Metabolic Response in Rabbit Urine to Occurrence and Relief of

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Metabolic Response in Rabbit Urine to Occurrence and Relief of Unilateral Ureteral Obstruction Zhenzhao Wang, Rui Xu, Guiping Shen, and Jianghua Feng J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00304 • Publication Date (Web): 19 Jul 2018 Downloaded from http://pubs.acs.org on July 24, 2018

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Journal of Proteome Research

Metabolic Response in Rabbit Urine to Occurrence and Relief of Unilateral Ureteral Obstruction

Zhenzhao Wang1, Rui Xu1, Guiping Shen1, Jianghua Feng1*

1.Department of Electronic Science, Fujian Provincial Key Laboratory of Plasma and Magnetic Resonance, Xiamen University, Xiamen, 361005, China

* Corresponding author Tel: +86-592-2182459; Fax: +86-592-2189426; E-mail: [email protected]

1

ACS Paragon Plus Environment

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Abstract Ureteral obstruction will lead clinically to hydronephrosis, which may further develop into partial or complete loss of kidney function and even cause permanent histological damage. However, there is little knowledge of metabolic responses during the obstructed process and its recoverability. In this study, a complete unilateral ureteral obstruction (CUUO) model was established in rabbit, and 1H NMR-based metabolomic analysis of urine was used to reveal the metabolic perturbations in rabbits caused by CUUO and the metabolic recovery after the CUUO was relieved. Univariate and multivariate statistical analyses were used to identify metabolic characteristics. The gradually decreased levels of 3-hydroxykynurenine, 3-methylhistidine, creatinine, guanidoacetate, meta- & para-hydroxyphenylacetate and phenylacetylglycine and the gradually increased levels of acetate, alanine, citrate, glycine, lactate and methionine in urine could be regarded as the potential biomarkers for the occurrence and severity of ureteral obstruction. And the reduced levels of 3-methylhistidine, creatinine, guanidoacetate, hippurate, meta-hydroxyphenylacetate and methylguanidine and the elevated levels of 2-aminoisobutyrate, acetylcholine, citrate, lactate, lysine, valine and α-ketoglutarate in urine compared with the obstructed level could characterize the metabolic recovery of ureteral obstruction. Our results depicted the disturbed biochemical pathways involved in ureteral obstruction and demonstrated the practicability of recovering renal functions for the patients with severe hydronephrosis in clinical practice by removing causes for obstruction.

Keywords

NMR;

Metabonomics;

Complete

unilateral

ureteral

Hydronephrosis; Rabbit

2


obstruction

(CUUO);

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1 Introduction Hydronephrosis is a common nephrology disease caused by urinary tract obstruction that brings about a tremendous influence on the renal function and then on the standard of living for human especially the antenatal, neonates and pregnant woman.1-3 Urinary tract obstruction is usually caused by congenital factors, however, some other postnatal factors, such as inflammation, tumors, stone and trauma, can also lead to urinary system obstruction and further caused hydronephrosis.4 Hydronephrosis caused by urinary tract obstruction is the main cause of renal failure. If the obstruction is not relieved in time, thus complicated infection occurs, hydronephrosis would become more difficult to treat and even cause more problems. In a vicious circle, the infection would accelerate the obstruction for the kidneys. The serious patient would develop to pyonephrosis and even endanger their lives. There are some traditional detection methods to test the occurrence and functional recovery of hydronephrosis, such as type-B ultrasonic, diuretic nephrogram, urography, imaging examination and glomerular filtration rate (GFR).5 At the same time, some literatures described the histological changes of the fibrosis after obstruction6-9 and correlated the development of hydronephrosis with the increased sympathetic nerve activity, oxidative stress and hypertension.10 Some researchers also identified certain metabolites as potential biomarkers for hydronephrosis. Johri et al. discussed the changes of calcium, oxalate, uric acid, and citric acid levels in hydronephrosis caused by stones.11 Maril et al. applied magnetic resonance imaging to compare the gradient of sodium distribution in normal rats and obstructed rats.12 Madsen et al. researched epidermal growth factor and monocyte chemotactic peptide-1 as potential biomarkers of urinary tract obstruction in children with hydronephrosis.13 However, these studies usually lack an overall description of metabolic profiles of hydronephrosis and 3



the corresponding molecular mechanism remains unclear, and there are few reports on the detailed metabolic phenotype of hydronephrosis occurrence and functional recovery. Consequently, it is necessary to establish the better diagnosis of urinary tract obstruction and metabolomic identification according to the severity of hydronephrosis. The measurable parameters from metabolomic analysis could serve as a qualitative or quantitative prognostic indicator for obstruction, and they also could be used to determine the severity of obstruction. The manifestation of the kidney disease in rabbits and human is similar.14 Besides, the establishment of animal models has high reproducibility and better specificity, and is easy to be realized. Using the animal models of rabbits, human renal diseases can be authentically simulated and monitored. As an emerging subject, metabonomics offers the potential to illustrate a number of physiological disorders caused by disease. Its primary purpose is to screen the metabolites with statistical and biological significance for the diseases from the identified metabolites, and further elucidate the metabolic process and change to respond to organism physiological status.15 In many cases, there is a certain correlation between the expression and the corresponding expression pattern of identified metabolites, such as the correlation of the metabolites in the same metabolic pathway. Therefore, we could obtain the interaction between the different metabolites through metabolic pathways. According to early reports,16-19 the changes of urinary metabolomes can better reflect the renal damage caused by the experimental hydronephrosis. In this study, we used rabbit model of complete unilateral ureteral obstruction (CUUO) to investigate the occurrence, development, and functional recovery of hydronephrosis. From the 1H NMR-based metabolomic investigation of urinary response through multivariate and univariate statistical analyses, the change pattern reflecting the abnormalities 4


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of tissue and cellular functions of CUUO were determined, and the corresponding recovery and repair patterns over time were also derived.

