Kr Chandan

Rakesh Barik

Yongxin Ganguly

Cédric Li

Michèle Przybylski

Weng Kee Salmain

Leong

Barik

Rakesh Ganguly

Yongxin Li

Cedric Przybylski

Michele Salmain
email: michele.salmain@sorbonne-universite.fr.

Weng Kee Leong

Cedric Orcid

Przybylski

Embedding a Ruthenium-Based Structural Mimic of the [Fe]-Hydrogenase Cofactor into Papain

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Three distinct classes of hydrogenases are known, referred to as the [NiFe]-, [FeFe]-, and [Fe]-hydrogenases, and they all catalyze the reversible oxidation of dihydrogen into protons. [START_REF] Stephenson | Hydrogenase: A Bacterial Enzyme Activating Molecular Hydrogen[END_REF][START_REF] Vignais | Classification and Phylogeny of Hydrogenases[END_REF][START_REF] Frey | Hydrogenases: Hydrogen-Activating Enzymes[END_REF] The redox-inactive [Fe]-hydrogenase, or H 2 -forming methylene-tetrahydromethanopterin dehydrogenase (Hmd), takes part in the conversion of carbon dioxide to methane in many methanogenic archaea. [START_REF] Thauer | Reactions with Molecular Hydrogen in Microorganisms: Evidence for a Purely Organic Hydrogenation Catalyst[END_REF] In particular, it catalyzes an intermediary step, which is the reversible and stereoselective reduction of a unique biological carbocationic substrate, N 5 ,N [START_REF] Tard | Structural and Functional Analogues of the Active Sites of the [Fe]-[NiFe]-, and [FeFe]-Hydrogenases[END_REF] methenyltetrahydromethanopterin (methenyl-H 4 MPT + ), with dihydrogen to produce neutral methylenetetrahydromethanopterin (methylene-H 4 MPT) and a proton (Scheme 1). [START_REF] Thauer | Reactions with Molecular Hydrogen in Microorganisms: Evidence for a Purely Organic Hydrogenation Catalyst[END_REF][START_REF] Corr | Amidine Dications as Superelectrofiles[END_REF][START_REF] Kalz | Functional Model for the [Fe]-Hydrogenase Inspired by the Frustrated Lewis Pair Concept[END_REF] Crystallographic elucidation of the [Fe]-hydrogenase active site and its apoenzyme have been achieved, and the former comprises an iron(II) atom with pseudo-octahedral geometry; [START_REF] Lyon | Carbon Monoxide as an Intrinsic Ligand to Iron in the Active Site of the Iron-Sulfur-Clusterfree Hydrogenase H 2 -Forming Methylenetetrahydromethanopterin Dehydrogenase as Revealed by Infrared Spectroscopy[END_REF][START_REF] Shima | The Crystal Structure of [Fe]-Hydrogenase Reveals the Geometry of the Active Site[END_REF][START_REF] Shima | Mossbauer Studies of the Iron-Sulfur Cluster-free Hydrogenase: The Electronic State of the Mononuclear Fe Active Site[END_REF][START_REF] Tard | Structural and Functional Analogues of the Active Sites of the [Fe]-[NiFe]-, and [FeFe]-Hydrogenases[END_REF] it is bound to one cysteine residue, two mutually cis CO ligands, and a 2-acetyl-6-hydroxypyridinol ligand. The identity of the sixth ligand, trans to the acyl group, has not been confirmed, although it is most probably a water molecule (Figure 1a). [START_REF] Hiromoto | The Crystal Structure of C176A Mutated [Fe]-Hydrogenase Suggests an Acyl-Iron Ligation in the Active Site Iron Complex[END_REF][START_REF] Shima | A Third Type of Hydrogenase Catalyzing H 2 Activation[END_REF][START_REF] Lyon | UV-A/blue-light Inactivation of the 'Metal-free' Hydrogenase (Hmd) from Methanogenic Archaea[END_REF] A number of Fe-based structural models of the active site have been reported over the years, including some which are also functional models. 14 In particular, Hu et al. recently showed that reconstitution of the entire [Fe]hydrogenase using the apoenzyme produced in E. coli and an Fe-based model complex could activate dihydrogen. [START_REF] Shima | Reconstitution of [Fe]-Hydrogenase using Model Complexes[END_REF] That study points to a role for the protein in [Fe]-hydrogenase for the dihydrogen activation and that the pyridinyl cofactor most likely activates dihydrogen via coordination at the site trans to the acyl group. Such investigations are, nevertheless, hampered by the air-sensitivity of organoiron compounds.

We recently reported on the first ruthenium and osmium structural mimics for the [Fe]-hydrogenase active site. [START_REF] Barik | Structural Mimics of the [Fe]-Hydrogenase: A Complete Set for Group VIII Metals[END_REF] Ruthenium complexes are generally more air-stable than their iron counterparts, and many are known to activate dihydrogen. [START_REF] Rohmann | Hydrogenation of CO 2 to Formic Acid with a Highly Active Ruthenium Acriphos Complex in DMSO and DMSO/Water[END_REF][START_REF] Chen | Enhanced hydrogen generation from formic acid by a biomimetic ruthenium complex with a covalently bonded phosphine ligand[END_REF] We therefore hypothesized that a reproduction of the [Fe]-hydrogenase using a more stable ruthenium model complex and an appropriate protein scaffold may serve as a useful model for studying aspects of its chemistry. Such metalloenzymes are also of interest as possibly possessing unique catalytic properties. [START_REF] Chevalley | Artificial metalloenzymes derived from bovine β-lactoglobulin for the asymmetric transfer hydrogenation of an aryl ketone-synthesis, characterization and catalytic activity[END_REF] We chose papain as protein scaffold as it is readily and cheaply available, and its tertiary structure and chemistry are well-understood. In addition, it has a single free sulfhydryl (Cys25) moiety located in a pocket forming the enzyme's active site, which may allow binding to the ruthenium center in an analogous way to that present in native [Fe]-hydrogenase. Our investigations into this are presented here.

RESULTS AND DISCUSSION

Synthesis. The ruthenium-based structural mimics 3 can be synthesized in two steps from the corresponding ionic carbamoyl ruthenacycles 1 by following a recently reported procedure (Scheme 2). [START_REF] Barik | Structural Mimics of the [Fe]-Hydrogenase: A Complete Set for Group VIII Metals[END_REF] Unlike the iron analogue, thiolation of 2 requires the addition of a base. 14d The mononuclear thiolato complexes 3 could be isolated only when the pyridine ligand is substituted at the 6-position, together with a bulky thiolate (3bB); dimerization occurs otherwise (4aA, 4aB, and 4bA). All the complexes 2-4 have been characterized spectroscopically and crystallographically; ORTEP plots of a representative of each are given in Figure 2.

The complexes 4 are sparingly soluble in dimethyl sulfoxide but not in other common organic solvents. All three are symmetrical Ru II -Ru II homodimers bridged by two thiolato ligands, but while 4aA and 4aB are centrosymmetric, the structure of 4bA has approximate C 2 symmetry with the two tolyl groups (as are the two pyridyl groups) on the same side of the slightly puckered Ru 2 S 2 ring. Isomerism. Steric effects of the 6-substituent on the pyridine ring are apparent in the bond parameters of 2a and 2b. These include an increasing angle between the pyridine ring and the acetonitrile and a longer Ru-N1 bond in moving from R = H (2a) to Me (2b); ∠N1-Ru-N2 = 92.73(8)°and 101.6(1)°, and d(Ru-N1) = 2.110(2) and 2.163(3) Å, respectively. There is also greater distortion of the fivemembered metallacycle, which does not lie directly above the Ru-N2 bond, in 2b; the N(2)-Ru-N(1)-R torsion angles are -2.43°and -11.40°in 2a and 2b, respectively. One chemical consequence of this steric interaction is that 2b is present as one isomer in solution, while 2a exists as a mixture of two isomers; the IR spectrum of 2b shows only two bands for the CO stretches, while that for 2a shows a split in the lower frequency band (vide infra). Similarly, the 1 H NMR spectrum of 2a shows two sets of resonances, in a 55:45 ratio, but only one for 2b. The observation of two isomers in 2a is also in agreement with an earlier report on an iron analogue. 14d A series of VT 1 H NMR spectra of 2a shows broadening and coalescence at higher temperatures, indicating isomerization at the NMR time scale (Figure 3).

The exchange rate and activation energy barrier estimated from the coalescence of the doublet at ∼8.35 ppm are 12 s -1 and 75 kJ mol -1 , respectively, at the coalescence temperature of 70 °C. In contrast, the VT 1 H NMR spectra of 2b show no significant broadening of the resonances, except for the NH resonance (Figure S9), consistent with the presence of essentially one isomer.

A computational study carried out on all seven possible stereoisomers of 2a and 2b (Tables S6 andS7) shows that the energy gap between the two lowest energy isomers (Figure 3 and Table S8) is about 1 and 8 kJ mol -1 , which corresponds to an isomeric ratio of 60:40 and 96:4 for 2a and 2b, respectively. These values are in very good agreement with those from NMR integration. The computed values for the CO vibrations also point to a larger difference (∼10 cm -1 ) in the lower frequency band between the isomers (Table S6), in agreement with the observed splitting of this band. The major isomer for both 2a and 2b from the computational study (isomer 1) corresponds to that observed in the solid-state structure; thus, the minor isomer in 2a is presumably isomer 2. The relative stability of the isomers can be rationalized on the basis of the trans influence: NHCO > CO > Br -> MeCN > Py. [START_REF] Ishii | Universal Spectrochemical series of Six-coordinate Octahedral Metal Complexes for Modifying the Ligand Field Splitting[END_REF] Thus, the strongly π-accepting CO and the strongly σ-donating carbamoyl prefer to be trans to weak π-acceptors or-donors such as Br -, MeCN, or Py. The larger isomer energy gap in 2b may be attributed to steric interaction between the bulky bromide ligand and the methyl substituent of the pyridine ring.

Dissolution in Polar Solvents. Complex 3bB is a ruthenium-based structural mimic of the [Fe]-hydrogenase active site, with acetonitrile in place of the unknown sixth ligand. 14d The infrared spectrum (in acetonitrile) shows two intense absorption bands at 2047 and 1978 cm -1 (Δν = 69 cm -1 ) for the cis-dicarbonyl ligands which are blue-shifted from that for the previously reported iron analogues (∼20 cm -1 ) 14d and the [Fe]-hydrogenase (∼15 cm -1 ), [START_REF] Hiromoto | The Crystal Structure of C176A Mutated [Fe]-Hydrogenase Suggests an Acyl-Iron Ligation in the Active Site Iron Complex[END_REF] consistent with ruthenium being less electron-rich. [START_REF] Bruce | Iron versus Ruthenium: Dramatic Changes in Electronic Structure Result from Replacement of One Fe by Ru in [{Cp*(dppe)Fe}-CC-CC-{Fe(dppe)Cp*}] n+ (n = 0, 1, 2)[END_REF][START_REF] Eskeńazi | Tuning of reactivity in epoxidation of alkenes by iron and ruthenium complexes associated to non-porphyrinic ligands[END_REF] Although complexes 2 and 3 are not very soluble in most of the common organic solvents, their solubility in acetone and tetrahydrofuran increased upon the addition of small amounts of water. It is also noted that the CO vibrations of 2b in acetone, for example, are blue-shifted with increasing amounts of water added (Figure S4), and a similar shift is observed for 3bB in acetonitrile (Figure S5). These shifts are indicative of changes in composition, and in the case of 2b in acetonitrile, may be attributed to the formation of a new species upon the addition of water, which has tentatively been identified as a cationic species in which both the Br -and MeCN ligands have been replaced by water molecules; the experimental ν CO values at 2086 and 2027 cm -1 are in good agreement with the values of 2089 and 2028 cm -1 calculated for this species (Figure 4).

