelines for the care and use of laboratory animals and approved by the French Poitou-Charentes ethical committee of animal experimentation (protocol no. CE2012-29). We used 3-month-old Large White pigs weighing 40  4 kg (IBiSA, INRA Magneraud, France). The procedure was performed as previously described.14 Briefly, after anesthesia, the kidneys were accessed through a midline abdominal incision. The left and right renal vascular pedicles were atraumatically dissected. Clamping the kidney pedicles for 60 minutes induced warm ischemia. The clamps were then removed to observe kidney reperfusion. Kidney biopsy was performed at day 14 to calculate the inflammation score. Animals were monitored for 90 days after reperfusion. Two groups were studied: the vehicle group (vehicle injection without soludactone) and the soludactone group (soludactone in vehicle injection) (n ¼ 6 animals per group). Vehicle or soludactone was injected i.v. (7 mg/kg) at 48 hours, 24 hours, and 30 minutes before ischemia induction plus 2 injections at 24 and 48 hours after the IR procedure. No additional MR antagonist dosing was performed thereafter until killing (D90). Plasma creatinine was measured with a Cobas bioanalyzer (RocheDiagnostics, Meylan, France). Kidney International (2018) 93, 1344–1355

AUTHOR CONTRIBUTIONS

JB-C, SML, PK, TH, and FJ conceived of and designed the experiments. JB-C, GRE, SML, SG, SEM, and SK performed the experiments. JB-C, GRE, SML, PK, TH, and FJ analyzed the data. PK, TH, and FJ contributed reagents, materials, and analysis tools. JB-C, SML, and FJ wrote the paper. JB-C, GRE, SML, SG, SEM, SK, PK, TH, and FJ approved the final version of the manuscript. SUPPLEMENTARY MATERIAL Figure S1. Experimental protocol for the mouse experiments with bilateral renal ischemia and vehicle or finerenone administration. Figure S2. Finerenone administration partially protects against 22.5 minutes of bilateral renal ischemia. Renal function was determined by measuring the plasma levels of (A) creatinine and (B) urea 24 hours after reperfusion. n ¼ 6 per group. *P < 0.05, ***P < 0.001. Figure S3. Finerenone protects against chronic injury induced by a severe ischemic injury. Left kidney ischemia (30 minutes) was induced and the mice were monitored for 30 days. Representative Sirius Red staining is shown for (A) sham-treated, (B) untreated unilateral ischemia-reperfusion (IR), and (C) unilateral IR plus finerenone treatment. (D) The fibrosis score was blindly quantified on 8 fields per mouse from the Sirius Red–stained slides. (E) Urinary protein excretion. n ¼ 5 per group. Bar ¼ 100 mm. *P < 0.05, ***P < 0.001. Figure S4. Mineralocorticoid receptor (MR) deficiency in smooth muscle cells does not prevent the transition from acute kidney injury

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to chronic kidney disease. Renal function was determined by quantifying the plasma levels of (A) creatinine. We determined the kidney mRNA levels of (B) transforming growth factor b1 and (C) fibronectin by real-time polymerase chain reaction as markers of kidney fibrosis. (D) The fibrosis score was blindly quantified on 8 fields per mouse. Representative Sirius Red–stained images for the (E) sham MRf/f, (F) ischemia-reperfusion (IR) MRf/f, (G) sham MRSMCKO, and (H) IR MRSMCKO groups. White bars indicate sham, and black bars indicate IR. n ¼ 6 per group. Two-way analysis of variance was performed. Bar ¼ 100 mm. *P < 0.05,** P < 0.01, *** P < 0.001. Figure S5. Effect of finerenone on inflammatory cell populations 24 hours after ischemia-reperfusion (IR). (A) Percentages of macrophages: F4/80þ, CD11bþ cells. (B) CD80 expression in macrophages. (C) Percentage of macrophages expressing the inflammatory macrophage marker LyGC at low, medium, or high levels. (D) CD206 expression in macrophages. White bars represent sham, black bars IR, and gray bars the IR þ finerenone (Fine) group. n ¼ 6 per group. One-way analysis of variance was performed. *P < 0.05. Figure S6. Myeloid mineralocorticoid receptor (MR) knockout model. (A) Schematic representation of the strategy to obtain the mice deficient of MR in myeloid cells (MRMyKO mice). We crossed mice with exon 3 of the MR gene flanked by loxP sites (MRf/f mice) with mice expressing the Cre recombinase under the activity of the LysM promoter. (B) MR protein expression was analyzed in isolated macrophages from the MRf/f and the MRMyKO mice. Figure S7. Mineralocorticoid receptor (MR) deficiency in myeloid cells does not protect against the acute injury induced by ischemiareperfusion (IR). Renal function was determined by measuring the levels of (A) plasma creatinine and (B) plasma urea. Representative hematoxylin and eosin–stained images for (C) IR MRf/f and (D) IR MRMyKO groups. (E) The percentage of injured tubules was quantified. n ¼ 5 per group. Bar ¼ 100 mm. Figure S8. Mineralocorticoid receptor (MR) deficiency in myeloid cells does not affect the infiltration of the kidney by T-cells, dendritic cells, or granulocytes to the kidney after ischemia-reperfusion (IR). The percentage of infiltrating (A) CD3þ, (B) CD8þ, and (C) CD4þ cells, (D) dendritic cells, and (E) granulocytes was determined by flow cytometry. Figure S9. The percentage of macrophages expressing the (A) M1 marker CD80, (B) the M2 marker CD206, or (C) the inflammatory marker Ly6C was determined 24 hours after reperfusion in kidneys from MRf/f (white bars) and MRSMCKO mice (black bars). Figure S10. Mineralocorticoid receptor (MR) inhibition facilitates macrophage polarization to an M2 phenotype. Peritoneal macrophages were cultured and the expression levels of (A) the M2 marker CD206 were determined in nontreated macrophages, and macrophages treated with finerenone (Fine) (10 mM), lipopolysaccharide (100 ng/ml), or lipopolysaccharide þ finerenone by flow cytometry. (B) The effect of interleukin-4 (IL-4; 5 ng/ml) and IL-4 þ finerenone on CD206 expression was also assessed. n ¼ 5 per group. One-way analysis of variance or the Student t-test was performed. *P < 0.05, **P < 0.01, ***P < 0.001. Table S1. List of primers used in the study. Supplementary material is linked to the online version of the paper at www.kidney-international.org. REFERENCES 1. Hill NR, Fatoba ST, Oke JL, et al. Global prevalence of chronic kidney disease–a systematic review and meta-analysis. PLoS One. 2016;11: e0158765. 2. Chawla LS, Eggers PW, Star RA, et al. Acute kidney injury and chronic kidney disease as interconnected syndromes. N Engl J Med. 2014;371: 58–66. 3. Zuk A, Bonventre JV. Acute kidney injury. Annu Rev Med. 2016;67:293–307.

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