2 Experimental Section 2.1 Experimental animals This study was performed in accordance with the local Principles of Laboratory Animal Care, and the protocols experimental were approved by the Ethical and Research Committee of Xiamen University. A total of 30 adult male New Zealand rabbits (weighing 2.0-2.2 kg) in clean grade were obtained from the Experimental Animal Center, Renmin Hospital of Wuhan University. All animals were randomly divided into 5 groups (n = 6 each group):

control group (as group C), groups at 1st

week (as group O1) and at 2nd week (as group O2) after CUUO, and groups at 1st week (as group P1) and at 2nd week (as group P2) after the relief of a two-week CUUO.

2.2 Establishment of CUUO animal model The CUUO animal model was established according to the protocol.20 Rabbits were anesthetized using 1% pentobarbital sodium (30 mL/kg body weight) by intraperitoneal injection and placed in prone position. After the skin preparation and sterilization, the left ureter of the rabbit was exposed through a longitudinal 5-cm mid-abdominal incision. The ureteral was ligated with 1-0 silk suture for twice with a 5-mm interval between the two ties. After the incision was closed, animals received intramuscular injections of the antibiotic to relieve pain and prevent postoperative infection. All rabbits in this study underwent the same surgical procedure. The groups O1, O2, P1 and P2 underwent the operation of CUUO under aseptic condition, while the control group was sham operated, in which the left ureter was only exposed but not ligated. All animals were housed individually in metabolic cages and allowed free access to water and standard food ad libitum. At 7th and 14th day postoperatively, the 5



urine specimens were drawn from the bladders and hydronephrosis of the obstructed kidney in groups O1 and O2 with a 2-mL syringe, respectively. At 15th day postoperatively, groups P1 and P2 were relieved from CUUO by removing the silk suture. At 7th and 14th day post-relief of CUUO, the urine specimens were also drawn from the bladders and hydronephrosis of the obstructed kidney in groups P1 and P2, respectively. Control groups animals only tapped the urine samples from the bladder under asepsis. Following the collection of urine samples, the animals were sacrificed under anesthesia. The collected urine samples consist of 9 groups: C (control groups), O1 and O2 (obstructed side of CUUO for one and two weeks), P1 and P2 (relieved obstructed side of CUUO one and two weeks post-relief), O1-C and O2-C (contralateral side of CUUO for one and two weeks), P1-C and P2C (contralateral side of relieved CUUO one and two weeks post-relief). And all urine specimens were collected into 2.0 mL EP tubes with 0.1% NaN3 to prevent bacterial growth and froze in liquid nitrogen for 30 minutes, and then stored at -80 ºC until use.

2.3 Urinary sample preparation and 1H-NMR data acquisition Each urine sample was thawed and individually analyzed by NMR spectroscopy. All the urine samples were prepared by mixing 400 µL of urine with 200 µL of deuterated phosphate buffer (0.2 M, pH 7.4, containing 0.5% sodium 3-(trimethylsilyl) propionate-2,2,3,3-d4 (TSP)). The mixture was then centrifuged at 11,000 rpm for 5 minutes at 4 ºC. Finally, the supernatant (550 µL) of the buffered sample was transferred into a 5-mm NMR tube and stored at 4 ºC until the 1H NMR data acquisition. TSP was used as an internal reference for chemical shift. All 1H-NMR spectra of urine samples were acquired at 298 K with a Bruker AV-600 NMR spectrometer (Bruker BioSpin, Germany) operating at 600.13 MHz. All samples were kept inside of the NMR probe for 5 minutes in order to maintaining the stability of the temperature before the spectral 6


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data acquisition. For each sample, the standard one-dimensional (1D) NMR spectrum was acquired by a NOESY (Nuclear Overhauser Effect Spectroscopy) pulse sequence (RD-90o-t1-90o-tm-90o-Acq) with water suppression (NOESYPR1D). The 90° pulse width was adjusted to approximately 10 µs. The spectra were recorded with 32 scans into 32 K data points, a spectral width of 20 ppm (12019.2Hz), and a relaxation delay of 2 s. Spectra processing was performed with the software MestReNova (Version 9.0.1, Mestrelab Research S. L., Spain). All the collected free induction decays were zero-filled to 128 K data points and multiplied by an exponential function of 1.0 Hz line-broadening factor for increasing noise-signal ratio before the regular Fourier transformation. The pure absorption peaks were obtained by manually phasing and the undistorted spectra were achieved by manually baseline correct. The chemical shifts were internally referenced to the TSP-d4 peak at 0.0 ppm. All spectra were also peak aligned to overcome peak shift due to the pH variation of the urine samples. The spectral regions of residual water resonance (5.05-4.55 ppm) and urea resonance (6.20-5.45 ppm) were removed to eliminate baseline effects of water and urea signals. Each spectrum was then binned into 1550 segments with intervals of 0.005 ppm across the range 9.5-0.5 ppm. To account for overall variations in sample concentration, each spectrum was normalized to its total integrated area. The assignments of various metabolites were gained according to previous study21 and confirmed by public NMR databases (Human Metabolome Database, see http://www.hmdb.ca).

