Claire Grison
email: claire.grison@u-psud.fr

Brice-Loïc Renard

Claude Grison

Claire M Grison

Claude Grison

A simple

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

Introduction

High ulosonic acids are well known to play an important structural role in the bacterial population. They are essential a-ketoacids such as 2-keto-3-deoxy-D-manno-octonic acid (KDO), which is mostly found in the lipopolysaccharide (LPS) outer membrane of Gram negative bacteria and such as 2-keto-3-deoxy-D-arabinoheptonic acid (DAH), which plays a key role in the shikimic pathway of bacteria and plants. Therefore their chemical syntheses have often been studied to design new antibacterial agents for inhibiting their own biosynthetic pathway [1].

However, a little known ulosonic acid, 2-keto-3-deoxy-D-erythro-hexonic acid (or 2-keto-3-deoxy-D-gluconic acid, KDG), is the key intermediate metabolite in the Entner Doudoroff (ED) glycolytic pathway of certain microbial species, especially Gram negative bacterial species living in a stressful environment [2,3]. Two novel heavy-metal resistant bacterial species were found to be of particular interest, Rhizobium metallidurans and Mesorhizobium metallidurans [4,5]. They had been discovered growing on a highly polluted mining site of Les Avinières (Saint-Laurent-Le-Minier, Gard, South of France) in symbiosis with the Zn hyperaccumulating legume Anthyllis vulneraria (Hyperaccumulating rate = 154 mg kg À1 of zinc) [6]. The novel strains had been found where the pollution is the highest, on the tailing basins (Zn: 160 g kg À1 , Pb: 84 g kg À1 Cd: 1.3 g kg À1 of soil) and where the vegetal and microbial diversity is the poorest. The ability to grow on a heavy-metal enriched soil devoid of carbon nutriment is a challenge. In such conditions, we assumed that their carbon metabolism is based on the ED pathway as the only glucose degradation way instead of the glycolytic pathway. This adaptation could be explained by the high selective pressure of a stressful environment [3,7]. KDG uptake would prove the presence and functionality of the ED pathway in bacterial strains.

Therefore a rapid and efficient synthesis of KDG was needed. However, few stereoselective chemical syntheses of KDG had been described in the literature. In 1991, Plantier-Royon et al. [8] reviewed the first chemical routes of KDG and chose to improve the most promising way. However KDG was obtained with an overall yield of 35% in 6 steps from D-glucose, using a Wittig-Horner phosphonate reagent. During this period of years, Ramage et al. [9] also reported a complicated chemical route to KDG with a poor yield (<7% in 4 steps) from dioxalan-4-one, with a Wittig reagent. Therefore a new chemical synthesis to KDG is needed. We have previously reported a general synthesis of a-ketoesters from carbonyl compounds, by using potassium alkyl dichloroacetates. Applied to a protected D-mannose, the method is convenient for the total synthesis of KDO [10]. This strategy is also very suitable to prepare the protected KDO under isopropyl carboxylic ester form, which is better for bacterial cell wall penetration [11]. We propose here an original but simple and enantiomerically pure synthesis of KDG isopropyl ester and microbiological assays of KDG isopropyl ester uptake, as a selective bacterial growth stimulator.

Experimental protocols

All starting materials were commercially available researchgrade chemicals and used without further purification. Reactions were monitored by TLC (Merck -5535 -Kieselgel 60-F 254 ), detection being carried out by UV, by iodine vapor or by spraying solution of H 2 SO 4 15% in ethanol followed by heating. NMR spectra were recorded on a Bruker DRX-300. Chemical shifts are expressed as parts per million downfield from the internal standard tetramethylsilane for 1 H and 13 C. Multiplicities are indicated by s (singlet), d (doublet), dd (dedoubled doublet), t (triplet), q (quadruplet), m (multiplet), bs (broadened singlet); coupling constants are reported in Hertz (Hz). IR spectra were recorded on a PerkinElmer Spectrum 100 FT-IR spectrometer, in ATR mode, and are given in cm À1 . High-resolution mass spectrometry (HRMS) data were recorded using the electrospray ionization technique in positive mode (ESI+) with a tandem Q-TOF analyser (Bruker, 2009). Optical rotations were measured using a 10 cm quartz cell (Jasco, P-1010). Values for ½/ T D were obtained with the D-line of sodium at the indicated temperature T, using solutions of concentration (c) in units of gÁ100 mL À1 . Column chromatography was performed on silica gel (MN Kieselgel 60, 0.063-0.2 mm/70-230 mesh, Macherey-Nagel). All primers were purchased from Invitrogen (Saint-Aubin, France).

2.1. Chemical synthesis of 2-keto-3-deoxy-D-erythro-hexonic acid isopropyl ester 8

2.1.1. Synthesis of 4-O-tert-butyldimethylsilyl-2,3-O-isopropylidene- D-threitol 2
To a suspension of NaH (60% in oil, 0.18 g, 6.63 mmol, 1 equiv) in THF (10 mL) was added, over 15 min under a stream of nitrogen at 5 °C, a solution of 2,3-O-isopropylidene-D-threitol (1.30 g, 6.63 mmol, 1 equiv) in THF (12 mL). The suspension was stirred for 45 min at 5 °C. Then tert-butyldimethylsilyl chloride (1 g, 6.63 mmol, 1 equiv) in 2 mL of THF was added. After stirring for 16 h, the reaction mixture was washed with 30 mL of saturated NaHCO 3 and extracted three times with diethylether (3 Â 20 mL). The organic layer was dried over MgSO 4 , filtered and concentrated under reduced pressure. The residue was purified by silica chromatography (3:1 hexane/EtOAc) to give 1.75 g of compound 2 as yellow oil (98% yield).

2: Rf (4:1 hexane/EtOAc) = 0.25; (4 mL). Then compound 2 (1.80 g, 6.63 mmol, 1 equiv) in 10 mL of CH 2 Cl 2 was added dropwise. After stirring for 20 min, triethylamine (3.35 g, 33.15 mmol, 5 equiv) was added and the reaction was allowed to warm to 20 °C over a period of 1 h. The heterogeneous mixture was extracted with water, diluted in (1:1 hexane/ether) and extracted three more times with water (3 Â 25 mL). Then the combined aqueous fractions were extracted with (1:1 hexane/ether) and the combined organic layers were dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. After coevaporation with toluene, the crude aldehyde 3 (Rf (5:1 hexane/EtOAc) = 0.5) was used immediately in the synthesis of compound 5.

Synthesis of isopropyl dichloroacetate 4

To 150 mL of anhydrous isopropanol (117.90 g, 1.961 mol, 13 equiv) were added dichloroacetic acid (19.35 g, 150 mmol, 1 equiv) and 2 mL of sulfuric acid (36 N). The solution was stirred for 26 h at 100 °C. Isopropanol was then evaporated under reduced pressure. The remaining yellow oil was dissolved in 60 mL of diethylether, washed with an aqueous solution of NaHCO 3 8% until pH = 8 and extracted three times with diethylether. The organic layer was dried over MgSO 4 , filtered and concentrated under reduced pressure to give 24 g of compound 4 as yellow oil (96% yield). The analytical data were consistent with literature [10].

2.1.4. Synthesis of 2,3-anhydro-2-C-chloro-6-O-[(1,1- dimethylethyl)dimethylsilyl]-4,5-O-isopropylidene-D-erythro-hexonic acid 1-methylethyl ester 5
A suspension of solid potassium (0.52 g, 13.2 mmol, 2 equiv) in 41 mL of isopropanol under nitrogen was stirred until complete dissolution of potassium. After addition of 8 mL of diethylether, the solution was cooled to 0 °C and a mixture of 3 (6.63 mmol, 1 equiv) and isopropyl dichloroacetate 4 (2.26 g, 13.2 mmol, 2 equiv) in 32 mL of diethylether, was added dropwise for 30 min. The mixture was stirred for 2 h, neutralized by addition of saturated HCl solution in diethylether and centrifuged (1000 rpm, 15 min). The supernatant was taken off, concentrated and purified by silica chromatography (5:1 hexane/EtOAc) to give 2,21 g of compound 5 as brownish oil (71% yield).

5a (60%): Rf (5: To a suspension of solid magnesium (0.08 g, 3.25 mmol, 1.3 equiv) in 22 mL of diethylether was added grinded iodine (0.83 g, 3.25 mmol, 1.3 equiv). After stirring for 3 h at 35 °C in the dark, compound 5 (1 g, 2.50 mmol, 1 equiv) in 14 mL of diethylether was added dropwise. The solution was refluxed for 2 h to give 6a and 6b. A fraction of the reaction mixture was isolated to characterize 6a and 6b. 6a and 6b were not enough stable to obtain 13 C NMR data. 6a, 6b: Rf (5:1 hexane/EtOAc) = 0.57; 1 After cooling, a freshly prepared aqueous solution of Na 2 S 2 O 5 8% was added to 6a/6b and the resulting mixture was stirred for 30 min at 20 °C, extracted with diethylether. The organic layer was dried over Na 2 SO 4 , filtered and concentrated under reduced pressure to give 0.59 g of compound 7 (64% yield). To a suspension of solid magnesium (0.13 g, 5.25 mmol, 2.1 equiv) in 5 mL of diethylether was added grinded iodine (0.63 g, 2.5 mmol, 1 equiv). After stirring for 1 h at 35 °C in the dark, compound 5 (1 g, 2.50 mmol, 1 equiv) in 8 mL of diethylether was added dropwise at 20 °C. The solution was refluxed for 2 h and stirred for 15 h at 20 °C, then extracted with diethylether. The organic layer was filtered on silica before, dried over Na 2 SO 4 and concentrated under reduced pressure. 0.72 g of compound 7 was obtained as pale yellow oil (78% yield).

1 hexane/EtOAc) = 0.5; 1 H RMN (300 MHz, CDCl 3 ) d 0.09 (s, 6 H, Si-H 3 ), 0.91 (s, 9 H, Si-tBu), 1.43-1.44 (s, 6 H, C-CH 3 ), 1.46-1.48 (m,
7: Rf (5:1 hexane/EtOAc) = 0.61; 100 mL of an aqueous solution of formic acid 50% was added to compound 7 (0.20 g, 0.53 mmol), the solution was stirred for 24 h at 20 °C and concentrated under reduced pressure to give 0.1 g of 8 (87% yield). After persilylation 0.20 g of compound 9 were obtained as yellow oil (100% yield). 9: Rf (5:1 hexane/EtOAc) = 0.25; Soil samples were collected on the mining site of Saint-Laurent-Le-Minier. 1 kg of soil samples were extracted from the rhizosphere of 15 plants of A. vulneraria. They were stored at 4 °C until analysis in the laboratory within 4 days. Total DNA was isolated by a Powersoil DNA isolation kit as recommended by the producer (MOBIO, Carlsbad, Canada). DNA concentration was checked by Nanodrop measurement. Each combined DNA isolate was precipitated by sodium acetate (12.5%) and isopropanol to be concentrated.

