Strain development for the production of D(−)-β-hydroxyisobutyric acid

Candida rugosa BR-120, which cannot catabolize D(−)-β-hydroxyisobutyric acid (D-HIBA), was developed fromCandida rugosa IFO 0750 by UV irradiationC. rugosa BR-120 could not assimilate propionic acid as a carbon source. It is presumed thatC. rugosa BR-120 lacks HIBA dehydrogenase.C. rugosa BR-120 produced D-HIBA from isobutyric acid (IBA) with a high yield about 94%, whileC. rugosa IFO 0750 did it with about 42% on 5 days batch cultivation. On 5 days fed-batch cultivation,C. rugosa BR-120 andC. rugosa IFO 0750 produced 12.45 g/L and 5.22 g/L of D-HIBA, respectively. The degradation rate of D-HIBA by the resting cell ofC. rugosa IFO 0750 was 31.95%, butC. rugosa BR-120 was 3.08% on 2 days incubation.     

Reaction engineering studies for the production of 2-hydroxyisobutyric acid with recombinant Cupriavidus necator H 16

Recombinant Cupriavidus necator H 16 with a novel metabolic pathway using a cobalamin-dependent mutase was exploited to produce 2-hydroxyisobutyric acid (2-HIBA) from renewable resources through microbial fermentation. 2-HIBA production capacities of different strains of C. necator H 16 deficient in the PHB synthase gene and genetically engineered to enable the production of 2-HIBA from the intracellular PHB precursor (R)-3-hydroxybutyryl-CoA were evaluated in 48 parallel milliliter-scale stirred tank bioreactors (V = 11 mL). The effects of media composition, limitations, pH, and feed rate were studied with respect to the overall process performances of the different recombinant strains. 2-HIBA production was at a maximum at nitrogen limiting conditions and if the pH was controlled between 6.8 and 7.2 under fed-batch operating conditions (intermittent fructose addition). The final concentration of 2-HIBA was 7.4 g L⁻¹ on a milliliter scale. Best reaction conditions identified on the milliliter scale were transferred to a laboratory-scale fed-batch process in a stirred tank bioreactor (V = 2 L). Two different process modes for the production of 2-HIBA, a single-phase and a dual-phase fermentation procedure, were evaluated and compared on a liter scale. The final concentration of 2-HIBA was 6.4 g L⁻¹ on a liter scale after 2 days of cultivation.     

Neutral amino acid transport by marine fish intestine: role of the side chain

This work was devoted to the study of the structure-affinity relationships in neutral amino acid transport by intestinal brush border of marine fish (Dicentrarchus labrax). The effects of the length of the side chain on kinetics of glycine, alanine, methionine and amino isobutyric acid were investigated. In the presence of K⁺ two components were characterized: one is saturable by increased substrate concentrations, whereas the other can be described by simple diffusion mechanism. Simple diffusion, a passive, non-saturable, Na⁺-independent route, contributes largely to the transport of methionine and to a much lesser extend to alanine, glycine or alphaaminoisobutyric acid uptakes. If a branched chain is present, as in the case of amino isobutyric acid, diffusion is low. A Na⁺-independent, saturable system has been fully characterized for methionine, but not for branched amino acids such as amino isobutyric acid. In the presence of Na⁺ saturable components were shown. Two distinct Na⁺-dependent pathways have been characterized for glycine uptake, with low and high affinities. For alanine and methionine only one Na⁺-dependent high affinity system exists with the same half-saturation concentration and the same maximum uptake at saturable concentrations. Glycine high affinity system has the same half-saturation concentration as methionine or alanine uptake, whereas maximum uptake is lower. The substitution of the hydrogen by a methyl group results in a severe decrease of uptake (aminoisobutyric acid). Mutual inhibition experiments indicate that the same carriers could be responsible for methionine and alanine uptakes and probably glycine Na⁺-dependent uptake. The influence of Na⁺ concentrations (100^-1 mol·l^-1) on amino acid uptake was examined. Glycine, alanine, methionine and amino isobutyric acid transport can be described by a hyperbolic function, with a saturation uptake which is highly increased for methionine. However, the half-saturation concentration does not seem to be strongly affected by the amino acid structure. The effect of Na⁺ concentration (25 and 100 mmol·l^-1) on the kinetics of methionine uptake have been also examined. The maximum uptake of the saturable system clearly shows a typical relationship with concentration.Abbreviations [AA] amino acid concentration - AIB aminoisobutyric acid - [I] Inhibitor amino acid concentration - J _i uptake in the presence of inhibitor - J _o uptake without inhibitor - K _d passive diffusion constant - K _i inhibitor constant - K _t concentration of test amino acid for half-maximal flux - MES 2[N-morpholino]ethanesulphonic acid - V _max maximum uptake at saturable amino acid concentrations - V _tot total amino acid uptake     