2.4 Statistical analysis The NMR spectral data obtained by normalization were imported into software SIMCA (version14.1, Umetrics AB, Umea Sweden) for analysis by multivariate statistical methods including principal component analysis (PCA), partial least squares discrimination analysis (PLS-DA) and 7



orthogonal PLS-DA (OPLS-DA) to understand the internal metabolic change in the data set. And the results were visualized by using the scores plots, where each individual sample was represented as a point. PCA could help to get the overviews of the samples distribution and identify possible outliers. Following, PLS-DA and OPLS-DA were performed to examine metabolic differences between groups. The quality of model was assessed by the cross-validation parameter Q2, indicating predict ability of the model, and R2, indicating the total explained variances. The OPLS-DA models were also cross-validated with the permutation test (200 permutations). The correlation coefficients （r） values and the variable importance for projection (VIP) values from OPLS-DA models were used to determine metabolites with significant changes. Meanwhile, for better reliability of characteristic metabolites’ screening, the relative concentration of each metabolite was quantified from the integral area of the corresponding signals. In order to avoid the influences of the spectral overlapped peaks, we chose those characteristic peaks with the least overlapping for the corresponding metabolites of interest. The results of statistical analyses were assessed by the methods of fold-change and the Student’s t-test. The values of Student’s t-test were transformed to p-values and used to obtain metabolites with statistically significant changes in metabolomes. The values of fold-change were calculated by the ratio of concentrations between pair-wise groups and were used to evaluate and determine the change of significant metabolites. In this study, we used the volcano-plot as a comprehensive and effective way to summarize and identify potential biomarker from both multivariate and univariate statistical analyses .Generally, it is an enhanced four-dimensional scatter plot of -log10(p-value) against log2(fold-change). And the absolute correlation coefficient

values and the VIP values from the OPLS-DA models were brought

as two additional variables in the volcano plot and were represented by color and circles size (warmer 8


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color to higher |r|, larger circle size corresponds to larger VIP value), respectively. In our study, a triple-standard was used to screen out the potential biomarkers for CUUO and its recovery. The thresholds of │r│ values, VIP value and p-value were set as more than 0.750, at the top of 10% of all VIP scores, and less than 0.05, respectively. Only when all of the three standards were met, the corresponding metabolites were screened out as the potential biomarkers.

2.5 Pathway analysis To obtain a global view of the effect of CUUO on metabolic correlations and biochemical pathways perturbation, a holistic metabolic correlation analysis and pathway analysis by utilizing Kyoto Encyclopaedia of Genes and Genomes (KEGG, see http://www.kegg.jp) and MetaboAnalyst3.0 online service (see http://www.metaboanalyst.ca/) were carried out on the differential metabolites derived from different pair-wise comparison models.

3 Results and discussion 3.1 1H NMR spectral profiles of rabbit urine following occurrence and relief of CUUO Figure 1 shows the mean 1H NMR spectra of rabbit urine following the establishment of CUUO model and its relief. There are large quantities of isolated and overlapped peaks in the spectra, suggesting a variety of metabolic end products and wastes excreted in rabbit urine due to daily metabolism. A total of 63 metabolites were identified from the 1H NMR spectra of rabbit urine with reference to the related literature21 and public metabolite NMR databases. The corresponding numbers of the metabolites were marked in 1H NMR spectra and the detailed assignment information was given in Table 1. Compared with the control group, the urine from the obstructed side of rabbits (groups O1 and O2) demonstrated the characteristic spectral profiles of occurrence and development of CUUO, while the obvious metabolic recovery could be observed from the urinary spectra following CUUO was 9



relieved (groups P1 and P2) by a visual comparison.

Figure 1. Mean1H NMR spectra (0.50-9.50 ppm) of rabbit urine obtained from groups C, O1, O2, P1 and P2. C, control rabbits; O1 and O2, the groups at 1st and 2nd week after CUUO; P1 and P2, the groups at 1st and 2nd week after the relief of a two-week CUUO in rabbit models. The spectral regions of 0.50-2.90 ppm, 4.55-6.50 ppm and 6.50-9.50 ppm (in the dashed boxes) were respectively vertically expanded3, 3 and 5 times compared with the spectral region of 2.90-4.55 ppm for the purpose of clarity. The keys for the numbers of assigned metabolites were given in Table 1.

Table 1. The identified metabolites from the NMR spectra of rabbit urine No.

Abbr

Metabolites

1

1

AIB

2-Aminoisobutyrate

1.50(sa)

2

HIB

2-Hydroxyisobutyrate

1.37(s)

3

MHM

3-Methoxy-4-hydroxymandelate

6.96(d), 7.00(d)

4

3-HB

3-Hydroxybutyrate

1.22(d),4.16(m)

5

HK

3-Hydroxykynurenine

6.72(m),7.06(d)

6

3-MH

3-Methylhistidine

7.68(s)

7

AD

Acetamide

1.98(s)

8

Ace

Acetate

1.93(s)

9

AA

Acetoacetate

2.30(s)

H chemical shift (ppm)(multiplicity)

10


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10

Ach

Acetylcholine

3.23(s)

11

Ala

Alanine

1.49(d)

12

Alt

Allantoate

5.25(m)

13

All

Allantoin

5.37(s)

14

AH

Aminohippurate

7.71(d)

15

Asc

Ascorbate

4.52(d)

16

Bet

Betaine

3.27(s), 3.90(s)

17

BA

Bile acid

0.66(m),0.71(m)