Computational studies on both 2b and 3bB also suggest that replacement of the acetonitrile ligand with water will lead to a decrease in the frequency difference between the two CO vibrational modes, as observed in the experimental spectra (Table S5). The weak Ru-OH 2 bond is corroborated by the mass spectrum; the molecular ion peak for 2b can be observed at ambient temperature but not at higher temperatures. The resonance at 2.10 ppm for the metal-bound CH 3 CN ligand in the 1 H NMR spectrum (in acetone-d 6 ) shifts to 2.05 ppm, corresponding to free acetonitrile, upon addition of water (Figure S12). [START_REF] Fulmer | NMR Chemical Shifts of Trace Impurities: Common Laboratory Solvents, Organics, and Gases in Deuterated Solvents Relevant to the Organometallic Chemist[END_REF] A density functional theory (DFT) calculation gives a ΔG°of -0.1 kJ mol -1 for the reaction: 2b + H 2 O → 2b•H 2 O + CH 3 CN (in acetone). All of these indicate that the sixth (acetonitrile) ligand in 2 and 3 is very weakly bound, reminiscent of the situation in the [Fe]-hydrogenase. [START_REF] Shima | The Crystal Structure of [Fe]-Hydrogenase Reveals the Geometry of the Active Site[END_REF][START_REF] Tard | Structural and Functional Analogues of the Active Sites of the [Fe]-[NiFe]-, and [FeFe]-Hydrogenases[END_REF] Bioconjugation to Papain. That complexes 2 can be thiolated suggests that they may also be incorporated into a protein via reaction with a free sulfhydryl moiety. Papain contains a free sulfhydryl moiety borne by Cys25 which, together with His159, are the two essential amino acids responsible for its proteolytic activity. [START_REF] Chevalley | Artificial metalloenzymes derived from bovine β-lactoglobulin for the asymmetric transfer hydrogenation of an aryl ketone-synthesis, characterization and catalytic activity[END_REF] Conjugation to the sulfhydryl group of Cys25 can thus be readily monitored via a fall in its enzymatic activity. As histidine residues such as His159 may also be involved in coordination owing to their imidazole group, a preliminary assessment of the reactivity of 2b with imidazole was first carried out.

We found that 2b reacts with imidazole to give 2b•Im, and this can in turn react with N-acetyl-L-cysteine-methyl ester (C) to afford the dimer 4bC; the latter can also be obtained directly from the reaction of 2b and C (Scheme 3). The complex 3bB reacts similarly with imidazole to form 3bB•Im. Both the imidazole complexes 2b•Im and 3bB•Im, however, slowly convert back to 2b and 3bB, respectively, on standing in acetonitrile (Figures S7 andS8). These results suggest that conjugation of 2b to a cysteine side chain should be favored over histidine.

Conjugation of 2b to papain was assessed through measurement of its effect on the hydrolysis rate of N-αcarbobenzoxy-glycine p-nitrophenyl ester (ZGlyONP). [START_REF] Kirsch | The Kinetics of the Papain-Catalyzed Hydrolysis of Esters of Carbobenzoxyglycine. Evidence for an Acyl-Enzyme Intermediate[END_REF] The activity dropped to almost zero, suggesting that Cys25 was affected (Figure S16). After a gel filtration step to remove excess complex, bioconjugate 5 was analyzed by several spectroscopic techniques. The UV-visible spectrum of 5 (Figure S17) shows two peaks at 279 and ∼318 nm ascribable to papain [START_REF] Haquette | Cysteine-Specific, Covalent Anchoring of Transition Organomettalic Complexes to the Protein Papain from Carica papaya[END_REF] and the ruthenium center, respectively. The IR spectrum of 5 shows that the characteristic carbonyl bands are red-shifted (2064 and 2000 cm -1 ) from that of 2b (Figure 5). This red shift is similar to the shift in comparing 2b with 3bB, and the smaller frequency gap between the two stretches (64 cm -1 ) is also consistent with thiolation. Indeed, the frequency gap is the same as that computed for 3bB•H 2 O (corresponding to replacement of the acetonitrile ligand in 3bB with water), suggesting that 5 contains thiolato and water ligands (Table S5).

While the electrospray ionization-high resolution mass spectrum (ESI-HRMS) of papain reveals a charge states distribution from 22+ (m/z 1065.69) to 12+ (m/z 1952.82), centered around 18+ (m/z 1302.28) and 17+ (m/z 1378.82) (Figure 6A), that for 5 shows charge states ranging from 22+ (m/z 1078.81) to 12+ (m/z 1980.15) (Figure 6B). After deconvolution and deisotoping, the mass spectrum of papain showed a major peak at a monoisotopic mass of 23 407.52205, in good agreement with the theoretical value (23 407.52457 g• mol -1 ; 0.2 ppm) (Figure S15A). Another species at a mass of 23 437.52205, corresponding to an amino acid variant with Thr in place of an Ala residue (mass shift of +30.01056) was also observed. In comparison, that for 5 showed two prominent additional species at 23 699.47624 and 23 729.48682, corresponding to an exact mass shift of 291.95417 (Figure S15B). This value matches very well with the conjugation of 2b to papain by substitution of the bromide ligand by a thiolate (and loss of MeCN). Interestingly, no other species at higher mass was observed, indicating monometalation of the protein.

CONCLUSION

In this study, we showed that organoruthenium complexes of the general formula [Ru(2-NHC(O)C 5 H 3 NR)(CO) 2 (X)(L)] (where R = H, Me, X = Br or SR′, and L = H 2 O or NCCH 3 ) exhibited chemistry that is relevant to understanding the chemistry of the [Fe]-hydrogenase: the lability of the so-called sixth ligand and the importance of steric bulk in preventing dimerization via thiolate bridges through this coordination site. We believe that embedding of the metal cofactor in a protein cavity is how nature achieves this and showed that this can be readily achieved with the complexes reported here. The extension of this idea to the construction of metalloenzymes is ongoing.

EXPERIMENTAL SECTION

General Procedures. All experiments were performed under an argon atmosphere using standard Schlenk techniques except for the bioconjugation experiments. Chemicals were obtained from Sigma-Aldrich and used as received. Papain was obtained from Calbiochem. [START_REF] Kirsch | The Kinetics of the Papain-Catalyzed Hydrolysis of Esters of Carbobenzoxyglycine. Evidence for an Acyl-Enzyme Intermediate[END_REF][START_REF] Riener | Quick measurement of protein sulfhydryls with Ellman's reagent and with 4,4'-dithiodipyridine[END_REF] Compounds 1 were synthesized as previously described. [START_REF] Barik | Structural Mimics of the [Fe]-Hydrogenase: A Complete Set for Group VIII Metals[END_REF] Solvents used for reactions were distilled over the appropriate drying agents under argon before use. ESI-HRMS for compounds were recorded on a Waters UPLC-Q-Tof MS mass spectrometer, while that for papain and bioconjugate 5 were performed on a LTQ-Orbitrap XL. 1 H and 13 C{ 1 H} NMR spectra were obtained on a JEOL 400 MHz spectrometer at room temperature unless stated otherwise. Chemical shifts for 1 H and 13 C{ 1 H} were referenced with respect to the residual resonances of the respective deuterated solvents. Infrared spectra were recorded on a Bruker Alpha FT-IR spectrometer in an IR cell with NaCl windows and a path length of 0.1 mm at a resolution of 2 cm -1 ; for the bioconjugation experiments, the spectra were recorded after deposition of the solutions onto nitrocellulose membrane and airdried. Absorbance spectra were recorded using a Varian Cary 50 spectrometer. Elemental analyses were performed on a PerkinElmer 2400 Series II CHN/O Elemental Analyzer in house.

Preparation (5 mL). The resulting solution was then stirred for 5 min at room temperature. Slow evaporation of the solvent at room temperature afforded 3bb as yellow crystals. Yield: 13 mg, 0.03 mmol, 55%. IR (MeCN, cm -1 ): ν CO 2047 (vs), 1978 (s). [START_REF] Stephenson | Hydrogenase: A Bacterial Enzyme Activating Molecular Hydrogen[END_REF] ] 2 (4aA). Triethylamine (5 μL, 0.05 mmol) and 4-methylbenzenethiol (6 mg, 0.05 mmol) were added into a solution of 2a (20 mg, 0.05 mmol) in acetonitrile (5 mL) at room temperature. A white precipitate was formed immediately, and the solution was left to stir for 2 h. The white precipitate was separated by filtration and washed with acetonitrile (20 mL) to afford analytically pure 4aA. Yield: 25 mg, 0.03 mmol, 60%. IR (MeCN, cm -1 ): ν CO 2053 (m), 2037 (s), 1991 (m), 1970 (s). [START_REF] Stephenson | Hydrogenase: A Bacterial Enzyme Activating Molecular Hydrogen[END_REF] H NMR ((CD 3 ) 2 SO): δ 10.70 (br, 1H, NH), 8.35-8.34 (d, 1H, J = 5.04 Hz, PyH), 8.00-7.95 (m, 1H, PyH), 7.47-7.45 (d, 2H, J = 8.72 Hz, PhH), 7.16-7.14 (d, 1H, J = 8.24 Hz, PyH), 7.05-7.03 (d, 2H, J = 5.04 Hz, PhH), 6.93-6.89 (m, 1H, J = 5.04 Hz, PyH), 2.25 (s, 3H, Me) ppm. [START_REF] Lyon | UV-A/blue-light Inactivation of the 'Metal-free' Hydrogenase (Hmd) from Methanogenic Archaea[END_REF] (4bC). Triethylamine (3 μL, 0.02 mmol) and Nacetyl-L-cysteine-methyl ester (4.3 mg, 0.02 mmol) were added into a solution of 2b (10 mg, 0.02 mmol) in methanol (5 mL). The resulting mixture was stirred for 30 min, and then the volume was reduced under vacuum to approximately 1 mL. After standing for 3 d, complex 4bC (9.0 mg, 0.01 mmol, 48%) was obtained as a yellow crystalline solid. IR (MeOH, cm -1 ): 2052 (s), 2038 (s), 1991 (s), 1978(s). [START_REF] Stephenson | Hydrogenase: A Bacterial Enzyme Activating Molecular Hydrogen[END_REF] Purification of Papain by Affinity Chromatography. [START_REF] Funk | Affinity Chromatographic Purification of Papain a Reinvestigation[END_REF] The peptide GGYR (Bachem) was attached to CNBr-activated Sepharose 4B (Sigma) via its terminal NH 2 group. The resin (20 mL) was packed into a column (XK 16, GE Healthcare) and conditioned with EDTA (20 mM) and mercaptoethanol (10 mM) pH 4.3, at 1 mL/ min. Papain (200 mg solid, Calbiochem) was dissolved in EDTA (25 mM), mercaptoethanol (30 mM) pH 4.3 (20 mL), and the solution was incubated for 30 min. The solution (18 mL) was injected into the column, and the resin was washed successively with conditioning buffer (2 volumes) then with washing buffer (5 mM EDTA, pH 6.0, 2 volumes). Papain was finally eluted with water (2 volumes). Protein concentration was calculated from the absorbance at 280 nm taking an extinction coefficient of 56 000 M -1 •cm -1 .

Enzymatic Assay. Affinity-purified papain (1.6 mL of a 3.2 μM solution in water; final concentration = 2.56 μM) was mixed with 50 mM phosphate buffer pH 7.8 and 2b (20 μL of a 10 mM solution; final concentration = 100 μM). Twenty microliters of the mixture was used to measure the hydrolytic activity of ZGlyONP (50 μL) in 430 μL of phosphate buffer pH 6.5 at various times. The remaining mixture was incubated at RT for 18 h and submitted to gel filtration on a dextran desalting column (10 mL, Thermo scientific). Species were eluted with water. One milliliter fractions were collected, and their absorbance at 280 nm was measured with a spectrophotometer. The fractions containing the protein were pooled, and the solution (3 mL; 1.25 μM) was concentrated by ultrafiltration on Amicon Ultra 4 (10 kDa cutoff, Millipore) to a final volume of 0.22 mL. A control experiment using papain was also carried out to measure the hydrolytic activity of ZGlyONP.

