Embed Size (px)
Citation preview
This journal is c The Royal Society of Chemistry 2011 Mol. BioSyst., 2011, 7, 2181–2188 2181
Cite this: Mol. BioSyst., 2011, 7, 2181–2188
Alterations in urinary metabolites due to unilateral ureteral obstruction
in a rodent modelwzDawn L. MacLellan,*
acDiane Mataija,
bAlan Doucette,
bWeei Huang,
c
Chantale Langlois,aGreg Trottier,
aIan W. Burton,
dJohn A. Walter
dand
Tobias K. Karakachd
Received 25th February 2011, Accepted 19th April 2011
DOI: 10.1039/c1mb05080j
Urinary tract obstruction (UTO) results in renal compensatory mechanisms and may progress to
irrecoverable functional loss and histologic alterations. The pathophysiology of this progression is
poorly understood. We identified urinary metabolite alterations in a rodent model of partial and
complete UTO using 1H nuclear magnetic resonance (1H-NMR) spectroscopy. Principal
component analysis (PCA) was used for classification and discovery of differentiating metabolites.
UTO was associated with elevated urinary levels of alanine, succinate, dimethylglycine (DMG),
creatinine, taurine, choline-like compounds, hippurate, and lactate. Decreased urinary levels of
2-oxoglutarate and citrate were noted. The patterns of alteration in partial and complete UTO
were similar except that an absence of elevated urinary osmolytes (DMG and hippurate) was
noted in complete UTO. This pattern of metabolite alteration indicates impaired oxidative
metabolism of the mitochondria in renal proximal tubules and production of renal protective
osmolytes by the medulla. Decreased production of osmolytes in complete obstruction better
elucidates the pathophysiology of progression from renal compensatory mechanisms to
irrecoverable changes. Further confirmation of these potential biomarkers in children with UTO
is necessary.
Introduction
Congenital hydronephrosis is a common birth anomaly that has
a substantial impact on the quality of life and renal function of
approximately 1% of children.1 One of the main causes of
congenital hydronephrosis is UTO which can range from mild
to moderate and severe. Children with UTO are carefully
followed with invasive radiological examinations at least
annually. While some cases of UTO will resolve spontaneously
over a period of several years, others will develop loss of renal
function or symptoms and require corrective surgery.
Congenital anomalies of the urinary tract are the cause of renal
failure in more than 40% of children.2 Currently, no prognostic
markers or radiologic investigations are able to determine
which children will deteriorate or require surgical intervention.
The description of progressive pathophysiology as well as
molecular processes and mechanisms leading to end-stage—
loss of renal function—remain largely deficient.2 Consequently,
there is concerted effort to establish better methods for
diagnosis and molecular characterization of ureteral obstruc-
tion and its severity. Some of these efforts include studies of
infants with severe obstruction that have shown evidence of
glomerulosclerosis.3 Others4,5 have shown that UTO affects
the molecular regulatory mechanisms of the kidney through
compensatory responses that play a protective role.6 Under
these circumstances, if the degree of compensatory response is
not appropriate it may become pathological over time and
progress to glomerulosclerosis, fibrosis, and poor renal
function.7 Unfortunately, the series of events leading from a
physiological to pathophysiological compensatory response
have yet to be defined. Moreover, there is a need to develop
measurable parameters that can either serve as qualitative or
quantitative prognostic indicators of the obstruction or
parameters that can be used to determine the severity of the
obstruction.
A number of rodent models of UTO exist, including
complete obstruction and partial obstruction. The latter more
closely mimics the human condition. Varying degrees of
partial UTO (mild to severe) can be reliably reproduced. These
models are able to provide a snapshot of the cellular dynamics
aDepartment of Urology, Dalhousie University, Canada.E-mail: [email protected]
bDepartment of Chemistry, Dalhousie University, CanadacDepartment of Pathology, Dalhousie University, CanadadNational Research Council of Canada, Institute for MarineBiosciences (NRC-IMB), Canada
w Funding: IWK Health Centre, Dalhousie University Faculty ofMedicine and Department of Urology, contributions in-kind fromNational Research Council Institute of Marine Biosciences.z Electronic supplementary information (ESI) available. See DOI:10.1039/c1mb05080j
MolecularBioSystems
Dynamic Article Links
www.rsc.org/molecularbiosystems PAPER
Publ
ishe
d on
06
May
201
1. D
ownl
oade
d by
Uni
vers
ity o
f B
irm
ingh
am o
n 31
/10/
2014
03:
59:4
9.