18

Bu

Butyrate

0.91(t),2.19(m)

19

Cho

Choline

3.20(s)

20

Ci

Citrate

2.56(d),2.69(d)

21

Cn

Creatinine

3.05(s),4.06(s)

22

DG

Deoxyguanosine

6.25(t)

23

Eth

Ethanol

1.19(t)

24

EA

Ethanolamine

3.16(t),3.90(t),

25

For

Formate

8.46(s)

26

Fum

Fumarate

6.53(s)

27

Glu

Glutamate

2.07(m),2.09(m)

28

Gly

Glycine

3.56(s)

29

Gla

Glycolate

3.93(s)

30

GA

Guanidoacetate

3.80(s)

31

Hip

Hippurate

3.97(d),7.55(t),7.64(t),7.84(d)

32

IL

Indole-3-lactate

7.18(d),7.75(d)

33

IB

Isobutyrate

1.09(d)

34

Ile

Isoleucine

0.94(t), 1.02(d)

35

Lac

Lactate

1.34(d),4.12(q)

36

Leu

Leucine

0.97(t)

37

Lys

Lysine

1.72(m),1.91(m)

38

Mal

Malate

4.30(m) 11



a

39

M

Malonate

3.11(s)

40

m-HPA

meta-Hydroxyphenylacetate

3.48(s),6.79(m),6.86(d),7.26(t)

41

Mol

Methanol

3.36(s)

42

Met

Methionine

2.14(s), 2.65(t)

43

MA

Methylamine

2.61(s)

44

MG

Methylguanidine

2.82(s)

45

DMG

N,N-Dimethylglycine

2.94(s)

46

NAA

N-Acetylalanine

2.04(s)

47

NP

Neopterin

8.65(d)

48

OA

Oxaloacetate

2.34(s)

49

p-HPA

para-Hydroxyphenylacetate

3.45(s), 7.17(d)

50

PAG

Phenylacetylglycine

3.68(s),3.76(d),7.29(m),7.36(m),7.43(m)

51

PA

Picolinate

7.50(d),7.91(d),8.59(d)

52

Py

Pyruvate

2.37(s)

53

Sar

Sarcosine

2.72(s),3.60(s)

54

Ser

Serotonin

6.91(m),7.13(d)

55

Suc

Succinate

2.41(s)

56

Tau

Taurine

3.27(t),3.43(t)

57

Tri

Trigonelline

4.44(s),8.04(m),8.83(t),9.12(s)

58

TMA

Trimethylamine

2.89(s)

59

TMAO

Trimethylamine N-oxide

3.30(s)

60

Uc

Urocanate

6.36(d),7.88(s)

61

Val

Valine

1.00(d),1.05(d)

62

Xan

Xanthine

7.94(s)

63

KG

α-Ketoglutarate

2.45(t), 3.01(t)

Multiplicity: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublets; m, multiplet.

The relative concentrations of the metabolites, thus the fold changes between the different experimental groups and the control group, could be calculated from the integral area of the 12


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corresponding signals. Combined with the corresponding heat-map of the alterations of urinary metabolites during the experiment (Figure S1 in the supplementary information), the NMR spectra could intuitively reflect the dynamic metabolic changes in the process of CUUO and the relief. The obstruction

was

highlighted

by

the

decreased

levels

of

3-hydroxykynurenine

3-methoxy-4-hydroxymandelate (MHM), 3-methylhistidine (3-MH), acetoacetate aminohippurate

(AH),

creatinine,

meta-hydroxyphenylacetate

deoxyguanosine

(m-HPA),

(DG),

methylamine

ethanol, (MA),

(HK),

, allantoin,

guanidoacetate

(GA),

oxaloacetate

(OA),

para-hydroxyphenylacetate (p-HPA), phenylacetylglycine (PAG), serine, allantoate, ethanolamine, fumarate, hippurate, pyruvate and trigonelline accompanied with the increased levels of acetamide, acetate, alanine, bile acid, citrate, N,N-dimethylglycine (DMG), glutamate, glycine, isoleucine, lactate, methionine, methanol, N-acetylalanine (NAA), 2-Aminoisobutyrate (AIB), 3-hydroxybutyrate, acetylcholine, butyrate, formate, glycolate, leucine, lysine, malate, neoptertin, trimethylamine22 and valine in rabbit urine. Among them, the severity of the obstruction could be characterized by the gradual decrease of HK, MHM, 3-MH, AA, allantoin, AH, creatinine, DG, ethanol, GA, m-HPA, MA, OA, p-HPA and PAG and the gradual increase of acetamide, acetate, alanine, bile acid, citrate, DMG, glutamate, glycine, isoleucine, lactate, methionine, methanol and NAA with the time of obstruction. However, some metabolites, including ascorbate, betaine, choline, indole-3-lactate (IL), malonate, picolinate, sarcosine, taurine, trimethylamine N-oxide (TMAO), urocanate and xanthine, demonstrated the inconsistent changes during the period of CUUO. HK, MHM, 3-MH, allantoin, AH, creatinine, DG, ethanol, p-HPA, PAG, serine, fumarate, hippurate, acetamide, acetate, alanine, citrate, DMG, glutamate, isoleucine, lactate, methionine, methanol, NAA, AIB, leucine, lysine and TMA demonstrated metabolic recovery with the relief of the 13