High Resolution Mass Spectrometry of Papain Samples. ESI-HRMS experiments were carried out using a LTQ-Orbitrap XL from Thermo Scientific (San Jose, CA, United States) and operated in positive ionization mode, with a spray voltage at 4.5 kV. Native papain or bioconjugate 5 in a water/acetonitrile/formic acid (49:50:1, v/v/v) mixture was continuously infused using a 250 μL syringe at 5 μL/min flow. Applied voltages were 40 and 100 V for the ion transfer capillary and the tube lens, respectively. The ion transfer capillary was held at 275 °C. Sheath and auxiliary gas were set at a flow rate of 45 and 15 arbitrary units (a.u.), respectively. Detection was achieved in the Orbitrap with a resolution set to 100 000 (at m/z 400) and a m/z range between 200 and 3000 in profile mode. Spectrum was analyzed using the acquisition software XCalibur 2.1 (Thermo Fisher Scientific, Courtaboeuf, France) without smoothing and background subtraction. The automatic gain control (AGC) allowed accumulation of up to 2 × 10 5 ions for FTMS scans. Maximum injection time was set to 500 ms, and 1 μscan was acquired.

Crystallographic Studies. Diffraction-quality crystals were grown from acetonitrile (2a, 2b, 3bB, 4aA, 4aB, and 4bA) mounted on quartz fibers. X-ray diffraction data were collected at 103(2) K on a Bruker X8 APEX system, using Mo Kα radiation, with the SMART suite of programs. 28 Data processing and correction were made for Lorentz and polarization effects with SAINT, 29 and adsorption effects with SADABS. 30 Twinning was observed for the crystals of 4bA; the twin laws and cell parameters were determined using the program CELL_NOW, [START_REF] Sheldrick | version 2008/1): Program for Obtaining Unit Cell Constants from Single Crystal Data[END_REF] and the reflection data processed and corrections applied using the program TWINABS. [START_REF] Sheldrick | SHELXTL, version 5.1[END_REF] Structural solution and refinement were carried out with the SHELXTL suite of programs. 33 The structures were solved by direct methods to locate the heavy atoms, followed by successive difference maps for the light, nonhydrogen atoms. Disorder of a carbonyl and the bromide ligand was found for 2b, which were modeled with two sites each, with occupancies summed to unity. All nonhydrogen atoms were refined with anisotropic displacement parameters in the final model. All organic hydrogen atoms were placed in calculated positions and refined with a riding model. The crystallographic data are summarized in Tables S1 andS2.

Computational Studies. The computational studies were carried out using DFT, utilizing the hybrid functional of Truhlar and Zhao (M06). [START_REF] Zhao | The M06 suite of density functionals for main group thermochemistry, thermochemical kinetics, noncovalent interactions, excited states, and transition elements: two new functionals and systematic testing of four M06-class functionals and 12 other functionals[END_REF] The basis set used was def2TZVP for ruthenium [START_REF] Ehlers | A Set of f-polarization Functions for Pseudo-potential basis sets of the Transition Metals Sc-Cu, Y-Ag and La-Au[END_REF] and 6-311+G(2d,p) for the light atoms. For calculations involving solvated species, the polarized continuum model was employed. Spin-restricted calculations were used for structural optimization, harmonic frequency calculations and to evaluate zero-point energy corrections. Optimized geometries were characterized as equilibrium structures with all real frequencies. All calculations were performed using the Gaussian 09 suite of programs. 36 The coordinates for parent complexes were taken from the crystal structure, and each of the other isomers was modified from this.

■ ASSOCIATED CONTENT

* S Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b01835.

NMR spectra showing presence of isomers, crystallographic tables, and optimized geometry of computed structures, experimental procedure and high-resolution mass spectra of papain and bioconjugate (PDF)

Scheme 1 .Figure 1 .

 11 Scheme 1. Reversible Reduction of Methenyl-H 4 MPT +

  Their 1 H NMR spectra in d 6 -DMSO show only one set of peaks; this is similar to the dimeric iron compound [{Fe(COMe)(CO) 2 (μ 2 -SPy-Me)} 2 ]. 14b As in the case of the iron analogues, attempts to cleave the Ru-S bridges with phosphines (PMe 3 and PPh 3 ) failed. 14d

Scheme 2 .

 2 Scheme 2. Synthesis of 2 and 3

Figure 2 .

 2 Figure 2. ORTEP plots showing the molecular structures of (a) 2b, (b) 3bB, and (c) 4aB (left) and 4bA (right). Thermal ellipsoids are drawn at the 50% probability level, and all hydrogen atoms were omitted for clarity.

Figure 3 .

 3 Figure 3. Top: Computed free energies (in kJ/mol) of the two lowest energy isomers of 2a and 2b relative to the most stable isomer (isomer 1). Bottom: VT 1 H NMR spectra of 2a in d 3 -acetonitrile.

Figure 4 .

 4 Figure 4. (Top) IR spectra of 2b in acetonitrile (black solid line) and various compositions of acetonitrile/water (v/v): 10:1 (red dashed line), 5:2 (purple dashed line), and 1:2 (green dashed-dot line). (Bottom) Proposed exchange process. Free energies are given in kJ/ mol for the forward reactions.

Scheme 3 .Figure 5 .

 35 Scheme 3. Reaction of 2b and 3bB with Imidazole and N-Acetyl-L-cysteine-methyl Ester

Figure 6 .

 6 Figure 6. ESI-HRMS mass spectra of (A) papain and (B) bioconjugate 5, both at 2.15 μM in water/acetonitrile/formic acid (49:50:1, v/v/v).

  of [RuBr(2-NHC(O)C 5 H 4 N)(CO) 2 (NCMe)] (2a). A sample of 1a (200 mg, 0.38 mmol) was dissolved in acetonitrile (50 mL). Slow evaporation of the solvent at room temperature afforded white crystal of 2a. Yield: 100 mg, 0.26 mmol, 68%. mp 343-344 °C. IR (acetonitrile, cm -1 ): ν CO 2061 (vs), 1999 (s), 1989 (s), 1622 (m). 1 H NMR (CD 3 CN): δ 9.10-9.09 (d, 1H, J = 5.5 Hz, PyH), 8.71-8.69 (br, 1H, NH), 8.54-8.552 (br, 1H, NH), 8.37-8.36 (d, 1H, J = 5.5 Hz, PyH), 7.89-7.80 (m, 2H, PyH), 7.06-7.00 (m, 4H, PyH), 1.96 (s, 6H, Me) ppm. 13 C{ 1 H} NMR (CD 3 CN): 198.8 (CO), 198.2 (CO), 195.8 (CO), 195.6 (CO), 195.4 (NHCO), 191.2 (NHCO), 158.0, 157.5, 148.6, 147.5, 141.2, 140.6, 116.8, 116.5, 109.3, 109.5 ppm. HRMS. Calcd for [M] + : m/z: 399.8871. Found: m/ z 399.8876. Anal. Calcd for C 8 H 5 N 2 O 3 BrRu (M-CH 3 CN): C 26.83, H 1.41, N 7.82. Found C 26.90, H 1.86, N 7.21. Complex 2b was similarly prepared from 1b (213 mg, 0.38 mmol). Yield: 113 mg, 0.27 mmol, 72%. mp 281-282 °C. IR (acetonitrile, cm -1 ): ν CO 2061 (vs), 1988 (s), 1619 (m). 1 H NMR (CD 3 CN): δ 8.52-8.50 (br, 1H, NH), 7.67-7.63 (t, 1H, J = 7.8 Hz, PyH), 6.

	88-
	6.86 (t, 1H, J = 7.8 Hz, PyH), 6.82-6.80 (d, 1H, J = 8.2 Hz, PyH),
	2.65 (s, 3H, Me), 1.98 (s, 3H, Me) ppm. 13 C{ 1 H} NMR
	((CD 3 ) 2 CO): 195.1 (CO), 191.8 (CO), 159.5 (NHCO), 158.4, 140.2, 117.2, 116.6, 106.9, 25.5 ppm. HRMS. Calcd for [M + H] + : m/
	z: 413.9027. Found: m/z 413.9021. Anal. Calcd for
	C 11 H 10 N 3 O 3 BrRu: C 31.98, H 2.44, N 10.17. Found C 31.70, H
	2.40, N 10.48.
	Preparation of [Ru(2-NHC(O)C 5 H 3 NMe)(SC 6 H 3 Me 2 )(CO) 2 (NCMe)] (3bB). Triethylamine (7 μL, 0.05 mmol) and 2,6-dimethylbenzene-
	thiol (5 μL, 0.05 mmol) were added into a solution of 2b (21 mg,
	0.05 mmol) in acetonitrile

  H NMR (CD 3 CN): δ 8.45 (br, 1H, NH), 7.64-7.60 (t, 1H, J = 7.4 Hz, PyH), 6.92-6.90 (d, 2H, J = 7.8 Hz, PyH), 6.83-6.75 (m, 3H, PyH), 2.44 (s, 3H, Me), 2.24 (s, 6H, Me), 1.96 (s, 3H, Me) ppm. 13 C{ 1 H} NMR (CD 3 CN): 159.5, 143.4, 139.6, 129.9, 123.3, 126.3, 123.5, 116.4, 106.4, 22.2, 20.9 ppm. HRMS. Calcd for [M + H] + : m/z: 472.0520. Found: m/z 472.0539. Anal. Calcd for C 19 H 19 N 3 O 3 SRu: C 48.50, H 4.07, N 8.93, S 6.81. Found C 48.47, H 4.46, N 9.21, S 6.73.

	Preparation of [Ru(2-NHC(O)C 5 H 4 N)(SC 6 H 4 Me)(CO) 2

  C{[START_REF] Stephenson | Hydrogenase: A Bacterial Enzyme Activating Molecular Hydrogen[END_REF] H} NMR ((CD 3 ) 2 SO): 201.9, 201.4, 193.2, 158.5, 148.1, 141.3, 135.2, 134.6, 132.3, 129.5, 118.6, 110.5, 21.0 ppm. HRMS. Calcd for [M + H] + : m/z: 804.9312. Found: m/z 804.9329. Complexes 4aB and 4bA were prepared in similar yields following an analogous procedure. 4aB. IR (KBr, cm -1 ): ν CO 2041 (s), 1979 (s). HRMS. Calcd for [M + H] + : m/z: 832.9625. Found: m/z 832.9624. MS. Calcd for [M + Na] + : m/z: 853.7. Found: m/z 853.8. Anal. Calcd for C 32 H 28 N 4 O 6 S 2 Ru 2 : C 46.26, H 3.40, N 6.74, S 7.72. C{ 1 H} NMR (d 6 -DMSO): 202.3, 199.7, 194.1, 160.7, 159.4, 141.1, 136.8, 134.4, 131.4, 129.5, 118.6, 108.1, 28.3, 20.9 ppm. HRMS. Calcd for [M + H] + : m/z: 832.9625. Found: m/z 832.9626. Preparation of [RuBr(2-NHC(O)C 5 H 3 NMe)(CO) 2 (C 3 H 4 N 2 )] (2b•Im). A sample of imidazole (1.6 mg, 0.02 mmol) was added into a solution of 2b (10 mg, 0.02 mmol) in methanol (5 mL). The resulting mixture was then stirred for 30 min at ambient temperature. Slow evaporation of the solvent afforded 2b•Im as a crystalline solid. Yield: 3 mg, 0.01 mmol, 34%). IR (MeOH, cm -1 ): 2062 (s), 1998 (s). 1 H NMR (d 6acetone): δ 11.95 (br, 1H, NH), 9.66 (br, 1H, NH), 8.24 (s, 1H, CH), 7.71-7.67 (t, 1H, J = 7.8 Hz, PyH), 7.25-7.20 (d, 2H, J = 10.5 Hz, CH), 6.93-6.87 (m, 2H, PyH), 3.06 (s, 3H, Me) ppm. 13 C{ 1 H} NMR (d 6 -acetone): 202.2, 197.4, 196.1, 162.0, 158.9, 140.4, 139.4, 129.0, 117.7, 117.0, 106.6, 25.0 ppm. HRMS. Calcd for [M + H] + : m/ z: 440.0828. Found: m/z 440.0812. Anal. Found C 34.37, H 2.80, N 13.59; Calculated for C 12 H 11 N 4 O 3 BrRu.C 2 H 3 N: C 34.94, H 2.93, N 14.55. Preparation of [Ru(2-NHC(O)C 5 H 3 NMe)(SCH 2 CH(NHCOMe)-CO 2 Me)(CO) 2 ] 2

	Found C
	46.33, H 3.33, N 6.98, S 7.72. 4bA. IR (KBr, cm -1 ): ν CO 2051 (m), 2038 (s), 1988 (m), 1975 (s). 1 H NMR (d 6 -DMSO): δ 10.50 (br, 1H, NH), 7.80-7.77 (t, 1H, J =
	8.2 Hz, PyH), 7.52-7.51 (d, 2H, J = 8.2 Hz, PhH), 7.03-7.01 (d, 2H,
	J = 7.8 Hz, PhH), 6.90-6.91 (d, 1H, J = 4.9 Hz, PyH), 6.82-6.80 (d,
	1H, J = 8.2 Hz, PyH), 2.49 (s, 3H, Me), 2.24 (s, 3H, Me) ppm.