View Article Online / Journal Homepage / Table of Contents for this issue
2182 Mol. BioSyst., 2011, 7, 2181–2188 This journal is c The Royal Society of Chemistry 2011
of human UTO. Molecular level studies, such as metabolomic
analysis, have the potential to elucidate the disruption of
cellular physiology brought about by many diseases.8–10 If
such studies are conducted in a non- or less-invasive manner,
translation to the diagnosis and management of human
congenital hydronephrosis brought about by UTO will be
greatly enhanced. Following recent advancements in analytical
technology in conjunction with advanced methods for multi-
dimensional data analysis, context dependent small molecules
have been isolated from the blood and urine of such disease
model rodents in a bid to establish biological markers of renal
injury or nephrotoxicity.11–14
Several examples of metabolomic techniques have
established urinary profiles for different renal pathologic
states. For example, using liquid chromatography and mass
spectrometry (LC/MS) in conjunction with PCA and partial
least squares discriminant analysis (PLS-DA), Yoshioka et al.11
analyzed the phospholipid profiles of rat urine exhibiting
unilateral ureteral obstruction and found differences in the
concentration of various lipid classes. Serkova et al.12 evaluated
potential metabolic markers for mild and severe ischemia/
reperfuson injury in rat kidney transplants by analyzing serum
and kidney extracts using 1H nuclear magnetic resonance
(1H-NMR) spectroscopy. Significant change in the levels of
polyunsaturated fatty acids and allantoin were found to
characterize kidney extracts from study animals, while
trimethylamine-N-oxide (TMAO) was found to characterize
the serum extracts from the experimental group. Lenz et al.13,14
administered different model nephrotoxins to rats and studied
fluctuations in metabolite profiles during the course of time
over which the organism rid itself of the toxin. Significant
perturbations were observed in the urinary metabolite profiles
of the rats using 1H NMR and LC/MS.
In this paper, we report on the work done to reproducibly
and non-invasively elucidate molecular signatures of partial
and complete unilateral ureteral obstruction by obtaining1H-NMR spectra of urine in a weanling rat model of UTO.
The primary goal of our study was to test whether subtle
perturbations in renal physiology resulting from variable
partial UTO could be manifested via alterations in urinary
metabolic profiles measured by1H NMR spectroscopy. This
goal is driven by the knowledge that NMR spectra of urine
have been used to identify metabolite alterations in other
rodent models of renal injury and by the fact that a well
established rodent model of UTO exists. The second objective
of our study was to compare the urinary metabolite alterations
that result from partial and complete UTO.
Results
Clinical chemistry
Standard clinical chemistry measurements of renal function
such as urine osmolality and serum creatinine were taken to
provide an indication of ‘renal status’ of the animals. This data
is shown in Table 1. Table 1 also highlights the experimental
groups. Group 1 includes a comparison of controls (n = 6)
and partial UTO (n = 6) in female weanling rats. Group 2
includes a comparison of controls (n=10) and complete UTO
(n = 10) in male weanling rats. Group 3 consists of a
validation experiment performed 1 year later on controls
(n = 7) and partial UTO (n = 8) in male weanling rats. Urine
osmolality was not significantly different between control rats
and those with partial UTO. The urine osmolality of rats
subjected to complete UTO was significantly lower than that
of control rats and rats with partial obstruction (18 degrees of
freedom (df) and, p-value = 0.0012). Serum creatinine levels
of the control rats did not differ significantly from partially
obstructed rats whereas rats with complete obstruction
exhibited significantly higher serum creatinine than controls
(df = 17, p-value = 0.0035).
