Martin Gillard

Guillaume Piraux

Martin Daenen

Michaël Abraham

Ludovic Troian-Gautier

Laure Bar

Hugues Bonnet

Frédérique Loiseau

Hélène Jamet

Jérôme Dejeu

Eric Defrancq

Benjamin Elias

Photo-Oxidizing Ruthenium(II) Complexes with Enhanced Visible-Light Absorption and G-quadruplex DNA Binding Abilities

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Introduction

Ruthenium(II) metal complexes offer many appealing assets in the design of photosensitizers for cancer phototherapy thanks to their high stability, strong visible light absorption and long-lived excited states. [1] Ruthenium(II) compounds containing polypyridyl ligands have been the most explored frameworks since the report of the DNA light switch effect of [Ru(bpy)2dppz] 2+ (bpy = 2,2′-bipyridine, dppz = dipyrido[3,2-:2′,3′-]phenazine) in the early 90's. [2] Many analogues were developed aiming to enhance the photo-oxidizing properties and to improve the accumulation of the compounds into cancer cells. The enhancement of the photo-oxidizing power of these complexes has been mainly achieved through the addition of nitrogen atoms in the ligands structure. Notably the use of TAP (TAP = 1,4,5,8-tetraazaphenanthrene) or bpz (bpz = 2,2′-bipyrazine) ancillary ligands leads to strong photo-oxidizing complexes able to trigger oxidative electron transfer with DNA (type I photooxidation). [3] Developing highly photo-oxidizing sensitizers constitutes interesting alternatives in cancer phototherapy when the type II pathway (ROS photoproduction) is prevented by hypoxia. [4] In order to favour accumulation of the photosensitizers into cancer cells, the association to DNA secondary structures that are present in higher prevalence in many cancer cell lines such as G-quadruplex DNA (G4 DNA) and DNA mismatches has been extensively studied. [5] Therefore, numerous compounds including ruthenium(II) complexes have been designed to bind selectively G4 DNA or DNA mismatches over double stranded DNA. [6] Telomeric G4 DNA has emerged as a promising target for cancer therapy since the discovery of its implication in the cancer cell immortalization process in addition to the major role it plays in the development of the disease. [7] Recently, we reported on the design of [Ru(phen)2dph] 2+ (1) (phen = 1,10-phenanthroline, dph = dipyrazino [2,3-a:2',3'h]phenazine), which showed high selectivity towards G4 over duplex DNA in addition to strong visible light absorption. This was attributed to the extended conjugation of the dph ligand. [8] Based on these interesting results, three analogues (Figure 1) were prepared to (i) enhance the conjugation of the extended dph ligand in the [Ru(phen)2bpph] 2+ (2) complex (bpph = benzo[a]pyrazino [2,3-h]phenazine) and to (ii) increase the photo-oxidizing power of the compounds by replacing the phen ancillary ligands by TAP in [Ru(TAP)2dph] 2+ (3) and [Ru(TAP)2bpph] 2+ (4). E-mail: Benjamin.Elias@uclouvain.be [b] Dr. L. Bar, H. Bonnet, Prof. F. Loiseau, Dr. H. Jamet, Dr. J. Dejeu and Prof E. Defrancq Université Grenoble Alpes (UGA) Département de Chimie Moléculaire, UMR CNRS 5250 CS 40700 -38058 Grenoble (France).

The synthesis of compounds 2-4 as well as their photophysical and photochemical properties are reported herein. The interaction of complexes 1-4 with G4 DNA and duplex DNA as well as their excited-state reactivity was investigated by bio-layer interferometry, luminescence titrations, nanosecond transient absorption and computational studies. Strikingly, such weak structural changes between the different analogues led to considerable changes both to the photophysics and the affinity of the compounds towards G4 over duplex DNA.

Results and Discussion

Synthesis

The pyrazine core synthesis of the dph ligand was previously developed by our group. It consists in the coupling of two 6aminoquinoxaline thanks to an optimized Chichibabin-like reaction (i.e. through an addition-oxidation-elimination mechanism). [8] The dissymmetric bpph ligand was prepared through the dione-diamine condensation of 1,2-naphtoquinone and 5,6-diaminoquinoxaline, as previously reported. [9] Phen-and TAP-based Ru II mononuclear complexes 1-4 (Figure 1) were synthesized through the direct chelation of the dph or bpph ligands onto [Ru(phen)

Electrochemical study

The electrochemical characterization of compounds 1-4 allowed to better understand their photophysical and photochemical properties. The data were recorded in acetonitrile containing 0.1 M tetrabutylammonium perchlorate (Bu4NClO4) (Table 1). Complex Eox [a] [V] [b] Ered [a] [V] [b] E*red [a] [V] Concerning the oxidation, the observed process can be attributed to the oxidation of the metallic centre. It should be noted that complexes 1-2 are less easily oxidized compared to the reference complex [Ru(phen)3] 2+ . Furthermore, no oxidation process could be monitored for TAP analogues (i.e. complexes 3 and 4) at a potential below 1.8 V, which is consistent with the data reported for the reference complex [Ru(TAP)3] 2+ . [10] Concerning the ligand-centred reduction processes, compounds 1 and 2 are notably easier to reduce than [Ru(phen) Ag/AgCl, respectively). This suggests that those compounds should trigger photo-induced electron transfer (PIET) in the presence of a guanine residue (Eox(dGMP) = +1.10 V vs. Ag/AgCl), the most oxidizable DNA nucleobase. [11]

Light absorption

The absorption and emission spectra of complexes 1-4 recorded in acetonitrile are shown in Figure 2. The corresponding photophysical data along with those of reference compounds [Ru(phen)3] 2+ and [Ru(TAP)3] 2+ are gathered in Table 2. [10] The absorption spectra show a strong peak at 265 nm for 2 and at 315 nm for its TAP analogue 4, which is attributed to LC transitions localized on the ligands. Strong absorption bands were also observed in the visible region (in the range 400-500 nm) which are attributed to MLCT transitions. A strong hypsochromic shift of the MLCT transition was observed for complex 2 compared to 1 (Table 2) which is consistent with the replacement of two non-chelating nitrogen by carbon atoms in the bpph, accounting for an increase of the LUMO orbital energy level. The large bathochromic shifts (> 40 nm) of the MLCT transitions observed for the complexes 1-4 compared to the reference complexes [Ru(phen)3] 2+ and [Ru(TAP)3] 2+ are of great interest for the proposed applications and most likely arise from the high conjugation of the dph and bpph ligands.

[ns]

[c] Φ [d] kr [10 [ Ru(phen)

Light emission

The four complexes 1-4 display strong visible light emission with quantum yields, excited state lifetimes and large kr values (> 10 3 s -1 ) (Table 2) that suggest a 3 MLCT-type emitting state as already reported for similar Ru II compounds. [8] Complexes 1-4 exhibit large bathochromic shifts of their emissions (>40 nm) compared to the reference compounds, which could be due to the high conjugation of the dph and bpph ligands. Interestingly, bpph containing complexes 2 and 4 show emission at higher energies than the corresponding dph containing compounds 1 and 3, which can be explained by the low-lying LUMO of the dph containing complexes. In addition, the fact that dph containing compounds 1 and 3 possess longer luminescence lifetimes than the corresponding bpph containing analogues 2 and 4 can be related to a smaller contribution of the non-radiative decay on the overall emission process in the dph complexes. Complexes 3 and 4 bearing TAP ancillary ligands exhibit strongly blue shifted emission compared to their phen analogues. The recorded emission wavelengths for compounds 3 and 4 are similar to those reported for other complexes containing TAP ancillary ligands. The luminescence lifetimes of the TAP containing compounds 3 and 4 are also noticeably longer than those of their respective phen analogues 1 and 2, as anticipated.

dGMP photo-oxidation

As suggested by the electrochemical and photophysical studies, the presence of two TAP ancillary ligands in compounds 3 and 4 leads to complexes with enhanced photo-oxidation capacities compared to 1 and 2. This was highlighted by the assessment of their reduction potentials in the excited state (Table 1).

Therefore, the ability of complexes 3 and 4 to oxidize deoxyguanosine monophosphate (dGMP) under light irradiation was explored by luminescence quenching experiments. As depicted in Figure 3, a dramatic luminescence decrease was observed through the gradual addition of dGMP to a buffered solution of compounds 3 and 4. 2+ 4 (λexc = 482 nm) in the presence of increasing concentrations of dGMP, while the complex concentration was kept at 5 µM.. Measurements were made in 50 mM Tris•HCl, 50 mM NaCl, pH = 7.4, under ambient air conditions. Insets: Stern-Volmer plots obtained upon the addition of dGMP to each complex.

Gibbs free energy variations (ΔG) corresponding to the electron transfer process (ET) from a guanine moiety to the excited complexes 3 and 4 were estimated from voltametric and photophysical data. The spontaneity of the ET process was thereby confirmed with calculated ∆GET ranging between -0.15 and -0.19 eV. The ∆GET were also calculated against a guanine rich Poly(dG-dC)2 sequence to assess the impact of the guanine stacking, which is known for making the photo-oxidation process even more favoured (Table 3). The efficiency of the luminescence quenching (kq) was determined thanks to Stern-Volmer relationship (Figure 3 -insets). The linearity of the Stern-Volmer plot indicates a pure dynamic quenching of the excited state of the two complexes by dGMP and the obtained kq values (Table 3) shows a diffusion rate limited process. [12] This efficient luminescence quenching is in agreement with a PIET process occurring between excited TAP containing complexes 3 and 4 and the guanine base. It also points out the ability of dph and bpph TAP-based complexes for DNA photo-oxidation. [a] Gibbs free energy variation corresponding to the ET process from guanine to the excited complex estimated as ΔGET = -nF(ERu2+* /Ru+ -EG•+/G), with EG•+/G = +1.10 V for dGMP or +0.85 V for Poly(dG-dC)2 and ERu2+* /Ru+ = E*red values in Table 1. [b] Quenching rate constant (kq) obtained using the Stern-Volmer equation I0/I = 1 + kqτ0[dGMP]. I0/I (where I0 is the luminescence of the complex in the absence of a quencher, here dGMP, and I is the luminescence in the presence of dGMP) as a function of the quencher concentration; τ0, the excited state lifetime of the complex in the absence of dGMP. 4B). This is fully consistent with the metal-to-ligand charge transfer (MLCT) nature of the excited state. The excitedstate reactivity with dGMP was then investigated using pulsed 480 nm light excitation. The transient absorption spectra recorded 200 ns after the laser pulse were fully consistent with excited-state electron transfer, generating the monoreduced complex and oxidized dGMP. Similar experiments were then conducted using G4. The spectral signatures observed 500 ns after the laser pulse matched those obtained using dGMP, indicative of the efficient photo-induced excited-state electron transfer between [Ru(TAP)2bpph] 2+ * and G4. Note that the signal for that experiment presents larger signal/noise ration below 400 nm. This is a consequence of the experimental setup as a volume of 300 µL was used in a 0.3x0.3cm cuvette. Nevertheless, the transient absorption features unambiguously confirm the photo-induced oxidative electron transfer from dGMP or guanine residue in G4 to the excited complex.

DNA interaction studies

Luminescence titrations, bio-layer interferometry analysis (BLI) and computational studies were implemented to study the interaction of complexes 1-4 towards duplex and G4 DNA. Overall, these experimental methods allowed to measure the affinity and selectivity of the compounds for G4 DNA secondary structure, in addition to characterize the interactions occurring with both duplex and G4 DNA. For DNA titrations, calf thymus DNA (CT-DNA) and wtTel23 were chosen as duplex and G4 DNA models respectively. For BLI, biotinylated versions of GCrich duplex and wtTel23 sequence were chosen (see Figure 5 A and 5 B). It must be noted that while calf thymus DNA contains an equivalent molar amount of the four DNA bases, the GC-rich hairpin model is richer in guanine and cytosine.