[START_REF] Lyon | UV-A/blue-light Inactivation of the 'Metal-free' Hydrogenase (Hmd) from Methanogenic Archaea[END_REF]

This work was supported by the Nanyang Technological University and a research grant from the Agency for Science, Technology and Research (A*STAR) (Research Grant M4070244). C.K.B is grateful to the university for the Research Scholarships.

. These data can be obtained free of charge via www.ccdc.cam.ac.


Mathieu Buonafine

Ernesto Martinez-Martinez

Frédéric Jaisser

More than a simple biomarker : Role of NGAL in cardiovascular and renal diseases

Neutrophil Gelatinase-Associated Lipocalin (NGAL) is a small circulating protein that is induced in a wide variety of pathological situations, making it an interesting biomarker of various disease states. It is considered as one of the best markers of acute kidney injury, since it is rapidly released after tubular damage. However, a growing body of evidence points toward an implication of NGAL beyond its role as a biomarker. Indeed, numerous studies have demonstrated a role for NGAL in the mediation of both cardiovascular and renal diseases. In the present review, we summarize what is known about NGAL involvement in cardiovascular and renal diseases and discuss the different mechanisms underlying its pathological implications.

INTRODUCTION

Neutrophil Gelatinase-Associated Lipocalin (NGAL) (also known as lipocalin-2, 24p3, siderocalin or uterocalin) is a small secreted glycoprotein of 25kDa. NGAL has been initially identified in mature neutrophil granules (1) but has since been described in many other cell types. NGAL is expressed in renal [START_REF] Liu | High expression of neutrophil gelatinaseassociated lipocalin (NGAL) in the kidney proximal tubules of diabetic rats[END_REF], endothelial [START_REF] Hamzic | Immune-Induced Expression of Lipocalin-2 in Brain Endothelial Cells: Relationship with Interleukin-6, Cyclooxygenase-2 and the Febrile Response[END_REF], liver [START_REF] Borkham-Kamphorst | Induction of lipocalin-2 expression in acute and chronic experimental liver injury moderated by pro-inflammatory cytokines interleukin-1β through nuclear factor-κB activation: LCN2 induction in acute and chronic experimental liver injury[END_REF], and smooth muscle cells (SMCs) [START_REF] Eilenberg | Neutrophil Gelatinase-Associated Lipocalin (NGAL) is Associated with Symptomatic Carotid Atherosclerosis and Drives Pro-inflammatory State In Vitro[END_REF] as well as in cardiomyocytes [START_REF] Latouche | Neutrophil gelatinase-associated lipocalin is a novel mineralocorticoid target in the cardiovascular system[END_REF], neurons [START_REF] Naudé | Neutrophil gelatinase-associated lipocalin: A novel inflammatory marker associated with latelife depression[END_REF], and in different populations of immune cells such as macrophages [START_REF] Eilenberg | Neutrophil Gelatinase-Associated Lipocalin (NGAL) is Associated with Symptomatic Carotid Atherosclerosis and Drives Pro-inflammatory State In Vitro[END_REF][START_REF] Flo | Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron[END_REF] and dendritic cells [START_REF] Floderer | Dendritic Cell-Secreted Lipocalin2 Induces CD8+ T-Cell Apoptosis, Contributes to T-Cell Priming and Leads to a TH1 Phenotype[END_REF].

Several roles have been ascribed to NGAL, including iron trafficking [START_REF] Bao | Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex[END_REF], chemotactic [START_REF] Schroll | Lipocalin-2 ameliorates granulocyte functionality: Innate immunity[END_REF][START_REF] Shao | Increased Lipocalin-2 Contributes to the Pathogenesis of Psoriasis by Modulating Neutrophil Chemotaxis and Cytokine Secretion[END_REF] and bacteriostatic [START_REF] Goetz | The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition[END_REF] effects as well as activities as differentiation, proliferation and growth factor [START_REF] Schmidt-Ott | Dual Action of Neutrophil Gelatinase-Associated Lipocalin[END_REF]. NGAL effects are mediated through two different receptors, 24p3R and megalin, depending on the tissue. NGAL is used as a biomarker of renal injury because it is rapidly released in response to tubular damage [START_REF] Grande | Neutrophil gelatinase-associated lipocalin: a novel biomarker for the early diagnosis of acute kidney injury in the emergency department[END_REF][START_REF] Parikh | New biomarkers of acute kidney injury[END_REF]. However, numerous studies indicate that NGAL is more than a simple biomarker, and that it plays an important role in the pathophysiology of renal as well as cardiovascular diseases. Moreover, NGAL has been involved in various deleterious process such as inflammation and fibrosis.

The lipocalin family

NGAL is part of the lipocalin protein family. This family includes many small proteins, the majority of which act as transporters, mainly for lipophilic substances. However, other roles for these proteins have been discovered, such as regulation of cell division, differentiation, cell-cell adhesion and cell survival [START_REF] Chakraborty | The multifaceted roles of neutrophil gelatinase associated lipocalin (NGAL) in inflammation and cancer[END_REF]. Unlike most protein families whose members are identified on the basis of similarities in their amino acid sequences, members of the lipocalin family share a common three-dimensional structure necessary for their transport function: the lipocalin fold. This structure is composed of eight anti-parallel sheets forming a cup-shaped cavity that serve as ligand binding site (Figure 1) [START_REF] Chakraborty | The multifaceted roles of neutrophil gelatinase associated lipocalin (NGAL) in inflammation and cancer[END_REF][START_REF] Flower | Beyond the superfamily: the lipocalin receptors[END_REF]. Differences in amino acid sequences between members of the lipocalin family can be very large (up to 80%), allowing a wide variety of ligands within the family [START_REF] Chakraborty | The multifaceted roles of neutrophil gelatinase associated lipocalin (NGAL) in inflammation and cancer[END_REF][START_REF] Flower | Beyond the superfamily: the lipocalin receptors[END_REF]. However, three particular domains are very well preserved and distinguish two branches of lipocalins: the "Kernel", that have three preserved domains (such as NGAL) and the "Outliers" that only have two. (Figure 1) [START_REF] Chakraborty | The multifaceted roles of neutrophil gelatinase associated lipocalin (NGAL) in inflammation and cancer[END_REF][START_REF] Flower | Beyond the superfamily: the lipocalin receptors[END_REF].

Structure of NGAL

Like other members of the lipocalin family, NGAL has a three-dimensional barrel structure. Its binding site, however, has two particularities: it is polar and wide enough to bind to certain proteins [START_REF] Goetz | The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition[END_REF]. In addition, the presence of a cysteine residue in position 87 allows NGAL to form a disulfide bridge with a specific ligand, the Matrix Metalloproteinase 9 (MMP-9 also known as gelatinase B) [START_REF] Yan | The High Molecular Weight Urinary Matrix Metalloproteinase (MMP) Activity Is a Complex of Gelatinase B/MMP-9 and Neutrophil Gelatinase-associated Lipocalin (NGAL): MODULATION OF MMP-9 ACTIVITY BY NGAL[END_REF], a protein whose enzymatic action allows the degradation of certain extracellular matrix (ECM) components and is therefore involved in tissue remodeling mechanisms [START_REF] Yabluchanskiy | Matrix Metalloproteinase-9: Many Shades of Function in Cardiovascular Disease[END_REF]. The binding of MMP-9 to NGAL does not modify its activity but stabilizes the protein and decreases its degradation [START_REF] Yan | The High Molecular Weight Urinary Matrix Metalloproteinase (MMP) Activity Is a Complex of Gelatinase B/MMP-9 and Neutrophil Gelatinase-associated Lipocalin (NGAL): MODULATION OF MMP-9 ACTIVITY BY NGAL[END_REF]. The cysteine 87 residue present in humans is absent in rodents, suggesting that NGAL/MMP-9 interaction is not possible in rodents. However, some studies suggest the opposite. Indeed, NGAL was detected in a complex with MMP-9 in the supernatant of a culture of rat SMCs [START_REF] Bu | Induction of Neutrophil Gelatinase-Associated Lipocalin in Vascular Injury via Activation of Nuclear Factor-?B[END_REF] and co-localized with MMP-9 in mouse atheroma plaques [START_REF] Hemdahl | Expression of neutrophil gelatinase-associated lipocalin in atherosclerosis and myocardial infarction[END_REF]. In addition, in a mouse breast cancer model, mice genetically invalidated for NGAL exhibited reduced plasma MMP-9 activity compared to Wild Type (WT) mice, illustrating a functional relationship between NGAL and MMP-9 [START_REF] Leng | Inhibition of Lipocalin 2 Impairs Breast Tumorigenesis and Metastasis[END_REF].

Apart from MMP-9, NGAL is also able to interact with other ligands, in particular with certain bacterial siderophores (Figure 2).

Roles of NGAL

Role of NGAL in iron binding and modulation

As mentioned above, NGAL is involved in antibacterial defense through iron sequestration. Iron is an essential element for the development of bacteria, but is present in very small quantities in the body. To capture iron from the host, bacteria release proteins with high affinity for iron, i.e. siderophores. In vitro studies have shown that NGAL is able of binding to bacterial siderophores, thus playing a bacteriostatic role in reducing the availability of iron for bacteria [START_REF] Goetz | The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition[END_REF]. In addition, a study using a mouse model with genetic invalidation of NGAL (NGAL KO) showed that these mice had increased susceptibility to bacterial infections [START_REF] Berger | Lipocalin 2-deficient mice exhibit increased sensitivity to Escherichia coli infection but not to ischemiareperfusion injury[END_REF].

NGAL is also able to bind endogenous siderophores present in humans, i.e. the catechols. This suggests a role of NGAL in iron homeostasis, even in the absence of bacterial infection [START_REF] Bao | Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex[END_REF]. When NGAL binds to an iron-coupled siderophore (holo-NGAL form), it will transport iron within the cell and thus increase the cytosolic iron concentration (Figure 3A).

Conversely, when it is free (apo-NGAL form), it will allow the capture of intracellular iron and its transport to extracellular space, thus inducing a decrease in intracellular iron concentration (Figure 3B) [START_REF] Devireddy | A Cell-Surface Receptor for Lipocalin 24p3 Selectively Mediates Apoptosis and Iron Uptake[END_REF]. This role of NGAL in iron homeostasis could have a significant impact on pathology since iron levels are important in various deleterious mechanisms such as oxidative stress [START_REF] Wang | Iron-Induced Cardiac Damage: Role of Apoptosis and Deferasirox Intervention[END_REF], inflammation [START_REF] Kim | The Role of Iron Metabolism in Lung Inflammation and Injury[END_REF], apoptosis [START_REF] Wang | Iron-Induced Cardiac Damage: Role of Apoptosis and Deferasirox Intervention[END_REF][START_REF] Whittaker | Histopathological Evaluation of Liver, Pancreas, Spleen, and Heart from Iron-Overloaded Sprague-Dawley Rats* 1, 2[END_REF] and fibrosis [START_REF] Whittaker | Histopathological Evaluation of Liver, Pancreas, Spleen, and Heart from Iron-Overloaded Sprague-Dawley Rats* 1, 2[END_REF].