Effect of excitatory amino acid neurotransmitters on acid secretion in the rat stomach

Excitatory amino acids (EAAs), in particular,L-aspartate (L-Asp) neurons and their processes, were localized in the rat stomach using a immunohistochemical method with specific antibodies against eitherL-Asp or its synthesizing enzyme, aspartate aminotransferase (AAT). Myenteric ganglia and nerve bundles in the circular muscle and in the longitudinal muscle were found to be AAT-orL-Asp-positive. In addition, AAT- orL-Asp-positive cells were also found in the muscle layer and the deep mucosal layer. The distribution of AAT- orL-Asp-positive cells in both the mucosal and muscle layers was heterogeneous in the stomach. In addition,L-Asp at 10^–6 M negligibly influenced acid secretion in an everted preparation of isolated rat stomach. However, according to our results,L-Asp markedly inhibited the histamine-stimulated acid secretion, but not the oxotremorine- or the pentagastrin-stimulated acid secretion. Furthermore,L-Asp also inhibited histamine-induced elevation of cAMP.L-Asp itself did not affect the cAMP level although it elevated the cGMP level in the stomach. Moreover, either (+)2-amino-5-phosphonovaleric acid or (±)3-(2-carboxy-piperazin-4-yl)prophyl-1-phosphonic acid, i.e. two specific antagonists for N-methyl-D-aspartic acid (NMDA) receptors, blocked the inhibitory effect ofL-Asp on histamine-stimulated acid secretion or histamine-induced elevation of cAMP. Since cAMP has been strongly implicated as the second messenger involved in histamine-induced acid secretion, we believe thatL-Asp regulates acid secretion in the stomach by inhibiting histamine release through the NMDA receptors, subsequently lowering the level of cAMP and ultimately reducing acid secretion.     

Crystal state conformation of three model monomer units for the β-bend ribbon structure

The molecular and crystal structures of three compounds, representing the repeating units of the β-bend ribbon (an approximate 3₁₀-helix, with an intramolecular hydrogen-bonding donor every two residues), have been determined by x-ray diffraction. They are Boc-Aib-Hib-NHBzl, Z-Aib-Hib-NHBzl, and Z-L -Hyp-Aib-NHMe (Aib, α-aminoisobutyric acid; Bzl, benzyl; Boc, t-butyloxycarbonyl; Hyp, hydroxyproline Hib, α-hydroxyisobutyric acid; Z, benzyloxycarbonyl). The two former compounds are folded in a β-bend conformation: type III (III′) for Boc-Aib-Hib-NHBzl, while type II (II′) for the Z analogue. Conversely, the structure of Z-L -Hyp-Aib-NHMe, although not far from a type II β-bend, is partially open.     

How water-soluble saccharides drive the metabolism of lactic acid bacteria during fermentation of brewers' spent grain

We proposed a novel phenomic approach to track the effect of short-term exposures of Lactiplantibacillus plantarum and Leuconostoc pseudomesenteroides to environmental pressure induced by brewers' spent grain (BSG)-derived saccharides. Water-soluble BSG-based medium (WS-BSG) was chosen as model system. The environmental pressure exerted by WS-BSG shifted the phenotypes of bacteria in species- and strains-dependent way. The metabolic drift was growth phase-dependent and likely underlay the diauxic profile of organic acids production by bacteria in response to the low availability of energy sources. Among pentosans, metabolism of arabinose was preferred by L. plantarum and xylose by Leuc. pseudomesenteroides as confirmed by the overexpression of related genes. Bayesian variance analysis showed that phenotype switching towards galactose metabolism suffered the greatest fluctuation in L. plantarum. All lactic acid bacteria strains utilized more intensively sucrose and its plant-derived isomers. Sucrose-6-phosphate activity in Leuc. pseudomesenteroides likely mediated the increased consumption of raffinose. The increased levels of some phenolic compounds suggested the involvement of 6-phospho-β-glucosidases in β-glucosides degradation. Expression of genes encoding β-glucoside/cellobiose-specific EII complexes and phenotyping highlighted an increased metabolism for cellobiose. Our reconstructed metabolic network will improve the understanding of how lactic acid bacteria may transform BSG into suitable food ingredients.     