Luminescence titrations

While enhanced luminescence was observed for phen containing complexes 1 and 2 in the presence of increasing concentration of CT and G4 DNA (Figure 6A), a decrease of the luminescence was observed for TAP containing complexes 3 and 4 (Figure 6B). The luminescence increase observed for both complexes 1 and 2 upon addition of DNA can be related to a light switch type effect as reported for [Ru(bpy)2dppz] 2+ . [1] This behaviour is explained by an efficient protection of the 3 MLCT excited state from non-radiative decay sources such as solvent molecules and dioxygen when interacting with DNA. The strong decrease of luminescence intensity for both complexes 3 and 4 show that complexes 3 and 4 are potential photoreactive equivalents of complexes 1 and 2, respectively. Binding affinity constants were estimated using a modified McGhee-von Hippel equation (lines and dashed lines in Figure 6A and6B), expressing the luminescence intensity as a function of the ratio of Ru II compound per DNA site (I/I0 vs. [Site]/ [Ru]), with I0, the intensity of luminescence in the absence of DNA and [Site] referring either a base-pair or a G4 equivalent (Table 4). Interestingly, higher affinities towards CT-DNA were observed when the two non-chelating nitrogen of dph (complexes 1 and 3) were removed (complexes 2 and 4). 2+ 4 (grey) by increasing G4-DNA (full diamonds) or ds-DNA (empty diamonds) ratio, while the complex concentration was kept at 5 µM. The fitted curves are obtained using a modified McGhee-von Hippel equation (see the SI) and drawn as solid and dashed curves for G4 and ds-DNA respectively. One site in ds-DNA corresponds to one base pair and to one quartet in G4 structures and I0, the photoluminescence of the complex in the absence of DNA. λexc = 450 nm; λEm = 773 (1), 727 (2), 667 (3) and 650 nm (4).

Bio-layer interferometry

Bio-layer interferometry (BLI) was implemented to gain access to the more accurate dissociation equilibrium constant of the interactions (KD) via the measurements of kinetics of association (kon) and dissociation (koff). Two DNA structures were used as models: the intramolecular G-quadruplex Figure 5A (HTelo sequence, in equilibrium between the different topologies), and the duplex DNA model Figure 5B (hairpin structure). These structures have been already used for the study of interactions of putative ligands with G4 and duplex DNA. [6b, 8] The binding constants for complexes 1-4 are reported in Table 5. The measured KD value for the interaction of complex 1 with G4 DNA is in the micromolar range (9 µM) while no equilibrium constant could be calculated for the interaction with the duplex DNA. This is in agreement with the previously reported results obtained from surface plasmon resonance analyses and confirms the selectivity of complex 1 towards G4 over double-stranded DNA structures. [8] Replacing the phen ancillary ligands of complex 1 by TAP in complex 3 led to a dramatic decrease of the affinity towards G4 DNA (KD = 66 µM) which could be attributed to both a slower association (factor 2) and a faster dissociation (factor 4) according to the kinetic constants (Figure 7 and Table 5).

However, the selectivity towards G4 vs. duplex DNA structures remains as no equilibrium constants could be measured for the interaction with the duplex DNA hairpin. These results were concordant with the luminescence titrations.

The removal of two non-chelating nitrogen atoms from the dph ligand in complex 1 to form the bpph ligand in complex 2 also led to a huge impact on the interactions with G4 and duplex DNA. Indeed, a decrease of the affinity towards G4 was observed (KD = 32 µM) while a weak affinity for duplex DNA could be measured (KD = 102 µM) thus leading to a partial loss of the selectivity towards G4 DNA. Nevertheless, it is noteworthy that the measured KD value for duplex DNA being far beyond the studied concentration range (due to solubility problems), its accuracy must be taken with care. For complex 4 which contains both bpph and tap ligands, the selectivity towards G4 strongly decreased due to a higher affinity of the compound for duplex DNA.

The comparison of the kinetic parameters measured for the interactions of bpph containing complexes 2 and 4 showed a slower dissociation rate when interacting with G4 compared to duplex DNA (0.5 s -1 versus 1.4-1.8 s -1 ). However, for complex 4 a higher association rate was observed for duplex versus G4 DNA (8.1 10 4 M -1 s -1 and 2.7 10 4 M -1 s -1 , respectively) thus leading to the partial loss of selectivity. The affinity distribution can be better visualized in Figure 6. It emerges from these results that the number of non-chelating nitrogen atoms and their position have a considerable impact on the interactions with both G4 and duplex DNA. This parameter appears to play a determining role on the interaction strength and on the selectivity of the compounds for G4 versus duplex DNA structures. In this context, we decided to perform a computational study of the interaction with the aim to explain the observed differences. Table 5: Equilibrium dissociation constants (KD) for the interaction of complexes 1-4 with G4 and duplex DNA measured from bio-layer interferometry analysis.

Complex G4 DNA Duplex DNA kon [10 4 M -1 .s -1 ] koff [s -1 ] KD [µM] [a] kon [10 5 M -1 .s -1 ] koff [s -1 ] KD [µM] [a] [Ru(phen)2dph] 2+ 1 6.6 ± 1.3 0.6 ± 0.2 9 ± 2 n.d. [b]
n.d. [b] n.d.

[b]

[ Ru(phen) [a] Equilibrium dissociation constants were deduced from the kinetic rate constants. [b] Due to the very low binding of the complex with duplex DNA, the kinetics of the interaction could not be determined (n.d.) in the studied concentration range. Measurements were performed using a concentration range from 5 μM to 40 μM of the complexes in 10 mM HEPES pH 7.5, 50 mM NaCl, 100 mM KCl and 0.05% v/v P20 surfactant. The interactions arising between complexes 1-4 and their DNA targets, namely duplex and G-quadruplex, were investigated through docking experiments. The choice of the structures used for the targets is explained in the experimental computational part. Docking studies were performed using the AutoDock 4.0 software package. [13] Different positions were generated and ranked in a histogram according to a scoring function correlated to the interactions. The analysis of the best-ranked docked positions gives similar results for all the four complexes.

Computational studies

Regarding the hairpin, two binding modes emerge: a more stable one where the complex interacts within the minor groove, and a slightly less stable one in which the extended planar ligand dph or bpph is inserted inside the loop. Figure 8a shows these two binding modes for complex 1 with their scoring function.

In the G4 structure, two positions were also obtained: a more stable interaction involving the insertion of the extended planar dph or bpph ligand inside one of the grooves, and a less stable one where the extended planar dph or bpph ligand is stacked over the guanine tetrad (Figure 8b for complex 1). Interestingly, the mean binding energies of the more stable interactions obtained by docking are consistently lower for the G-quadruplex than for the hairpin, suggesting that favored for the G4 over the hairpin structure. This observation is consistent with the luminescence titrations and BLI analysis results that gave lower KD values for the G4 for each complex assessed. Furthermore, theoretical dissociation constants were calculated upon introduction of the mean binding energies in with T = 298.15K, giving KD values in the order of magnitude of 100 µ M, in agreement with the BLI experiments. Finally, the interactions were visualized through a non-covalent interaction (NCI) analysis in the quadruplex, highlighting the regions associated with specific interactions of various strengths; from steric repulsion (red), van der Waals interactions (green), to strong attractions (blue). Thus, the NCI analysis for the docked position inside the G4s groove showed that the main interactions arise from vdW interactions mostly located around the embedded extended planar ligand. As for the stacked position, the NCI analysis confirms a large π-π stacking interaction as well as some vdW interactions with one of the ancillary ligands facing towards the G-tetrads (Figure 9). However, no significant difference was observed amongst the four complexes and no additional interactions were found between the two additional nonchelating nitrogen's in the dph containing compounds 1 and 3 compared to their bpph analogs 2 and 4, so the NCI analysis cannot account for the variations of the KD observed experimentally.

Conclusion

In this paper, the synthesis of three new Ru(II) complexes 2-4 is reported. That includes the interesting dissymmetric bpph ligand and two photo-oxidizing TAP analogues. The designed compounds photophysical properties were studied by electrochemistry, UV/Vis absorption and emission spectroscopy.

The results show the compatibility between the dph and bpph ligands and the presence of TAP ancillary ligands to provide strongly photo-oxidizing compounds. It also appeared that Ru(II) complexes 3 and 4 gather high photo-oxidizing power (E*1/2red > 1.2 V) and strong visible light absorption (ε = 2800 and 5200 M -1 cm -1 at 480 nm) thanks to a rational ligand design. Compounds 3 and 4 can photo-oxidize dGMP under visible light irradiation (λexc = 480 nm) via PIET, as evidenced by nanosecond transient absorption spectroscopy. This PIET occurred with high quenching constant values (kq > 10 9 M -1 s -1 ). Luminescence titrations and BLI analysis allowed to gain access to the kinetic and thermodynamic parameters of the interaction. The two implemented techniques gave concordant results that highlight the substantial influence of the non-chelating nitrogen numbers and position on the interaction with both G4 and duplex DNA.

The dph analogues 1 and 3 appeared to be slightly more selective towards G4 vs. duplex DNA compared to their respective bpph analogues 2 and 4. Computational studies describing the interactions between the four complexes and the two targets revealed two binding modes and confirmed higher interactions with the G4 compared to the hairpin structures.

Experimental Section

Materials and instrumentation [Ru(phen)2Cl2], [Ru(TAP)2Cl2], [Ru(phen)2dph] +2 1 and bpph were synthesized according to previously described literature protocols. [8][9] All solvents and reagents for the synthesis were of reagent grade and were used without any further purification. All solvents for the spectroscopic and electrochemical measurements were of spectroscopic grade. Water was purified with a Millipore Milli-Q system. Calf thymus DNA Type I (CT-DNA) was purchased from Sigma-Aldrich. 1 H and 13 C NMR experiments were performed in CDCl3, CD3OD or CD3CN on a Bruker AC-300 Advance II (300 MHz) or on a Bruker AM-500 (500 MHz) at 20°C. The chemical shifts (given in ppm) are measured vs the residual peak of the solvent as the internal standard. High-resolution mass spectrometry (HRMS) spectra were recorded on a Q-Extractive orbitrap from ThermoFisher using reserpine as internal standard. Samples were ionized by electrospray ionization (ESI; capillary temperature = 320°C, vaporizer temperature = 320°C, sheath gas flow rate = 5 mL/min). UV-vis absorption spectra were recorded on a Shimadzu UV-1700. Room temperature luminescence spectra were recorded on a Varian Cary Eclipse instrument. Luminescence intensity at 77 K was recorded on a FluoroLog3 FL3-22 from Jobin Yvon equipped with an 18V 450 W Xenon Short Arc lamp and an R928P photomultiplier, using an Oxford Instrument Optistat DN nitrogen cryostat controlled by an Oxford Intelligent Temperature Controller (ITC503S) instrument. Luminescence lifetime measurements were performed after irradiation at λ = 400 nm obtained by the second harmonic of a Titanium:Sapphire laser (picosecond Tsunami laser spectra physics 3950-M1BB+39868-03 pulse picker doubler) at an 80 kHz repetition rate. The Fluotime 200 from AMS technologies was used for the decay acquisition. It consists of a GaAs microchannel plate photomultiplier tube (Hamamatsu model R3809U-50) followed by a time-correlated single photon counting system from Picoquant (PicoHarp300). The ultimate time resolution of the system is close to 30 ps. Luminescence decays were analysed with FLUOFIT software available from Picoquant. Cyclic voltammetry was carried out in a one-compartment cell, using a glassy carbon disk working electrode (approximate area = 0.03 cm 2 ), a platinum wire counter electrode, and an Ag/AgCl reference electrode. The potential of the working electrode is controlled by an Auto lab PGSTAT 100 potentiostat through a PC interface. The cyclic voltammograms were recorded with a sweep rate of 300 mV s -1 , in dried acetonitrile (Sigma-Aldrich, HPLC grade). The concentration of the complexes was 8.10 -4 mol/L, with 0.1 mol/L tetrabutylammonium perchlorate as supporting electrolyte. Before each measurement, the samples were purged by nitrogen. Redox potentials were controlled by comparison with ferrocene, added at the end of the measurement. [Ru(phen)2bpph] 2+ 2. [Ru(TAP)2Cl2] (17 mg, 0.0317 mmol) and (Benzo[a]pyrazino [2,3-h]phenazine) (9 mg, 0.0318 mmol) were dissolved ethylene glycol (2 mL) and heated at 120 °C for 5h. After cooling to room temperature, a solution of NH4PF6 (aq) (2 mL) was added and the mixture was left over night at 5 °C. The resulting mixture was centrifuged and the solid was washed successively with H2O, EtOH and diethylether, which gave the final product as a red powder. 1 H NMR (300 MHz, CD3CN) δ 9.51 (d, J = 7.8 Hz, 1H), 8.84 (d, J = 2.9 Hz, 1H), 8.81 (d, J = 9.4 Hz, 1H), 8.73 -8.65 (m, 3H), 8.62 (d, J = 9.4 Hz, 1H), 8.54 (dd, J = 8.3, 1.3 Hz, 1H), 8.31 (d, J = 1.6 Hz, 2H), 8.28 (dd, J = 5.3, 1.3 Hz, 1H), 8.27 -8.17 (m, 2H), 8.14 (dd, J = 5.3, 1.3 Hz, 1H), 8.00 -7.86 (m, 4H), 7.78 -7.65 (m, 4H), 7.64 -7.49 (m, 3H), 7.02 (d, J = 9.7 Hz, 1H). HR-MS Calcd for C42H26N8PF6Ru: 889.09679 Da, found 889.09687 Da.