Chemotactic Role of NGAL

NGAL has pro-inflammatory and chemotactic roles. The migration of neutrophils in culture is induced by treatment with recombinant NGAL [START_REF] Schroll | Lipocalin-2 ameliorates granulocyte functionality: Innate immunity[END_REF][START_REF] Shao | Increased Lipocalin-2 Contributes to the Pathogenesis of Psoriasis by Modulating Neutrophil Chemotaxis and Cytokine Secretion[END_REF]. In addition, neutrophils from NGAL KO mice have reduced chemotactic properties and adhesion capabilities [START_REF] Schroll | Lipocalin-2 ameliorates granulocyte functionality: Innate immunity[END_REF]. In a mouse model of psoriasis (inflammatory skin disease), neutrophil infiltration into the dermis was reduced in mice treated with an anti-NGAL antibody and increased by in vivo treatment with recombinant NGAL [START_REF] Shao | Increased Lipocalin-2 Contributes to the Pathogenesis of Psoriasis by Modulating Neutrophil Chemotaxis and Cytokine Secretion[END_REF]. The recruitment of immune cells in the heart of mice subjected to an ischemia/reperfusion (I/R) episode was blunted in NGAL KO mice [START_REF] Aigner | Lipocalin-2 Regulates the Inflammatory Response During Ischemia and Reperfusion of the Transplanted Heart[END_REF]. When hearts from NGAL KO mice were transplanted into WT recipients, a significant decrease in granulocyte infiltration was observed compared to a WT to WT heart transplant [START_REF] Aigner | Lipocalin-2 Regulates the Inflammatory Response During Ischemia and Reperfusion of the Transplanted Heart[END_REF].

Numerous experiments carried out in cellular and murine cancer models have also demonstrated a role of NGAL in cell migration and invasion [START_REF] Ding | Over-expression of lipocalin 2 promotes cell migration and invasion through activating ERK signaling to increase SLUG expression in prostate cancer: LCN2/ERK/SLUG Axis Promotes PCa Aggression[END_REF][START_REF] Du | Lipocalin 2 promotes the migration and invasion of esophageal squamous cell carcinoma cells through a novel positive feedback loop[END_REF][START_REF] Leung | Lipocalin2 Promotes Invasion, Tumorigenicity and Gemcitabine Resistance in Pancreatic Ductal Adenocarcinoma[END_REF].

Role of NGAL in differentiation, proliferation and as growth factor

NGAL has also been shown to promote differentiation and proliferation, acting as a growth factor [START_REF] Schmidt-Ott | Dual Action of Neutrophil Gelatinase-Associated Lipocalin[END_REF]. Indeed, NGAL stimulates the proliferation and epithelial differentiation of rat embryonic kidney cells and is able to induce the tubular organization of mouse epithelial cells in culture [START_REF] Gwira | Expression of Neutrophil Gelatinase-associated Lipocalin Regulates Epithelial Morphogenesis in Vitro[END_REF]. NGAL also induces the proliferation of human vascular SMCs [START_REF] Wang | Activation of the phosphatidylinositol 3kinase/Akt pathway is involved in lipocalin-2-promoted human pulmonary artery smooth muscle cell proliferation[END_REF] and cardiac fibroblasts (Buonafine et al. [unpublished]). The role of NGAL in cell proliferation has also been demonstrated in gastric and thyroid cancer models [START_REF] Iannetti | The neutrophil gelatinase-associated lipocalin (NGAL), a NF-κB-regulated gene, is a survival factor for thyroid neoplastic cells[END_REF][START_REF] Koh | HGF mediated upregulation of lipocalin 2 regulates MMP9 through nuclear factor-κB activation[END_REF]. NGAL participates to the epithelial-mesenchymal transition (EMT) in vivo in a pulmonary adenocarcinoma model in mice [START_REF] Mongre | Epigenetic induction of epithelial to mesenchymal transition by LCN2 mediates metastasis and tumorigenesis, which is abrogated by NF-κB inhibitor BRM270 in a xenograft model of lung adenocarcinoma[END_REF] and in vitro in prostate [START_REF] Ding | Over-expression of lipocalin 2 promotes cell migration and invasion through activating ERK signaling to increase SLUG expression in prostate cancer: LCN2/ERK/SLUG Axis Promotes PCa Aggression[END_REF] and breast cancer cells [START_REF] Yang | Lipocalin 2 promotes breast cancer progression[END_REF]. In these models, NGAL promoted the motility, invasiveness and metastatic capacities of cancer cells.

NGAL receptors 24p3 Receptor

The 24p3 receptor (24p3R) is one of the two receptors currently described for NGAL.

24p3R is an endocytic receptor with a strong affinity for NGAL which allows it to penetrate inside the cells. It participates to the control of iron homeostasis by allowing NGAL to enter the cells, thus modulating the amount of intracellular iron. The expression of this receptor has been identified in different tissues and notably in the heart [START_REF] Devireddy | A Cell-Surface Receptor for Lipocalin 24p3 Selectively Mediates Apoptosis and Iron Uptake[END_REF]. Under inflammatory conditions, 24p3R is expressed in the whole cardiac tissue and, in particular, on the surface of cardiomyocytes [START_REF] Ding | Lipocalin-2/Neutrophil Gelatinase-B Associated Lipocalin Is Strongly Induced in Hearts of Rats With Autoimmune Myocarditis and in Human Myocarditis[END_REF]. In addition, 24p3R is expressed in the renal distal nephron [START_REF] Langelueddecke | Lipocalin-2 (24p3/Neutrophil Gelatinase-associated Lipocalin (NGAL)) Receptor Is Expressed in Distal Nephron and Mediates Protein Endocytosis[END_REF] and is involved in albumin endocytosis and activation of pro-inflammatory and pro-fibrotic signaling pathways of NF-B and TGF- [START_REF] Dizin | Albuminuria induces a proinflammatory and profibrotic response in cortical collecting ducts via the 24p3 receptor[END_REF]. 24p3R expression was found to be increased on the surface of neutrophils of patients with psoriasis [START_REF] Shao | Increased Lipocalin-2 Contributes to the Pathogenesis of Psoriasis by Modulating Neutrophil Chemotaxis and Cytokine Secretion[END_REF]. Finally, the use of an anti-24p3R siRNA has also highlighted the important role of this receptor in the activation of neutrophils by NGAL in culture [START_REF] Shao | Increased Lipocalin-2 Contributes to the Pathogenesis of Psoriasis by Modulating Neutrophil Chemotaxis and Cytokine Secretion[END_REF].

Megalin

The other known receptor for NGAL is megalin (or low-density lipoprotein receptorrelated protein 2, LRP2). Megalin is a multi-ligand endocytic receptor which is expressed in various epithelia and in particular in epithelia with high absorptive capacity such as the epithelium of the renal tubule, ileum, or choroid plexus in the brain [START_REF] Moestrup | Megalin-and Cubilin-Mediated Endocytosis of Protein-Bound Vitamins, Lipids, and Hormones in Polarized Epithelia[END_REF]. Megalin has also been detected in cardiomyocytes cultured in vitro [START_REF] Van Dijk | Intravenous clusterin administration reduces myocardial infarct size in rats: CLUSTERIN ADMINISTRATION REDUCES INFARCT SIZE[END_REF] as well as in different types of immune cells such as T-cells, B-cells, granulocytes and monocytes/macrophages [START_REF] Miharada | Lipocalin 2mediated growth suppression is evident in human erythroid and monocyte/macrophage lineage cells[END_REF]. Megalin belongs to the family of low-density lipoprotein receptors [START_REF] Saito | Complete cloning and sequencing of rat gp330/" megalin," a distinctive member of the low density lipoprotein receptor gene family[END_REF] and has been shown to bind to various lipocalins [START_REF] Flower | Beyond the superfamily: the lipocalin receptors[END_REF][START_REF] Leheste | Megalin knockout mice as an animal model of low molecular weight proteinuria[END_REF]. However, its affinity for NGAL is higher than for other lipocalins [START_REF] Hvidberg | The endocytic receptor megalin binds the iron transporting neutrophil-gelatinase-associated lipocalin with high affinity and mediates its cellular uptake[END_REF]. The pathophysiological role of the NGAL-megalin complex is still not well described.

Involvement of NGAL in renal pathologies

NGAL has been described as an acute renal lesion biomarker because it is rapidly released in response to tubular damage [START_REF] Grande | Neutrophil gelatinase-associated lipocalin: a novel biomarker for the early diagnosis of acute kidney injury in the emergency department[END_REF][START_REF] Parikh | New biomarkers of acute kidney injury[END_REF]. NGAL is a secreted protein and can be quantified in plasma or serum. In healthy humans, plasma concentration of NGAL is approximately 70 ng/mL [START_REF] Axelsson | Studies of the release and turnover of a human neutrophil lipocalin[END_REF] and about 100 ng/mL in mice [START_REF] Flo | Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron[END_REF][START_REF] Mori | Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury[END_REF]. NGAL levels analysis is also possible in urine [START_REF] Mishra | Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery[END_REF]. These elements, combined with its good stability and resistance to proteases, make it a biomarker of choice for clinical use [START_REF] Grande | Neutrophil gelatinase-associated lipocalin: a novel biomarker for the early diagnosis of acute kidney injury in the emergency department[END_REF][START_REF] Parikh | New biomarkers of acute kidney injury[END_REF]. Creatinine plasma levels are commonly used to evaluate renal function. However, an ever-growing number of studies describe NGAL as a better marker of acute kidney injury (AKI).

NGAL in AKI

In animals, ischemia/reperfusion (I/R) induces a massive increase in renal NGAL levels within 3 hours after ischemia, while the increase in serum creatinine is still mild [START_REF] Mishra | Identification of Neutrophil Gelatinase-Associated Lipocalin as a Novel Early Urinary Biomarker for Ischemic Renal Injury[END_REF].

In addition, serum creatinine concentration increases after severe bilateral ischemia but remains unchanged after mild bilateral or unilateral ischemia [START_REF] Mishra | Identification of Neutrophil Gelatinase-Associated Lipocalin as a Novel Early Urinary Biomarker for Ischemic Renal Injury[END_REF]. In AKI, NGAL levels are increased up to 300 times in blood (0.1 to 30 µg/mL) and 1000 times in urine (0.04 to 40 mg/mL) [START_REF] Mori | Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury[END_REF][START_REF] Mishra | Identification of Neutrophil Gelatinase-Associated Lipocalin as a Novel Early Urinary Biomarker for Ischemic Renal Injury[END_REF].

In patients with AKI, the relative level of serum NGAL was correlated with the severity of renal damage and high levels of serum NGAL were associated with an increased risk of mortality [START_REF] Kümpers | Serum neutrophil gelatinase-associated lipocalin at inception of renal replacement therapy predicts survival in critically ill patients with acute kidney injury[END_REF]. In addition, urinary and serum NGAL concentrations have been described as a sensitive, specific and highly predictive markers of AKI after cardiac surgery [START_REF] Mishra | Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery[END_REF]. Several studies have also suggested NGAL as a marker of renal damage in broader pathological contexts than renal ischemia such as glomerulonephritis and IgA nephropathy [START_REF] Bolignano | Pathological and Prognostic Value of Urinary Neutrophil Gelatinase-Associated Lipocalin in Macroproteinuric Patients with Worsening Renal Function[END_REF][START_REF] Ding | Urinary neutrophil gelatinaseassociated lipocalin (NGAL) is an early biomarker for renal tubulointerstitial injury in IgA nephropathy[END_REF]. Another suggested source of urinary NGAL, especially in non-renal diseases, could be circulating NGAL from extra renal origin, being released in the systemic circulation at sites of inflammation, notably by immune cells [START_REF] Kanda | An AKI biomarker lipocalin 2 in the blood derives from the kidney in renal injury but from neutrophils in normal and infected conditions[END_REF], and then filtered by the renal glomerulus. The majority of NGAL would then be reabsorbed by the proximal tubule, which expresses megalin, and the remaining NGAL would be excreted in the urine. This last point is illustrated, for example, by a study showing that mice deficient in megalin show a leakage of NGAL in the urine [START_REF] Hvidberg | The endocytic receptor megalin binds the iron transporting neutrophil-gelatinase-associated lipocalin with high affinity and mediates its cellular uptake[END_REF].