ureteral obstruction. Among them, the reduced levels of MHM, 3-MH, creatinine, DG, p-HPA, PAG and fumarate and the elevated levels of acetamide, acetate, glutamate, isoleucine, lactate, methionine, NAA, leucine and lysine showed an obvious time-response relationship. Especially, the concentrations of DG, fumarate, hippurate and TMA ethanolamine almost came back to the basal levels two weeks after the relief. It was noteworthy that formate, malate, neoptertin and picolinate kept in an increased level while acetoacetate, aminohippurate, ethanol, oxaloacetate and pyruvate kept in an decreased level during the whole experimental period even when the obstruction was relieved. Some metabolites, including 2-hydroxyisobutyrate, isobutyrate, methylguanidine, succinate and α-ketoglutarate, seem to be unaffected by the occurrence and relief of CUUO. However, the visual comparison cannot provide the quantitative information, and more detailed statistical analysis could help to obtain the specific biological effects of CUUO and its metabolic recovery to respond to the relief of the obstruction.

3.2 The metabolic trajectory with obstruction time and the relief of CUUO Principal component analysis (PCA) scores plots based on the NMR data from urinary samples obtained from the difference rabbit groups reveal the metabolic differences between CUUO model and control group with the obstruction time and the relief of obstruction in obstructed side (Figure S2A), while the affected metabolic functions could be observed in the contralateral side of CUUO model (Figure S2B). PCA is used for the detection of outliers and suspect points. In our case, no obvious outlier was identified. Although a few samples seem to be suspected (those samples who are out of the 95% confidence limit in Figs. S2A and S2B), they were not identified as outliers but as the large-individual-metabolic-difference samples according to confirmation of their original NMR spectra. 14


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PCA scores plots demonstrated the metabolic evolution following the occurrence and development of CUUO (Fig. S2A), while the metabolic recovery could be observed following CUUO was relieved (Fig. S2B). The PLS-DA scores plots highlight the metabolic pattern of the obstruction and relief of CUUO (Figure 2). With the obstruction time, the gradually significant metabolic differences were demonstrated between the obstructed side and the control group (Fig. 2A), indicating CUUO model exerted a progressively great effect on the biological function of the animals. When the obstruction was relieved, the obvious metabolic recovery was observed in the obstructed side (Fig. 2A), implying the CUUO is remediable and even curable. Meanwhile, the scores plots show that contralateral side of obstruction was keeping away from control group with the obstruction time, indicating that the obstruction indeed affected the corresponding contralateral side although it did not undergo unilateral ureteral obstruction (Fig. 2B). While contralateral side of obstruction more quickly recuperated the normal range than the obstructed side when the obstruction was relieved (Fig. 2B).

Figure 2. PLS-DA scores plots obtained from the 1H NMR data of urine samples from corresponding rabbit groups reveal (A) the metabolic trend with CUUO time and the metabolic recovery post-relief of CUUO in obstructed side and( (B) the affected metabolic function and its recovery post-relief of CUUO in the contralateral side of CUUO. C, control groups; O1 and O2, 15



obstructed side of CUUO for one and two weeks; P1 and P2, relieved obstructed side of CUUO one and two weeks post-relief; O1-C and O2-C, contralateral side of CUUO for one and two weeks; P1-C and P2C, contralateral side of relieved CUUO one and two weeks post-relief.

3.3 The metabolic responses to CUUO To get further the detailed metabolites changes with CUUO time, the orthogonal partial least squares discrimination analysis (OPLS-DA) was constructed with the relevant NMR data from the pair-wise groups (including C-O1, and C-O2) (Figs. 3A&3B). The corresponding permutation tests were constructed for the purpose of evaluating the validity of these models (middle panels in Fig. 3). And the volcano plots (right panels in Fig. 3) were employed to identify the specific metabolites which contributed to the inter-group separation (left panels Fig.3). A series of differential metabolites were obtained and marked on the volcano plots' homologous dots, and the detailed model parameters and information were listed in Table S1 in the supporting information. The results of those volcano plots indicated the metabolomic variations of the rabbits at different periods. For groups C-O1 (Fig. 3A), the OPLS-DA scores plot indicated good separation between the control rabbits and the obstructed side of obstruction for one week. Moreover, the permutation tests also confirmed that there remained indeed metabolic differences between the two groups. Considerable differential metabolites corresponding to the early stage of CUUO were identified from the volcano plots. According to the volcano plots, the early stage of CUUO of rabbits was characterized by the decreased levels of hippurate, DG, HK, betaine, urocanate, m-HPA, PAG, allantoate, ethanolamine, picolinate and methylamine and the increased level of TMA, 3-hydroxybutyrate, methionine, sarcosine, lactate, trigonelline, malate, fumarate and glutamate. Among them, hippurate, DG, HK and betaine showed the most drastic changes. 16


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Figure 3. OPLS-DA scores plots (left panels), the corresponding permutations plots (middle panels) and volcano plots (right panels) derived from 1H NMR data of urine samples from corresponding rabbit groups. A, B, C, D, E and F represent six comparison about different pair-wise groups C-O1, C-O2, C-P1, C-P2, O1-P1, and P2-P2C, respectively. O1 and O2 indicate the groups at 1st and 2nd week after CUUO; P1 and P2 indicate the groups at 1st and 2nd week after the relief of a two-week CUUO; P2-C, contralateral side of relieved CUUO two weeks post-relief. The keys of the metabolites were showed in Table 1, and the detailed information on p-values, R values and VIP values was showed in Table S1 in the supplementary information. 17