Synthesis

[Ru(TAP)2dph] 2+ 3. [Ru(TAP)2Cl2] (20 mg, 0.0373 mmol) and dipyrazino [2,3-a:2',3'-h]phenazine (12.8 mg, 0.0451 mmol) were dissolved in EtOH/H2O (5 mL/ 1:1) and heated at reflux for 24h. After cooling to room temperature and addition of CH3CN (2 mL) and H2O (2 mL), the mixture was centrifuged. NH4PF6 was added and the organic solvents were evaporated under vacuum. The resulting mixture was centrifuged and the solid was washed successively with H2O, EtOH and diethylether to give the crude product. Purification over preparative chromatography on silica (CH3CN/H2O/NH4Clsat 4/4/1, v/v/v) gave the final product as a red powder. 1 H NMR (500 MHz, CD3CN) δ 9.25 (d, J = 2.0 Hz, 1H), 9.15 (d, J = 2.0 Hz, 1H), 9.10 (d, J = 2.8 Hz, 1H), 9.00 (d, J = 2.9 Hz, 1H), 8.87 (d, J = 2.8 Hz, 1H), 8.75 (d, J = 9.5 Hz, 1H), 8.71 (d, J = 9.4 Hz, 1H), 8.69 (d, J = 9.4 Hz, 1H), 8.62 (d, J = 9.4 Hz, 1H), 8.56 (d, J = 9.4 Hz, 1H), 8.50 (d, J = 2.8 Hz, 1H), 8.45 (d, J = 2.8 Hz, 1H), 8.06 (d, J = 2.9 Hz, 1H), 8.00 (d, J = 9.9 Hz, 2H), 8.00 (d, J = 2.7 Hz, 1H), 7.87 (d, J = 2.8 Hz, 1H), 7.35 (d, J = 9.9 Hz, 1H). HR-MS Calcd for C36H20N14PF6Ru: 895.06807 Da, found 895.06822 Da.

[Ru(TAP)2bpph] 2+ 4. [Ru(TAP)2Cl2] (17 mg, 0.0317 mmol) and (Benzo[a]pyrazino [2,3-h]phenazine) (9 mg, 0.0318 mmol) were dissolved ethylene glycol (2 mL) and heated at 120 °C for 16h. After cooling to room temperature and the addition of a solution of NH4PF6 (aq) (2 mL), the mixture was left over night at 5°C. The resulting mixture was centrifuged and the solid was washed successively with H2O, EtOH and diethylether to the final product as a red powder. 1 H NMR (300 MHz, CD3CN) δ 9.51 (d, J = 7.7 Hz, 1H), 9.08 (d, J = 2.8 Hz, 1H), 8.95 (t, J = 2.5 Hz, 3H), 8.89 -8.84 (m, 2H), 8.75 -8.65 (m, 3H), 8.62 -8.52 (m, 2H), 8.49 (dd, J = 2.8, 1.1 Hz, 2H), 8.04 -7.92 (m, 5H), 7.88 -7.82 (m, 2H), 6.99 (d, J = 9.6 Hz, 1H).

Electrochemical studies

Cyclic voltammetry was carried out in a one-compartment cell, using a glassy carbon disk working electrode (approximate area 0.03 cm 2 ), a platinum wire counter electrode, and an Ag/AgCl reference electrode. The potential of the working electrode was controlled by an Autolab PGSTAT 100 potentiostat through a PC interface. Cyclic voltammograms were recorded at a sweep rate of 100 mVs -1 from solutions in dry acetonitrile (Sigma-Aldrich, HPLC grade). The concentration of the complexes was 8x10 -4 mol L -1 , with 0.1 mol L -1 tetrabutylammonium perchlorate as supporting electrolyte. Before each measurement, the samples were purged with nitrogen. Redox potentials were determined by comparison with ferrocene, added at the end of the measurement. Luminescence titration experiments with ODNs (GC-rich hairpin or wtTel23 G-quadruplex DNA) were conducted by recording spectra from solutions in 10 mM HEPES, 50 mM NaCl, 100 mM KCl (pH 7.4) buffer on a Varian Cary Eclipse instrument. Titrations were performed by starting from the highest DNA concentration and progressively decreasing it, whilst the concentration of the complex (5 µm) was kept constant.

Photophysical measurements and luminescence titrations

UV/Vis absorption spectra were recorded on a Shimadzu UV-1700 spectrophotometer. Room temperature luminescence spectra were recorded on a Varian Cary Eclipse instrument. Luminescence intensity at 77 K was recorded on a FluoroLog 3 FL3-22 from Jobin Yvon equipped with an 18 V 450 W short-arc xenon lamp and an R928P photomultiplier, using an Oxford Instruments Optistat DN nitrogen cryostat controlled by an Oxford Intelligent Temperature Controller (ITC503S). Quantum yields were obtained using [Ru(bpy)3] 2+ as a reference. [14] Luminescence lifetime measurements were performed after irradiation at λ=450 nm obtained as the second harmonic of a titanium: sapphire laser (picosecond Tsunami laser Spectra at a repetition rate of 80 kHz. A Fluotime 200 instrument from AMS Technologies was used for the decay acquisition. It consists of a GaAs microchannel plate photomultiplier tube (Hamamatsu model R3809U-50) followed by a time-correlated single-photon counting system from Picoquant (PicoHarp300). The ultimate time resolution of the system is close to 4 ps. Luminescence titration experiments with ODNs (GC-rich hairpin or wtTel23 Gquadruplex DNA) were conducted by recording spectra from solutions in 10 mM HEPES, 50 mM NaCl, 100 mM KCl (pH 7.4) buffer on a Varian Cary Eclipse instrument. Titrations were performed by starting from the highest DNA concentration and progressively decreasing it, whilst the concentration of the complex (5 µm) was kept constant.

Nanosecond transient absorption

Nanosecond transient absorption measurements were recorded on a previously described apparatus. [15] Briefly, a LP920-K spectrometer from Edinburgh Instruments equipped with an iCCD detector from Andor was used. The excitation source was a tunable Nd:YAG Laser NT342 Series from EKSPLA. The third harmonic (355 nm) at 150 mJ was directed into an optical parametric oscillator (OPO) to enable wavelength tuning starting from 410 nm. The laser power at 480 nm was then attenuated to reach appreciable signal/noise (typically 10 mJ/pulse) and the integrity of the samples was verified by UV-Vis measurements. All samples were recorded in argon-purged buffered aqueous (either 10 mM HEPES, pH 7.5, 50 mM NaCl, 100 mM KCl or 50 mM Tris•HCl, 50 mM NaCl, pH = 7.4) at room temperature.

Bio layer interferometry (BLI)

BLI sensors coated with streptavidin (SA sensors) were purchased from Forte Bio (PALL). Prior to use, they were immerged 10 minutes in buffer before functionalization to dissolve the sucrose layer. Then the sensors were dipped for 15 minutes in 100 nM DNA containing solutions (biotinylated systems A-B) and rinsed in buffer solution (10 mM HEPES pH 7.4, 50 mM NaCl, 100 mM KCl and 0.05% v/v surfactant P20) for 10 minutes. The functionalized sensors were next dipped in different ruthenium(II) complex solution at different concentrations for 2 minutes interspersed by a rinsing step in the buffer solution during 4 minutes. Reference sensors without DNA immobilization were used to subtract the non-specific adsorption on the SA layer. The sensorgrams were fitted using a heterogeneous model. The reported values are the means of representative independent experiments, and the errors provided are standard deviations from the mean. Each experiment was repeated at least two times.

Computational studies

The geometry of all complexes were optimized at the B3LYP/6-31 g* level using Gaussian 09 software. [16] Although the chirality of the complexes might play a role in the binding association, enantiomeric resolution is beyond the scope of the present study. Therefore, for ease of interpretation, the  enantiomers of the complexes were chosen for the computational studies. For the targets we used for the quadruplex, the crystal structure of parallel quadruplexes from human telomeric DNA (PDB entry: 1KF1) whereas we had to construct an hairpin sequence similar to the one used for BLI experiments. First, an available hairpin sequence (PDB entry 2VAH) was modified by removing the nitrogenous base of the nucleotides differing from the BLI hairpin sequence. The abasic structure was then completed using xleap from the AMBER 12 software package, [17] solvated in a TIP3P water box. Sodium cations were added until the global charge was neutral and long-range electrostatic interactions were computed using the particlemesh Ewald method with a cut-off value of 10 . After minimization and heating, an MD simulation was run at 300 K for 10 ns with the time step set at 1 fs. Among the different hairpin conformations obtained, with the thymine residues moving in and out of the hairpin's T-loop, an "open" structure was selected to account for any possible insertion of the complexes inside the loop.

For docking calculations , we used the AutoDock 4.0 software package. [13] A grid of 80*80*80 points with a spacing of 0.5 Ả between these points was used; non-polar hydrogen atoms were merged and Gasteiger-Huckel charges were added on both the ligand and the target. The parameters for the Ru atom were set at r=2.96 Ả, q= +2.0 and the van der Waals well depth was 0.056 kcal.mol -1 . The docking calculations involved a genetic algorithm search generating 50 docked structures. A default protocol was applied, with an initial population of 150 randomly placed individuals, a maximum number of 2.5*10 5 energy evaluations, a maximum number of 2.7*10 4 generations, a mutation rate of 0.02, and a crossover rate of 0.8. Results differing by less than 2 in positional root-mean square deviation (RMSD) were clustered together.

For the two docked positions of complex 1 against the quadruplex, NCI computations were performed using the NCI-Plot package [18] with the promolecular densities, using only the upper tetrad of 1KF1. For this analysis, since no parameter exists for the ruthenium atom, an iron atom was used.

Figure 1 :

 1 Figure 1: Structures of complexes 1-4.

  , G. Piraux, M. Daenen, M. Abraham, Dr. L. Troian-Gautier and Prof. B. Elias Université catholique de Louvain (UCLouvain) Institut de la Matière Condensée et des Nanosciences (IMCN), Molecular Chemistry, Materials and Catalysis (MOST) Place Louis Pasteur 1, bte L4.01.02, 1348 Louvain-la-Neuve (Belgium)

  Data were measured at room temperature in MeCN with 0.1 M Bu4NClO4 as supporting electrolyte, with a concentration of complexes of 0.8 mM[b] Potentials are given vs. Ag/AgCl. Excited-state potentials estimated from the equations: E*1/2red = E1/2red + E0-0. The energy of the excited state, E0-0, was estimated by Franck-Condon line-shape analysis of the emission spectra at 298 K in MeCN.