Beyond its role as a biomarker, NGAL is actively involved in the mechanisms underlying kidney damages. NGAL has been shown to play a protective role in AKI after an episode of I/R [START_REF] Mori | Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury[END_REF][START_REF] Mishra | Amelioration of Ischemic Acute Renal Injury by Neutrophil Gelatinase-Associated Lipocalin[END_REF]. During an I/R episode, the release of large amounts of iron at the time of ischemia promotes oxidative stress and induction of tissue damage. In addition, the subsequent reperfusion further increases the amount of iron, exacerbating the damage caused by oxidative stress [START_REF] Choi | Inhibition of Oxidative Stress in Renal Ischemia-Reperfusion Injury[END_REF][START_REF] Sponsel | Effect of iron on renal tubular epithelial cells[END_REF]. In animal models, the use of iron-binding protein [START_REF] Bernardi | Nacetylcysteine and deferrioxamine protects against acute renal failure induced by ischemia/reperfusion in rats[END_REF][START_REF] De Vries | Reduction of circulating redox-active iron by apotransferrin protects against renal ischemia-reperfusion injury[END_REF], including the injection of recombinant NGAL [START_REF] Mori | Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury[END_REF][START_REF] Mishra | Amelioration of Ischemic Acute Renal Injury by Neutrophil Gelatinase-Associated Lipocalin[END_REF], has been shown to limit early damage caused by I/R or rejection of the graft during renal transplantation [START_REF] Ashraf | Exogenous Lipocalin 2 Ameliorates Acute Rejection in a Mouse Model of Renal Transplantation[END_REF]. A study also showed that intravenous injection of macrophages over-expressing the anti-inflammatory cytokine IL-10 was able to protect against renal ischemia in rats and improve cell regeneration and tissue repair through the induction of NGAL [START_REF] Jung | Infusion of IL-10expressing cells protects against renal ischemia through induction of lipocalin-2[END_REF]. When rats were treated with an anti-NGAL antibody, the protective role of the adoptive transfer of macrophages over-expressing IL10 was lost [START_REF] Jung | Infusion of IL-10expressing cells protects against renal ischemia through induction of lipocalin-2[END_REF], underlying a crucial role of NGAL. More recently, a study showed that the infusion of macrophages overexpressing NGAL could improve renal fibrosis in a unilateral ureteral obstruction model in mice [START_REF] Guiteras | Macrophage Overexpressing NGAL Ameliorated Kidney Fibrosis in the UUO Mice Model[END_REF].

NGAL in CKD

In contrast to its protective effect in AKI, NGAL is described to be harmful in the long term. NGAL is considered as a pro-inflammatory factor that promotes the progression toward CKD [START_REF] Pawar | Neutrophil gelatinase-associated lipocalin is instrumental in the pathogenesis of antibody-mediated nephritis in mice[END_REF][START_REF] Viau | Lipocalin 2 is essential for chronic kidney disease progression in mice and humans[END_REF]. A high level of urinary NGAL was associated with a higher risk of CKD [START_REF] Viau | Lipocalin 2 is essential for chronic kidney disease progression in mice and humans[END_REF][START_REF] Wu | Urinary neutrophil gelatinase-associated lipocalin: A potential biomarker for predicting rapid progression of drug-induced chronic tubulointerstitial nephritis[END_REF]. In CKD patients [START_REF] Viau | Lipocalin 2 is essential for chronic kidney disease progression in mice and humans[END_REF][START_REF] Nickolas | NGAL (Lcn2) monomer is associated with tubulointerstitial damage in chronic kidney disease[END_REF] or in mouse models of CKD [START_REF] Pawar | Neutrophil gelatinase-associated lipocalin is instrumental in the pathogenesis of antibody-mediated nephritis in mice[END_REF][START_REF] Viau | Lipocalin 2 is essential for chronic kidney disease progression in mice and humans[END_REF], NGAL levels were increased and correlated with the severity of renal injury.

The role of NGAL in CKD has been investigated in animal models, particularly using NGAL KO mice. In an anti-glomerular basement membrane antibody-induced glomerulonephritis, NGAL gene invalidation protected mice from proteinuria and tubular lesions while the addition of recombinant NGAL exacerbated kidney disease and decreased survival [START_REF] Pawar | Neutrophil gelatinase-associated lipocalin is instrumental in the pathogenesis of antibody-mediated nephritis in mice[END_REF]. Similarly, in a subtotal nephrectomy-induced CKD model, NGAL KO mice exhibited less apoptosis, less renal lesions (glomerulosclerosis, tubular atrophy, interstitial fibrosis, immune infiltration), less proteinuria and better renal function than WT mice [START_REF] Viau | Lipocalin 2 is essential for chronic kidney disease progression in mice and humans[END_REF].

Interestingly, this study also showed that NGAL was an effector of the proliferative effects of EGFR (Epidermal Growth Factor Receptor) [START_REF] Viau | Lipocalin 2 is essential for chronic kidney disease progression in mice and humans[END_REF] which is known to play an important role in the progression of CKD [START_REF] Zeng | The role of the EGF family of ligands and receptors in renal development, physiology and pathophysiology[END_REF].

In conclusion, the role of NGAL in kidney disease seems to depend on the associated pathological mechanisms: after an acute kidney injury, NGAL plays a protective role, notably via its iron-modulating effects, whereas in a more chronic context, its pro-inflammatory and proliferative effects make it a harmful actor.

Involvement of NGAL in cardiovascular diseases

Besides its role as a biomarker of kidney lesion and its implication in the pathophysiology of renal diseases, NGAL has also been shown to be involved in the development of cardiovascular (CV) diseases.

NGAL in myocardial infarction and heart failure

Several studies have documented elevated circulating NGAL levels in patients with CV disease [START_REF] Damman | Urinary neutrophil gelatinase associated lipocalin (NGAL), a marker of tubular damage, is increased in patients with chronic heart failure[END_REF][START_REF] Shrestha | Association Between Systemic Neutrophil Gelatinase-Associated Lipocalin and Anemia, Relative Hypochromia, and Inflammation in Chronic Systolic Heart Failure: NGAL and anemia in chronic heart failure[END_REF]. In patients with acute myocardial infarction (MI) or chronic heart failure (HF), serum NGAL levels are higher than in healthy subjects [START_REF] Damman | Urinary neutrophil gelatinase associated lipocalin (NGAL), a marker of tubular damage, is increased in patients with chronic heart failure[END_REF][START_REF] Yndestad | Increased systemic and myocardial expression of neutrophil gelatinase-associated lipocalin in clinical and experimental heart failure[END_REF]. Plasma NGAL levels are higher in MI patients than in patients with stable coronary heart disease [START_REF] Sahinarslan | Plasma neutrophil gelatinase-associated lipocalin levels in acute myocardial infarction and stable coronary artery disease[END_REF]. Some studies suggest that NGAL may have prognostic value in HF patients because high levels of plasma or urinary NGAL are associated with more renal complications [START_REF] Damman | Urinary neutrophil gelatinase associated lipocalin (NGAL), a marker of tubular damage, is increased in patients with chronic heart failure[END_REF][START_REF] Yndestad | Increased systemic and myocardial expression of neutrophil gelatinase-associated lipocalin in clinical and experimental heart failure[END_REF][START_REF] Mortara | Neutrophil gelatinase-associated lipocalin predicts worsening of renal function in acute heart failure: methodological and clinical issues[END_REF] or mortality [START_REF] Bolignano | Increased plasma neutrophil gelatinase-associated lipocalin levels predict mortality in elderly patients with chronic heart failure[END_REF][START_REF] Van Deursen | Prognostic value of plasma neutrophil gelatinase-associated lipocalin for mortality in patients with heart failure[END_REF][START_REF] Villacorta | Prognostic value of plasma neutrophil gelatinase-associated lipocalin in patients with heart failure[END_REF]. In a 10-year follow-up study of healthy subjects, a higher basal level of NGAL was associated with an increase in the proportion of adverse cardiac events and overall mortality from all causes [START_REF] Lindberg | Plasma Neutrophil Gelatinase-Associated Lipocalinin in the General Population Association With Inflammation and Prognosis[END_REF]. These high levels of NGAL may be explained in part by the renal failure observed in a large number of HF patients [START_REF] Berardinis | Comparison between admission natriuretic peptides, NGAL and sST2 testing for the prediction of worsening renal function in patients with acutely decompensated heart failure[END_REF]. However, several studies have showed that NGAL was a predictor of CV incident even in the absence of renal dysfunction [START_REF] Daniels | Plasma Neutrophil Gelatinase-Associated Lipocalin Is Independently Associated With Cardiovascular Disease and Mortality in Community-Dwelling Older Adults[END_REF][START_REF] Katagiri | Serum neutrophil gelatinase-associated lipocalin concentration reflects severity of coronary artery disease in patients without heart failure and chronic kidney disease[END_REF][START_REF] Wu | Elevated circulating lipocalin-2 levels independently predict incident cardiovascular events in men in a population-based cohort[END_REF].

Circulating NGAL levels have also been described as predictors of CV complications in patients with CKD [START_REF] Hasegawa | Plasma Neutrophil Gelatinase-Associated Lipocalin as a Predictor of Cardiovascular Events in Patients with Chronic Kidney Disease[END_REF][START_REF] Solak | Serum neutrophil gelatinase-associated lipocalin is associated with cardiovascular events in patients with chronic kidney disease[END_REF].

In animals, NGAL production is increased in the heart and aorta after MI [START_REF] Hemdahl | Expression of neutrophil gelatinase-associated lipocalin in atherosclerosis and myocardial infarction[END_REF][START_REF] Yndestad | Increased systemic and myocardial expression of neutrophil gelatinase-associated lipocalin in clinical and experimental heart failure[END_REF].

NGAL production is also increased in isolated rat cardiomyocytes in response to stimulation by different pro-inflammatory molecules such as endothelin 1, IL-1 and TNF- [START_REF] Yndestad | Increased systemic and myocardial expression of neutrophil gelatinase-associated lipocalin in clinical and experimental heart failure[END_REF]. In a recent study from our laboratory, we demonstrated an important role of NGAL in the pathophysiological mechanisms of MI. NGAL KO mice showed decreased cardiac fibrosis and inflammation as well as preserved cardiac function 3 months after MI [START_REF] Martínez-Martínez | Aldosterone Target NGAL (Neutrophil Gelatinase-Associated Lipocalin) Is Involved in Cardiac Remodeling After Myocardial Infarction Through NFκB Pathway[END_REF][START_REF] Bauersachs | Mineralocorticoid Receptor-Dependent Adverse Remodeling After Myocardial Infarction Mediated by uNGALant Activation of NFκB[END_REF]. A recent study from Sung et al. using a similar MI model showed that NGAL KO mice exhibited an increase in autophagy associated with decreased apoptosis and preserved cardiac function after infarction [START_REF] Sung | Lipocalin-2 (NGAL) Attenuates Autophagy to Exacerbate Cardiac Apoptosis Induced by Myocardial Ischemia[END_REF].

NGAL in atherosclerosis

In arteries of atherosclerotic patients, NGAL expression was observed in endothelial cells, SMCs and macrophages and was detected at a higher rate in patients with symptomatic carotid stenosis compared to asymptomatic patients [START_REF] Eilenberg | Neutrophil Gelatinase-Associated Lipocalin (NGAL) is Associated with Symptomatic Carotid Atherosclerosis and Drives Pro-inflammatory State In Vitro[END_REF]. The level of circulating NGAL was also associated with plaque vulnerability [START_REF] Eilenberg | NGAL and MMP-9/NGAL as biomarkers of plaque vulnerability and targets of statins in patients with carotid atherosclerosis[END_REF]. In a mouse atherosclerosis model, NGAL expression was co-localized with macrophages and MMP-9 in the atherosclerotic plaques, suggesting a possible role of NGAL in MMP-9-mediated remodeling [START_REF] Hemdahl | Expression of neutrophil gelatinase-associated lipocalin in atherosclerosis and myocardial infarction[END_REF].