16S rDNA library from Anthyllis vulneraria rhizosphere

A region of approximately 1500 bp of 16S rDNA genes was amplified using the primers 27f (5 0 -AGAGTTTGATCMTGGCT CAG-3 0 ) and 1492r (5 0 -ACCTTGTTACGACTT-3 0 ) and cloned before sequencing. [12] The sequenced full-legnth 16S rDNA genes were well suited for phylogenetic analysis. The analyses were based on around 800 nucleotides. [13] The similarity searches for sequences were carried out by the BLASTN program of the National Center of Biotechnology Information, MD, USA.

The 16S rDNA gene library was kept in 20% glycerol (v/v) at À80 °C.

KDG isopropyl ester-enriched medium

Growth test was performed on glucose minimal medium (Na 2 HPO 4 (120 mM), KH 2 PO 4 (55 mM), NaCl (20 mM), NH 4 Cl (45 mM), MgSO 4 (20 mM), CaCl 2 (2 mM) and glucose (20 mM)) and KDG isopropyl ester minimal medium (Na 2 HPO 4 (120 mM), KH 2 PO 4 (55 mM), NaCl (20 mM), NH 4 Cl (45 mM), MgSO 4 (20 mM), CaCl 2 (2 mM) and KDG (20 mM)).

Results and discussion

Synthesis of KDG isopropyl ester

According to our previous chemical syntheses of superior ulosonic acid analogs (KDO, DAH) [11] we designed a rapid strategy based on the use of alkyldihalogenoacetate in chain extension of an aldose with a a-ketoester unit. It constituted a [n + 2] methodology which led to an enantiopure ulosonic acid. Since configuration of KDG is D-erythro, protected D-erythrose would be the saccharide to use. However no protected D-erythrose precursor is commercially available because of its instability. Evans et al. [14] had described an access to 4-O-tert-butyldimethylsilyl-2,3-O-isopropylidene-D-erythrose. But this synthesis was tricky and hard to reproduce.

Therefore D-threo compound was chosen as the carbohydrate precursor. Thus KDG synthesis was based on a key step of epimerization of the D-threose b-position. The epimerization was induced by a Swern oxidation of protected D-threitol and a diastereoselective Darzens type condensation [15].

A potassic enolate anion derived from isopropyl dichloroacetate was a synthetic equivalent of ('' À C(O)COO À '') synthon. It was added on a judiciously protected D-threose. This last was prepared from commercial 2,3-O-isopropylidene-D-threitol 1 (Scheme 1).

According to Condon et al. [16], it was selectively monosilylated into 2 with tert-butyldimethylsilyl chloride and one equivalent of strong base NaH. Swern oxidation was performed in basic conditions which partially epimerized the b-position of 2 as expected [17]. It led to a mixture of protected D-erythrose and D-threose 3a/3b, which were not isolated and directly engaged into the following step. Darzens modified reaction was also carried out in basic conditions on the crude aldehydes 3a/3b, with the potassic anion of isopropyl dichloroacetate 4 in alcohol-ether solvent. The proportion of solvents (isopropanol/ether: 50/50) played an important role. Isopropanol was used as a protic solvent to stabilize the alcoolate formed during the early stage of the reaction. Purification of the crude product by chromatography over silica gel afforded a diastereoisomeric mixture of a-chloroglycidic esters 5 in high yield.

It is noteworthy that Darzens reaction leads to the formation of two new stereogenic centers, C-2 and C-3 positions. Since Darzens modified reaction had been conducted on two aldehyde epimers, eight diastereoisomers could be obtained. But we have previously shown that the carboxylic ester group was only in trans position of the carbon chain [10,11,15], so only four diastereoisomers could be obtained.

In some cases, we have shown that Darzens modified reaction proceeded in total epimerization in carbohydrate series on C-4 positions. For example, the sequence Swern oxidation/Darzens modified reaction of protected D-mannose led to chloro epoxy ester derived from D-glucose [11]. We observed the same tendency with D-threose. Only two epimers were observed in 1 H NMR and 13 C NMR. So we could conclude that the epimerization into C-4 position was complete. The ratio of the two epimers 5a/5b was established by the integration of H-6 of (60/40). Previously, we have described the mild opening of a-halogenoepoxy ester with Lewis acids to introduce directly a b-halogeno-aketo ester moiety on a carbonyl compound [15]. In the present case the application of this strategy to the suitable protected a-chloroglycidic esters 5a/5b should give the required b-iodo-a-ketoester precursor 6. 5a/5b were treated with one equivalent of magnesium iodide in ether. It led to the rearrangement of the chloroepoxy ring into a mixture of the b-iodo-a-ketoester diastereoisomers 6a/6b with an excellent yield (Scheme 2).

Subsequent reduction of 6a/6b gave the protected target molecule 7. Two were investigated.

The first was the reduction of C-I bond with an aqueous solution of NaHSO 3 which yielded to the enol form of 7.

A proper one-pot strategy of isomerization-reduction was also performed. It was based on the preparation of the enolate derived from an a-ketoester from the reaction between the b-iodo-a-ketoester precursors 6 and active magnesium (like to prepare the corresponding Grignard reagent) (Scheme 1) [18]. Magnesium was intentionally used in a ratio Mg/I 2 = 2/1 in the preparation of MgI 2 to completely transform in situ the a-chloroglycidic esters into iodomagnesium enolates 6 0 (Scheme 2). These enolates were hydrolyzed to give the enol form of 7.

The a-enolester was observed because of the presence of a stabilizing intramolecular hydrogen bond between the alcohol from the enol function and the carbonyl from the carboxylic acid function. After a simple filtration on silica, it gave the a-ketoester 7.

Despite the fragility of erythro derivatives, we found that the two methodologies of isomerization-reduction of 5 were very convenient and directly afforded the ketoester 7 in good yields.

A final acid hydrolysis by formic acid (50% in water) was achieved for simultaneous removal of the isopropylidene and O-silyl group to give the compound 8. The isopropyl ester was kept to allow protected KDG 8 penetration through Gram negative cellwall [19,20]. The product 8 was persilylated into 9 to allow a better characterization by NMR, IR and GCMS. The silylation involved a green method based on the use of hexamethyldisilazane catalyzed by Zn hyperaccumulating plants as we have previously described [21]. In these conditions, 8 directly afforded the target molecule entirely silylated 9 (quantitative yield). In 1 H NMR, each hydrogen of CH 2 group in 9 appeared as a doublet of doublet at 2.8 and 3.1 ppm. These data were typical of pyruvic ester moiety. The IR data confirmed the assignment. We observed notably the presence of absorption bands at 1711 cm À1 for the C@O bond of the ketone and 1739 cm À1 for the C@O bond of the ester group, characteristic of the a-ketocarboxylic ester.

Microbiological tests of competitive growth

KDG is the key intermediate metabolite of the ED pathway (Scheme 3). It is specific of this pathway and cannot be metabolized in another glycolytic pathway. It can be used as a marker of a functional ED pathway. Moreover, it might be used to stimulate specifically the growth of bacteria using the ED pathway. It would avoid competition to glucose.

In Saint-Laurent-Le-Minier, carbon source availability is quite low because of the high pollution and thus the low abundance of vegetation. A vast program of revegetation started with the hyperaccumulating plant and R. metallidurans host, A. vulneraria [START_REF] Losfeld | Phytoextraction et biodégradation dynamisée: une approche interdisciplinaire inventive au service de l'environnement[END_REF]. R. metallidurans living in symbiosis with the legume, promotes its growth by producing ammonium from atmospheric N 2 fixation. Thereby stimulating selectively R. metallidurans growth among the total microbial population from Saint-Laurent-Le-Minier soil would stimulate A. vulneraria growth.

The microbial diversity of the polluted soil from Saint-Laurent-Le-Minier had been investigated. A cultur-independent approach was chosen to create a 16S rDNA library of 303 clones from the rhizosphere of A. vulneraria. According to searches similarity on BLASTN, 93% of the clones are novel species. We chose their most closely cultured related strains (>95% of similarity) to compare their growth with R. metallidurans on a KDG minimal medium. Bacterial population evolution could not be measured in solution, because most of the strains produce extracellular polysaccharides whilst forming aggregates, which is characteristic of biofilm formation. The comparison of growth was carried out on solid media with glucose as the sole source of carbon, for a positive growth control, and with KDG as the sole source of carbon, highlighting the functionality of the ED pathway (Table 1).

75% of the most closely related strains were not able to grow on KDG minimal medium. Therefore KDG promoted only 25% of the most closely related strains within the R. metallidurans target by avoiding the substrate uptake competition. We assumed that they were able to uptake KDG isopropyl ester and hydrolyze it before metabolizing it within the cell, through the ED pathway; as we had previously shown for an analog of KDO [11].

It was noticed that the growth-promoted strains by KDG are all capable of producing ammonium by fixing atmospheric N 2 , which should enrich the polluted soil and promote the growth of hyperaccumulating plants.

Conclusion

We described here an enantiomerically pure and original synthesis of 3-deoxy-D-erythro-hexulosonic acid isopropyl ester with good yield. The chiral configuration of the final product was controlled through a Swern oxidation followed by a diastereoselective Darzens type condensation leading to the epimerization of the KDG b-position. The presented results constituted a novel efficient synthesis of partially or completely protected KDG. Partially protected KDG uptake was tested on the total microbial diversity from Saint-Laurent-Le-Minier soil by growth assays. Only 25% of the total microbial population growth was promoted by 3-deoxy-D-erythro-hexulosonic acid isopropyl ester. These strains, which are all capable of producing ammonium, constitute huge benefits to agriculture, especially in a nutrient-poor soil. This microbial biotechnology promoted by KDG isopropyl ester feeding constitutes a promising green process to restore polluted soil by stimulating a revegetation project.