  3] 2+ . The first reduction wave is attributed to the reduction of the dph or bpph ligand. Both TAP analogues 3 and 4 are the easiest to reduce displaying more positive reduction potentials than [Ru(TAP)3] 2+ . These results indicate that the HOMO of complexes 1-4 is centred on the metal core while their LUMO orbital is localized on the ligands, which is in good agreement with the occurrence of a MLCT transition as suggested by light absorption (vide infra) and literature data. The excited-state reduction potentials were estimated based on the ground-state reduction potentials and the energy stored in the excited state, E0-0. As expected, complexes 3 and 4 display strong photo-oxidizing power (E*red = +1.21 and +1.25 V vs.

Figure 2 .

 2 Figure 2. Absorption (solid line) and emission (dashed line) spectra of [Ru(phen)2dph] 2+ 1 (orange), [Ru(phen)2bpph] 2+ 2 (blue), Ru(TAP)2dph] 2+ 3 (green) and [Ru(TAP)2bpph]2+ 4 (red) in acetonitrile at room temperature.

Figure 3 :

 3 Figure 3: Evolution of the luminescence spectra of (A) [Ru(TAP)2dph] 2+ 3 (λexc = 479 nm), (B) [Ru(TAP)2bpph]2+ 4 (λexc = 482 nm) in the presence of increasing concentrations of dGMP, while the complex concentration was kept at 5 µM.. Measurements were made in 50 mM Tris•HCl, 50 mM NaCl, pH = 7.4, under ambient air conditions. Insets: Stern-Volmer plots obtained upon the addition of dGMP to each complex.

  [c] ΔGET values with Poly(dG-dC)2 are given for comparison purpose in order to indicate the effect of the guanine stack.Time-resolved spectroscopies were then used to obtain further information about the light-induced quenching processes. The experiments were carried out in argon-purged buffered solutions using [Ru(TAP)2bpph] 2+ . The excited-state quenching with dGMP was first repeated as the excited-state lifetime of[Ru(TAP)2bpph] 2+ in 50 mM Tris•HCl, 50 mM NaCl and pH = 7.4 was determined as 3.1 µs. Upon the addition of dGMP, a drastic decrease of the excited-state lifetime was observed (Figure4A). The corresponding Stern-Volmer plot was linear and allowed to determine the quenching rate constant, kq = 2.45 x10 9 M -1 s -1 . This agrees with the quenching rate constant determined via steady-state spectroscopy. We then turned to nanosecond transient absorption spectroscopy. The excited state absorption features of [Ru(TAP)2bpph] 2+ were in line with prototypical Ru(II) complexes, i.e. a strong bleaching observed in the visible part of the spectrum, corresponding to light oxidation of Ru(II) to Ru(III) and positive absorption features in the UV and red region of the spectrum, in agreement with the formation of a reduced ligand (Figure

Figure 4 :

 4 Figure 4: (A) Time-resolved excited-state quenching of [Ru(TAP)2bpph] 2+ with increasing amount of dGMP. The inset shows the corresponding Stern-Volmer plot from which kq = 2.45 x10 9 M -1 s -1 was determined. (B) Transient absorption spectroscopy of [Ru(TAP)2bpph] 2+ (red), [Ru(TAP)2bpph] 2+ with 10 mM of dGMP (black) and [Ru(TAP)2bpph] 2+ with 43 µM G4 (red) recorded 200 ns (integrated for 100 ns) following pulsed 480 nm light excitation. The experiments using G4 were recorded in 10 mM HEPES, pH 7.5, 50 mM NaCl, 100 mM KCl under argon whereas the other experiments were recorded in 50 mM Tris•HCl, 50 mM NaCl, pH = 7.4, under argon.

Figure 5 :

 5 Figure 5: Biomolecular models used for bio-layer interferometry studies: A, intramolecular G-quadruplex structure; B, duplex DNA hairpin.

Figure 6 :

 6 Figure 6: Steady state photoluminescence titration of (A) [Ru(phen)2dph] 2+ 1 (purple) and [Ru(phen)2bpph] 2+ 2 (grey) and (B) [Ru(TAP)2dph] 2+ 3 (purple) and [Ru(TAP)2bpph]2+ 4 (grey) by increasing G4-DNA (full diamonds) or ds-DNA (empty diamonds) ratio, while the complex concentration was kept at 5 µM. The fitted curves are obtained using a modified McGhee-von Hippel equation (see the SI) and drawn as solid and dashed curves for G4 and ds-DNA respectively. One site in ds-DNA corresponds to one base pair and to one quartet in G4 structures and I0, the photoluminescence of the complex in the absence of DNA. λexc = 450 nm; λEm = 773 (1), 727 (2), 667 (3) and 650 nm (4).

Figure 7 :

 7 Figure 7: Isoaffinity kinetic plot of rate constants measured by BLI. The dashed diagonals depict the equilibrium binding constants and are shown to help with the visualization of the affinity distribution. Complexes 1 and 3 are plotted in the presence of G4 DNA (G4) and complexes 2 and 4 both in presence of G4 DNA and duplex DNA hairpin (HP).

Figure 8 :

 8 Figure 8: The main docked positions of complex 1 (a) against a hairpin structure, inside the T-loop (orange, scoring function -4.45 kcal/mol) or in the minor groove (red, scoring function -4.25 kcal/mol), (b) against a human Gquadruplex (PDB entry 1KF1), stacked above a G quartet (orange, scoring function -4.45 kcal/mol) or embedded in the groove (red, scoring function -6.22 kcal/mol ).

Figure 9 :

 9 Figure 9: NCI surfaces obtained for the complex 1 (red) in interaction with the human G-quadruplex (PDB entry 1KF1), the gradient cut-off is 0.45 u.a. and the colour scale set to <0.07 u.a.; (a) interaction within the G-quadruplex groove, (b) π-stacked position over the G-tetrad.

Table 1 :

 1 Oxidation

(Eox)

and reduction (Ered) potentials of complexes 1-4 in acetonitrile. Estimated reduction potentials (E*red) of complexes 1-4 in acetonitrile.

Table 2 :

 2 Absorption and emission data in acetonitrile for complexes 1-4 at 298 K.

	Complex	λAbs	λEm
		[nm]	[nm] [b]
		(ε) [a]

Table 3 :

 3 Photo-induced ET from guanine-based moieties to 3 and 4.

			∆GET [eV] [a]
	Compound	dGMP	Poly(dG-dC)2 [c]	kq [M -1 s -1 ] [b]
	[Ru(TAP)2dph] 2+ 3	-0.15	-0.40	2.20 x 10 9
	[Ru(TAP)2bpph] 2+ 4	-0.19	-0.44	3.02 x 10 9

Table 4 :

 4 Equilibrium dissociation constants (KD) for the interaction of complexes 1-4 with G4 and duplex DNA estimated from steady state luminescence titrations.

		G4 DNA (wtTel23)	Duplex DNA (CT-DNA)
	Compound	KD (µM)	I/I0 max	KD (µM)	I/I0 max
	[Ru(phen)2dph] 2+ 1	18.9	4.9	652	1.8
	[Ru(phen)2bpph] 2+ 2	14.6	3.7	91	3.1
	[Ru(TAP)2dph] 2+ 3	31.7	0.36	198	0.28
	[Ru(TAP)2bpph] 2+ 4	19.1	0.32	31	0.14

Binding constants are obtained using a McGhee-von Hippel type equation; the binding site corresponds to base pair or G-quartet (best fit). Errors estimated to 5%.

Acknowledgements: M. G., G.P., M.D., M.A. and B. E. gratefully acknowledge the Université catholique de Louvain (UCLouvain), and the Prix Pierre et Colette Bauchau for financial support. This work was supported by the Fonds de la Recherche Scientifique (F.R.S.-FNRS) under grant no. U.N021.21 (B.E.) and the Collaborateur Scientifique fellowship (L.T.-G). This work was partially supported by the "Agence National de la Recherche": LabEx ARCANE and CBH-EUR-GS (ANR-17-EURE-0003), and the r gion Auvergne-Rh ne-Alpes. The NanoBio-ICMG platforms (UAR 2607) are acknowledged for their support Keywords: G-quadruplex DNA • Ruthenium complexes • Photooxidation• Doking experiments • Bio-layer interferometry


Marceau Quatredeniers

Frank Bienaimé

Giulia Ferri

Pierre Isnard

Esther Porée

Katy Billot

Eléonore Birgy

Manal Mazloum

Salomé Ceccarelli

Flora Silbermann

Simone Braeg

Thao Nguyen-Khoa

Rémi Salomon

Marie-Claire Gubler

E Wolfgang Kuehn

Sophie Saunier

Amandine Viau
email: amandine.viau@inserm.fr

The renal inflammatory network of nephronophthisis

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

INTRODUCTION

The primary cilium is a solitary antenna-like structure protruding from the apical surface of renal epithelial tubular cells, that is believed to act as complex macromolecular sensors and signal transducers [START_REF] Reiter | The base of the cilium: Roles for transition fibres and the transition zone in ciliary formation, maintenance and compartmentalization[END_REF][START_REF] Satir | CILIA: Before and after. CILIA: Before and after[END_REF].

Renal ciliopathies are inherited disorders caused by mutations in genes encoding proteins that localize to primary cilia [START_REF] Braun | Ciliopathies[END_REF][START_REF] Devlin | Renal ciliopathies[END_REF]. These renal ciliopathies share common features, including tubular dilation, interstitial fibrosis and loss of tubular cell differentiation. Yet, the spectrum of renal ciliopathies encompasses genetically distinct and phenotypically heterogeneous diseases that manifest from early-childhood to late-adulthood eventually leading to kidney failure.

Autosomal dominant polycystic kidney disease (ADPKD), the most common ciliopathy manifesting in adulthood, is characterized by the development of multiple renal cysts resulting in progressive kidney enlargement and kidney function decline. Most ADPKD cases are caused by inactivating mutations in PKD1, which encodes polycystin 1 (PC1), that localizes to the ciliary membrane. By contrast, nephronophthisis (NPH), the second most prevalent renal ciliopathy, is a rare autosomal recessive disorder that manifests in children and young adults by enuresis due to impaired urine concentrating ability and progressive kidney function decline.

Although some cysts are observed in NPH, cyst burden remains marginal compared to ADPKD, and the kidneys mostly appear small and fibrotic. NPH is a genetically heterogeneous disorder caused by mutations in more than 20 genes identified so far [START_REF] Srivastava | Many genes-one disease? Genetics of nephronophthisis (NPHP) and NPHP-associated disorders. Many genes-one disease? Genetics of nephronophthisis (NPHP) and NPHP-associated disorders[END_REF]. NPHP1 mutations are the most common genetic cause, accounting for 40-50% of identified causative mutations in NPH [START_REF] König | Phenotypic spectrum of children with nephronophthisis and related ciliopathies[END_REF].

Most of the proteins encoded by NPHP genes assemble in functional modules that control cilia morphology and gate protein entry and exit to and from the cilia [START_REF] Sang | Mapping the NPHP-JBTS-MKS protein network reveals ciliopathy disease genes and pathways[END_REF][START_REF] Toriyama | The ciliopathy-associated CPLANE proteins direct basal body recruitment of intraflagellar transport machinery[END_REF]. Yet, how disruption of these complexes translate into kidney damage is poorly understood.

Primary cilia of renal tubular cells are required for proper kidney development and maintenance [START_REF] Lin | Kidney-specific inactivation of the KIF3A subunit of kinesin-II inhibits renal ciliogenesis and produces polycystic kidney disease[END_REF]. Yet, none of the mutations reported in renal ciliopathies abolish ciliogenesis. While renal tubular cells remain ciliated in renal ciliopathies, they may display either increased or decreased cilia length [START_REF] Hopp | Functional polycystin-1 dosage governs autosomal dominant polycystic kidney disease severity[END_REF][START_REF] Davis | TTC21B contributes both causal and modifying alleles across the ciliopathy spectrum[END_REF]. It is believed that the genetic defects involved in renal ciliopathies adversely affect cilia composition and signaling which results in modifications of tubular cell behavior that, in turn, alter kidney morphology and function. Even outside cilia, NPHP regulate cellular junctions and epithelialization which may also contribute to NPH-associated tubular defects [START_REF] Delous | Nephrocystin-1 and nephrocystin-4 are required for epithelial morphogenesis and associate with PALS1/PATJ and Par6[END_REF].