For groups C-O2 (Fig. 3B), the obvious distinction between the controls and the obstructed side of obstruction was also observed two week post-obstruction. Furthermore, the permutation tests results (middle panel in Fig. 3B) and the model parameters, R2X, R2Y and Q2 (Table S1), indicated the severity of obstruction. According to the volcano plots (right panel in Fig. 3B), the metabolic consequences due to a two-week CUUO were highlighted by the decreased levels of hippurate, HK, m-HPA, p-HPA, PAG, creatinine, 3-methylhistidine, guanidoacetate and methylguanidine and the increased levels of 2-aminoisobutyrate, acetylcholine, α-ketoglutarate, betaine, lactate, lysine, acetate, picolinate, methionine, alanine, valine, DMG, citrate and glycine. Compare with the one-week CUUO, some metabolites demonstrated the discriminatory variations due to regulating function and stress reaction of the organism, for example, the levels of hippurate, betaine and picolinate raised again after a fall, and the contents of betaine and picolinate even get an increase compared with the controls. The levels of methionine and lactate gradually increased and the levels of HK, m-HPA and PAG gradually decreased with the obstruction time. The rebound of hippurate level came from the surfeit of glycine, which was forced to transform into hippurate.23 The growth of the level of betaine and picolinate was related to amino acid metabolism.24, 25 Combined the observation from Fig.S1, the decreased levels of HK, 3-MH, creatinine, GA, m- & p-HPA and PAG and the increased levels of acetate, alanine, citrate, glycine, lactate and methionine in urine could be regarded as the potential biomarkers for ureteral obstruction.

3.4 The metabolic recovery corresponding to the relief of CUUO In order to understand the metabolic changes with the relief of CUUO, the pair-wise groups including C-P1, C-P2, O2-P1 and P2-P2C were compared by the corresponding OPLS-DA models (Figs. 3C, 3D, 3E and 3F and Table S1). From groups C-P1 (Fig. 3C), we could find that the number of 18


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discrepant metabolites began to fall away when the CUUO was released. Of course, some metabolic changes still existed in group P1 when compared with the controls. The levels of 3-hydroxybutyrate, butyrate, methionine, DMG, neopterin, succinate and α-ketoglutarate were increased and the levels of acetylcholine, choline, GA, PAG, picolinate, sarcosine and taurine were decreased. But, the metabolic recovery already started. According to the comparison of group P2 and the control (Fig. 3D), the metabolic differences still existed between the controls and the relieved obstructed side two week post-relief but the influence degree was getting weak as demonstrated by the fewer discriminating metabolites in the volcano plots. When the obstruction was relieved for two weeks, the obstructed side still showed the decreased levels of indole-3-lactate, aminohippurate, HK, picolinate and oxaloacetate accompanied with the increased levels of PAG, methionine, DMG, butyrate and acetate. Such results indicate that CUUO damaged the normal biological function of the animals, and the relief will lead to the metabolic recovery but its recovery needs a long-term process. From the comparison of groups O2 and P1 (Fig. 3E), the information of metabolic changes could be obtained when the CUUO was relieved. The clear metabolic distinction could be observed in the obstructed side between two-week obstruction and one-week relief, implying some biological changes happening, possibly metabolic regression. The volcano plot showed that there are a series of noteworthy differential metabolites including the reduced levels of glycine, betaine, acetylcholine, citrate, DMG, alanine, choline, α-ketoglutarate and taurine and the elevated levels of methylguanidine, formate, 2-hydroxyisobutyrate, ethanol, picolinate, m-HPA, p-HPA, 3-hydroxybutyrate, PAG, trigonelline and ethanolamine. Together with the results of C-O1 and C-O2, we suppose that most differential metabolites gradually restored to the basal level. It is noteworthy that trigonelline and 19



picolinate exhibited a different change. Compare with C-O2, the level of picolinate descended again after a rise. The change of physical function, the relief of CUUO, brought about the picolinate’s increase.25 Trigonelline functions as repairing kidney damage.26 Experimental hydronephrosis caused obstructive nephropathy, and the organism tries to repair the damage, thus leading to the keep-growing level of trigonelline even if obstruction was relieved. From the comparison of groups P2 and P2-C (Fig. 3F), no significant metabolic differences were observed between the obstructed side and the contralateral side of relieved CUUO two weeks post-relief. And the OPLS-DA model parameters (R2X=61.5%, R2Y=0.543 and Q2=0.329) and permutation tests also confirmed that there remained only tiny differences between the two groups. These results implied that the urinary metabonome of the CUUO rabbits will return at least to the level of unobstructed side in two weeks when the obstruction was relieved. Generally speaking, the operation of complete unilateral ureteral obstruction not only would impair normal renal functions of the obstructed side but also make an impact on the unobstructed side. However, when the obstruction was relieved, the impaired function could be recovered though it would take a longer time. It seems that the metergasis happened during the obstruction as demonstrated as the similar reflection of urinary metabolites in the obstructed side and the corresponding contralateral side. Considering the obstructed side of CUUO could not produce urine, we guess that the urine of the obstructed side may come from the contralateral side of the obstruction, while the kidney in the obstructed side will reabsorb and secrete, thus resulting in the metabolic difference between the obstructed side and the contralateral side of the obstruction. From the comparison of groups C-O2 and C-P2 and the results in Fig. S1, the decreased levels of 20


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3-MH, creatinine, GA, hippurate, m-HPA and methylguanidine and the increased levels of AIB, acetylcholine, citrate, lactate, lysine, valine and α-ketoglutarate in urine compared with the obstructed level could characterize the metabolic recovery of ureteral obstruction.