  6 H, CH-CH 3 ), 3.50-3.52 (d, 1 H, CH-O), 3.80-3.90 (2dd, 2 H, CH 2 -OTBDMS), 4.05-4.30 (m, 2 H, 2 CH-O), 5.09-5.18 (h, 1 H, O-CH-); 13 C NMR (75 MHz, CDCl 3 ) d À5.3 (Si-C(CH 3 ) 3 ), 16.7 (C(CH 3 ) 3 ), 21.5 (C(CH 3 ) 3 ), 25.7 (CH(CH 3 ) 2 ), 26.2 (C(CH 3 ) 2 ), 63.2 (CH-O(CCl)), 65.2 (CH 2 -O), 65.9 (Cl-C-O), 66.5 (CH-O), 67.8 (CH-O), 73.4 (CH(CH 3 ) 2 ), 111.5 (C(CH 3 ) 2 ), 163.7 (C@O). 5b (40%): Rf (5:1 hexane/EtOAc) = 0.5; 1 H RMN (300 MHz, CDCl 3 ) d 0.09 (s, 6 H, Si-CH 3 ), 0.91 (s, 9 H, Si-tBu), 1.31-1.32 (s, 6 H, C-CH 3 ), 1.46-1.48 (m, 6 H, CH-CH 3 ), 3.51 (d, 1 H, CH-O), 3.60-3.88 (2dd, 2 H, CH 2 -OTBDMS), 4.05-4.30 (m, 2 H, 2 CH-O), 5.09-5.18 (h, 1 H, O-CH-); 13 C NMR (75 MHz, CDCl 3 ) d À5.3 (Si-C(CH 3 ) 3 ), 16.7 (C(CH 3 ) 3 ), 21.5 (C(CH 3 ) 3 ), 25.7 (CH(CH 3 ) 2 ), 26.2 (C(CH 3 ) 2 ), 62.4 (CH-O(CCl)), 64.6 (CH 2 -O), 65.9 (Cl-C-O), 66.5 (CH-O), 67.8 (CH-O), 73.4 (CH(CH 3 ) 2 ), 111.2 (C(CH 3 ) 2 ), 163.7 (C@O). HRMS (ESI+) Cacld. for C 18 H 33 ClNaO 6 Si [M+Na] + : 431.1633, found 431.1635. 2.1.5. One-pot sequential synthesis of 3-deoxy 6-O-[(1,1dimethylethyl)dimethylsilyl]-4,5-O-(1-methylethylylidene)-D-erythrohexulosonic acid 1-methylethyl ester 7

2. 1 . 6 .

 16 One-pot synthesis of 3-deoxy-6-O-[(1,1dimethylethyl)dimethylsilyl]-4,5-O-(1-methylethylylidene)-D-erythrohexulosonic acid 1-methylethyl ester 7

2. 1 . 7 .

 17 Synthesis 3-deoxy-D-erythro-hexulosonic acid 1-methylethyl ester 8 and 3-deoxy-3,4,5,6-O-tetrasilyl-D-erythro-hexulosonic acid 1-methylethyl ester 9

Scheme 3 .

 3 Scheme 3. Non-phosphorylative Entner Doudoroff pathway.

  H RMN (300 MHz, CDCl 3 ) d 0.08 (s, 12 H, Si-CH 3 ), 0.91 (s, 18 H, Si-tBu), 1.34-1.40 (m, 24 H, C-CH 3 , CH-CH 3 ), 3.67-3.72 (2dd, 2 H, CH CH 2 -I), 5.1-5.2 (h, 2 H, O-CH-); HRMS (ESI+) Cacld. for C 18 H 34 IO 6 Si [M+H] + : 501.1169, found 501.1171.

2 -OTBDMS), 3.78-3.83 (m, 2 H, CH-O), 3.84-3.89 (2dd, 2 H, CH 2 -OTBDMS), 4.05-4.30 (d, 1 H, CH-O), 4.17-4.25 (d, 1 H, CH-O), 4.35-4.42 (d, 2 H,

  1 H RMN (300 MHz, CDCl 3 ) d 0.12 (s, 6 H, Si-CH 3 ), 0.92 (s, 9 H, Si-tBu), 1.23-1.40 (m, 12 H, CH-CH 3 + C-CH 3 ), 2.70-2.80 (d, 1 H, CH 2 ), 3.67-3.91 (2dd, 2 H, CH 2 -OTBDMS), 4.19-4.44 (m, 2 H, 2 CH-O), 5.23-5.40 (h, 1 H, O-CH-); 13 C NMR (75 MHz, CDCl 3 ) d À5.7 (Si-(CH 3 ) 2 ), 15.3 (Si-C(CH 3 ) 3 ), 21.3 (C(CH 3 ) 3 ), 21.6 (CH(CH 3 ) 2 ), 25.7-25.9 (C(CH 3 ) 2 ), 29.7 (CH 2 ), 64.6 (CH 2 -O), 65.8 (CH-O), 71.9 (CH(CH 3 ) 2 ), 109.6 (C(CH 3 ) 2 ), 164 (C@O), 191.9 (C@O); HRMS (ESI+) Cacld. for C 18 H 34 NaO 6 Si [M+Na] + : 397.2022, found 397.2017; ½/ 29 D À19.3°(c 1.0, CHCl 3 ).

Table 1

 1 Growth comparison of most closely related strains of the library clones on glucose minimal medium and KDG minimal medium; +, growth; À, no growth; w, weak growth; ND, not determined; * Rhizobium metallidurans had been found in the soil in addition to the nodules of Anthyllis vulneraria; ** Staphylococcus aureus was not present in the soil, it represented a negative control.

	Sugar-enriched medium	Number of	Glucose KDG
		related clones		isopropyl ester
	Rhizobium metallidurans Ã	2	+	+
	Staphylococcus aureus ÃÃ	0	+	À
	Arenimonas metalli	1	+	À
	Azotobacter beijerinckii	1	+	+
	Bradyrhizobium canariense	3	+	w
	Brevibacillus brevis	4	+	À
	Burkholderia sp.	2	+	+
	Gluconacetobacter diazotrophicus 1	+	À
	Limnobacter litoralis	3	À	À
	Nitrospira sp.	7	+	À
	Nitrosospira multiformis	6	+	À
	Rubrobacteridae	1	N D	N D
	Segetibacter koreensis	3	+	À
	Sphingomonas elodea	2	+	+
	Thermobaculum terrenum	1	+	À
	Xanthomonas campestris	2	+	À

Acknowledgments

We are very grateful to Eddy Petit, Institut Européen des Membranes, Montpellier, for material support and Dr. Stephen Jackson and Pr. Alan Dobson, Environmental Research Institute, Cork, for the fruitful discussions on the microbiological part. We also would like to thank ANR and FEDER for financial supports.


Pierre Letourneau
email: pierre.letourneau@chu-lyon.fr

Stanislas Bataille
email: bataille.stanislas@sfr.fr

Philippe Chauveau
email: ph.chauveau@gmail.com

Denis Fouque
email: denis.fouque@chu-lyon.fr

Laetitia Koppe
email: laetitia.koppe@chu-lyon.fr

Source and Composition in Amino Acid of Dietary Proteins in the Primary Prevention and Treatment of CKD

Keywords: chronic kidney disease, amino-acids, plant-based diet, low-protein diet

Nutrition is a cornerstone in the management of chronic kidney disease (CKD). To limit urea generation and accumulation, a global reduction in protein intake is routinely proposed. However, recent evidence has accumulated on the benefits of plant-based diets and plant-derived proteins without a clear understanding of underlying mechanisms. Particularly the roles of some amino acids (AAs) appear to be either deleterious or beneficial on the progression of CKD and its complications. This review outlines recent data on the role of a low protein intake, the plant nature of proteins, and some specific AAs actions on kidney function and metabolic disorders. We will focus on renal hemodynamics, intestinal microbiota, and the production of uremic toxins. Overall, these mechanistic effects are still poorly understood but deserve special attention to understand why low-protein diets provide clinical benefits and to find potential new therapeutic targets in CKD.

Introduction

Chronic kidney disease (CKD) affects nearly 800 million people worldwide. The kidney has a major role in nutritional homeostasis and partly regulates the amino acids (AAs) pool via their synthesis and degradation. CKD is characterized by a reduction in nitrogenous waste excretion and the accumulation of many organic solutes called uremic toxins (UTs). Many of these UTs are produced by the degradation of dietary proteins and AAs by gut microbiota and appear to accelerate CKD progression and promote a variety of abnormalities, including endothelial dysfunction and insulin resistance. Thus, the composition and balance of gut microbiota in the role of the production of UTs has elicited a recent and important research [START_REF] Koppe | Probiotics and Chronic Kidney Disease[END_REF][START_REF] Koppe | The Role of Gut Microbiota and Diet on Uremic Retention Solutes Production in the Context of Chronic Kidney Disease[END_REF].

Experimentally, Brenner et al. [START_REF] Brenner | Dietary Protein Intake and the Progressive Nature of Kidney Disease: The Role of Hemodynamically Mediated Glomerular Injury in the Pathogenesis of Progressive Glomerular Sclerosis in Aging, Renal Ablation, and Intrinsic Renal Disease[END_REF] showed that a high protein intake induced marked kidney hypertrophy, an increase in intra-glomerular pressure, and hyperfiltration leading to progressive glomerular sclerosis. All observational studies in CKD patients have shown that a high protein intake was associated with CKD progression [START_REF] Metzger | Association of a Low-Protein Diet With Slower Progression of CKD[END_REF][START_REF] Ko | The Effects of High-Protein Diets on Kidney Health and Longevity[END_REF]. Based on these observations, a reduction in protein intake can be expected to preserve renal function and is highly recommended by international guidelines for patients with advanced CKD [START_REF] Ikizler | KDOQI Clinical Practice Guideline for Nutrition in CKD: 2020 Update[END_REF].

Although dietary adaptation is a cornerstone of CKD treatment, the mechanistic roles of a low-protein diet (LPD) in CKD pathogenesis and treatment have been partially explored. The interest in the AAs composition of the various animal and plant proteins has emerged after the second world war at a time when the world faced a food shortage [START_REF] Block | The Amino Acid Composition of Proteins and Foods[END_REF]. The first aim was to choose proteins so that they become mutually supplementary, and thus to ensure a diet that contains the qualitative and quantitative requirements of essential amino acids (EAAs). However, in industrialized countries, humans rarely face protein/AAs insufficient intake, and for the first time in human history, nutritional excesses and overweight people outnumbers the underweight people. The consumption of protein is around 1.2 to 1.4 g/kg/day, mostly from animal sources, according to the National Health and Nutrition Examination Survey (NHANES) [START_REF] Moore | The Mean Dietary Protein Intake at Different Stages of Chronic Kidney Disease Is Higher than Current Guidelines[END_REF]. If a high-protein diet and the keto diet have been suggested to fight against obesity and diabetes, these diets have not been as effective as expected for glycemic control or weight loss in randomized controlled trials (RCT) [START_REF] Athinarayanan | Long-Term Effects of a Novel Continuous Remote Care Intervention Including Nutritional Ketosis for the Management of Type 2 Diabetes: A 2-Year Non-Randomized Clinical Trial[END_REF][START_REF] Joshi | Plant-Based Diets for Prevention and Management of Chronic Kidney Disease[END_REF]. This calls for a reconsideration of AAs functions in nutrition, now based upon health-related criteria to limit the negative effect on kidney health over time across populations with or at-risk for CKD.

Therefore, recent studies have focused on the quality and diversity of proteins more than on quantity for the prevention and management of CKD. In addition, the increasing consumption of plant foods and vegan/vegetarian diets has remotivated the scientific community to identify its impact on health in the context of CKD [START_REF] Carrero | Plant-Based Diets to Manage the Risks and Complications of Chronic Kidney Disease[END_REF][START_REF] Chauveau | Vegetarian Diets and Chronic Kidney Disease[END_REF]. In the present article, we review the recent evidence suggesting that the source of proteins (animal versus plant) and composition of AAs specifically affect CKD progression and complications. Because various new functions of AAs on gut microbiota have been recently uncovered, we will focus on recent protein metabolism data on the host/intestinal microbiota balance.