Lessons gathered from acquired chronic kidney disease (CKD) models indicate that progressive renal scarring involves interplay between tubular, immune and mesenchymal cells that prompts immune cell infiltration, fibroblast activation and nephron loss [START_REF] Andrade-Oliveira | Inflammation in renal diseases: New and old players[END_REF][START_REF] Bienaimé | Stat3 Controls Tubulointerstitial Communication during CKD[END_REF][START_REF] Kitagawa | Blockade of CCR2 ameliorates progressive fibrosis in kidney[END_REF].

Concordant evidence has pinpointed an important role of renal inflammation in the progression of ADPKD. Indeed, macrophages infiltrate the kidney of ADPKD patients and mice and promote cyst growth [START_REF] Karihaloo | Macrophages Promote Cyst Growth in Polycystic Kidney Disease[END_REF]. Cilia signaling seems to be directly involved in this process as cilia ablation prevents macrophage recruitment in an orthologous mouse model of ADPKD [START_REF] Viau | Cilia-localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney[END_REF].

Mechanistically, cilia positively regulate the expression of the macrophage chemoattractant CCL2 in Pkd1-deficient tubular cells both in vivo and in vitro. Consistently, kidney specific disruption of Ccl2 or inhibition of the CCL2 receptor reduce cyst burden in Pkd1 mutant mice [START_REF] Viau | Cilia-localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney[END_REF][START_REF] Cassini | Mcp1 Promotes Macrophage-Dependent Cyst Expansion in Autosomal Dominant Polycystic Kidney Disease[END_REF].

In contrast to ADPKD, the role of immune cells in NPH has received little attention. Recent evidence suggests that renal inflammation may be involved in the disease. First, the inactivation of TLR2, a critical receptor of the immune system, prevents renal damage in an orthologous mouse model of a rare form of NPH caused by a mutation in Glis2/Nphp7 (Glis2 lacZ/lacZ mice) [START_REF] Attanasio | Loss of GLIS2 causes nephronophthisis in humans and mice by increased apoptosis and fibrosis[END_REF][START_REF] Jin | Innate Immune Signaling Contributes to Tubular Cell Senescence in the Glis2 Knockout Mouse Model of Nephronophthisis[END_REF]. Besides, we previously reported that LKB1, a ciliary kinase involved in the control of cell size and metabolism, interacts with NPHP proteins including NPHP1 [START_REF] Viau | Cilia-localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney[END_REF]. In human, heterozygous mutation in STK11, the gene encoding LKB1, does not lead to NPH but to Peutz-Jeghers syndrome, characterized by benign tumors of the skin and intestine, as well as cancer.

Homozygous mutations in LKB1 have not been reported in humans, while mice with bi-allelic inactivation of Lkb1 die at mid-gestation [START_REF] Ollila | The tumor suppressor kinase LKB1: Lessons from mouse models[END_REF]. Yet, mice bearing a selective inactivation of Lkb1 in renal tubules (Lkb1 Tub mice) develop a NPH-like phenotype recapitulating the consequences of NPHP1 loss in humans including impaired urine concentration, thickened tubular basement membranes and fibrosis [START_REF] Viau | Cilia-localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney[END_REF]. The relevance of this mouse model to human NPH is not only sustained by the interaction of LKB1 with NPH proteins but also by parallel functions of NPHP1 and LKB1 in tubular cells. Indeed, in vitro, both NPHP1 and LKB1 repress the expression of CCL2, while CCL2 upregulation and macrophage recruitment are observed in vivo at an early time point of the development of the disease in Lkb1 Tub mice [START_REF] Viau | Cilia-localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney[END_REF]. Importantly, CCL2 dependent recruitment of immune cells has been repeatedly shown to promote renal fibrosis in different acquired CKD models [START_REF] Kitagawa | Blockade of CCR2 ameliorates progressive fibrosis in kidney[END_REF][START_REF] Ollila | The tumor suppressor kinase LKB1: Lessons from mouse models[END_REF]. In contrast to these observations made in NPH mice models, most of the histopathologic studies performed in human NPH did not report immune cells infiltration as a notable feature of the disease [START_REF] Srivastava | Many genes-one disease? Genetics of nephronophthisis (NPHP) and NPHP-associated disorders. Many genes-one disease? Genetics of nephronophthisis (NPHP) and NPHP-associated disorders[END_REF]. Yet, these studies have limitations: they are rare, included a limited number of patients (≤9 patients) and lacked standardized quantitative assessment of renal inflammation.

Considering the paucity and the inconstancy of the data regarding renal inflammation in NPH, we decided to explore the interplay between immune and tubular cells in NPH through a translational approach combining data from transgenic animals and human patients.

RESULTS

Nephronophthisis is associated with macrophage recruitment and enhanced CCL2 expression.

To elucidate the contribution of immune cell recruitment in the phenotype of NPH, we first aimed to assess whether, similar to ADPKD, macrophage infiltration occurs in human NPH.

Histology inspection of kidney tissues from NPH patients mostly bearing NPHP1 mutations, revealed significant infiltration by immune cells (Figure 1A). Immunolabelling identified macrophages (CD68-positive cells) as an important contingent of cells infiltrating the kidneys from NPH patients as compared to control individuals (Figure 1B-C). As CCL2 has been shown to be the major chemokine responsible for macrophage recruitment in ADPKD, we analyzed primary tubular epithelial cells derived from urine (UREC) of patients bearing NPHP1 mutations and controls, including both age-matched controls and healthy relatives. UREC derived from NPHP1 patients showed decreased expression of NPHP1 mRNA associated with increased expression of LCN2 transcript, a marker of tubular injury known to correlate with the progression of CKD (Supplemental Figure 1A-B). Tubular cells derived from NPHP1 patients also showed enhanced expression of CCL2 transcript (Figure 1D and Supplemental Figure 1C). Consistently, we observed that NPHP1 patients display a higher urinary excretion rate of CCL2 than controls (Figure 1E). Of note, in NPHP1 patients, CCL2 urinary excretion was not correlated with eGFR (Pearson correlation P=0.218, R 2 =0.183) and no difference was observed according to the patient genotype (homozygous deletion vs heterozygous compound mutations). Collectively, these data revealed that, similarly to ADPKD, NPH patients show increased renal tubular expression of CCL2 associated with macrophage recruitment.

Tubule-specific Ccl2 inactivation does not prevent nephronophthisis-like phenotype.

Deletion of Lkb1 in the distal tubule of mouse kidney cells results in an NPH phenotype associated with CCL2 upregulation and macrophage recruitment [START_REF] Viau | Cilia-localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney[END_REF][START_REF] Braga | CCR2 contributes to the recruitment of monocytes and leads to kidney inflammation and fibrosis development[END_REF]. To determine if the deletion of Ccl2 together with Lkb1 ameliorates the NPH-like renal disease, we crossed tubulespecific Lkb1 tub mice with mice bearing Ccl2 floxed alleles and compared mice with distal tubular inactivation of Lkb1 alone (Lkb1 tub ), Ccl2 alone (Ccl2 tub ) or Lkb1 and Ccl2 (Lkb1 tub ; Ccl2 tub ) with littermates controls. At 10 weeks of age, quantitative RT-PCR revealed that Ccl2 inactivation in distal tubules drastically blunted Ccl2 induction in Lkb1 deficient kidneys (Figure 2A). Macroscopic inspection revealed irregular kidneys in both Lkb1 tub and Lkb1 tub ; Ccl2 tub mice associated with reduced kidney size, which was even more pronounced in Lkb1 tub ; Ccl2 tub mice (Figure 2B-C and Supplemental Figure 2A-B). Lkb1 tub and Lkb1 tub ; Ccl2 tub mice displayed similar urine concentration defect and loss of kidney function (Figure 2D-F). Renal histology revealed similar tubular and glomerular basement membranes thickening, interstitial inflammation, fibrosis, and tubular dilation in Lkb1 tub and Lkb1 tub ; Ccl2 tub mice. Consistently, Ccl2 inactivation neither affected the upregulation of the tubular injury marker Lcn2, nor the extracellular matrix deposition markers Col1a1 and Tgfb1, in Lkb1 deficient kidneys (Figure 2G-L). Ccl2 tub animals had normal renal function and histology (Supplemental Figure 2C-J). Unexpectedly, contrary to what was observed in Pkd1 deficient mice [START_REF] Viau | Cilia-localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney[END_REF][START_REF] Cassini | Mcp1 Promotes Macrophage-Dependent Cyst Expansion in Autosomal Dominant Polycystic Kidney Disease[END_REF], Ccl2 tubular inactivation did not prevent macrophage infiltration of Lkb1 deficient kidneys as judged by quantification of both F4/80 immunolabelling and Adgre1 transcript (Figure 2M-O). Collectively, these results demonstrate that, while distal tubules represent the major source of renal CCL2 in Lkb1 tub mice, CCL2 induction in those cells does not mediate macrophage infiltration of the kidney nor kidney damage.

Early NPH renal disease in mice is associated with a prominent immune signature.

So far, our findings revealed macrophage infiltration in NPH but, as opposed to ADPKD, this recruitment appears independent of tubular CCL2. To approach the mechanisms underlying renal inflammation in NPH in an unbiased manner, we compared microarray-based transcriptomes from Lkb1 Tub and Glis2 lacZ/lacZ kidneys [START_REF] Viau | Cilia-localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney[END_REF][START_REF] Attanasio | Loss of GLIS2 causes nephronophthisis in humans and mice by increased apoptosis and fibrosis[END_REF] to controls. Both mouse models recapitulated the features of human NPH: polyuria followed by progressive interstitial fibrosis, tubular basement membrane thickening, tubular dilations and immune cell infiltration [START_REF] Viau | Cilia-localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney[END_REF][START_REF] Attanasio | Loss of GLIS2 causes nephronophthisis in humans and mice by increased apoptosis and fibrosis[END_REF][START_REF] Han | Deletion of Lkb1 in renal tubular epithelial cells leads to CKD by altering metabolism[END_REF]. Transcriptome datasets were obtained at an early time point of disease development (5 and 4 weeks, respectively) when histological lesions were sparse and renal function decline mild [START_REF] Viau | Cilia-localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney[END_REF][START_REF] Attanasio | Loss of GLIS2 causes nephronophthisis in humans and mice by increased apoptosis and fibrosis[END_REF]. Gene overlap analysis identified 1,262 genes that were commonly deregulated in the two models (Figure 3A-B and Supplemental Table 3). Gene set enrichment analysis (GSEA) of these genes identified a total of 72 enriched pathways (FDR < 0.05). Among those pathways, 8 were downregulated, consisting mostly of metabolic processes, while 64 biological processes were upregulated (Supplemental Tables 45). Among the latter, the GSEA revealed a high enrichment in biological processes linked to immune response and inflammation (Figure 3C).

Network analysis showed a marked association of the common regulated genes with immune pathways (Figure 3D and Supplemental Figure 3). This unbiased analysis identified renal inflammation as an early and prominent phenomenon in the course of NPH mouse models.

Lkb1 tubular inactivation activates multiple chemotactic pathways and drives the recruitment of distinct immune cell populations independently of CCL2.

To identify specific cytokines that might illuminate CCL2 independent immune cell recruitment in NPH, we matched the common upregulated genes between Lkb1 Tub and Glis2 lacZ/lacZ kidneys with UniProt database. This analysis retrieved 17 pro-inflammatory cytokines, other than CCL2 (Supplemental Table 6) that have been implicated in macrophage, neutrophil, T cell and/or dendritic cell chemotaxis. Quantifying these transcripts in kidneys from Lkb1 tub , Lkb1 tub ; Ccl2 tub and control mice, we observed that Ccl2 disruption had no impact on the up-regulation of these immune mediators in Lkb1 deficient kidneys (Figure 4A and Supplemental Figure 4). In line with the different immune cell populations attracted by these cytokines, immunolabelling demonstrated enhanced neutrophils (Ly-6B.2-positive cells) and T cells (CD3-positive cells) recruitment in both kidneys from Lkb1 tub and Lkb1 tub ; Ccl2 tub mice as compared to control mice (Figure 4B-F). These results uncover a CCL2 independent inflammatory network in NPH-like renal disease.