NGAL in cardiovascular ischemia

The role of NGAL has also been shown in the cardiac lesions induced by I/R [START_REF] Cheng | Lipocalin-2 Promotes M1 Macrophages Polarization in a Mouse Cardiac Ischaemia-Reperfusion Injury Model[END_REF][START_REF] Yang | Improved functional recovery to I/R injury in hearts from lipocalin-2 deficiency mice: restoration of mitochondrial function and phospholipids remodeling[END_REF].

After an episode of I/R, in a model of isolated-perfused heart, NGAL KO heart exhibited a reduced infarct size and better cardiac contractile function than the WT heart [START_REF] Yang | Improved functional recovery to I/R injury in hearts from lipocalin-2 deficiency mice: restoration of mitochondrial function and phospholipids remodeling[END_REF]. In addition, increasing NGAL circulating levels of NGAL KO mice using an NGAL encoding adenovirus treatment resulted in impaired cardiac functional recovery and decreased mitochondrial function of the isolated-perfused hearts [START_REF] Yang | Improved functional recovery to I/R injury in hearts from lipocalin-2 deficiency mice: restoration of mitochondrial function and phospholipids remodeling[END_REF]. In a mouse model of I/R after heart transplantation, the use of anti-NGAL antibodies reduced the infiltration of macrophages and neutrophils into the ischemic zone, suppressed the M1 polarization of macrophages, and blunted the I/R-induced cardiac lesions [START_REF] Cheng | Lipocalin-2 Promotes M1 Macrophages Polarization in a Mouse Cardiac Ischaemia-Reperfusion Injury Model[END_REF].

In a rat model of cerebrovascular ischemia, a large increase of NGAL and MMP-9 was found in the intima of the common carotid artery [START_REF] Bu | Induction of Neutrophil Gelatinase-Associated Lipocalin in Vascular Injury via Activation of Nuclear Factor-?B[END_REF]. In this model, blocking the NF-B pathway in vivo led to an almost total suppression of NGAL and MMP-9 expression, suggesting a central role of NF-B signaling in NGAL and MMP-9 transcriptional regulation in this context [START_REF] Bu | Induction of Neutrophil Gelatinase-Associated Lipocalin in Vascular Injury via Activation of Nuclear Factor-?B[END_REF]. In vitro, expression of NGAL in cultured SMCs was also induced in an NF-B dependent manner by treatment with IL-1 [START_REF] Bu | Induction of Neutrophil Gelatinase-Associated Lipocalin in Vascular Injury via Activation of Nuclear Factor-?B[END_REF].

NGAL in abdominal aortic aneurysm

Biopsy analysis of patients with abdominal aortic aneurysm (AAA) identified the expression of the NGAL/MMP-9 complex in the vascular wall and in the thrombus present in the blood vessel lumen [START_REF] Folkesson | Presence of NGAL/MMP-9 complexes in human abdominal aortic aneurysms[END_REF]. Neutrophils were the major source of NGAL expression in this context. In an AAA model in mice, the absence of NGAL (in NGAL KO mice) or its blockade (with anti-NGAL antibodies) had the same protective effects against AAA development, with a decrease in neutrophil infiltration and MMP activity [START_REF] Tarín | Lipocalin-2 deficiency or blockade protects against aortic abdominal aneurysm development in mice[END_REF].

NGAL in cardiometabolic disorders

In obesity in humans and animals, circulating NGAL levels are increased and correlated with increases in blood pressure and insulin resistance [START_REF] Wang | Small lipid-binding proteins in regulating endothelial and vascular functions: focusing on adipocyte fatty acid binding protein and lipocalin-2: Lipid chaperones in cardiovascular diseases[END_REF]. A recent study from our laboratory also reported an increase in plasma levels of the NGAL/MMP-9 complex in a cohort of obese patients correlated with an increase in circulating fibrosis markers [START_REF] Tarjus | Neutrophil Gelatinase-Associated Lipocalin, a Novel Mineralocorticoid Biotarget, Mediates Vascular Profibrotic Effects of Mineralocorticoids[END_REF].

In animal models of obesity, NGAL KO mice were protected against hypertension [START_REF] Song | Deamidated Lipocalin-2 Induces Endothelial Dysfunction and Hypertension in Dietary Obese Mice[END_REF], inflammation [START_REF] Song | Deamidated Lipocalin-2 Induces Endothelial Dysfunction and Hypertension in Dietary Obese Mice[END_REF][START_REF] Law | Lipocalin-2 Deficiency Attenuates Insulin Resistance Associated With Aging and Obesity[END_REF] as well as endothelial [START_REF] Liu | Lipocalin-2 deficiency prevents endothelial dysfunction associated with dietary obesity: role of cytochrome P450 2C inhibition: Lipocalin-2 and endothelial dysfunction[END_REF] and cardiometabolic dysfonctions [START_REF] Yang | Improved functional recovery to I/R injury in hearts from lipocalin-2 deficiency mice: restoration of mitochondrial function and phospholipids remodeling[END_REF][START_REF] Law | Lipocalin-2 Deficiency Attenuates Insulin Resistance Associated With Aging and Obesity[END_REF][START_REF] Liu | Lipocalin-2 deficiency prevents endothelial dysfunction associated with dietary obesity: role of cytochrome P450 2C inhibition: Lipocalin-2 and endothelial dysfunction[END_REF] induced by a high fat diet. Song et al. reported that the deamidation of NGAL (by linoleic acid in particular) improved its stability and promoted its accumulation [START_REF] Song | Deamidated Lipocalin-2 Induces Endothelial Dysfunction and Hypertension in Dietary Obese Mice[END_REF]. The combined administration of recombinant NGAL and linoleic acid in WT mice promoted oxidative stress, endothelial dysfunction, inflammation and hypertension [START_REF] Song | Deamidated Lipocalin-2 Induces Endothelial Dysfunction and Hypertension in Dietary Obese Mice[END_REF].

NGAL and iron metabolism in cardiomyopathies

NGAL participated to iron transport by binding a siderophore and can increase or decrease the amount of intracellular iron, thus participating in the maintenance of iron homeostasis. Both iron overload and iron deficiency have been linked to cardiomyopathies: iron overload has been associated with increased oxidative stress and iron deficiency with mitochondrial dysfunction, impaired cardiac function [START_REF] Jankowska | Iron deficiency and heart failure: diagnostic dilemmas and therapeutic perspectives[END_REF], hyper-coagulation and oxidative stress related to anemia [START_REF] Lapice | Iron Deficiency and Cardiovascular Disease: An Updated Review of the Evidence[END_REF]. The involvement of NGAL in iron homeostasis could therefore play an important role in the development of some cardiomyopathies. Indeed, NGAL has also been shown to participate to cardiomyocyte apoptosis resulting from intracellular iron accumulation (100). The apoptosis of cardiomyocytes could therefore influence the remodeling process involved in the development of certain cardiac pathologies, underlying a potential implication of NGAL in this process.

Involvement of NGAL in inflammatory mechanisms

NGAL and pro-inflammatory cytokines expression

In heart failure and various other inflammatory pathologies, serum or plasma levels of NGAL have been shown to correlate with those of other inflammatory markers such as TNF-, CRP, IL-6 or leukocytes number (101,102).

NGAL expression can be induced by various pro-inflammatory stimuli, such as LPS (103), IL-1 (104), IL-6 (3), IL-17 (105), IFN- (106) and TNF- (72) depending on cell type. Furthermore, NGAL expression is positively regulated by the activation of the NF-B pathway [START_REF] Bu | Induction of Neutrophil Gelatinase-Associated Lipocalin in Vascular Injury via Activation of Nuclear Factor-?B[END_REF] which is known for its role in pro-inflammatory mechanisms. Conversely, NGAL is able to activate the NF-B pathway (107,108) and to induce the expression of diverse pro-inflammatory molecules such as IL-8 [START_REF] Eilenberg | Neutrophil Gelatinase-Associated Lipocalin (NGAL) is Associated with Symptomatic Carotid Atherosclerosis and Drives Pro-inflammatory State In Vitro[END_REF][START_REF] Shao | Increased Lipocalin-2 Contributes to the Pathogenesis of Psoriasis by Modulating Neutrophil Chemotaxis and Cytokine Secretion[END_REF], IL-6 (5,12), IL1- ( 12), TNF- [START_REF] Shao | Increased Lipocalin-2 Contributes to the Pathogenesis of Psoriasis by Modulating Neutrophil Chemotaxis and Cytokine Secretion[END_REF] and MCP-1 [START_REF] Eilenberg | Neutrophil Gelatinase-Associated Lipocalin (NGAL) is Associated with Symptomatic Carotid Atherosclerosis and Drives Pro-inflammatory State In Vitro[END_REF]. Finally, it has been shown that NGAL was involved in the polarization of macrophages towards the pro-inflammatory phenotype M1, in vitro and in vivo [START_REF] Cheng | Lipocalin-2 Promotes M1 Macrophages Polarization in a Mouse Cardiac Ischaemia-Reperfusion Injury Model[END_REF]109).

Altogether these data suggest the existence of a vicious circle in which NGAL is overexpressed in inflammatory conditions and capable, in turn, to potentiate inflammation by inducing the expression of pro-inflammatory mediators (Figure 4).

NGAL and inflammatory diseases

NGAL is proposed to be involved in chronic inflammation and autoimmune diseases.

Urinary levels of NGAL were increased in patients with lupus nephropathy and were associated with the severity of the disease (110). In a rat autoimmune myocarditis model, NGAL was strongly expressed in cardiomyocytes, vascular SMCs, fibroblasts and neutrophils [START_REF] Ding | Lipocalin-2/Neutrophil Gelatinase-B Associated Lipocalin Is Strongly Induced in Hearts of Rats With Autoimmune Myocarditis and in Human Myocarditis[END_REF]. In addition, increased NGAL levels were particularly important during the active phase of myocarditis and closely followed cardiac and plasma IL-1 levels [START_REF] Ding | Lipocalin-2/Neutrophil Gelatinase-B Associated Lipocalin Is Strongly Induced in Hearts of Rats With Autoimmune Myocarditis and in Human Myocarditis[END_REF]. In an acute antibody-induced skin inflammation model, NGAL KO mice showed a 50% reduction in inflammation associated with reduced immune infiltration compared to WT mice (111). The same benefit was found by injecting WT mice with anti-NGAL antibodies while inflammation was restored by treatment with recombinant NGAL (111).

NGAL expression by immune cells

NGAL expression was first described in neutrophils (1) and later observed in other immune cell types such as macrophages [START_REF] Eilenberg | Neutrophil Gelatinase-Associated Lipocalin (NGAL) is Associated with Symptomatic Carotid Atherosclerosis and Drives Pro-inflammatory State In Vitro[END_REF][START_REF] Flo | Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron[END_REF] and dendritic cells [START_REF] Floderer | Dendritic Cell-Secreted Lipocalin2 Induces CD8+ T-Cell Apoptosis, Contributes to T-Cell Priming and Leads to a TH1 Phenotype[END_REF]. Recent studies suggest that the expression of NGAL by these immune cell types could be important in the regulation of inflammatory processes. It has been shown, for instance, that NGAL secreted by dendritic cells was involved in the activation and polarization of T-cells toward a pro-inflammatory phenotype (9). Gilet et al. showed that aldosterone was able to induce the production of NGAL by human neutrophils in complex with MMP-9 (112). Aigner et al. identified neutrophils as the primary source of NGAL released at the time of immune infiltration in the mouse heart after an episode of I/R [START_REF] Aigner | Lipocalin-2 Regulates the Inflammatory Response During Ischemia and Reperfusion of the Transplanted Heart[END_REF].