Clinical Evidence of Plant-Based Diets in CKD Incidence, Progression, and Complications

Plant-based diets -including vegan and vegetarian diets (typically including dairy products and eggs, fruits, plants, grains, and legumes) have recently been considered beneficial for human health [START_REF] Melina | Position of the Academy of Nutrition and Dietetics: Vegetarian Diets[END_REF]. Numerous observational studies in the general population have reported a higher incidence of all-cause mortality, cardiometabolic diseases, and cancer in people who consume greater quantities of red meat [START_REF] Sinha | Meat Intake and Mortality: A Prospective Study of over Half a Million People[END_REF][START_REF] Haring | Dietary Protein Sources and Risk for Incident Chronic Kidney Disease: Results from the Atherosclerosis Risk in Communities (ARIC) Study[END_REF][START_REF] Oosterwijk | High Dietary Intake of Vegetable Protein Is Associated with Lower Prevalence of Renal Function Impairment: Results of the Dutch DIALECT-1 Cohort[END_REF], which has been recently challenged. Indeed, in a recent meta-analysis, there was low-or very-low-certainty evidence suggesting that dietary patterns with less red and processed meat intake may result in reductions in adverse cardiometabolic and cancer outcomes [START_REF] Vernooij | Patterns of Red and Processed Meat Consumption and Risk for Cardiometabolic and Cancer Outcomes: A Systematic Review and Meta-Analysis of Cohort Studies[END_REF].

In the context of CKD, another recent meta-analysis of cohort studies linked the daily consumption of plant-based diets and plant proteins with a lower risk of CKD prevalence, incidence, and estimated glomerular filtration rate (eGFR) decline over time [START_REF] Kalantar-Zadeh | Plant-Dominant Low-Protein Diet for Conservative Management of Chronic Kidney Disease[END_REF][START_REF] Kelly | Healthy Dietary Patterns and Risk of Mortality and ESRD in CKD: A Meta-Analysis of Cohort Studies[END_REF]. However, the review was based on low power RCTs existing in this field with an observation time greater than six months. Most of these trials were conducted in patients with preexisting diseases that predispose to CKD [START_REF] Haring | Dietary Protein Sources and Risk for Incident Chronic Kidney Disease: Results from the Atherosclerosis Risk in Communities (ARIC) Study[END_REF][START_REF] Kamper | Long-Term Effects of High-Protein Diets on Renal Function[END_REF] as summarized in Tables 1 and2. It is important to mention that diet investigations are associated with several biases, and it is important to consider that the association between socio-economic level and animal-protein intakes might influence the results of observational studies.

Metabolic acidosis, eGFR decline and CVD risk factors

Fruit and plant diet are as effective as oral bicarbonate to correct metabolic acidosis (p < 0.01), eGFR decline (-10.0, 95% CI -10.6 to -9.4 mL/min/1.73 m2 versus -18.8, 95% CI -19.5 to -18.2 mL/min/1.73 m2 in usual care group), p < 0.01.) and was better than bicarbonate to reduce systolic blood pressure (p < 0.01) It was more effective to lower low-density lipoprotein and increase serum vitamin K1 Only randomized controlled trials were selected in PubMed database. Abbreviations: APD, animal protein diet; CRP: C reactive protein; CVD, Cardiovascular disease; CKD, chronic kidney disease; ESKD, end-stage kidney disease; eGFR, estimated glomerular filtration rate; FGF23, fibroblast growth factor 23; NaHCO3, sodium bicarbonate; VPD, plant protein diet; HR, hazard ratio.

Therefore, the new international guidelines of the Kidney Disease Outcomes Quality Initiative (KDOQI) did not recommend plant protein over animal protein in CKD due to lack of evidence and because overall data strength was found to be low. Yet, they do recommend a plant-based pattern in CKD patients for its potential benefits on body weight, blood pressure, and net acid production but not for renal protection [START_REF] Ikizler | KDOQI Clinical Practice Guideline for Nutrition in CKD: 2020 Update[END_REF].

Presumed Mechanisms of Plant-Diets Beneficial Effects on CKD Progression

Preliminary experimental and human studies reported that independently of quantity, plant proteins have different renal effects from animal protein.

There appears to be a beneficial effect on renal hemodynamics with a reduced glomerular hyperfiltration and a decrease in albumin clearance during an equal protein intake regardless of the presence of associated fiber. The mechanism is not yet elucidated but could involve insulin-like growth factor 1 (IGF1) and/or prostaglandins [START_REF] Kontessis | Metabolic and Hormonal Responses to Ingestion of Animal and Vegetable Proteins[END_REF] effects on glomerular filtration rate (GFR). The progression of chronic nephropathy in older rats was markedly delayed by replacing casein with soy protein [START_REF] Iwasaki | The Influence of Dietary Protein Source on Longevity and Age-Related Disease Processes of Fischer Rats[END_REF]. The different AA content between plant and meat could be the explanation of the observed benefit and will be discussed below.

There is extensive literature on the benefits of plant-diet in CKD progression and complications [START_REF] Carrero | Plant-Based Diets to Manage the Risks and Complications of Chronic Kidney Disease[END_REF][START_REF] Chauveau | Vegetarian Diets and Chronic Kidney Disease[END_REF][START_REF] Kalantar-Zadeh | Plant-Dominant Low-Protein Diet for Conservative Management of Chronic Kidney Disease[END_REF]. Based on these studies, we have summarized on Figure 1 the mechanisms of plant-based diets on CKD progression. However, not all pathways are fully understood and the effect of plant based-diet on metabolic parameters such as insulin sensitivity or hypertension must be consolidated in large cohorts. Indeed, only few adequate trials have investigated the benefits of plant-based diets on metabolic complications. For example, acidosis improvement is well demonstrated but the effects of plant diets on potassium, uremic toxins or bone parameters need further assessment."

Limitations of Plant-Diets in CKD

LPD and plant-based diets share possible caveats or preconceived ideas that may limit their implementation. Firstly, plant-diet regimen brings several beneficial nutrients. It includes a higher fiber intake, which induces a better weight control and improves serum lipid profile [START_REF] Bozzetto | Dietary Fibre as a Unifying Remedy for the Whole Spectrum of Obesity-Associated Cardiovascular Risk[END_REF][START_REF] Anderson | Postprandial Serum Glucose, Insulin, and Lipoprotein Responses to High-and Low-Fiber Diets[END_REF]. A plant-based diet is also associated with higher consumption of mono or polyunsaturated fatty acids, an improvement in lipid profile [START_REF] Ferdowsian | Effects of Plant-Based Diets on Plasma Lipids[END_REF], and a weight reduction [START_REF] Austel | Weight Loss with a Modified Mediterranean-Type Diet Using Fat Modification: A Randomized Controlled Trial[END_REF]. There is also a better control of metabolic acidosis, equivalent to oral sodium bicarbonate supplements on the preservation of eGFR, and a decrease in systolic blood pressure [START_REF] Goraya | A Comparison of Treating Metabolic Acidosis in CKD Stage 4 Hypertensive Kidney Disease with Fruits and Vegetables or Sodium Bicarbonate[END_REF][START_REF] Goraya | Treatment of Metabolic Acidosis in Patients with Stage 3 Chronic Kidney Disease with Fruits and Vegetables or Oral Bicarbonate Reduces Urine Angiotensinogen and Preserves Glomerular Filtration Rate[END_REF][START_REF] Goraya | Fruit and Vegetable Treatment of Chronic Kidney Disease-Related Metabolic Acidosis Reduces Cardiovascular Risk Better than Sodium Bicarbonate[END_REF][START_REF] Goraya | Dietary Acid Reduction with Fruits and Vegetables or Bicarbonate Attenuates Kidney Injury in Patients with a Moderately Reduced Glomerular Filtration Rate Due to Hypertensive Nephropathy[END_REF][START_REF] Wesson | Acid Retention during Kidney Failure Induces Endothelin and Aldosterone Production Which Lead to Progressive GFR Decline, a Situation Ameliorated by Alkali Diet[END_REF][START_REF] Wesson | Endogenous Endothelins Mediate Increased Acidification in Remnant Kidneys[END_REF]. Blood pressure control and volume overload are also improved by these diets because they generally contain a lower sodium intake [START_REF] Sacks | Effects on Blood Pressure of Reduced Dietary Sodium and the Dietary Approaches to Stop Hypertension (DASH) Diet. DASH-Sodium Collaborative Research Group[END_REF]. Concerning phosphorus, these diets decrease serum phosphorus on the one hand because phosphate of plant origin is present as phytate and is less absorbed in the gut [START_REF] Moe | Vegetarian Compared with Meat Dietary Protein Source and Phosphorus Homeostasis in Chronic Kidney Disease[END_REF][START_REF] Moorthi | The Effect of a Diet Containing 70% Protein from Plants on Mineral Metabolism and Musculoskeletal Health in Chronic Kidney Disease[END_REF], and on the other hand because they avoid processed food industry products enriched in inorganic phosphate. A plant-based regimen is also associated with a decrease in inflammation and the risk of mortality in CKD patients [START_REF] Xu | Dietary Fiber, Kidney Function, Inflammation, and Mortality Risk[END_REF][START_REF] Krishnamurthy | High Dietary Fiber Intake Is Associated with Decreased Inflammation and All-Cause Mortality in Patients with Chronic Kidney Disease[END_REF].