Histopathology
All control kidneys exhibited normal histopathology. The left
kidney of the rats subjected to partial UTO had either normal
histology (n = 6) or changes consistent with mild UTO
(n = 4). The left kidney of rats subjected to complete UTO
demonstrated histology corresponding to moderate UTO (n=3)
or severe UTO (n = 7). Fig. 1 shows representative images of
the histological classification of normal, mild, moderate to
severe obstruction. In mild obstruction (Fig. 1B), hydrone-
phrosis with focal mildly attenuated parenchyma and preserved
cortical architecture were observed while in moderate obstruction
(Fig. 1C) we observed hydronephrosis with focal moderately
attenuated parenchyma exhibiting a disorganized cortical
architecture, chronic interstitial inflammation, tubular atrophy
and cystic dilation whereas the rest of parenchyma was largely
preserved. In contrast, histological exams of kidneys with
severe obstruction (Fig. 1D) exhibited markedly attenuated
parenchyma with diffuse interstitial fibrosis, chronic interstitial
inflammation, tubular atrophy and dropout.
Nuclear magnetic resonance spectroscopy
NMR Spectra
Fig. 2 shows a typical 1H NMR spectrum of rat normal urine
divided into: (A) aliphatic and (B) aromatic. This a spectrum
of an inhomogeneous complex mixture of low molecular
weight compounds that comprise rat urine.
Principal component analysis
Spectra of urine from female rats with partial UTO
exhibited the following characteristics (Group 1). First, PCA
(see Experimental section) depicted two clear groups separated
on the basis of the pathology along PC1, and smaller groups
along PC2 that depict differences among study subjects. This
observation is consistent for all data analyzed and attention
will be paid primarily to the separation along PC1 which
depicts the phenotype under investigation (unless clearly
stated). Fig. 3(a) shows a scores plot of the first vs. second
principal component (PC1 vs. PC2) with separation along
PC1. The first four PC’s accounted for 49.9% of the total
variance in the data while the first two PCs accounted for more
than 32%. A plot PC2 vs. PC3 or any of the further principal
components did not show any degree of separation in the data.
The scores plot (Fig. 3A) depicts the separation of control and
partially obstructed rat urine profiles. Whereas the metabolite
Publ
ishe
d on
06
May
201
1. D
ownl
oade
d by
Uni
vers
ity o
f B
irm
ingh
am o
n 31
/10/
2014
03:
59:4
9.
View Article Online
This journal is c The Royal Society of Chemistry 2011 Mol. BioSyst., 2011, 7, 2181–2188 2183
profiles of the control population appear more homogenous in
principal component space, those of partial UTO subjects are
to the left of PC1 and appear more dispersed. There is a clear
separation of the two groups along PC1. The corresponding
loadings plot shown in Fig. 3B show variables with positive
loadings as important for describing corresponding positive
scores in Fig. 3A. Thus, positive peaks correspond to meta-
bolites that are at higher concentration in the control samples
while the reverse is also true. On the right hand side of Fig. 3B
are expansions of an original urine spectrum around regions
that exhibit signal crowding to demonstrate chemical shift
multiplicities that are otherwise obscured.
From Fig. 3B it is possible to infer that partial UTO rats
exhibited a relative increase in the concentrations of acetate,
creatinine, taurine, hippurate, dimethylglycine and choline-
like compounds such as betaine, trimethylamine-N-oxide
(TMAO) or phosphocholine. At this exploratory stage, we
did not attempt spike experiments for unique identification of
the particular type of choline-like compound from the signal at
3.27 ppm. In contrast the concentration of citrate and
2-oxoglutarate exhibited a notable relative decrease in
experimental animals compared to controls. A summary of
the urinary metabolite changes in all experiments is presented
in Table 2. There was no significant difference in body weight
for rats with obstruction compared to controls over the course
of the experiment.
Principal components scores and loadings plots showing the
analysis of complete UTO samples (Group 2) are included in
the supplementary material and depict each scores plot with
its respective loading plot (Fig. SM1 (A and B)z). Changesin metabolite profiles are similar to those observed in
partial UTO.