3.5 The involved metabolic pathways during CUUO and its recovery All of the above results indicated that CUUO significantly brought about the metabolic disturbance in rabbit urine as time went on. Subsequently, the repair function of the organism obviously also resulted in the metabolites changes in urine when the obstruction was relieved. The changes generally related to carbohydrate metabolism, energy metabolism, lipid metabolism and amino acid metabolism. A monolithic metabolic pathway analysis was built on the differential metabolites derived from different comparison groups to obtain an integral view on the effect of obstruction and relief on metabolic pathways (Fig.4).

21



Figure 4. The involved metabolic pathways with the establishment (top panel) and the relief (bottom panel) of CUUO in rabbits. The increased, decreased and no significantly changed levels of the metabolites are represented by red, green and black boxes. The different metabolic pathways are indicated by the different font colors.

3.5.1 Carbohydrate metabolism 22


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The content of urinary citrate increased with the obstruction. Citrate is an important intermediary metabolite in tricarboxylic acid (TCA) cycle. The main metabolites involved in TCA cycle include malate, oxaloacetate, citrate, α-ketoglutarate, succinate and fumarate. Among them, the levels of citrate,

α-ketoglutarate, fumarate and malate engendered the discrepant increase in the period of obstruction. In TCA cycle, citrate links oxaloacetate and iso-citric acid and α-ketoglutarate, and it is synthesized by catalyzing of citrate synthase from oxaloacetate and acetyl CoA. Alpha-ketoglutarate is the key node of the carbon-nitrogen metabolism in the cell.27 The increase of these metabolites suggested the disorders of TCA cycle. TCA cycle, an irreversible reaction, is carried out in the mitochondria, and the abnormality of TCA cycle indicated the mitochondria malfunction. Mitochondria played an important role in the apoptosis and differentiation of cells.28 The rise of the urinary metabolites such as citrate,

α-ketoglutarate, fumarate and malate suggested that obstruction would bring out mitochondrial dysfunction and further caused apoptosis of cells by the disorder of TCA cycle. TCA cycle also affected a legion of metabolic pathways in carbohydrate metabolism including pentose phosphate, polysaccharide and monosaccharide metabolisms. A considerable number of differential metabolites appeared in these metabolic pathways, such as methylamine, trimethylamine, butyrate, acetate, lactate, 3-hydroxybutyrate and some other amino acids. Methylamine and trimethylamine are the key metabolites in methylamine metabolism. Methylamine is an important class of compounds used to mediate the osmotic pressure29 by the dynamic balance of body fluids. The changes in methylamine and trimethylamine implied the dynamic balance of body fluids was broken. The excessive accumulation of acetate inhibited the growth and metabolism of cells and further affected the growth state of the whole cell.30 The increase of the levels of 3-hydroxybutyrate, acetate 23



and lactate suggested the raise of anaerobic glycolysis,31 and it was obvious related to the establishment of CUUO.

3.5.2 Energy metabolism As TCA cycle intermediates, the increased levels of citrate, α-ketoglutarate, fumarate and malate in the period of obstruction revealed the accelerated TCA cycle. The acceleration of TCA cycle was due to the low energy provided by glycolysis in relation to aerobic metabolism. Obstruction caused a certain degree of hypoxia on the obstructed side, while glycolysis is the important route of glucose utilization in the condition of hypoxia.32 Subsequently, as the end-product of glycolysis, the level of lactate increased. Glycolysis produced less ATP, and thus requiring the accelerated TCA cycle for providing more ATP. At the same time, another pathway of providing ATP (TCA cycle) was also accelerated. According to Fig. S1, the level of methanol was gradually increased while the level of ethanol was gradually decreased with the obstruction. Alcohol dehydrogenase（ADH）metabolism transformed methanol into formate and transformed ethanol into acetate.33 Excess methanol occupied ADH metabolism, which caused methanol poisoning, further brought about kidney impairment. Meanwhile, formate was associated with one-carbon compound metabolism. Responsively, the levels of methanol and ethanol were recovering when the CUUO was relieved though not to the normal levels. Pyruvate is the hub of carbohydrate metabolism, lipid metabolism and amino acid metabolism and the central segment linking glycolysis and TCA cycle.28 As a dynamic intermediate point, pyruvate could be easily converted to other compounds. Taurine is excreted from the urine, and the kidneys would regulate its excretion according to the content of taurine in body. It is a biomarker for the changing in intrarenal environment.9 Therefore, as 24


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demonstrated in Fig.S1, the content of taurine changed both in the early stage of the obstruction and the relief. The elevated levels of alanine and glycine in rabbit urine maybe responded to acceleration of glycolysis and the protein digestion and absorption.32

3.5.3 Lipid metabolism Lipid metabolism included the biosynthesis and degradation of fatty acids, steroid and related substances. Besides as an important substance in the energy storage and energy supply for the body, lipid is also a significant constituent of biomembranes. The level of choline climbed up when CUUO occurred and then declined with the relief of CUUO, while the level of ethanolamine possessed the opposite trend of change. Ethanolamine is an intense inhibitor of some cholinergic metabolic enzymes that controlled the changes in the content of choline through interrelated metabolism pathways.34 Betaine could regulate the osmotic pressure and inhibit renal fibrosis, at the meantime, plays an important role in continuous protein synthesis when the osmotic pressure germinated upheaval.24 Therefore, the decreased content of betaine in the early stage of obstruction and relieving obstruction was a responsive action. Choline catalyzed the synthesis of betaine by using related dehydrogenase and oxidase, and betaine further turned into methionine for the synthesis of body protein and participation in a carbon metabolism as a methyl donor. As a significant component of biofilm and regulated cell apoptosis, the increase of choline content might damage cell membrane permeability and even cell structure. That further may cause renal fibrosis. The synthesis of acetylcholine required a certain amount of choline, and the increase of acetylcholine may be the reason of the declined choline in the late stage of obstruction. Meanwhile, 25



lysine could promote that the renal cortex assimilate choline,35 and the increase of lysine maybe keep choline decline after the obstruction was relieved. The main effect of bile acid is to promote the absorption and digestion of lipid nutrients. Bile acid metabolism imbalance and bile acid deposition would occur under the pathological conditions. That maybe was the main reason of the increased bile acid under the obstruction.