Secondly, in plant diets, protein quantities are usually reduced and can be assimilated with an LPD that has been associated with a delay in dialysis start [START_REF] Garneata | Ketoanalogue-Supplemented Vegetarian Very Low-Protein Diet and CKD Progression[END_REF][START_REF] Hahn | Low Protein Diets for Non-Diabetic Adults with Chronic Kidney Disease[END_REF]. Some of the major UTs are produced by intestinal microbiota from diet AAs: Indoles as indoxyl sulfate (IS) and indole-3-acetic acid (IAA) from tryptophane, p-cresyl sulfate (PCS) from tyrosine or phenylalanine, trimethyl amine oxidase (TMAO) from choline or L-carnitine. One of the hypothetical mechanisms of reducing protein intake and increasing plant-based diet in CKD is the capacity to reduce UTs production. Indeed a relationship has been reported between the quantity of protein intake and plasma levels of UTs (such as IS and PCS) and urea have been observed [START_REF] Poesen | The Influence of Dietary Protein Intake on Mammalian Tryptophan and Phenolic Metabolites[END_REF]. In this condition, a vegetarian diet (with less precursor of UTs) showed a two-fold reduction in major gut-derived UTs such as PCS and IS in adults with normal renal function [START_REF] Patel | The Production of P-Cresol Sulfate and Indoxyl Sulfate in Vegetarians versus Omnivores[END_REF] and consistent results in patients on maintenance hemodialysis [START_REF] Marzocco | Very Low Protein Diet Reduces Indoxyl Sulfate Levels in Chronic Kidney Disease[END_REF]. A short-term (12-week) randomized crossover trial reported that patients with CKD who followed a vegetarian very low-protein diet exhibited a decrease in these UTs and a modification of intestinal microbiota composition [START_REF] Di Iorio | Nutritional Therapy Reduces Protein Carbamylation through Urea Lowering in Chronic Kidney Disease[END_REF][START_REF] Di Iorio | Nutritional Therapy Modulates Intestinal Microbiota and Reduces Serum Levels of Total and Free Indoxyl Sulfate and P-Cresyl Sulfate in Chronic Kidney Disease (Medika Study)[END_REF]. The increased intake of fibers in plant-diets allows a modification of colic microbiota towards bacteria that decrease inflammation, bacterial translocation, and the production of gut-derived UTs [START_REF] Sirich | Effect of Increasing Dietary Fiber on Plasma Levels of Colon-Derived Solutes in Hemodialysis Patients[END_REF][START_REF] Xie | Effects of Fermentable Dietary Fiber Supplementation on Oxidative and Inflammatory Status in Hemodialysis Patients[END_REF][START_REF] Xu | Excess Protein Intake Relative to Fiber and Cardiovascular Events in Elderly Men with Chronic Kidney Disease[END_REF][START_REF] Rossi | Dietary Protein-Fiber Ratio Associates with Circulating Levels of Indoxyl Sulfate and p-Cresyl Sulfate in Chronic Kidney Disease Patients[END_REF][START_REF] Wu | Linking Long-Term Dietary Patterns with Gut Microbial Enterotypes[END_REF].

Limitations of Plant-Diets in CKD

LPD and plant-based diets share possible caveats or preconceived ideas that may limit their implementation.

Denutrition/Malnutrition

Nowadays, it is well established that LPD is beneficial for the preservation of kidney function and retarding the need for maintenance dialysis [START_REF] Ikizler | KDOQI Clinical Practice Guideline for Nutrition in CKD: 2020 Update[END_REF]. The main reluctance for its implementation is linked to a possible fear for increased protein-energy wasting (PEW) and sarcopenia. Concerning sarcopenia, Garibotto et al. shown that LPD and very LPD (VLPD) are safe in response to an adapted muscle protein metabolism [START_REF] Garibotto | Effects of Low-Protein, and Supplemented Very Low-Protein Diets, on Muscle Protein Turnover in Patients With CKD[END_REF]. Concerning PEW, a report of 16 controlled trials listed by Kalantar-Zadeh et al. did not show PEW [START_REF] Kalantar-Zadeh | Plant-Dominant Low-Protein Diet for Conservative Management of Chronic Kidney Disease[END_REF]. In a recent meta-analysis of randomized controlled trials over more than 2000 patients, Hahn et al. did not find a significant modification of nutritional measures when dietary management was performed by trained dietitians. Indeed, the mean final body weight was 1.4 kg higher in patients receiving a VLPD compared to a LPD. The risk for wasting during a VLPD was 0.6%, a magnitude that is not different from a normal diet (0.4%) [START_REF] Hahn | Low Protein Diets for Non-Diabetic Adults with Chronic Kidney Disease[END_REF].

The potential hazardous risk of a vegetarian diet is also discussed. This is partly based on beliefs that, as compared to animal proteins, proteins of plant origin would be of lower quality, less complete, poorly digested, and not sufficient to meet daily protein targets in hemodialysis patients. Numerous studies have confirmed the nutritional safety of vegetarian diets in non-dialysis CKD patients [START_REF] Soroka | Comparison of a Vegetable-Based (Soya) and an Animal-Based Low-Protein Diet in Predialysis Chronic Renal Failure Patients[END_REF][START_REF] Chauveau | Vegetarianism: Advantages and Drawbacks in Patients with Chronic Kidney Diseases[END_REF][START_REF] Wu | Nutritional Status of Vegetarians on Maintenance Haemodialysis[END_REF]. In addition, the same holds true for maintenance dialysis patients following LPD with plant protein source and higher caloric intakes compared to patients following a LPD with animal protein diet [START_REF] Soroka | Comparison of a Vegetable-Based (Soya) and an Animal-Based Low-Protein Diet in Predialysis Chronic Renal Failure Patients[END_REF]. A number of studies confirmed that is possible to achieve a target of 1-1.2 g protein/kg/day as recommended in hemodialysis patients with a vegetarian diet [START_REF] Chauveau | Vegetarianism: Advantages and Drawbacks in Patients with Chronic Kidney Diseases[END_REF][START_REF] Wu | Nutritional Status of Vegetarians on Maintenance Haemodialysis[END_REF].

Bone Disorder

Adequate calcium, protein, and vitamin D intakes are needed for optimal bone health. A plant-based diet is supposed to contain lower calcium with poorer bioavailability. It appears that the absorption of plant-calcium is as efficient or better than that of milk (i.e., 20-40%) if the plant does not have high oxalic acid nor phytic acid concentration. Some plants such as soy or beans can contain as much as 100 mg/100 g of calcium, but overall plants are less concentrated in calcium than dairy products [START_REF] Weaver | Calcium Bioavailability and Its Relation to Osteoporosis[END_REF][START_REF] Weaver | Dietary Calcium: Adequacy of a Vegetarian Diet[END_REF]. Therefore, ensuring an adequate calcium intake from plants or via supplementation is mandatory with a vegan diet but not in an ovo-lacto-vegetarian diet.

Plant-based diets tend to contain less aminoacids that could lead to osteoporosis. A systematic review confirmed this hypothesis by showing no adverse effects on bone mineral density and hip fracture occurrence with lower compared to higher total protein intake [START_REF] Shams-White | Dietary Protein and Bone Health: A Systematic Review and Meta-Analysis from the National Osteoporosis Foundation[END_REF]. One RCT comparing isoflavone-rich soy protein versus various animal-based proteins did not show abnormal bone health [START_REF] Shams-White | Animal versus Plant Protein and Adult Bone Health: A Systematic Review and Meta-Analysis from the National Osteoporosis Foundation[END_REF]. One prospective study found that plant diet improved bone mineral density in the general population [START_REF] Movassagh | Vegetarian-Style Dietary Pattern during Adolescence Has Long-Term Positive Impact on Bone from Adolescence to Young Adulthood: A Longitudinal Study[END_REF]. Furthermore, a cross-sectional study found an association between plant food consumption and improvement in bone mineralization markers [START_REF] Berg | Increased Consumption of Plant Foods Is Associated with Increased Bone Mineral Density[END_REF]. A meta-analysis including 18 studies found an inverse association between the consumption of fruit and the risk of postmenopausal osteoporosis [START_REF] Hu | Fruit and Vegetable Consumption and the Risk of Postmenopausal Osteoporosis: A Meta-Analysis of Observational Studies[END_REF]. Some authors suggested that these reassuring results can be explained through the reduction in BMI, an increase in beneficial phytochemicals, and microbiota modification due to plant dietary patterns [START_REF] Hsu | Plant-Based Diets and Bone Health: Sorting through the Evidence[END_REF]. This point has never been specifically explored in CKD patients and needs further studies. However, in the last meta-analysis, which included 17 RCTs and 1459 participants, it was observed that VLPD reduced serum phosphate and parathyroid hormone (PTH) levels suggesting an improved mineral and bone health [START_REF] Kieneker | Low Potassium Excretion but Not High Sodium Excretion Is Associated with Increased Risk of Developing Chronic Kidney Disease[END_REF].

Hyperkalemia

Another important question related to plant-based diets is the high potassium content for some of them [START_REF] Carrero | Plant-Based Diets to Manage the Risks and Complications of Chronic Kidney Disease[END_REF] and the potential risk of hyperkalemia in CKD patients. A recent international recommendation indicates a lack of robust studies assessing potassium intake requirements in CKD patients [START_REF] Ikizler | KDOQI Clinical Practice Guideline for Nutrition in CKD: 2020 Update[END_REF]. Authors highlighted the difficulty of conducting reliable studies due to several reasons such as treatments used (angiotensin-converting enzyme inhibitors, potassium binders), volume status, acid-base status, possible gastrointestinal symptoms, as well as the difficulty of assessing dietary potassium intakes and their poor correlation with serum potassium [START_REF] St-Jules | Nutrient Non-Equivalence: Does Restricting High-Potassium Plant Foods Help to Prevent Hyperkalemia in Hemodialysis Patients?[END_REF].

In the general population, subjects consuming more potassium develop less CKD [START_REF] Kieneker | Low Potassium Excretion but Not High Sodium Excretion Is Associated with Increased Risk of Developing Chronic Kidney Disease[END_REF], and less hypertension [START_REF] Chiu | Comparison of the DASH (Dietary Approaches to Stop Hypertension) Diet and a Higher-Fat DASH Diet on Blood Pressure and Lipids and Lipoproteins: A Randomized Controlled Trial123[END_REF]. Furthermore, Aburto and al [START_REF] Aburto | Effect of Increased Potassium Intake on Cardiovascular Risk Factors and Disease: Systematic Review and Meta-Analyses[END_REF] showed that increasing potassium intake reduces blood pressure in known hypertensive patients. In patients with CKD, there are also several studies in favor of a potassium-rich diet that tends to be similar to a plant and fruit diet. However, most of them are retrospective and based on surrogate criteria of 24-h kaliuresis to assess dietary potassium intake. Therefore, they do not establish a direct causal link between increased potassium intake and a reduction in the risk of progression of cardiovascular or renal diseases. Several studies suggest that increased kaliuresis is associated with a lower occurrence of dialysis initiation, a slower progression of CKD and proteinuria, and death [START_REF] Smyth | ONTARGET and TRANSCEND investigators The Relationship between Estimated Sodium and Potassium Excretion and Subsequent Renal Outcomes[END_REF][START_REF] Nagata | Association between 24h Urinary Sodium and Potassium Excretion and Estimated Glomerular Filtration Rate (EGFR) Decline or Death in Patients with Diabetes Mellitus and EGFR More than 30 Ml/Min/1.73m2[END_REF]. It should be noted that in these two studies, the beneficial effect seems to be less significant in patients with more severe CKD (i.e., <60 mL/min eGFR in Nagata et al. [START_REF] Nagata | Association between 24h Urinary Sodium and Potassium Excretion and Estimated Glomerular Filtration Rate (EGFR) Decline or Death in Patients with Diabetes Mellitus and EGFR More than 30 Ml/Min/1.73m2[END_REF] and <45 mL/min in Smyth et al. [START_REF] Smyth | ONTARGET and TRANSCEND investigators The Relationship between Estimated Sodium and Potassium Excretion and Subsequent Renal Outcomes[END_REF]). There are few interventional studies in advanced CKD patients. Tyson et al. reported no hyperkalemia episode during a two-week DASH regimen in patients with CKD stage 3-4 [START_REF] Tyson | Short-Term Effects of the DASH Diet in Adults with Moderate Chronic Kidney Disease: A Pilot Feeding Study[END_REF]. Goraya et al. showed no association between plant-based diet and hyperkalemia after a five-year follow-up in patients with CKD stage 3-4 [START_REF] Goraya | Fruit and Vegetable Treatment of Chronic Kidney Disease-Related Metabolic Acidosis Reduces Cardiovascular Risk Better than Sodium Bicarbonate[END_REF]. A study of with 11 patients with an eGFR <30 mL/min/1.73 m2 taking a vegan diet did not disclose an increase in serum potassium at three months [START_REF] Barsotti | A Low-Nitrogen Low-Phosphorus Vegan Diet for Patients with Chronic Renal Failure[END_REF]. On the other hand, plant-based diets, by its fibers content, improve gut transit, reduce constipation, leading to an increased potassium elimination into the feces [START_REF] Cupisti | Dietary Approach to Recurrent or Chronic Hyperkalaemia in Patients with Decreased Kidney Function[END_REF].