Optimized fed-batch fermentation of L-β-hydroxy isobutyric acid by Yarrowia lipolytica

Yarrowia lipolytica KCCM50506, which transforms isobutyric acid to L-#-hydroxy isobutyric acid (L-#-HIBA), was screened. Chemostat cultures were carried out in jar fermentors at dilution rates of 0.02 h^у to 0.12 h^у. L-#-HIBA fermentation-regulating factors were determined to be specific growth rate, and concentrations of glucose and isobutyric acid in fermentor from analysis of steady-state data. The specific productivity of L-#-HIBA increased as the specific growth rate increased, apparently as a growth-associated type of product formation. A fed-batch culture was carried out under optimum conditions where the concentrations of glucose and isobutyric acid in the fermentor were maintained at 23 g l^у and 9 g l^у, respectively. The concentrations of cells and L-#-HIBA obtained at the end of fermentation were 20 g l^у and 49 g l^у, respectively, corresponding to 2.0 and 2.7 times more than concentrations in batch culture.     

Regulation of amino acid transport in L6 muscle cells: I. Stimulation of transport system a by amino acid deprivation

The regulation of amino acid transport in L6 muscle cells by amino acid deprivation was investigated. Proline uptake was Na⁺-dependent, saturable and concentrative, and was predominantly through system A. Proline uptake was inhibited by alanine, α-amino isobutyric acid (AIB), and by α-methylamino isobutyric acid, but not by lysine or valine. At 25°C, Km of proline uptake was 0.5 mM. Amino acid-deprivation resulted in a progressive increase in the rate of proline uptake, reaching up to 6-fold stimulation after 6 hours. The basal and stimulated transport were equally Na⁺-dependent, and both were inhibited by competition with the same amino acids. Kinetic analysis showed that Km decreased by a factor of 2.4 and Vmax increased 1.9-fold in deprived cells. Amino acid-deprivation did not stimulate amino acid uptake through systems other than system A. This suggests that the higher Km in proline-supplemented cells is not due to release of intracellular amino acids into unstirred layers surrounding the cells. The presence of amino acids which are substrates of system A (including AIB) during proline-deprivation, prevented stimulation of proline uptake, whereas those transported by systems Ly⁺ or L exclusively were ineffective. The stimulation of the transport-rate in deprived cells could be reversed by subsequent exposure to proline or other substrates of system A. L6 cells, deprived of proline for 6 hours, retained the stimulation of transport after detachment from the monolayers with trypsin. Uptake rates were comparable in suspended and attached cells in monolayer culture. Thus, amino acid-depreivation of L6 cells results in an adaptive increase in proline uptake, which is not due to unstirred layers but appears to be mediated by other mechanisms of selective transport regulation.     

Biosynthesis of 2-hydroxyisobutyric acid (2-HIBA) from renewable carbon

Nowadays a growing demand for green chemicals and cleantech solutions is motivating the industry to strive for biobased building blocks. We have identified the tertiary carbon atom-containing 2-hydroxyisobutyric acid (2-HIBA) as an interesting building block for polymer synthesis. Starting from this carboxylic acid, practically all compounds possessing the isobutane structure are accessible by simple chemical conversions, e. g. the commodity methacrylic acid as well as isobutylene glycol and oxide. During recent years, biotechnological routes to 2-HIBA acid have been proposed and significant progress in elucidating the underlying biochemistry has been made. Besides biohydrolysis and biooxidation, now a bioisomerization reaction can be employed, converting the common metabolite 3-hydroxybutyric acid to 2-HIBA by a novel cobalamin-dependent CoA-carbonyl mutase. The latter reaction has recently been discovered in the course of elucidating the degradation pathway of the groundwater pollutant methyl tert -butyl ether (MTBE) in the new bacterial species Aquincola tertiaricarbonis. This discovery opens the ground for developing a completely biotechnological process for producing 2-HIBA. The mutase enzyme has to be active in a suitable biological system producing 3-hydroxybutyryl-CoA, which is the precursor of the well-known bacterial bioplastic polyhydroxybutyrate (PHB). This connection to the PHB metabolism is a great advantage as its underlying biochemistry and physiology is well understood and can easily be adopted towards producing 2-HIBA. This review highlights the potential of these discoveries for a large-scale 2-HIBA biosynthesis from renewable carbon, replacing conventional chemistry as synthesis route and petrochemicals as carbon source.     