The identified cytokine signature is not a general feature of renal ciliopathies.

At this point, our findings identified a set of pro-inflammatory cytokines that is expressed early on in the course of NPH renal disease in mice. We next asked whether the observed immune signature was consistent across renal ciliopathies. To address this question, we compared Lkb1 tub mice, mimicking human NPH, to an orthologous model of ADPKD, in which postnatal inactivation of Pkd1 (Pkd1 tub mice) results in slow-onset PKD, recapitulating human disease. We focused on early time point of disease development (5 weeks and 10 weeks, respectively). At 5 weeks of age, Lkb1 tub mice present scarce renal lesions while Pkd1 tub kidneys showed mild tubular dilations 6 weeks after the induction of Pkd1 recombination, leading to kidney enlargement (Figure 5A-B). Quantitative RT-PCR analysis confirmed a marked upregulation of the NPH inflammatory signature in the kidneys from Lkb1 tub mice at 5 week of age (Figure 5C and Supplemental Figure 5). In contrast, while Pkd1 deficient kidneys displayed a slight but significant induction of CCL2 (Supplemental Figure 5A), most of the other cytokines precociously triggered by Glis2 or Lkb1 deletion were not induced 6 weeks after the induction of Pkd1 recombination (Figure 5C and Supplemental Figure 5).

Reanalyzing RNA-seq data from a study involving Pkd2 tub mice, another orthologous rodent model of ADPKD [START_REF] Kim | Kruppel-Like Zinc Finger Protein Glis2 Is Essential for the Maintenance of Normal Renal Functions[END_REF], further confirmed these results (Supplemental Figure 6 and Supplemental Table 7).

Immunostaining and qPCR analysis demonstrated an infiltration of Lkb1 deficient kidneys by neutrophils, macrophages and T cells. In contrast, only rare and focal macrophage infiltrates surrounding expanding cysts were observed in Pkd1 tub mice (Figure 5D-H). Collectively, these data pinpoint important divergence in the inflammatory processes triggered by Pkd1 or Lkb1 deletion. While the inactivation of the former produced tubular dilations associated with focal and CCL2 dependent macrophage recruitment around expanding cysts, the latter is characterized by the up-regulation of multiple cytokines driving kidney infiltration by neutrophils, macrophages and T cells.

Identifying the inflammatory network associated with loss of NPHP1 function in humans.

Considering the distinct immune cell populations infiltrating the kidneys of Lkb1 tub mice, we aimed to determine if such phenomena were also found in NPH patients. CD15 and CD3 immunostaining revealed increased neutrophils and T cells recruitment to the renal parenchyma of NPH patients compared to controls (Figure 6A-B). In NPH patients, neutrophil infiltrate correlated with the severity of renal lesions evaluated by the interstitial fibrosis and tubular atrophy (IFTA) score (Pearson correlation P=0.02, R 2 =0.552) while no correlation was observed with T cells infiltrate (Pearson correlation P=0.69, R 2 =0.027). In contrast, these immune cell populations were less abundant in kidney biopsies from patients suffering from acquired non-immune chronic kidney disease (CKD) caused by diabetes or hypertension (Figure 6A-B), supporting the notion that a specific inflammatory network delineates human NPH.

To determine if the cytokine network that we identified as features of NPH-like mouse models was relevant to human NPH, we quantified the mRNA abundance of its components in UREC from NPHP1 patients and controls. As there are no human orthologs for CCL6, 9 and 12, we focused our analysis on the 14 other inflammatory mediators. Indeed, we found 8 transcripts upregulated in human tubular cells with NPHP1 mutations (CCL5, CXCL1, CXCL10, CXCL16, CXCL17, CX3CL1, IL1RN and LGALS9), while two were not differentially regulated (CCL19 and IL33) and 4 were not detected (CXCL9, CXCL12, CXCL14, IL34) (Figure 6C-L). No difference of these cytokines mRNA expression was observed between age-matched controls and relatives with normal kidney function (Supplemental Figure 7). In addition, the mRNA level of CXCL1, CXCL17, IL1RN and LGALS9 were significantly higher in UREC derived from NPHP1 patients than from patients suffering from other CKD. Of note, CXCL17 mRNA was significantly correlated with eGFR of NPHP1 patients (Supplemental Table 8). CX3CL1, CXCL10 and CXCL16 show the same trend, while not reaching statistical significance (Figure 6C-L). Although the molecular mechanisms leading to the upregulation of cytokine expression remain unclear, our in vitro data demonstrate that renal tubular cells are the source of cytokine expression in NPH. These data reveal that, in addition to previously described CCL2 upregulation and macrophage infiltration, NPH is characterized by a complex and specific cytokine signature, which is associated with kidney infiltration by neutrophils and T cells.

DISCUSSION

In sharp contrast with the abundant literature regarding the molecular mechanisms of disease progression in ADPKD, insights into the pathophysiology of NPH remain scarce. Despite a growing list of causative gene defects, it is unknown how dysfunction of ciliary NPHP complexes results in the unique renal manifestations of NPH. While some data point to a defect in renal development, a prominent feature is severe progressive fibrosis, resulting in kidney failure during the second decade of life. The study of the molecular pathogenesis of NPH is hampered by the lack of orthologous mouse models recapitulating the fibrotic disease observed in most NPH patients. Indeed, neither Nphp1 -/-nor Nphp4 -/-mice develop renal fibrosis [START_REF] Zhang | Cyclin-Dependent Kinase 1 Activity Is a Driver of Cyst Growth in Polycystic Kidney Disease[END_REF][START_REF] Jiang | Targeted disruption of Nphp1 causes male infertility due to defects in the later steps of sperm morphogenesis in mice[END_REF] and only few mouse models orthologous to rare forms of the disease phenocopy the human pathology [START_REF] Attanasio | Loss of GLIS2 causes nephronophthisis in humans and mice by increased apoptosis and fibrosis[END_REF][START_REF] Han | Deletion of Lkb1 in renal tubular epithelial cells leads to CKD by altering metabolism[END_REF][START_REF] Won | NPHP4 is necessary for normal photoreceptor ribbon synapse maintenance and outer segment formation, and for sperm development[END_REF]. In addition, since genetic testing has become more widely available, kidney biopsies are less and less performed in NPH, limiting availability of kidney tissues from patients and insights into the disease.

Renal inflammation has emerged as an important mediator of fibrosis in acquired chronic kidney disease [START_REF] Andrade-Oliveira | Inflammation in renal diseases: New and old players[END_REF]. In ADPKD, CCL2 dependent macrophage recruitment promotes disease progression [START_REF] Viau | Cilia-localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney[END_REF][START_REF] Cassini | Mcp1 Promotes Macrophage-Dependent Cyst Expansion in Autosomal Dominant Polycystic Kidney Disease[END_REF]. Having previously shown that both NPHP1 and LKB1 repress CCL2 expression in vitro and that Lkb1 inactivation in mice results in an NPH-like phenotype preceded by CCL2 upregulation and macrophage recruitment [START_REF] Viau | Cilia-localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney[END_REF], we first sought to determine if CCL2 induction and macrophage recruitment were also hallmarks of human NPH. In line with the data gathered from Lkb1 tub mice, we observed significant macrophage infiltration in kidney biopsies from NPH patients and enhanced CCL2 levels in the urine. We then assessed the role of CCL2 in the Lkb1 tub phenotype by inactivating Ccl2 specifically in tubular cells.

Contrary to its effect in ADPKD models [START_REF] Viau | Cilia-localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney[END_REF][START_REF] Cassini | Mcp1 Promotes Macrophage-Dependent Cyst Expansion in Autosomal Dominant Polycystic Kidney Disease[END_REF], Ccl2 invalidation had no impact on the NPH phenotype of Lkb1 tub mice and did not reduce macrophage infiltration, nor inflammatory cytokine expression in Lkb1 deficient kidneys. Strikingly, CCL2 inactivation even aggravated kidney size decrease and macrophage infiltration in Lkb1 tub mice, suggesting that CCL2 may even exert a limited protective effect on Lkb1 deficient kidneys.

Beyond ADPKD, CCL2 is instrumental to macrophage recruitment and kidney damage in a range of experimental renal diseases including unilateral ureteric obstruction, acute kidney injury, diabetic nephropathy, subtotal nephrectomy [START_REF] Airik | Renal-retinal ciliopathy gene Sdccag8 regulates DNA damage response signaling[END_REF] or in response to renal infection [START_REF] Akli Ayoub | Functional Interaction between Angiotensin II Receptor Type 1 and Chemokine (C-C Motif) Receptor 2 with Implications for Chronic Kidney Disease[END_REF].

Thus, the fact that renal inflammation and macrophage recruitment in Lkb1 tub mice is independent of tubular CCL2 suggests that the mechanisms driving renal inflammation in NPH are distinct from those implicated in most kidney diseases. Of note, as we deleted CCL2 only in Lkb1 deficient distal tubules, we cannot formerly exclude that CCL2 induction in other cell types may be involved in disease progression. Yet, our results clearly rule out the contribution of CCL2 to immune cell recruitment and disease progression in Lkb1 deficient cells. To get further insight into the nature of renal inflammation in NPH, we analyzed the population of immune cells infiltrating the kidneys in human NPH and Lkb1 tub mice. In both case, macrophage infiltration was associated with the recruitment of T cells and neutrophils, by opposition to ADPKD where macrophages are the prominent drivers of inflammation [START_REF] Karihaloo | Macrophages Promote Cyst Growth in Polycystic Kidney Disease[END_REF][START_REF] Viau | Cilia-localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney[END_REF][START_REF] Cassini | Mcp1 Promotes Macrophage-Dependent Cyst Expansion in Autosomal Dominant Polycystic Kidney Disease[END_REF].

To identify the mediators driving early NPH pathology, we analyzed common regulated genes in the transcriptome of Glis2 lacZ/lacZ and Lkb1 tub kidneys, two genetically distinct mouse models of NPH. Unbiased pathway enrichment analysis of their common upregulated genes revealed a striking preponderance of inflammatory processes, pinpointing that early inflammation is a prominent feature of experimental NPH. Indeed, we found a large number of soluble mediators of immune activation in urinary tubular cells derived from NPH patients. A subset of 7 pro-inflammatory cytokines were specifically upregulated in NPHP1 UREC and NPH mouse models as compared to urinary tubular cells derived from non-ciliopathy CKD patients: CX3CL1, CXCL1, CXCL10, CXCL16 and CXCL17, IL1RN and LGALS9. Thus, these cytokines appear more specific of the early renal inflammation observed in NPH.

CXCL1 induces the recruitment of various immune cell types, especially neutrophils, through its receptor CXCR2. The CXCL1/CXCR2/neutrophil axis plays an important role in the pathology of acute kidney inflammation in different mouse models [START_REF] Berry | Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone In Brief Sodium gradient guides the migration of innate immune cells in the kidney during infections. Article Renal Sodium Gradient Orchestrates a Dynamic Antibacterial Defense Zone[END_REF][START_REF] Liu | Inhibition of CXCL1-CXCR2 axis ameliorates cisplatin-induced acute kidney injury by mediating inflammatory response[END_REF][START_REF] Ranganathan | CXCR2 knockout mice are protected against DSS-colitis-induced acute kidney injury and inflammation[END_REF]. Interestingly, we observed concordant neutrophil infiltration in human NPH kidneys and mice, even at an early stage of the disease. In contrast, neutrophil recruitment does not occur in ADPKD [START_REF] Viau | Cilia-localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney[END_REF]. As neutrophils are known to promote renal fibrosis [START_REF] Brown | Toll-like receptor 4 ligation on intrinsic renal cells contributes to the induction of antibody-mediated glomerulonephritis via CXCL1 and CXCL2[END_REF] it is plausible that CXCL1 mediated neutrophil recruitment contributes to phenotypic changes in NPH.