Overall, it seems overrated to accuse plant-based diets for high potassium content since high potassium intake occurs as well with highly processed foods of animal origin [START_REF] Picard | Handouts for Low-Potassium Diets Disproportionately Restrict Fruits and Vegetables[END_REF]. Obviously it is possible to reduce the potassium content of foods of plant origin by appropriate cooking. An ongoing RCT of approximately 500 patients with CKD stage 3-4, who will receive either oral potassium supplementation with potassium chloride or potassium citrate versus placebo will answer the question whether potassium alone (i.e., not associated with a plant diet) can lead to better kidney outcomes [START_REF]Potassium Supplementation in CKD-Full Text View-ClinicalTrials[END_REF].

Does the AA Composition Influence the Progression of CKD and Its Complications?

For decades, various health benefits have been attributed to protein restriction in patients with CKD, such as favorable metabolic effects, reduction in proteinuria and uremic symptoms, and improvement in insulin sensitivity [START_REF] Koppe | The Role for Protein Restriction in Addition to Renin-Angiotensin-Aldosterone System Inhibitors in the Management of CKD[END_REF]. Beside the CKD area, new emerging studies have suggested that a LPD can increase longevity in many species and can retard age-related diseases such as cancer, type 2 diabetes [START_REF] Treviño-Villarreal | Dietary Protein Restriction Reduces Circulating VLDL Triglyceride Levels via CREBH-APOA5-Dependent and -Independent Mechanisms[END_REF], and dyslipidemia/fatty liver disease [START_REF] Maida | A Liver Stress-Endocrine Nexus Promotes Metabolic Integrity during Dietary Protein Dilution[END_REF]. Following these observations, several studies have tried to decipher which particular AAs are required to induce the systemic responses to a LPD. In addition, it is tempting to check if specific dietary AA restrictions (one or several AA) are both sufficient and necessary to improve the metabolic response to a LPD. The challenge to apply these specific diets during CKD appears important and complex because (1) diet must ensure that EAAs are sufficient if dietary protein intake is reduced; (2) the recommendation of EAAs requirement is not consensual (Table 3) and not specific for CKD patients; (3) the transposition of experimental to human studies must be carefully interpreted because of EAAs are species-specific and they differ between humans and rodents; (4) the analysis of actual dietary intakes is difficult in human and can be different than prescribed. Serving sizes and food selection vary, making it difficult to estimate single-nutrient and -AA intakes. (1) Mean nitrogen requirement of 105 mg nitrogen/kg per day (0.66 g protein/kg per day).

Plasma Concentrations of Amino Acids in the Context of Plants and Animal Diets

Both animal and plant proteins are made up by about 20 common AAs. Nine AAs-histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine-are not synthesized by mammals and are therefore dietarily essential or indispensable nutrients. These are commonly called essential AAs (EAAs). The branched-chain amino acids (BCAAs) leucine, isoleucine, and valine belong to the nine EAAs. The proportion of these AAs varies as a characteristic of a given protein, but all food proteins-except for gelatin-contain some of each. For example, EAAs content of plant-based protein isolates such as lupin (21%), oat (21%), and wheat (22%) are lower than animal-based proteins (casein 34%, milk 39%, whey 43%, and egg 32%) and muscle protein (38%). AAs profiles differ among plant-based proteins with leucine contents ranging from 13.5% in corn protein to 5.1% in hemp, compared to 7.0% in eggs, 9.0% in milk, and 7.6% in muscle protein. Methionine and lysine are typically lower in plant proteins such as rice and beans (1.0 and 3.6%) compared with animal proteins (2.5 and 7.0%) and muscle protein (2.0 and 7.8%, respectively) [START_REF] Gorissen | Protein Content and Amino Acid Composition of Commercially Available Plant-Based Protein Isolates[END_REF]. Furthermore, proteins of animal origin contain more aromatic amino acids (AAAs) (tyrosine, tryptophan, and phenylalanine).

Although plant and animal proteins differ in their AAs profiles, digestibility, and availability [START_REF] Carrero | Plant-Based Diets to Manage the Risks and Complications of Chronic Kidney Disease[END_REF], these differences do not seem to be clinically relevant for the healthy adult general population with a varied and sufficient diet [START_REF] Millward | Identifying Recommended Dietary Allowances for Protein and Amino Acids: A Critique of the 2007 WHO/FAO/UNU Report[END_REF]. Indeed, the plasma AAs profile and in particular EAAs [START_REF] Millward | The Nutritional Value of Plant-Based Diets in Relation to Human Amino Acid and Protein Requirements[END_REF][START_REF] Young | Plant Proteins in Relation to Human Protein and Amino Acid Nutrition[END_REF] of plant-based diets and animal-based diets is almost the same, except for a lower lysine-to-arginine ratio [START_REF] Schmidt | Plasma Concentrations and Intakes of Amino Acids in Male Meat-Eaters, Fish-Eaters, Vegetarians and Vegans: A Cross-Sectional Analysis in the EPIC-Oxford Cohort[END_REF]. However, CKD patients have strikingly different plasma concentrations of AAs from those of healthy subjects [START_REF] Schmidt | Plasma Concentrations and Intakes of Amino Acids in Male Meat-Eaters, Fish-Eaters, Vegetarians and Vegans: A Cross-Sectional Analysis in the EPIC-Oxford Cohort[END_REF][START_REF] Guebre-Egziabher | Short-Term Administration of a Combination of Recombinant Growth Hormone and Insulin-like Growth Factor-I Induces Anabolism in Maintenance Hemodialysis[END_REF][START_REF] Lim | Does Hemodialysis Increase Protein Breakdown? Dissociation between Whole-Body Amino Acid Turnover and Regional Muscle Kinetics[END_REF]. For example, BCAAs concentration is lower in CKD patients and non-EAAs are higher. These variations may be related to modified protein and AAs metabolism, to low energy and protein intakes, or to AAs and protein losses due to peritoneal dialysis or hemodialysis. Further specific studies are needed to determine if plant diets can affect, in the long term, the plasma concentration of AAs in CKD patients.

Influence of Specific Amino Acids on Renal Hemodynamics

Early mechanistic studies suggested that not all proteins and AAs equally induce glomerular hyperfiltration. This finding has been proposed because, in a preliminary study in which 10 healthy volunteers consumed equivalent amounts of animal or plant protein for three weeks, consumption of plant protein resulted in a reduction in renal plasma flow and increased renal vascular resistance [START_REF] Kontessis | Metabolic and Hormonal Responses to Ingestion of Animal and Vegetable Proteins[END_REF][START_REF] Kontessis | Metabolic, and Hormonal Responses to Proteins of Different Origin in Normotensive, Nonproteinuric Type I Diabetic Patients[END_REF]. No difference was found in plasma AAs levels following the two types of protein loads but glycine, alanine, methionine, and lysine were less abundant in plant proteins, suggesting a potential mechanism for the beneficial effects of plants diet [START_REF] Kontessis | Metabolic and Hormonal Responses to Ingestion of Animal and Vegetable Proteins[END_REF][START_REF] Kontessis | Metabolic, and Hormonal Responses to Proteins of Different Origin in Normotensive, Nonproteinuric Type I Diabetic Patients[END_REF]. In another seminal study, five adult volunteers were infused on separate occasions with BCAAs or a mixture of non-EAAs and EAAs. BCAAs infusion caused a moderate renal vasoconstriction, a slight increase in GFR, whereas the AAs mixture induced a significantly higher increase in GFR and a state of renal vasodilation [START_REF] Claris-Appiani | Lack of Glomerular Hemodynamic Stimulation after Infusion of Branched-Chain Amino Acids[END_REF]. An experimental study had 5/6 nephrectomy rats submitted for five weeks to either a control diet (18% casein protein) or diets containing 8% casein supplemented with 10% of a mix of BCAAs or AAAs. These AAs (BCAA and AAA) displayed seemingly opposite effects. The AAAs stimulated renal plasma flow and to a lesser extent GFR, possibly via an effect on the production of bioactive molecules such as 5'AMP-activated protein kinase (AMPK), which is a sensor of cellular energy homeostasis. The BCAAs supplementation was associated with a higher level of fibrosis in the kidney and an increase in plasma free fatty acids pointing to an abnormality at the level of energy metabolism [START_REF] Pillai | Differential Impact of Dietary Branched Chain and Aromatic Amino Acids on Chronic Kidney Disease Progression in Rats[END_REF]. BCAAs have been shown to impact metabolism by modulating phosphoinositide 3-kinase (PI3K)-AKT signaling and its downstream transcriptional regulation, in particular of the transcription factor KLF15 (Kruppel-like factor 15). In a CKD mice model, an invalidation of KLF15 was associated with an increased renal fibrosis [START_REF] Gao | Low-Protein Diet Supplemented with Ketoacids Reduces the Severity of Renal Disease in 5/6 Nephrectomized Rats: A Role for KLF15[END_REF]. The KLF15 downregulation and renal fibrosis are reversed with dietary protein restriction in mice. These results suggest that KLF15 may play a role in suppressing renal fibrosis and could contribute to the benefits reported during dietary protein restriction and BCAAs restriction. By contrast, we showed in a preliminary work that a specific restriction in aromatic AAs intake in CKD mice mitigated inflammation that plays a major role in the progression of renal damage [START_REF] Barba | A Low Aromatic Amino-Acid Diet Improves Renal Function and Prevents Kidney Fibrosis iN Mice with Chronic Kidney Disease[END_REF]. Therefore, studies investigating underlying mechanisms of AAs in renal progression are lacking and remain to be done.

Influence of Specific Amino Acids on Uremic Toxins Generation and Intestinal Microbiota

There is a growing recognition of the role of diet in modulating the composition and metabolic activity of the human gut microbiota, which in turn can impact health. Probably independently of fiber intake, the composition of AAs from plant origin may influence the production of UTs and potentially alleviate symptoms associated with the accumulation of UTs.