A recent study from our laboratory, using mice depleted from NGAL in their immune

Involvement of NGAL in hypertensive mechanisms

In patients with essential hypertension, plasma NGAL level were higher than in healthy subjects and correlated with blood pressure (113). In clinical studies, polymorphisms in the promoter of NGAL have been associated with changes in blood pressure (114).

The role of NGAL in hypertensive mechanisms was also demonstrated in animal models of obesity. NGAL KO mice were protected against hypertension, inflammation and cardiometabolic dysfunction induced by a high fat diet [START_REF] Song | Deamidated Lipocalin-2 Induces Endothelial Dysfunction and Hypertension in Dietary Obese Mice[END_REF][START_REF] Law | Lipocalin-2 Deficiency Attenuates Insulin Resistance Associated With Aging and Obesity[END_REF][START_REF] Liu | Lipocalin-2 deficiency prevents endothelial dysfunction associated with dietary obesity: role of cytochrome P450 2C inhibition: Lipocalin-2 and endothelial dysfunction[END_REF]. The direct role of NGAL in blood pressure control was further demonstrated by the combined administration of recombinant NGAL and linoleic acid in mice, which induced an increase in mouse blood pressure [START_REF] Song | Deamidated Lipocalin-2 Induces Endothelial Dysfunction and Hypertension in Dietary Obese Mice[END_REF]. Recently, a study from our laboratory demonstrated the crucial role of NGAL in the setting of aldosterone-mediated hypertension. Indeed, the global genetic inactivation of NGAL in mice prevented the increase in blood pressure induced by a mineralocorticoid challenge [START_REF] Tarjus | Neutrophil Gelatinase-Associated Lipocalin, a Novel Mineralocorticoid Biotarget, Mediates Vascular Profibrotic Effects of Mineralocorticoids[END_REF].

Involvement of NGAL in pro-fibrotic mechanisms

The role of NGAL in pro-fibrotic mechanisms was first suggested by the identification of its binding to MMP-9, a protein involved in extracellular matrix remodeling [START_REF] Yan | The High Molecular Weight Urinary Matrix Metalloproteinase (MMP) Activity Is a Complex of Gelatinase B/MMP-9 and Neutrophil Gelatinase-associated Lipocalin (NGAL): MODULATION OF MMP-9 ACTIVITY BY NGAL[END_REF]. Our laboratory demonstrated recently the crucial role of NGAL in the pro-fibrotic effects of a mineralocorticoid challenge [START_REF] Tarjus | Neutrophil Gelatinase-Associated Lipocalin, a Novel Mineralocorticoid Biotarget, Mediates Vascular Profibrotic Effects of Mineralocorticoids[END_REF] and in a model of MI [START_REF] Martínez-Martínez | Aldosterone Target NGAL (Neutrophil Gelatinase-Associated Lipocalin) Is Involved in Cardiac Remodeling After Myocardial Infarction Through NFκB Pathway[END_REF].

The mechanisms by which NGAL induces fibrosis are probably diverse. NGAL binding to MMP-9 may be one, although the existence of NGAL/MMP-9 in rodents remains controversial. It is also possible that NGAL binds to other molecules able of modulating fibrosis. For example, NGAL binds and negatively regulates the activity of HGF (Hepatocyte Growth Factor), a peptide known for its anti-fibrotic properties (115). We recently showed a direct pro-fibrotic role of NGAL in human cardiac fibroblasts ((85); Buonafine et al.

[unpublished]). The treatment of fibroblasts with aldosterone induced the expression of NGAL and collagen I. However, inhibition of NGAL expression by siRNA resulted in the loss of induction of collagen I expression by aldosterone. This suggests that NGAL could act as a mediator of the pro-fibrotic effects of aldosterone in vivo [START_REF] Martínez-Martínez | Aldosterone Target NGAL (Neutrophil Gelatinase-Associated Lipocalin) Is Involved in Cardiac Remodeling After Myocardial Infarction Through NFκB Pathway[END_REF].

In addition, our data indicate that NGAL has an effect on the proliferation of fibroblasts in vitro (Buonafine et al. [unpublished]). This proliferative effect of NGAL was also described in vivo in renal I/R models in which recombinant NGAL injection induced tubular cell proliferation [START_REF] Mori | Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury[END_REF][START_REF] Mishra | Amelioration of Ischemic Acute Renal Injury by Neutrophil Gelatinase-Associated Lipocalin[END_REF] while NGAL KO mice had reduced tubular proliferation in a mouse model of CKD [START_REF] Viau | Lipocalin 2 is essential for chronic kidney disease progression in mice and humans[END_REF]. Given the key role of proliferation (of myofibroblastes in particular) in the mechanisms of remodeling and fibrosis (116), the proliferative effects of NGAL could also be implicated in pathological organ remodeling.

Signaling pathways involved in the pathological effects of NGAL

Erk1/2 signaling pathway

Another signaling pathway modulated by NGAL is the ERK1/2 pathway. This signaling pathway is known for its involvement in cell proliferation and cell death processes (117). Aldosterone has been shown to induce NGAL/MMP-9 in human neutrophils in culture, and this effect was prevented by the use of an ERK1/2 inhibitor (112). The role of the ERK1/2 pathway in the induction of NGAL has also been described in intestinal epithelial cells in response to a bacterial toxin (118). It is interesting to note that expression of the NGAL receptor 24p3R was induced by IL-1 in human mesangial cells in culture and also involved activation of the ERK1/2 signaling pathway (119). Conversely, several studies have shown that NGAL is able to activate the ERK1/2 signaling pathway and that this pathway is involved in the cellular effects of NGAL. Treatment of cultured epithelial cells with NGAL induced activation of the ERK1/2 pathway and induced cell migration, which was lost through inhibition of this signaling pathway [START_REF] Gwira | Expression of Neutrophil Gelatinase-associated Lipocalin Regulates Epithelial Morphogenesis in Vitro[END_REF]. Similarly, the treatment of human neutrophils in culture with recombinant NGAL stimulated cell migration and proinflammatory cytokine expression via the 24p3R receptor and induction of the ERK1/2 signaling pathway [START_REF] Shao | Increased Lipocalin-2 Contributes to the Pathogenesis of Psoriasis by Modulating Neutrophil Chemotaxis and Cytokine Secretion[END_REF]. In models of esophageal [START_REF] Du | Lipocalin 2 promotes the migration and invasion of esophageal squamous cell carcinoma cells through a novel positive feedback loop[END_REF] or prostate cancer cells (120), overexpression of NGAL or treatment with recombinant NGAL activated the ERK1/2 pathway and blocking this pathway blocked the migration and invasive properties of the cells promoted by NGAL.

NFB signaling pathway

A large number of studies have shown that the NF-B pathway controls the expression of NGAL [START_REF] Borkham-Kamphorst | Induction of lipocalin-2 expression in acute and chronic experimental liver injury moderated by pro-inflammatory cytokines interleukin-1β through nuclear factor-κB activation: LCN2 induction in acute and chronic experimental liver injury[END_REF][START_REF] Bu | Induction of Neutrophil Gelatinase-Associated Lipocalin in Vascular Injury via Activation of Nuclear Factor-?B[END_REF][START_REF] Iannetti | The neutrophil gelatinase-associated lipocalin (NGAL), a NF-κB-regulated gene, is a survival factor for thyroid neoplastic cells[END_REF][START_REF] Koh | HGF mediated upregulation of lipocalin 2 regulates MMP9 through nuclear factor-κB activation[END_REF]. Conversely, some studies have shown that NGAL itself is capable of activating the NF-B pathway [START_REF] Mongre | Epigenetic induction of epithelial to mesenchymal transition by LCN2 mediates metastasis and tumorigenesis, which is abrogated by NF-κB inhibitor BRM270 in a xenograft model of lung adenocarcinoma[END_REF]107,108), which is of primary importance given the major involvement of this pathway in inflammatory and fibrotic mechanisms (121,122). In a cell model with NGAL overexpression, Wang et al. showed, for example, that NGAL activated the NF-B pathway via increased levels of intracellular iron and reactive oxygen species (108). Consistent with this, we recently showed that NGAL activated the NF-B pathway in cardiac cells, in vivo and in vitro [START_REF] Martínez-Martínez | Aldosterone Target NGAL (Neutrophil Gelatinase-Associated Lipocalin) Is Involved in Cardiac Remodeling After Myocardial Infarction Through NFκB Pathway[END_REF]. The treatment of cardiac fibroblasts with recombinant NGAL induced NF-B activation while blocking the NF-B pathway prevented NGALinduced production of collagen I (85). The NF-B pathway was activated in WT mice seven days after MI, but this was not the case in NGAL KO mice, demonstrating the importance of NGAL in activating this pathway in vivo [START_REF] Martínez-Martínez | Aldosterone Target NGAL (Neutrophil Gelatinase-Associated Lipocalin) Is Involved in Cardiac Remodeling After Myocardial Infarction Through NFκB Pathway[END_REF]. These findings highlight the importance of the NF-B pathway in mediating the direct pro-fibrotic effects of NGAL.

CONCLUSION

In conclusion, NGAL appears to be an important mediator of cardiac and renal diseases through its role in hypertensive, inflammatory and fibrotic processes. High levels of NGAL are reported in a wide variety of pathological situations, both in animals and in patients, and NGAL blockade using neutralizing antibodies or genetic inactivation is beneficial in animal models of cardiac or renal injuries. The role of NGAL in inflammation is of particular importance since NGAL is part of a pro-inflammatory amplification loop. NGAL is induced in inflammatory situations and is able to amplify inflammation, by the production of pro-inflammatory cytokines, the activation of pro-inflammatory pathways as well as the polarization of immune cells toward pro-inflammatory phenotypes. This pro-inflammatory role of NGAL could be important in the mediation of its hypertensive and fibrotic effects, since the interconnection of these pathological mechanisms is now well described (123-125).

Finally, NGAL production and secretion by immune cells themselves seem to play an important role in some cardiorenal pathological situations. The role of NGAL produced by specific immune cell populations should thus be further studied.

Given the importance of its pathological implications, NGAL could represent an interesting therapeutic target in CV and renal diseases and beyond, especially in pathologies involving inflammatory mechanisms.

  Using a mouse model expressing a bioluminescent reporter of NGAL expression, Paragas et al. have studied the origin of NGAL during AKI. They determined that during I/R, NGAL was synthetized by cells of the thick ascending limb of Henle's loop and of the collecting duct and only in ischemic areas of the kidney (55). In addition, by performing kidney transplants between WT and NGAL KO mice, Paragas et al. demonstrated that the increase in urinary NGAL concentration during renal ischemia was mainly due to the release of NGAL originating in the kidney and that the contribution of extra-renal NGAL to urinary NGAL was small (55).

  cells only (after bone marrow transplantation), allowed us to demonstrate the pivotal role of NGAL produced by immune cells in the cardiac and renal deleterious effects of a mineralocorticoid challenge (Buonafine et al. [unpublished]). In WT mice, this challenge induced systemic inflammation and an induction of NGAL expression by macrophages, dendritic cells, and peripheral blood mononuclear cells (PBMCs). The depletion of NGAL from immune cells protected the mice against cardiac and renal remodeling as well as inflammation induced by mineralocorticoid excess (Buonafine et al. [unpublished]). Of note, mice depleted of NGAL in their immune cells presented lower levels of cardiac NGAL compared to control mice, revealing immune cells as a major source of NGAL in the heart (Buonafine et al. [unpublished]).

FIGURESFigure 1 -

 1 FIGURES

Figure 2 -

 2 Figure 2 -Three-dimensional structure of the NGAL-siderophore-iron complex

Figure 3 -

 3 Figure 3 -Role of NGAL in iron trafficking

Figure 4 -

 4 Figure 4 -Implication of NGAL in a pro-inflammatory amplification loop

Funding

This work was funded by grants from the Institut National de la Santé et de la Recherche Médicale, ANR MRFOCUS (ANR-15-CE14-0032-02), the Fight-HF Avenir investment program (ANR-15-RHUS-0004). MB was supported by a PhD grant from Region Ile de France CORDDIM.

Disclosure

FJ received research grants from BAYER Pharma AG.