3.5.4 Amino acid metabolism The concentration of amino acids in urine of normal rabbits is usually relatively stable with little fluctuation. It was regulated by maintaining dynamic balance between tissue protein releasing amino acids and utilization of amino acids in tissue. When the obstruction occurred, the levels of amino acids (alanine, glutamate, glycine, lysine, methionine, valine, creatine, sarcosine and isoleucine) increased, breaking the balance between protein and amino acids (synthesis and decomposition) in the organism. Correspondingly, the amount of synthetic proteins, including all kinds of enzymes and hormones in the organism, decreased, thus further resulting in the metabolic disorders and kidney damage. Amino acid decomposition could produce α-ketoacids and amines, which were further converted into carbohydrates, lipids and other amino acids, however, the increase in the content of amino acids hindered such normal biological process. Lysine, methionine, isoleucine and valine are the essential amino acids in rabbits, and their increase in the period of obstruction was mainly due to the hindered nutrient biosynthesis after decomposition of the intake protein. The increase of alanine, sarcosine, glycine and glutamate was related to a variety of metabolism in the organism. DMG is a derivative of an amino acid and also an intermediate product of choline metabolism to glycine in the organism. It plays an important role as a methyl donor in one carbon metabolism, which is related to the synthesis of biological macromolecules.36 The inhibition of biological macromolecule 26


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synthesis dictated the increase of DMG. Deoxyguanosine is one of the basic forms of nucleotides, and its decrease was connected with oxidative DNA damage. The change of creatinine should be related to the flow of glycolysis pathway. When the glycolysis process was weakened, the content of creatinine was increased. Meanwhile, creatinine could prevent ATP from exhaustion and increase cell tolerance.37 Allantoin is a derivative of uric acid, which could promote cell growth and eliminate necrotic tissue. The decrease of creatinine and allantoin indicated that the renal function was further impaired. PAG, m- and p-HPA participated in the metabolism of microbial communities in rabbit body.38 Their decrease suggested the disorder of microbial colony metabolism. The dynamically growing of picolinate and hippurate maybe was resulted from the continuous impact of microbial metabolic disorders.23 Urocanate is intermediate products of histidine metabolism, and is also related to microbial metabolism. The fluctuation of urocanate may be the result of the synergistic action by both of them. Trigonelline could significantly improve carbohydrate metabolism and repair kidney damage.26 As a response, the level of trigonelline began to increase at the late stage of obstruction and kept growing even if the obstruction was relieved.

4 Conclusions In this study, the establishment of experimental hydronephrosis by CUUO caused serious renal damage in rabbits. 1H NMR-based investigation of metabolic response to severe hydronephrosis in rabbit identified the potential biomarkers that could characterize the occurrence and development of hydronephrosis caused by CUUO and the recovery after obstruction was relieved. The pathogenesis and prognosis of obstructive hydronephrosis were elucidated on the basis of the changes of metabolic trajectories of these biomarkers. Further research is necessary to confirm the metabolic similarity and 27



biomarkers in human hydronephrosis, and further to determine the clinical feasibility of the corresponding metabolic markers.

Conflict of Interest Declaration The authors declare no competing financial interest.

Author Contributions ZW and JF conceived and designed the study. ZW and RX conducted the experiments. ZW, GS and JF analyzed the data. ZW and JF wrote the paper. All the authors reviewed and approved the manuscript.

Acknowledgments This work is financially supported by the National Natural Science Foundation of China (No. 31671920) and the Natural Science Foundation of Fujian Province (2018Y0078). We thank the help from Mr. Yi Yao of Renmin Hospital of Wuhan University in the establishment of CUUO model.

Supporting information The following supporting information is available free of charge at ACS website http://pub.acs.org.

Figure S1. Dynamic alterations of urinary metabolites in concentration following the occurrence and relief of CUUO Figure S2. PCA scores plots obtained from the 1H NMR data of urine samples from corresponding rabbit groups reveal the metabolic differences. Table S1. The summary of metabolites statistical data from different pair-wise groups

Abbreviations ADH

alcohol dehydrogenase

CUUO

complete unilateral ureteral obstruction 28


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GFR

glomerular filtration rate

KEGG

Kyoto Encyclopaedia of Genes and Genomes

NMR

nuclear magnetic resonance

NOESY

Nuclear Overhauser Effect Spectroscopy

OPLS-DA

orthogonal partial least squares discrimination analysis

PCA

principal component analysis

PLS-DA

partial least squares discrimination analysis

TCA cycle

tricarboxylic acid cycle

TSP

3-(trimethylsilyl) propionate-2,2,3,3-d4

VIP

variable importance for projection

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For TOC only

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Metabolic Response in Rabbit Urine to Occurrence and Relief of

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