Validation
Principal component analysis. Fig. 4A and B show PCA
scores and loadings plots of validation experiments for partial
UTO respectively (Group 3). Validation studies were conducted
with new groups of animals as described in experimental
section except that the experiments were repeated one year
after the initial animal studies. It can be seen from these
experiments that the clusters, along treatment groups,
Table 1 Urine osmolality and serum creatinine of control and experimental animals on the 3rd week after surgery. The osmolality values arenormalized to the volume (mL) of urine per 100 g of body weight. * Samples were not available for all animals
Treatment Partial UTO (female rats) Complete UTO (male rats) Partial UTO (male rats)
n* Control = 6 Control = 10 Control = 7Obstructed = 6 Obstructed = 10 Obstructed = 8Osmolality per mLof urine per 100 gbody weight (mOsm)
Serum creatininelevel (mg/dL)
Osmolality per mLof urine per 100 gbody weight (mOsm)
Serum creatininelevel (mg dL�1)
Osmolality per mLof urine per 100gbody weight (mOsm)
Serum creatininelevel (mg dL�1)
Control 1296.2 � 468.6 44.90 � 13.9 1409.5 � 403.8 2.20 � 0.34 1735.0 � 124.2 2.97 � 0.41UTO 1010.8 � 336.0 37.9 � 17.5 835.3 � 243.19 1.33 � 0.73 1524.5 � 430.8 2.57 � 0.85Ho:%xcntrl = %xobst
Cannot reject Ho;p-value: 0.2534
Cannot reject Ho;p-value: 0.46534
Reject Ho:p-value = 0.0012
Reject Ho:p-value = 0.0035
Cannot reject Ho;p-value = 0.4579
Cannot reject Ho;p-value = 0. 0.4928
a = 95% df = 10 df = 10 df = 18 df = 17 df = 5 df = 5
Fig. 1 Representative histological images of (A) normal (100� magnification), (B) mild (20� magnification), (C) moderate (200� magnification)
and (D) severe kidney obstruction (100� magnification).
Publ
ishe
d on
06
May
201
1. D
ownl
oade
d by
Uni
vers
ity o
f B
irm
ingh
am o
n 31
/10/
2014
03:
59:4
9.
View Article Online
2184 Mol. BioSyst., 2011, 7, 2181–2188 This journal is c The Royal Society of Chemistry 2011
observed in the initial studies are maintained in this second,
independent study.
Discussion
Previous metabolomic studies of various nephrotoxins have
characterized the urinary metabolite alterations that are
specific to targeted kidney functions and therefore localize
the injury to precise areas of the kidney. One of the most
important functions of the kidney is the transfer of sodium
ions from the tubular fluid to the blood; energy demands for
this process are provided by mitochondrial aerobic and
anaerobic metabolism.15 Decreased urinary excretion of
Kreb’s cycle intermediates indicates impairment of this oxida-
tive metabolism16 perhaps suggesting injury to the proximal
tubule or loop of Henle.15,17,18 It has been reported that injury
to the renal medulla is characterized by the early appearance
of TMAO and dimethylamine, followed by increased excretion
of acetate and succinate.16 Given that renal papillary cells
regulate urine osmolality and interstitial solute concentration
by accumulating and releasing osmolytes,15 it is possible that
the increased accumulation of these metabolites in the urine of
UTO animals, as observed here, signifies an injury to the cells.
The observed elevation in urinary creatinine is expected in
partial and complete UTO because of the likely impairment of
renal function. Both rodent models of UTO (partial and
complete) investigated here appear to point to impairment of
mitochondrial oxidative metabolism and injury to the
proximal tubule, implied by decreased urinary levels of the
Kreb’s cycle intermediates, citrate and 2-oxoglutarate.13,16–19
A related study18 of renal injury in a cold ischemia model
demonstrated that elevated urinary levels of acetate and
lactate were indicative of acute tubular necrosis and damage
to proximal tubular metabolism. In the current study, elevated
levels of acetate and lactate were noted for rats subjected to
partial kidney obstruction that had normal or mild histologic
changes of UTO. Rats with complete UTO exhibited elevated
levels of urinary lactate, also supporting evidence of injury to
proximal tubular metabolism. Other specific metabolite
markers of proximal tubule damage have been determined
from nephrotoxins such as gentamicin and cyclosporin A20,21
which yielded characteristic increases in levels of alanine (seen
in complete UTO) and succinate (noted in partial and
complete UTO).
Renal tubules are thought to be the primary site of excretion
for hippurate22 and the renal metabolism of hippurate has
been identified as a hydroxyl trapping mechanism in a rodent
model.23 We identified elevated urinary levels of hippurate in
partial UTO and decreased levels in complete UTO. The
elevation of hippurate noted in partial UTO may result from
excretion by the renal tubules as a compensatory mechanism
to the oxidative stress resulting from UTO. Whereas, in
complete UTO this mechanism may be overwhelmed due to
the irreversible injury.