Anaerobic reduction of ferulic acid to dihydroferulic acid by Wolinella succinogenes from cow rumen

Summary Ferulic acid(FA)-modifying microflora from the rumen of cows were acclimated in an FA-containing medium, in which aromatic compounds (dihydroferulic acid, homovanillic acid, carboxymethylphenol and vanillic acid) and volatile fatty acids (acetic, butyric and isobutyric) were detected by gas chromatography and mass spectrometry. An anaerobic curved bacterium was isolated from the rumen microflora. This bacterium was characterized and identified as Wolinella succinogenes according to the method of Holdeman et al. (1977). It could only reduce FA to dihydroferulic acid in the absence of hydrogen acceptors such as nitrate or fumarate and under strictly anaerobic conditions. FA-reducing ability of the bacterium was inhibited to some extent at FA concentrations greater than 5 mM. The FA was reduced more effectively at pH values of 7.0–7.2 than at 6.8 and the reduction was enhanced by the addition of an Ruminococcus albus' culture supernatant.     

Demonstration of the biofumigation activity of <Emphasis Type="Italic">Muscodor albus</Emphasis> against <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> in soil and potting mix

This research demonstrates the role of antimicrobial volatiles produced by Muscodor albus in disease control in soil and potting mix. The volatiles controlled damping-off of broccoli seedlings when pots containing soil or soilless potting mix infested with Rhizoctonia solani were placed in the presence of active M. albus culture without physical contact in closed containers. Conversely, plugs of R. solani on potato dextrose agar were inhibited when they were placed in the presence of M. albus incorporated into garden soil or soilless potting mix. Gas chromatographic analysis with solid-phase micro extraction showed that isobutyric acid and 2-methyl-1-butanol were released from the treated substrates. There was a significant relationship between the production of isobutyric acid in soil and damping-off control (P = 0.0415). Production of isobutyric acid was short-lived in treated substrates, peaking at 24 h in potting mix and 48 h in soil. Amounts of isobutyric acid released from soil were several times higher than those released from potting mix. Also, higher rates of M. albus rye grain culture were required to control damping-off in potting mix than in soil. This suggests that the soil used in this study is a better environment than soilless potting mix for the biological activity or viability of M. albus and components from the potting mix might bind the volatiles. The release of volatiles from soil during the biofumigation process suggests that containment measures such as tarping could be used to improve the control of soil-borne diseases and reduce use rate of the biocontrol agent.     

Novel synthetic co-culture of Acetobacterium woodii and Clostridium drakei using CO2 and in situ generated H2 for the production of caproic acid via lactic acid

Acetobacterium woodii is known to produce mainly acetate from CO₂ and H₂, but the production of higher value chemicals is desired for the bioeconomy. Using chain-elongating bacteria, synthetic co-cultures have the potential to produce longer-chained products such as caproic acid. In this study, we present first results for a successful autotrophic co-cultivation of A. woodii mutants and a Clostridium drakei wild-type strain in a stirred-tank bioreactor for the production of caproic acid from CO₂ and H₂ via the intermediate lactic acid. For autotrophic lactate production, a recombinant A. woodii strain with a deleted Lct-dehydrogenase complex, which is encoded by the lctBCD genes, and an inserted D-lactate dehydrogenase (LdhD) originating from Leuconostoc mesenteroides, was used. Hydrogen for the process was supplied using an All-in-One electrode for in situ water electrolysis. Lactate concentrations as high as 0.5 g L^–1 were achieved with the AiO-electrode, whereas 8.1 g L^–1 lactate were produced with direct H₂ sparging in a stirred-tank bioreactor. Hydrogen limitation was identified in the AiO process. However, with cathode surface area enlargement or numbering-up of the electrode and on-demand hydrogen generation, this process has great potential for a true carbon-negative production of value chemicals from CO₂.     