Our findings add to the recently identified expression of CXCL17 in the kidney [START_REF] Wang | MMP-9-positive neutrophils are essential for establishing profibrotic microenvironment in the obstructed kidney of UUO mice[END_REF], the last identified member of the CXC chemokine family in mammals [START_REF] Conway | Kidney single-cell atlas reveals myeloid heterogeneity in progression and regression of kidney disease[END_REF][START_REF] Pisabarro | Cutting Edge: Novel Human Dendritic Cell-and Monocyte-Attracting Chemokine-Like Protein Identified by Fold Recognition Methods[END_REF]. CXCL17 attracts professional antigen presenting cells [START_REF] Weinstein | VCC-1, a novel chemokine, promotes tumor growth[END_REF], including macrophages, through its suspected receptor GPR35/CXCR8 [START_REF] Xiao | Mucosal chemokine CXCL17: What is known and not known. Mucosal chemokine CXCL17: What is known and not known[END_REF], even though a second unidentified G protein-coupled receptor may mediate CXCL17-dependent signaling [START_REF] Maravillas-Montero | Cutting Edge: GPR35/CXCR8 Is the Receptor of the Mucosal Chemokine CXCL17[END_REF]. Although very little is known about the role of CXCL17 in the kidney, single-cell RNA sequencing analysis revealed that its expression is enhanced at an early time point in murine tubulointerstitial fibrosis model [START_REF] Wang | MMP-9-positive neutrophils are essential for establishing profibrotic microenvironment in the obstructed kidney of UUO mice[END_REF]. It is consistent with the increased levels of CXCL17 observed in interstitial pulmonary fibrosis, where it may recruit immune cells that in turn produce pro-inflammatory cytokines [START_REF] Maravillas-Montero | Cutting Edge: GPR35/CXCR8 Is the Receptor of the Mucosal Chemokine CXCL17[END_REF]. Further research may determine if this axis could participate in the pathogenesis of NPH and its fibrotic features.

LGALS9, a mammalian -galactoside binding lectin (Gal-9), was first isolated from murine embryonic kidney [START_REF] Choreño-Parra | The protective and pathogenic roles of CXCL17 in human health and disease: Potential in respiratory medicine. The protective and pathogenic roles of CXCL17 in human health and disease: Potential in respiratory medicine[END_REF]. LGALS9 is known to participate in numerous cellular processes including the induction of apoptosis of different immune cells, particularly cytotoxic T lymphocytes when bound to its surface receptor TIM3/HAVCR2 [START_REF] Wada | Identification and characterization of galectin-9, a novel βgalactoside-binding mammalian lectin[END_REF]. The precise role of

LGALS9 in the kidney has not been described so far, however a recent study showed that anti-TIM3 antibody ameliorates kidney injury and decreased macrophage infiltration in ischemic mouse model [START_REF] Guo | Tim-3 exacerbates kidney ischaemia/reperfusion injury through the TLR-4/NF-κB signalling pathway and an NLR-C4 inflammasome activation[END_REF]. This is consistent with a concomitant study that showed increased levels of soluble TIM3 and soluble Gal-9 in the blood of patients with kidney transplantation-related renal dysfunction [START_REF] Guo | Tim-3 exacerbates kidney ischaemia/reperfusion injury through the TLR-4/NF-κB signalling pathway and an NLR-C4 inflammasome activation[END_REF]. In addition, upregulation of serum Gal-9 is closely related to glomerular filtration rate decrease in patients with type 2 diabetes [START_REF] Li | Soluble Tim-3 and Gal-9 are associated with renal allograft dysfunction in kidney transplant recipients: A cross-sectional study[END_REF]. Thus, circulating Gal-9 and TIM3 may be useful biomarkers to monitor GFR decline in nephronophthisis. Further studies are needed to assess their function and interplay in the disease. Besides, Gal-9-dependent signaling may participate in switching macrophages from pro-inflammatory M1 phenotype to its antiinflammatory M2 counterpart, raising the possibility that Gal-9/TIM3 is involved in the polarization of macrophages in NPH [START_REF] Kurose | Serum galectin-9 levels are elevated in the patients with type 2 diabetes and chronic kidney disease[END_REF][START_REF] Jung | Human galectin-9 on the porcine cells affects the cytotoxic activity of M1differentiated THP-1 cells through inducing a shift in M2-differentiated THP-1 cells[END_REF]. This work identified a specific network of commonly regulated cytokines that represent plausible mediators of immune cell recruitment to NPH kidneys beyond CCL2. We identified inflammation as the predominant signature in two independent models of NPH and we found that this holds true in human patients. As most of the NPH patients studied bear NPHP1 mutations, we cannot exclude that these findings are specific to NPHP1 patients. Using a nonorthologous mouse model of NPH based on genetic disruption of Lkb1 may be a limitation of the study. Yet, we confirmed the inflammatory signature identified in Lkb1 Tub mice in one of the rare murine orthologous model of NPH leading to renal fibrosis (Glis2 lacZ/lacZ mice) as well as in UREC derived from NPHP1 patients. Overall, the similar inflammatory profiles in NPHlike mouse models and in tubular cells derived from NPH patients strengthen our view that Lkb1 Tub mice represent a faithful model to study the pathophysiology of NPH. The parallelism between the inflammatory pathways activated in the early diseased kidneys from Lkb1 Tub or Glis2 lacZ/lacZ mice in one hand, and those enriched in the UREC from NPH patients in the other hand, along with the protective effect of TLR2/MYD88 inhibition in Glis2 lacZ/lacZ mice, suggest causality. However, we cannot exclude that chemokine expression and immune cell recruitment are innocent bystanders of an undefined process leading to kidney damage in NPH. Certain immune cells may also be protective, such as T lymphocytes in the context of ADPKD [START_REF] Zhang | Lipopolysaccharide mediates time-dependent macrophage M1/M2 polarization through the Tim-3/Galectin-9 signalling pathway[END_REF]. This work establishes renal inflammation as a prominent feature of human NPH and identifies specific mediators of this process that are common to NPH mouse models and patients.

Addressing the precise function of these mediators in NPH in the future will help to characterize the underlying processes responsible for renal deterioration in this orphan disease and to evaluate inflammation as a potential therapeutic target.

MATERIALS and METHODS

Human kidney tissue specimens

Renal kidney tissues from patients suffering from juvenile nephronophthisis (NPH) were collected from 1973 to 1998. Uneligible kidneys donated for transplantation or kidney from patient suffering from renal disease other than nephronophthisis with minimal histological lesions and collected over the same time period were used as controls. Renal biopsies from patients suffering from diabetic nephropathy or hypertensive nephrosclerosis were used to control for non-specific, chronic kidney disease (CKD) related inflammation. Interstitial fibrosis and tubular atrophy (IFTA) score was evaluated on renal biopsy by a trained pathologist in a blinded fashion. NPH and CKD patient groups were matched according to their IFTA score.

Patient data are listed in Supplemental Table 1.

Isolation of urine-derived renal epithelial cells (UREC)

Urine samples were collected from NPHP1 patients, healthy relatives and unrelated controls as well as CKD patients recruited at Necker Hospital (Paris, France) after written informed consent of the donor. Inclusion criteria for affected patients were to suffer from nephronophthisis with known genetic diagnosis. The relatives were the healthy relatives (father/mother/sister) of an included patient. Healthy controls were free from any chronic kidney disease or with normal renal function. CKD patients were suffering from CKD other than renal ciliopathy. Patient data are listed in Supplemental Table 1.

Urine-derived renal tubular epithelial cells (primary UREC) were isolated and cultured as previously described [START_REF] Ajzenberg | Noninvasive sources of cells with primary cilia from pediatric and adult patients[END_REF] with some modifications. Briefly, after centrifugation and washing steps, urine-derived cells were initially cultured for 4 days at 37°C in primary medium containing Dulbecco's Modified Eagle Medium : Nutrient Mixture F-12 (DMEM/F-12) supplemented with 10% fetal bovine serum (16000-036, Gibco), 10% Penicillin-Streptomycin (15140-122, Gibco), 10% Amphotericin B (15290026, Gibco) and 1X REGM TM SingleQuots TM kit (CC-4127, Lonza) to enhance cell survival and adherence. At day 4, primary medium was replaced by a growth medium containing REBM TM (Basal Medium, CC-3191, Lonza) supplemented with 2% fetal bovine serum, 10% Penicillin-Streptomycin, 10% Amphotericin B, 1X REGM TM SingleQuots TM kit and 10 ng/mL rhEGF (R&D system) and was then changed every 2 days. At 80% confluence (7-30 days), 2x10 4 cells were seeded for 7-24 days until confluence on 12 well plates (353043, Dutscher) for RNA analysis.

Mice

Mice were housed in a specific pathogen-free facility, fed ad libitum and housed at constant ambient temperature in a 12-hour day/night cycle. Breeding and genotyping were done according to standard procedures.

Lkb1 Tub mice were previously described [START_REF] Viau | Cilia-localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney[END_REF]. Ccl2-RFP flox/flox (B6.Cg-Ccl2tm1.1Pame/J, stock number: 016849, C57BL/6J genetic background) were purchased from The Jackson Laboratories and were backcrossed for 2 generations with Lkb1 Tub mice. The progeny was then intercrossed to generate mice with tubule-specific Lkb1 knockout (further referred to as Lkb1 Tub ), Ccl2 knockout (further referred to as Ccl2 Tub ) and Lkb1; Ccl2 double knockout (further referred to as Lkb1 Tub ; Ccl2 Tub ) on a common mixed C57BL/6J; C57BL/6N genetic background. Littermates lacking KspCre transgene were used as controls. Experiments were conducted on both females and males.

Pkd1 flox/flox mice (B6.129S4-Pkd1tm2Ggg/J, stock number: 010671, 129.B6 mixed genetic background) were purchased from The Jackson Laboratories and were crossed to Pax8rtTA (Tg(Pax8-rtTA2S*M2)1Koes) and TetOCre (Tg(tetO-cre)1Jaw) mice to generate an inducible tubule-specific Pkd1 knockout (further referred to as iPkd1 Tub ) as previously described [START_REF] Viau | Cilia-localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney[END_REF].

To induce recombination, mice received doxycycline (Abcam, ab141091) in drinking water (2mg/mL with 5% sucrose, protected from light) from post-natal day 28 (P28) to P42.

Littermates lacking either TetOCre or Pax8rtTA were used as controls. Experiments were conducted on males.

Quantitative PCR

Total RNAs were obtained from human UREC or mouse kidneys using RNeasy Mini Kit (Qiagen) and reverse transcribed using SuperScript II Reverse Transcriptase (Life Technologies) or High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer's protocol. Quantitative PCR were performed with iTaq™ Universal SYBR® Green Supermix (Bio-Rad) on a CFX384 C1000 Touch (Bio-Rad). Hprt, Ppia, Rpl13, Sdha and Tbp were used as normalization controls [START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF]. Each biological replicate was measured in technical duplicates. The primers used for qRT-PCR are listed in Supplemental Table 2.

CCL2 ELISA

For the quantification of urinary CCL2 excretion, urine specimens were collected and centrifuged at 1500 g for 10 minutes at 4°C within 4h of collection. The supernatants were collected and stored at -80°C. Frozen aliquots of urine supernatants were thawed at room temperature immediately before the ELISA. The samples were used with a 2-fold dilution and were tested in duplicates. CCL2 levels in urine specimens were quantified using Human CCL2/MCP-1 Quantikine® ELISA Kit (R&D systems, DCP00) according to the manufacturer's instructions. The plate was read using a Multiskan Sky plate reader. The optical densities were derived from 4-parameter logistic regression of the standard curve. Measurement of creatinine in urine was performed in the same samples using IDMS-standardized enzymatic method on C16000 Architect analyzer (Abbott Diagnostic). The results were normalized to the urinary creatinine level. Indeed, normalization by the urinary creatinine levels avoids the pitfall of concentration or dilution of urine.

Morphological Analysis

Human kidney biopsies were fixed in alcohol formalin and acetic acid and paraffin embedded, 4µm sections were stained with periodic acid-Schiff (PAS). PAS-stained full size images were recorded using a whole slide scanner Nanozoomer 560 (Hamamatsu) coupled to NDPview software (Hamamatsu).