The inner portion of the nephron (loops of Henle and
collecting duct) contains glycolytic and oxidative enzymes in
addition to a high amount of osmolyte transporters. As a
consequence there is a high oxygen demand and increased
osmolality in the medullary and papillary interstitium of the
kidney.15 Excessive increases in urine osmolality and inter-
stitial solute concentrations can harm renal papillary cells,
which release osmolytes to regulate their volume. Thus, our
finding of elevated urinary osmolytes in UTO, including
dimethylglycine, taurine, and betaine/TMAO/phosphocholine
Fig. 2 A representative 1H NMR spectrum of rat urine showing (A) the aliphatic and (B) the aromatic regions of signature chemical shifts
(and multiplicities) for major urinary metabolites. Abbreviations: Tau = taurine; TMAO = trimethylamine N-oxide; 2-OG = 2-oxoglutarate;
DMG= dimethylglycine; DMA= dimethylamine; Cit = citrate; NAA= N-acetyl-L-aspartic acid; oAc = acetate; Ala = alanine; d= chemical
shift
Publ
ishe
d on
06
May
201
1. D
ownl
oade
d by
Uni
vers
ity o
f B
irm
ingh
am o
n 31
/10/
2014
03:
59:4
9.
View Article Online
This journal is c The Royal Society of Chemistry 2011 Mol. BioSyst., 2011, 7, 2181–2188 2185
Fig. 3 (A) Principal component analysis scores plot depicting a separation of control and partially obstructed rat urine profiles (Group 1).
Whereas the metabolite profiles of the control population appear more homogenous in principal component space, the profiles of partial UTO
subjects are to the left of PC1 and appear more dispersed. There is a clear separation of the two groups along PC1. The ellipse does represent a
confidence interval but indicates the grouping for the control population. (B) Loadings plot corresponding to the scores plot shown in Fig. 3A. The
variables with positive loadings are important for describing corresponding positive scores in Fig. 3A. Thus, positive peaks correspond to
metabolites that are at higher concentration in the control samples. The reverse is true for partial UTO samples. On the right hand side, are
expansions of an original urine spectrum around regions that exhibit signal crowding. Abbreviations and symbols: * = controls; squares = partial
UTO; PC = principal component; dH = chemical shift
Table 2 Urinary metabolite alterations of rats subjected to partial and complete UTO. Abbreviations and symbols: d = chemical shift; ppm =parts per million; cpd = compound; s = singlet; d = doublet; t = triplet; q = quartet; * refers to TMAO/betaine/phosphocholine; Up arrows (m)indicate a relative increase in obstructed group compared to controls; Down arrows (k) indicate a relative decrease in obstructed groups comparedto controls
Metabolites 1H shift (d (ppm)) Multiplicity
Urinary tract obstruction protocol
Group 1 partial UTO Group 2 complete UTO Group 3 partial UTO
Acetate 1.91 s m mAlanine 1.48 d mSuccinate 2.43 s m m m2-Oxoglutarate 2.46, 3.02 t, t k k kCitrate 2.65 dd kk kk kkDimethylglycine 2.73 s m mCreatinine 3.06 s m mTaurine 3.28, 3.43 t, t m m mCholine-like cpd* 3.27 s mm mm mmHippurate 3.95, 7.54, 7.62, 7.81 s, t, t, d m kLactate 1.5, 4.10 d, q m m
Publ
ishe
d on
06
May
201
1. D
ownl
oade
d by
Uni
vers
ity o
f B
irm
ingh
am o
n 31
/10/
2014
03:
59:4
9.
View Article Online
2186 Mol. BioSyst., 2011, 7, 2181–2188 This journal is c The Royal Society of Chemistry 2011
(choline-like compounds) could be explained by increased
excretion of these metabolites by the papillary cells in response
to increased urine osmolality due to impaired sodium trans-
porting function of the proximal tubule.