Amino acid immobilization of fungal motile cells

Summary A 0.2 M mixture of L-leucine and L-lysine, a pair of amino acids which Machlis (1969) had shown could attract the zoospores of Allomyces in much lower concentrations, was found to immobilize zoospores by stopping flagellar motion. While the age of the spores does not affect the response to the amino acid mixture, the time for 100% immobilization does increase with increasing numbers of spores. Viability of the spores is not altered by treatment with the mixture of L-leucine and L-lysine and subsequent germling development is highly synchronized.Several other amino acid mixtures had a similar effect upon the Allomyces' flagellum. Indeed, L-lysine by itself seems to be the most effective compound tested. Immobilization of flagella in other fungi, algae, and one protozoan was also caused by treatment with L-leucine and L-lysine. Nothing is known of the mechanism of action of this amino acid treatment.     

Enhancement of root hydraulic conductivity by methyl jasmonate and the role of calcium and abscisic acid in this process

The role of jasmonic acid in the induction of stomatal closure is well known. However, its role in regulating root hydraulic conductivity (L) has not yet been explored. The objectives of the present research were to evaluate how JA regulates L and how calcium and abscisic acid (ABA) could be involved in such regulation. We found that exogenous methyl jasmonate (MeJA) increased L of Phaseolus vulgaris, Solanum lycopersicum and Arabidopsis thaliana roots. Tomato plants defective in JA biosynthesis had lower values of L than wild‐type plants, and that L was restored by addition of MeJA. The increase of L by MeJA was accompanied by an increase of the phosphorylation state of the aquaporin PIP2. We observed that MeJA addition increased the concentration of cytosolic calcium and that calcium channel blockers inhibited the rise of L caused by MeJA. Treatment with fluoridone, an inhibitor of ABA biosynthesis, partially inhibited the increase of L caused by MeJA, and tomato plants defective in ABA biosynthesis increased their L after application of MeJA. It is concluded that JA enhances L and that this enhancement is linked to calcium and ABA dependent and independent signalling pathways.     

Amino acid transport by choroid plexus in vitro

Choroid plexus from mongrel cats was incubated from 1 to 120 min in artificial cerebrospinal fluid containing α-amino[1-¹⁴C]isobutyric acid. The uptake of α-amino [1-¹⁴C]isobutyric acid occurred against a concentration gradient, was saturable, dependent on metabolic energy, and inhibited by natural amino acids. These results indicate that a transport mechanism is present in choroid plexus which could serve to regulate amino acid concentration in the cerebrospinal fluid of animals.     

Heterotrophic production of ascorbic acid by microalgae

An aerobic fermentation process has been developed for the production of L-ascorbic acid (vitamin C). After an extensive screening program for microorganisms capable of heterotrophically synthesizing L-ascorbic acid, a unicellular green microalga,Chlorella pyrenoidosa, was selected. This organism has a number of characteristics that recommend it as an industrial organism: (1) it can double every 3.5 h when growing aerobically in the dark on a glucose-minimal salts medium; (2) its small size and tough cell wall make it very insensitive to shear, allowing very high impeller velocities; (3) it can be grown to 100 g L^–1 cell dry weight; (4) it is readily mutable by classical mutagenesis techniques; and (5) it has efficient growth kinetics with respect to yield of cell mass on glucose and oxygen. Fermentation process development and classical strain improvement have resulted in a greater than 70-fold increase in intracellular ascorbic acid concentration compared to the parent strainC. pyrenoidosa UTEX 1663. The process is compatible with existing industrial fermentation technology and equipment and is described in U.S. Patent 5,001,059. Patents have been submitted for a process in which the ascorbic acid accumulates extracellularly.     

Conversion ofL-sorbose to 2-keto-L-gulonic acid by mixtures of immobilized cells ofGluconobacter melanogenus IFO 3293 andPseudomonas species

Summary L-Sorbose was converted to 2-keto-L-gulonic acid by mixtures ofGluconobacter melanogenus IFO 3293 andPseudomonas syringae NRRL B-865 entrapped simultaneously in polyacrylamide gel. Since the temperature optima of both enzymatic reactions involved differed, a two-stage process with cells immobilized separately seems to offer a more efficient method to prepare 2-keto-L-gulonic acid.For preceeding paper in this series see Martin & Perlman (1976).     