Aromatic Amino Acids (AAAs)

The transformation of tryptophan to indolic uremic toxins (UTs) IS and IAA requires bacterial tryptophanase from Citrobacter, Escherichia, and Proteus, among others. Indolic UTs (like IS and indole-3-acetic acid) are ligands of the transcription factor aryl hydrocarbon receptor (AhR), also known as the dioxin receptor. AhR activation is known to mediate cardiotoxicity, vascular inflammation, and renal dysfunction. Breakdown of tyrosine and phenylalanine by intestinal anaerobic bacteria, such as Bacteroides, Bifidobacterium, Lactobacillus, Enterobacter, and Clostridium generate phenols such as p-cresol. P-cresol is absorbed in the gut and metabolized to PCS in enterocytes and liver. In a preliminary work, we have shown that a specific restriction in the intake of AAAs in a CKD mice model lowered UTs concentrations as efficiently as a LPD [START_REF] Barba | A Low Aromatic Amino-Acid Diet Improves Renal Function and Prevents Kidney Fibrosis iN Mice with Chronic Kidney Disease[END_REF]. However, in a small cohort of hemodialysis patients, Brito et al. failed to show an association between dietary tryptophan intake and IS levels. A weakness of this study was the fact that the tryptophan intake was only evaluated by a 24-hr dietary recall performed on three different days [START_REF] De Brito | Is there a relationship between tryptophan dietary intake and plasma levels of indoxyl sulfate in chronic kidney disease patients on hemodialysis?[END_REF]. The amount of indolic UTs produced could not only be related to dietary tryptophan but also to the indole production by microbiota tryptophanases and through the liver transformation of indole into IS and IAA [100].

More recently, in a large cohort of 223 hemodialysis patients, using the characterization of the gut microbiome, serum, and fecal metabolome, permutational multivariate analysis of variance analysis revealed that the food categories (such as plant, red meat, fish, fruit, egg, etc.) did not have a significant impact on this UTs profile and microbiome. If the composition of diet was evaluated by a food frequency questionnaire of 117 items, the quality of protein was not evaluated, as well as the quantity of tyrosine or tryptophan [101]. Overall, the bacterial metabolism of AAAs is both multifaceted, interconvertible, and non-fully explored in CKD.

Sulfur-Containing Amino Acids (SAAs)

Although a number of sulfur compounds are metabolized by the gut bacteria, sulfide is generated into the human large intestine by two principal routes: By the action of sulfate-reducing bacteria on inorganic sulfur (sulfate and sulfite) and through the fermentation of sulfur-containing amino acids (SAAs). The chief sources of dietary sulfur are inorganic sulfate and the SAAs methionine, cysteine, cystine, and taurine [102]. Dietary SAAs intake has been reported to preserve CKD progression in patients and experimental disease models [103,104]. To date, it is thought that diet acts to alter the relative abundance (or diversity) of gut microbes, which can then lead to changes in gut microbial metabolite production. Indeed, sulfur compounds increase the abundance of sulfur-reducing bacteria, such as Escherichia coli and Clostridium spp., in the colon, which increases the generation of hydrogen sulfide (H 2 S). However, Lobel et al. [105] reported that diet can post-translationally modify the gut microbial proteome (S-sulfhydration), which can alter microbial metabolite production to drive changes in renal function. Interestingly, they demonstrated that SAAs can increase sulfahydrated of tryptophanase in Escherichia coli and induce a reduction of indole production in CKD mice. These results are in contradiction with the potential beneficial effect of plants because methionine and cysteine are proportionately slightly lower in legumes. Similarly, urea can contribute to a post-translational modification of proteins via increased O-glycosylation and could potentially modify bacterial activity [106]. Increased O-glycosylation of mucins strongly and negatively affects the mucus layer cohesive properties in the gut lumen [107]. Further, protein glycosylation is central to the physiology of intestinal bacteria like Bacterioides fragilis [108]. However, the production of urea is not linked to specific AAs. Finally, methionine could potentially influence methylation by providing methyl groups (CH3). Epigenetic alterations such as DNA methylation could potentially provide explanations for altered gene expressions in CKD and in bacterial metabolism [109]. Therefore, the interaction between diet, microbial metabolism, and posttranslational protein regulation must be explored in CKD.

L-carnitine and Choline

Dietary choline and L-carnitine are precursors of TMAO. Choline is metabolized by gut microbiota to trimethylamine (TMA), which is absorbed and oxidized by hepatic flavin monooxygenase-3 (FMO) into TMAO. A phosphatidylcholine challenge (ingestion of two hard-boiled eggs for example) also increases plasma TMAO in humans [110]. This response is suppressed by the administration of antibiotics, which modify the microbiota or by the use of a synthetic diet with a choline analog such as 3,3-dimethyl-1-butanol in mice [111].

The link between TMAO concentrations and renal function has been demonstrated by Tang et al. who observed in healthy male mice fed with a choline-supplemented diet an increase in collagen deposition and tubulointerstitial fibrosis [112]. By contrast, choline-deficient diets have also been associated with hepatic steatosis and disrupt the intestinal barrier [113].

In addition, L-carnitine, which can be produced via nutritionally derived lysine and methionine has been proposed as a therapeutic agent to improve mitochondrial energy metabolism. Carnitine transports free-fatty acids into the mitochondria to maintain mitochondrial function. A meta-analysis has demonstrated that L-carnitine can improve metabolic parameters and survival but the results are not conclusive in the context of CKD [114]. In vitro, cultured myoblast incubation with L-carnitine restored the IS-induced decrease in mitochondrial membrane potential. In a CKD mice model, L-carnitine did not affect plasma IS concentration, suggesting no effect on intestinal metabolism of tryptophan. However, L-carnitine can limit the induction of myostatin and atrogin-1 expression in CKD mice and improve exercise and mitochondrial status by increasing peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha expression (a transcriptional coactivator that is a central inducer of mitochondrial biogenesis) [115].

Furthermore, it seems that there is a much lower production of TMAO during plant-diet because it is mainly induced by a diet rich in red meat. It is also noted that changes in microbiota caused by a vegetarian/vegan diet induce resistance to the production of TMAO when these precursors (choline, L-carnitine) are ingested [111,[116][117][118][119].

Despite an overwhelming amount of evidence demonstrating correlations between choline and L-carnitine, and their metabolite TMAO, in terms of disease onset, there are conflicting data about their relationships [120]. Some inconsistencies occur about L-carnitine showing protective effects against metabolic diseases and therefore cast doubt on TMAO's involvement. First, the way l-carnitine is administered is important and may explain some of the contradictory results. L-carnitine administered intravenously could bypass gut microbial conversion of L-carnitine to TMAO and provide beneficial mitochondrial effects. Secondly, the knowledge of metabolism of choline and L-carnitine is rather limited. The development of a faster and simpler Ultra Performance Liquid Chromatograph-mass spectrometer (UPLC-MS)/MS method for the simultaneous determination of TMAO, TMA, and other choline metabolites in different types of biological samples may help establishing the effect of dietary factors on TMAO metabolism [121]. Finally, not only TMAO, but also TMA, have been described as deleterious, and the latter has been proposed to be involved in cardiovascular pathology, at least in vitro [122].

Amino Acids Composition and Metabolic Complications

Disorders of glucose homeostasis affect approximately 50% of CKD patients even in absence of diabetes. Over 30 years ago, experimental studies reported that treatment of uremic patients with an LPD improved insulin resistance [123]. Until today, there has been no identified picture of the specific role of AAs on metabolic perturbation in the general population and even less in CKD patients. On one hand, the restriction of EAAs such as the BCAAs [124] and SAAs [125] have been shown to be responsible for the systemic effects of a LPD. On the other hand, others have demonstrated that the altered non-EAA metabolism is necessary and sufficient for explaining the beneficial effects of LPDs [START_REF] Maida | A Liver Stress-Endocrine Nexus Promotes Metabolic Integrity during Dietary Protein Dilution[END_REF]. For example, the increase of SAAs and H2S generation on metabolic effect is unclear. H2S is a double-sided sword: At low levels, it exerts beneficial effects, but at higher concentrations it can damage pancreatic beta cells and glucose homeostasis, in animal models. By contrast, decreased levels of plasma H2S levels were reported in diabetic patients [126]. Feeding mice and humans with a diet specifically deprived of BCAAs was sufficient to improve glucose tolerance and body composition to the same extent as a LPD [127]. There are evidences that BCAAs and mostly leucine stimulates the expression of mitochondrial biogenesis genes like PGC1 and plays a leading role in mammalian target of rapamycin complex 1 (mTORC1) activation that is involved in the process of protein synthesis and energy metabolism. However, leucine-stimulated mTOR signaling is partly attenuated in skeletal muscle of chronically uremic rats [128]. In healthy older males, ingestion of 6 g BCAA increases myofibrillar protein synthesis rates during the early postprandial phase [129]. Using ketoacids that are produced as a result of BCAA degradation (removal of the amino group) by the enzyme branched-chain amino-acid aminotransferase can be an attractive strategy to efficiently stimulate muscle protein synthesis in CKD [130]. A systemic deprivation of threonine and tryptophan are both adequate and necessary to improve metabolic parameters as LPD in obesity context [131]. One proposed mechanism is that reduction of threonine and tryptophan and LPD promotes such metabolic remodeling and health by the liver-derived hormone fibroblast growth factor 21 (FGF21). However, the role that FGF21 plays in pathophysiology of CKD remains elusive. We have demonstrated that the metabolism of tyrosine, which is converted to PCS by the host, triggers insulin resistance [132]. However, in the other way, a decrease of AAAs in CKD mice is not able to restore insulin sensitivity [START_REF] Barba | A Low Aromatic Amino-Acid Diet Improves Renal Function and Prevents Kidney Fibrosis iN Mice with Chronic Kidney Disease[END_REF]. The contradictory results can be explained by the simultaneous increase in other non-protein/AA nutrients such as certain carbohydrates that are required for LPD effects [133].

Conclusions

In summary, the analysis of the literature presented in this review shows a large benefit in the adoption of LPD and plant-based diets in CKD patients. These diets may have an impact on CKD progression and also in the management of CKD-associated metabolic complications. The increase in fibers intake associated with plant-based diet might result in potentially reduced risk of cancer, metabolic and vascular diseases, and death in this population. However, additional intervention trials are required in particular to assess the risk of hyperkalemia even if there is no clear evidence to support limiting plant-based foods in normokalemic patients. The evolution of the society and the realization of clinical studies on the topic in the coming years will be helpful to validate and motivate CKD patients to adopt this diet in safe conditions.