Correlation of metabolite alterations with histologic and
functional changes
Statistically significant evidence of renal functional compro-
mise, characterized by decreased urine osmolality and elevated
serum creatinine, was demonstrated only in rats with complete
UTO. We were unable to demonstrate a correlation between
renal function and the histologic changes of mild, moderate,
and severe UTO in our study. It is interesting to note that
despite normal histology, rats subjected to partial UTO
exhibited functional changes evidenced by changes in the
metabolite profile of the urine. This demonstrates the
sensitivity of metabolomic analysis to the effects of kidney
obstruction on renal cellular metabolism. The lack of correla-
tion between function and histology in our study is not
surprising considering that several studies in children with
congenital UTO have demonstrated that renal histology does
not correlate with differential renal function as documented by
nuclear renal scans.24,25
The urine metabolite changes of rats with partial UTO and
complete UTO were identical with the exception of decreased
urinary hippurate in complete UTO (in contrast to elevated
levels in partial UTO) and the absence of a relative increase of
DMG. The absence of increased urinary levels of these two
osmolytes in complete UTOmay reflect a lack of ability for the
kidney cells to compensate because of irreversible injury.
Hippurate is a well known uremic toxin that accumulates in
renal failure and is thought to participate in the correction of
metabolic acidosis.26 DMG has been shown to accumulate in
the serum of humans with chronic renal failure and its’ serum
level correlates with declining renal function.27 Lack of
urinary secretion of the osmolytes, hippurate and DMG,
may indicate irrecoverable loss of function in this model.
The roles of these two potential biomarkers in UTO warrant
further investigation.
We were unable to correlate a specific metabolite pattern of
change with each grade (mild, moderate, and severe) of kidney
obstruction. This may have been due to the small numbers in
each group and to the fact that the uncertainty measurements
are composite values that include biological and technical
variability.28
Thus, it appears that the functional effects of UTO, as
reflected by urinary metabolite alteration, are similar in partial
and complete obstruction. This may indicate that the
metabolic changes arise from the compensating normal,
contralateral kidney since a completely obstructed kidney will
not contribute urine. An alternative explanation might be that
metabolites secreted in the urine from completely obstructed
kidneys are reabsorbed systemically and subsequently secreted
into the urine by the contralateral, normal kidney.
Experimental
Animal surgery and sample collection
All animal procedures were carried out in compliance with the
Canadian Council on Animal Care (protocol number 05-083).
Animals were maintained on standard chow. Anaesthesia was
preformed with either intraperitoneal ketamine/xylazine or
inhaled isoflurane. Experimental UTO was created in
Sprague-Dawley male and female weanling rats using a flank
incision in groups of 3 to 6 animals. Partial obstruction was
created by burying the left ureter in the psoas muscle.29
Complete obstruction was generated by complete ligation of
the left ureter with a 6.0 proline suture (Ethicon, Skillman, NJ).
The left ureter of control rats was exposed but not
Fig. 4 (A) PCA scores plot depicting a separation of control and
partially obstructed rat urine profiles for a validation study (Group 3).
Similar to earlier observations, the metabolite profiles of the control
population are more homogenous in principal component space while
those of obstructed subjects appear more dispersed. There is a clear
separation of the two groups along PC1. (B) PCA loadings plot
corresponding to the scores plot shown in Fig. 5(A). The variables
with positive loadings scale are important for describing corresponding
scores. Thus, positive peaks correspond to metabolites that are at
higher concentration in the obstructed samples. The reverse is true for
partial control samples. Abbreviations and symbols: * = controls;
arrowhead = partial UTO; PC = principal component; dH =
chemical shift; cpds = compounds.
Publ
ishe
d on
06
May
201
1. D
ownl
oade
d by
Uni
vers
ity o
f B
irm
ingh
am o
n 31
/10/
2014
03:
59:4
9.
View Article Online
This journal is c The Royal Society of Chemistry 2011 Mol. BioSyst., 2011, 7, 2181–2188 2187
manipulated. All animals were housed individually in meta-
bolic cages (Thermo Fisher Scientific, Rochester, NY) and
urine specimens were collected three times weekly for three
weeks, following which the animals were sacrificed. Urine
collection tubes were chilled and contained 0.1% NaN3 to
prevent bacterial growth. Urine samples were centrifuged at
1600 � g for 10 min at 4 1C and aliquots were separated and
stored at �80 1C until analysis was completed. Each urine
sample was thawed and individually analyzed by NMR
spectroscopy (see below). Kidneys were split longitudinally
and fixed in 10% formalin. In the control group, the left ureter
was exposed through a flank incision by receiving a sham
operation which exposed the ureter without manipulation. Six
females underwent partial UTO. The paired control group for
this experimental group included 6 females. Ten male weanling
rats were subjected to complete UTO with a matching set of
10 male control rats. A validation experiment completed one
year later included 7 control male rats and 8 male rats
subjected to partial UTO. See Table 1. The degree of UTO
(partial or complete) for animals in all protocols was
confirmed at the termination of the experiment (week 3) by
visual inspection along with the injection of methylene blue
into the renal pelvis with observation of its drainage beyond
the obstructing suture.