Mouse kidneys were fixed in 4% paraformaldehyde, embedded in paraffin, and 4µm sections were stained with PAS or Picrosirius Red. Stained full size images were recorded using a whole slide scanner Nanozoomer 2.0 (Hamamatsu) equipped with a 20x/0.75 NA objective coupled to NDPview software (Hamamatsu). Histology score was evaluated by an independent observer in a blinded fashion assessing the overall lesions of the whole kidney section stained with PAS.

Six scores were defined ranging from score 1 normal kidney architecture to score 6 associating tubular atrophy, tubular basement thickening and interstitial cell infiltration.

Urine and Plasma Analyses

8-hour urine samples were obtained from mice housed in individual boxes without access to water and food. Body weight and urine excretion were measured. Urine osmolality was measured with a freezing point depression osmometer (Micro-Osmometer from Knauer or OSMOMAT 3000basic from Gonotec). Retro-orbital blood was collected from anaesthetized mice. Plasma blood urea nitrogen (BUN) was measured using urea kit (LT-UR; Labor&Technik, Eberhard Lehmann GmbH) according to the manufacturer's instructions.

Immunohistochemistry

For human kidney sections An automated IHC stainer BOND-III (Leica Biosystems) was used. Briefly, 4µm sections of paraffin-embedded human kidney biopsies were submitted to the appropriate antigen retrieval.

Then, sections were incubated with CD68 (Dako, M0814, 1:3,000), CD15 (Beckam Coulter, NIM0165, 1:200) or CD3 (Dako, A0452, 1:200) antibodies. Peroxide blocking, post primary, DAB chromogen and hematoxylin counterstaining was performed automatically using Bond polymer refine detection kit (Leica Biosystems, DS9800). The degree of interstitial cell infiltration was determined using immunostaining targeting macrophages (CD68), neutrophils (CD15) and T lymphocytes (CD3). Slides were scanned with a whole slide scanner Nanozoomer 560 (Hamamatsu). Randomly selected microscopic fields (x200) representative of the entire cortical surface were scored. The degree of cell infiltrate was quantified using ImageJ software. For CD3 and CD68, the area of DAB staining was measured after color deconvolution and intensity thresholding of the images and visualized as the ratio of DAB surface to cortical surface on each microscopic field. For CD15, the number of CD15-positive interstitial cells was quantified manually in a blinded fashion and was expressed as a ratio per mm  .

For mouse kidney sections Macrophage staining: 4µm sections of paraffin-embedded mouse kidneys were incubated for 20 min at 95°C in citrate buffer (Zytomed, ZUCD28) followed by avidin/biotin blocking (Vector, SP-2001). Sections were incubated with F4/80 antibody (Clone Cl:A3-1, Bio-Rad, MCA497R, 1:100) followed by biotinylated antibody (Vector, BA-4001, 1:200), HRP-labeled streptavidin (Southern Biotech, 7100-05, 1:2,000) and 3-3′-diamino-benzidinetetrahydrochloride (DAB, Dako, K3468) revelation.

Neutrophil staining: 4µm sections of paraffin-embedded mouse kidneys were submitted to antigen retrieval for 20 min at 95°C in citrate buffer followed by incubation with Ly-6B.2 antibody (Abcam, ab53457, 1:100). Sections were incubated with HRP-labeled secondary antibody (Vector, PI-9400, 1:200) and DAB revelation.

T cell staining: 4µm sections of paraffin-embedded mouse kidneys were incubated for 20 min at 95°C in Tris-EDTA pH9 buffer followed by avidin/biotin blocking. Sections were incubated with CD3 antibody (Abcam, ab16669, 1:100) followed by biotinylated antibody (GE Healthcare, RPN1004V, 1:200), HRP-labeled streptavidin (Southern Biotech, 7100-05, 1:2,000) and DAB revelation.

Full size images were recorded using a whole slide scanner Nanozoomer 2.0 coupled to NDPview software. Stained area was measured with ImageJ software from full size kidney images and visualized as the ratio of stained DAB surface to total kidney section area. For neutrophil quantification in Figure 5, since neutrophils are rare and focal in 5 weeks old mice, we counted manually the number of foci in whole kidney section. The number of foci per kidney section was scaled to the surface of the section. Foci were defined as 4 or more neutrophils surrounding a tubule. For all quantification, glomerular and non-specific intra-tubular staining were removed from the analysis.

Comparative microarray data analysis

Processing of data was carried out using R v3.6.0, RStudio and R package dplyr v1.0.1. Renal transcriptomic datasets from 5 weeks old Lkb1 Tub and 4 weeks old Glis2 lacZ/lacZ mouse models [START_REF] Viau | Cilia-localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney[END_REF][START_REF] Attanasio | Loss of GLIS2 causes nephronophthisis in humans and mice by increased apoptosis and fibrosis[END_REF] were compared. While transcriptomic dataset from Lkb1 Tub mice was established by our team (GSE86011) [START_REF] Viau | Cilia-localized LKB 1 regulates chemokine signaling, macrophage recruitment, and tissue homeostasis in the kidney[END_REF], we downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/) the expression matrix of mRNAs expressions in the kidneys of Glis2 lacZ/lacZ mice under the accession number GSE6113 [START_REF] Attanasio | Loss of GLIS2 causes nephronophthisis in humans and mice by increased apoptosis and fibrosis[END_REF]. Probe IDs in the expression matrix were matched with the corresponding gene IDs in the lookup table (GPL2897) to identify the expression of each mRNA. To assess the common regulated genes between the two mouse models, Lkb1 Tub dataset was filtered out according to false discovery rate (FDR, Benjamini-Hochberg procedure) < 0.05 to obtain a list of 1991 differentially expressed genes (DEGs). Then, Glis2 lacZ/lacZ dataset (23957 genes) was matched with this DEGs list, and the genes whose expression varies in the same way were considered as the common regulated genes (Supplemental Table 3).

GSEA, networking and visualization

Data were expressed as means. Differences between groups were evaluated using Mann-Whitney test when only two groups were compared. When testing more comparisons, Kruskal-Wallis test was used when the groups do not meet normal distribution otherwise one-way ANOVA followed when significant (P < 0.05) by the Tukey-Kramer test was used. The statistical analysis was performed using GraphPad Prism V8 software. All image analysis and mouse phenotypic analysis were performed in a blinded fashion.

Study approval

The human kidney tissue specimens belonging to the Imagine Biocollection are declared to the

French

  Minister of Research under the number DC-2020-3994 and approved by the French Ethics committee for research at Assistance Publique-Hôpitaux de Paris (CERAPHP) under the IRB registration number #00011928. The urine study was approved by the French National Committee for the Protection of Persons (CPP) under the ID-RCB number 2016-A00541-50 and is kept in full accordance with the principles of the Declaration of Helsinki and Good Clinical Practice guidelines. All animal experiments were conducted according to the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals, as well as the German and French laws for animal welfare, and were approved by regional authorities (Regierungspräsidium Freiburg G-16/27 and Ministère de l'Enseignement, de la Recherche et de l'Innovation APAFIS#2020090715389782).

Figure 1 .

 1 Figure 1. Human nephronophthisis is associated with renal inflammation, macrophage

Figure 2 .

 2 Figure 2. Tubule specific Ccl2 inactivation does not prevent renal lesion development nor

Figure 3 .

 3 Figure 3. Comparative renal transcriptome analysis in Glis2 mutant mice and kidneyspecific Lkb1 deficient mice. (A) Venn diagram showing intersection between Glis2 lacZ/lacZ

Figure 4 .

 4 Figure 4. NPH cytokine signature and immune cell infiltrations occurred independently

Figure 5 .

 5 Figure 5. The early inflammatory signature of NPH is not a common feature of renal

Figure 6 .

 6 Figure 6. The immune signature of murine NPH is also prominent in human NPH. (A)

ACKNOWLEDGEMENTS

The authors thank Dr. Alexandre Benmerah for critical appraisal of the manuscript.

We thank the technicians from the mouse histology and breeding facilities (S.F.R Necker INSERM US24, Paris, France) and the department of Pathology (Necker Hospital, Paris, France) for technical assistance. We are grateful to the patients and their families for their participation. We thank Pauline Krug, Olivia Boyer, Nathalie Biebuyck, Saoussen Krid, Marina Charbit, Romain Berthaud and Guillaume Lezmi (Pediatric Nephrology, Necker Hospital, AP-HP, Paris, France), Aurélie Hummel (Adult Nephrology, Necker Hospital, AP-HP, Paris, France), Amélie Lezmi Ryckewaert and Sophie Taque (Pediatric Hematology and Oncology, Hôpital Universitaire, Rennes, France), Odile Boespflug-Tanguy (Centre de Compétence des Leucodystrophies et Leucoencéphalopathies de Cause Rare, Pôle Femme et Enfant, Hôpital Estaing, Centre Hospitalier Universitaire de Clermont-Ferrand, Clermont-Ferrand, France), Jérôme Harambat and Brigitte Llanas (Department of Pediatrics, Bordeaux University Hospital, Bordeaux, France), Bruno Ranchin (Pediatric Nephrology, Centre Hospitalier Universitaire de Lyon, Bron, France), Elodie Merieau (Centre Hospitalier Régional Universitaire de Tours, Tours, France), Marc Fila (Centre Hospitalier Universitaire de Montpellier, Montpellier, France) and Tory Kalman (Semmelweis University, Budapest, Hungary) who helped the follow-up of patients. We thank Corinne Antignac and Laurence Heidet (Department of Genetics, Necker Hospital, AP-HP, Paris, France) for genetic diagnostic. We thank the Department of Clinical Research at Imagine Institute with the sponsorship team that facilitates and structures the set-up of the clinical research projects and the investigation team that prepares and ensures the follow-up of the clinical trials. Marceau Quatredeniers and Amandine Viau were supported by a public grant "RHU-C'IL-LICO" overseen by the Agence Nationale de la Recherche (grant number: ANR-17-RHUS-0002), Frank Bienaimé was supported by EMBO (grant number: ALTF 927-2013), E.

Subsequently, Gene Set Enrichment Analysis (GSEA) approach was applied to the 1262 common regulated genes identified using GSEA software v4.0.0 (Broad Institute) set for 000 gene set permutations. The enrichment involved the « biological process » classification of the Gene Ontology downloaded from MSigDB v7.1 (C5-BP). Up-and down-regulated pathways are listed in Tables S4-S5. Pathways with FDR < 0.05 were considered significant. R package ggplot2 was used to present the more significant up-and down-regulated pathways. Cytoscape software v3.0 and Cytoscape applications EnrichmentMap v3.3.0 (edge cutoff threshold set to 0.5) and WordCloud v3.1.3 were used for pathway clustering and pathway network visualization [START_REF] Reimand | Pathway enrichment analysis and visualization of omics data using g:Profiler, GSEA, Cytoscape and EnrichmentMap[END_REF]. Pathway clusters have been hand-annotated.

Identification of common upregulated cytokines

To identify common upregulated cytokines, the 823 common upregulated genes were matched with lists of genes obtained from the UniProt database (https://www.uniprot.org/) using the following keywords: « cytokine », « secreted » and « immune/inflammation ». Genes that were linked to the 3 keywords are listed in Supplemental Table 6.

Heatmaps of the expression of the identified cytokines from 5 weeks old Lkb1 Tub and 10 weeks old Pkd2 Tub were compared. The Pkd2 Tub log-normalized expression matrix was downloaded from GEO under the accession number GSE149739 [START_REF] Kim | Kruppel-Like Zinc Finger Protein Glis2 Is Essential for the Maintenance of Normal Renal Functions[END_REF]. Differential expression testing between groups was performed using limma v3.42.2. Heatmaps were generated using the R package pheatmap v1.0.12 (note: heatmaps show Z-scores computed from the expression matrix for each gene, thus presenting expression levels with relatives equal means for each gene). Expression levels of the identified cytokines are listed in Supplemental Table 7. Finally, the heatmap in Figures 4c and5c displays Z-scores computed on the expression levels of the identified cytokines, measured by qPCR. The corresponding qPCR results are listed in Figures S4 andS5.

Statistical analysis

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no conflict of interest.

ABBREVIATIONS