The evolution of the concept of protein restriction to targeted restriction of AAs is now emerging. (Figure 1) A better understanding of the role of each AA in the progression of CKD will make it possible to give targeted nutritional advice and to open up new therapeutic avenues notably interacting with the intestinal microbiota. However; the human gut microbiota is highly variable and structurally distinct from experimental animals, setting up more challenges to the discovery and validation of novel microbial targets to reduce UTs production. Given that many metabolic pathways are maintained in microorganisms, it is possible to identify several functionally redundant yet phylogenetically distinct species, making it difficult to identify the bacterial origin for one specific UT. By contrast, the existence of certain genes does not necessarily mean that a downstream metabolite will be fully produced. The addition of post-translational modifications in the processes partially explains the difficulty to find ways to reprogram the metabolic output of the gut microbiome with diet. However, all of these challenges should not limit scientific efforts to deepen the relationships between the intestinal microbiota, diet, and renal function. On the contrary, the complexity of the interactions allows a larger possibility of new therapies on the microbiota. In particular, the emergence of new powerful bioinformatics analysis tools and using shotgun metagenomic sequencing allow characterization of non-bacterial components of the microbiota, provide information about function and open new hopes. Additionally, note that most of the data available on intestinal microbiota come from analysis of stool; however, there are several niches inside the gastrointestinal tract and stool is only one. Finally, much is yet to be learned and discovered and the coming years are likely to be highly informative and promising.

Figure 1 .

 1 Figure 1. Recommendation evolution of a dietary protein in the prevention and treatment of CKD. Abbreviations: AAA, aromatic amino acids; AGE, advanced glycation end-products; BCAA, branched-chain amino acids; FGF23, fibroblast growth factor 23; GFR, Glomerular filtration rate; IS, indoxyl sulfate; pCS, p-cresyl sulfate; P/S ratio FA, polyunsaturated/saturated ratio of fatty acids; SAA, sulfur-containing amino acids; TMAO, trimethylamine-N-oxide.

Figure 1 .

 1 Figure 1. Recommendation evolution of a dietary protein in the prevention and treatment of CKD. Abbreviations: AAA, aromatic amino acids; AGE, advanced glycation end-products; BCAA, branched-chain amino acids; FGF23, fibroblast growth factor 23; GFR, Glomerular filtration rate; IS, indoxyl sulfate; pCS, p-cresyl sulfate; P/S ratio FA, polyunsaturated/saturated ratio of fatty acids; SAA, sulfur-containing amino acids; TMAO, trimethylamine-N-oxide.

Table 1 .

 1 Observational studies analyzing plant protein or plant-based dietary pattern and chronic kidney disease (CKD) incidence or progression.

	Study	Type	Intervention/Assessment	Population	Follow Up	Outcomes		Results/Comments
							animal fat consumption was associated
							with	risk of microalbuminuria (OR, 1.51;
	Lin et al. 2010 [21]	Observational	FFQ: Salt, animal fat, several nutrients	n = 3348 General population	11 years	eGFR decline Microalbuminuria	95% CI, 1.01 to 2.26) β-carotene intake appeared protective
							against eGFR decline (OR, 0.56; 95% CI,
								0.40 to 0.78)
							risk of mortality with higher adherence
	Gutiérrez et al. 2014 [22]	Observational	FFQ: Plant-Based pattern score	n = 3972 CKD G3-G5	6 years	All-cause mortality Kidney failure	of plant-based diet (HR, 0.77; 95% CI; 0.61 to 0.97, p < 0.05) No negative association between plant diet
								and risk of ESKD
	Chen et al. 2016 [23]	Observational	Dietary interview: Plant protein-total protein ratio and total plant protein intake	n = 14,866 including 728 patients with CKD	6.2 to 8.6 years	All-cause mortality	plant protein-total protein ratio is associated with a significant risk of death in CKD population with eGFR < 60 mL/min/1.73 m 2 (HR, 0.77; 95% CI, 0.61 to 0.96)
								risk of developing CKD with
	Haring et al. 2017 [15]	Observational	FFQ: Plant protein intake	n = 11,952 General population	23 years	Incident CKD	consumption of nuts (HR, 0.75; 95% CI, 0.65 to 0.85, p < 0.001), low-fat dairy products, or legumes (HR, 0.83; 95% CI, 0.72 to 0.95,
								p = 0.03)
	Herber-Gast et al. 2017 [24]	Observational	FFQ: consumption of whole grains, fruit, and plants	n = 3787 General population	15 years	eGFR decline Albuminuria creatinine ratio	No association on multivariate model of fruit and plant intakes with changes in renal function or albuminuria creatinine ratio
	Asghari et al. 2018 [25]	Observational	FFQ: Lacto-vegetarian, traditional Iranian, and high fat, high sugar dietary pattern	n = 1630 General population	6.1 years	Incident CKD	CKD incidence (OR, 0.57; 95% CI, 0.41 to 0.8, p = 0.002) with adherence to the vegetarian dietary pattern

Table 1 .

 1 Cont.

	Study	Type	Intervention/Assessment	Population	Follow Up	Outcomes	Results/Comments
	Liu et al. 2019 [26]	Observational	FFQ: vegan, ovo-lacto vegetarian, or omnivore diets	n = 55,113 General population	Cross-sectional	Prevalence of CKD	CKD in vegan diet adherent (OR, 0.87; 95% CI, 0.75 to 0.97, p = 0.018) and ovo-lacto vegetarian diet adherent (OR, 0.84; 95% CI 0.77 to 0.88, p < 0.001)
							CKD incidence (HR, 0.86; 95% CI, 0.76 to
	Jhee et al. 2019 [27]	Observational	FFQ: nonfermented and fermented plant and fruit	n = 9229 General population	8.2 years	Incidence of CKD Incident proteinuria	0.98, p < 0.05) and proteinuria incidence (HR, 0.68; 95% CI, 0.59 to 0.78, p < 0.05) with highest versus lowest intake of
							nonfermented plants
							CKD incidence with higher adherence to
							the healthy plant-based diet (OR, 0.86; 95%
	Kim et al. 2019 [28]	Observational	FFQ: Healthy pro-vegetarian diet to less healthy	n = 14,686 General population	24 years	Incident CKD eGFR decline	CI, 0.78 to 0.96, p = 0.001) and pro-vegetarian diets (OR, 0.9; 95% CI, 0.82 to 0.99, p = 0.03) eGFR annual decline with healthy
							plant-based diet (OR, -1.46; 95% CI, -1.50
							to -1.43 p <0.001)
	Oosterwijk et al. 2019 [16]	Observational	FFQ: protein intake, including types and sources of protein	n = 420 Type 2 diabetes population	Cross-sectional	Prevalence of CKD	intake of vegetable protein is associated with prevalence of CKD in higher tertile (OR, 0.47: 95% CI, 0.23 to 0.98, p = 0.04)
							In the hemodialysis population,
				n = 8078			consumption of fruit and plant is associated
	Saglimbene et al. 2019 [29]	Observational	FFQ: fruit and plant intake	Adults on maintenance	2.7 years	Mortality	with 0.91, p = 0.002) and non-cardiovascular all-cause (OR, 0.8; 95% CI, 0.71 to
				hemodialysis			death (OR, 0.84; 95% CI, 0.70 to 1.00,
							p = 0.14)
	Studies were selected in PubMed database with a follow up over than 6 months. Abbreviations: CKD, chronic kidney disease; ESKD, end-stage kidney disease; eGFR, estimated glomerular
	filtration rate; FFQ, Food Frequency Questionnaire; OR, odd ratio; p for p-value.

Table 2 .

 2 Interventional studies analyzing the effect of plant protein or plant-based dietary pattern on CKD complications.

	Study	Type	Intervention/ Assessment	Population	Follow Up	Outcomes	Results/Comments
	INTERVENTIONAL
	STUDIES
	Fanti et al. 2006 [30]	Randomized controlled trial	Isoflavone-containing soy-based nutritional supplements (soy group) or isoflavone-free milk protein (control group)	n = 25 ESKD on chronic hemodialysis with systemic inflammation	8 weeks	Impact on inflammatory markers and nutrition markers	serum isoflavone levels associated with CRP (HR = -0.599, p = 0.02) and albumin (HR = 0.522, p < 0.05) No significant decrease of CRP between the two groups but a trend in the soy protein group
							Failed to find a difference between APD
	Soroka et al. 1998 [31]	Randomized cross-over trial	Plant protein diet versus animal protein diet	n = 9 CKD G3-G4	1 year	eGFR decline	versus VPD but it was underpowered and short trial A better degree of compliance with caloric,
							protein, and phosphate intakes
	Tabibi et al. 2009 [32]	Randomized controlled trial	Soy flour (14 g of soy protein) versus usual diet	n = 40 ESKD on peritoneal dialysis	8 weeks	Serum lipid profile	serum Lipoprotein A concentration in the soy protein group (p < 0.05)
	Moe et al. 2011 [33]	Cross-over trial	Vegetarian versus meat diet comparison	n = 8 CKD G3-G4	7 days	Impact on phosphorus homeostasis	phosphorus serum concentration (p = 0.02) and FGF23 (p = 0.008) in the vegetarian diet
	Goraya et al. 2013 [34]	Randomized controlled trial	Oral NaHCO3 compared with fruit and plant diet with a controlled arm	n = 106 CKD G4 with metabolic acidosis	1 year	Metabolic acidosis	Fruit and plant diet are as effective as oral bicarbonate to improve metabolic acidosis (19.9 versus 19.3 mM; p = 0.01), without an increase of hyperkaliemia risk.
	Goraya et al. 2014 [35]	Randomized controlled trial	Oral NaHCO3 compared with fruit and p diet with a controlled arm	n = 108 CKD G3 A > 1	3 years	Urine excretion of angiotensinogen eGFR decline	Fruit and plant diet are as effective as oral bicarbonate decrease angiotensinogen urine excretion (p < 0.05) and preserve eGFR (p < 0.01) versus usual care
	Goraya et al. 2019 [36]	Randomized controlled trial	Oral NaHCO3 compared with a controlled arm with fruit and plant diet	n = 108 Nondiabetic CKD G3-4 A > 1	5 years

Table 3 .

 3 Recommendation of estimated amino-acids requirement From WHO (2007)[START_REF] Gorissen | Protein Content and Amino Acid Composition of Commercially Available Plant-Based Protein Isolates[END_REF].

	Amino Acid	Requirements, mg/kg per day	mg/g Protein (1)
	Histidine	10	15
	Isoleucine	20	30
	Leucine	39	59
	Lysine	30	45
	Methionine	10	16
	Cysteine	4	6
	Phenylalanine plus tyrosine	25	38
	Threonine	15	23
	Tryptophan	4	6
	Valine	26	39
	Total	184	277

Nutrients 2020, 12, x FOR PEER REVIEW 7 of 21

Author Contributions: P.L. and L.K.; writing-original draft preparation, P.L., L.K., D.F., P.C., and S.B.; writing-review and editing. All authors have read and agreed to the published version of the manuscript.

Funding: This research received no external funding.

Conflicts of Interest:

The authors declare no conflict of interest.