L-Phenylalanine formation from acetamidocinnamic acid by newly isolated bacteria

Summary Bacteria with the ability to form L-phenylalanine from acetamidocinnamic acid were isolated from several soils. Among them, strain no. S-7 and strain no. N-7 were identified as Alcaligenus faecalis S-7 and Bacillus sphaericus N-7, respectively. The L-phenylalanine-forming enzyme systems in both bacteria were found to be inducible and intracellular. With intact cells of both bacteria and 40 mg/ml as wet base, 10 mg/ml acetamidocinnamic acid was utilized, and 7.7 mg/ml L-phenylalanine in a molar yield of 94% was produced after 72h incubation. The pathway of L-phenylalanine formation is considered to take the following course: acetamidocinnamic acid is deacetylated to -amino cinnamic acid; this is spontaneously changed to phenylpyruvic acid, and L-phenylalanine is formed by transamination.     

Effect of expressing polyhydroxybutyrate synthesis genes (phbCAB) in Streptococcus zooepidemicus on production of lactic acid and hyaluronic acid

Hyaluronic acid (HA) has been industrially produced using the gram-positive bacterium Streptococcus zooepidemicus. Large amount of lactic acid formation was one of the important factors that restricted cell growth and HA productivity and lowered the substrate to HA conversion efficiency in a fermentor. In this study, polyhydroxybutyrate (PHB) synthesis genes (phbCAB) of Ralstonia eutropha were cloned from the plasmid pBHR68 and were inserted into the plasmid pEU308, an expression vector for gram-positive bacteria. The plasmid was transformed into S. zooepidemicus by electroporation. β-Ketothiolase (PhbA), acetoacetyl-CoA reductase (PhbB), and polyhydroxyalkanoate (PHA) synthase (PhbC) activity assays were carried out to demonstrate the expression of these genes. The PhbA and PhbB activities were 3.13 and 1.23 U mg⁻¹, respectively. No PhbC activities were detected. In shake flask studies, there was no obvious difference between the wild-type and recombinant S. zooepidemicus harboring phbCAB genes in terms of lactic acid and HA formation. However, in fermentor studies, the recombinant produced only 40 g L⁻¹ lactic acid and 7.5 g L⁻¹ HA, whereas the wild type produced 65 g L⁻¹ lactic acid and 5.5 g L⁻¹ HA. These results suggested that expression of phbCAB genes in S. zooepidemicus could help regulate HA production metabolism. Because the lactic acid formation in S. zooepidemicus was sensitive to cellular oxidation/reduction potential, it is proposed that the PHB synthesis pathway could act as a regulator to adjust the cellular oxidation/reduction potential. This is the first study demonstrating that PHA synthesis related to energy and carbon metabolism could be employed as a pathway to regulate other cellular metabolism and possibly to regulate the production of other metabolic products.     

Urinary liver-type fatty acid binding protein as a useful biomarker in chronic kidney disease

Background: We reported that urinary L-FABP reflected the progression of chronic kidney disease (CKD). This study is aimed to evaluate the clinical significance of urinary liver type fatty acid binding protein (L-FABP) as a biomarker for monitoring CKD. Methods: Urinary L-FABP was measured using human L-FABP ELISA kit (CMIC.Co., Ltd., Tokyo, Japan). The relations between urinary L-FABP and clinical parameters were evaluated in non-diabetic CKD (n = 48) for a year. In order to evaluate the influence of serum L-FABP derived from liver upon urinary L-FABP, both serum and urinary L-FABP were simultaneously measured in patients with CKD (n = 73). Results: For monitoring CKD, the cut-off value in urinary L-FABP was determined as 17.4 μg/g.cr. by using a receiver operating characteristics (ROC) curve. Renal function deteriorated significantly more in patients with ‘high’ urinary L-FABP (n = 36) than in those with ‘low’ L-FABP (n = 12). The decrease in creatinine clearance was accompanied by an increase in urinary L-FABP, but not in urinary protein. Serum L-FABP in patients with CKD was not correlated with urinary L-FABP. Conclusion: Urinary excretion of L-FABP increases with the deterioration of renal function. Serum L-FABP did not influence on urinary L-FABP. Urinary L-FABP may be a useful clinical biomarker for monitoring CKD.