Histology
Formalin-fixed kidney specimens were paraffin-embedded and
sectioned. Slides were subsequently stained with hemotoxylin
and eosin and periodic acid-Schiff (PAS) stain (Fisher Scientific
Limited, Nepean, ON, Canada). Slides were reviewed in
a blinded fashion by a pathologist (W.H.) and graded as
normal, mild, moderate, or severe obstruction (please see
results section for full description).
Clinical chemistry
Urine osmolality was measured by freezing point depression.
Serum samples were assayed for creatinine according to the
manufacturer’s instructions using Quanichromt Creatinine
Assay Kit (Bioassay Systems, Hayward, CA). Statistical
analysis was carried out with students 2-tailed T-test, assuming
unequal variances.
Sample analysis and 1D 1H NMR spectroscopy
A 500 mL of urine sample was mixed with 200 mL NaH2PO4
pH 7.3 and 50 mM sodium 3-trimethylsilyl-2,2,3,3-d4-propionate
(TSP) and diluted in D2O. A 600-mL aliquot was added to a
5-mm-od NMR tube and analyzed on a BrukerAvance DRX
500 spectrometer using a triple axis gradient inverse detection
probe at 293.21K and operating at 500 MHz 1H frequency.
For each urine sample, a standard 1D spectrum was acquired
using a standard pulse sequence for water peak suppression
[RD(cw)-901-acquired free induction decay (FID)], with a
selective irradiation of water peak applied during the relaxa-
tion delay. A total of 128 transients were acquired into 32 k
data points using a spectral width of 6 kHz (12 ppm) and an
acquisition time of 2.73 s. Prior to Fourier transformation, the
data were zero filled to 64 k points and an exponential line
broadening function of 0.3 Hz applied to the FID. A manual
zero and first order phasing was then applied to the data
followed by a first order polynomial baseline correction
using XWINNMR (Version 3.1; Bruker). The TSP signal at
0.00 ppm was used as a chemical shift reference.
Data reduction and exploratory analysis
After manual phasing and baseline correction, the spectra
were converted to an appropriate format for subsequent
multivariate analyses using MATLABs 7.1 software
(MathWorks, Natick, MA) using an in-house written code.
Each spectrum was segmented into 0.005-ppm chemical shift
(bin size) between 0.200 and 10.000 ppm, and the spectral area
within each bin integrated. Bins between 4.500–5.980 ppm
containing residual water and urea resonances were removed.
Variations in chemical shift induced by the pH, often observed
at the citrate ranges of 2.660–2.740 ppm and 2.500–2.600 ppm
were mitigated by compressing these chemical shifts into single
bins centered around 2.700 ppm and 2.550 ppm respectively.
This localized each of the resonances that exhibit pH-induced
chemical shift variability into single bins. Prior to PCA, the
data were pre-processed to identify outliers using Hotelling’s
T2 values, Q residuals30 and plots of Studentized residuals vs.
residual leverage.31 Any spectrum which was flagged as an
outlier was inspected to determine the cause for its outlying
tendency and if found to be experimental, that sample was
prepared again and a new spectrum acquired for it. All
biological replicates (3 per rat) between day 15 to day 21 were
treated as individual measurements. Analysis of the data was
carried out using PCA models constructed for the data
consisting of an NMR spectrum for each urine sample. Each
urine spectrum was normalized to unit sum and centered on
the mean for the spectra of rat urine in each week. Scores plots
for principal component (PC) and their respective loadings
plots were generated and assessed for classification and meta-
bolite identification.
Using TSP as an internal standard, the relative concentra-
tions of metabolites of interest were determined. The data
were also analyzed using the Chenomx NMR Suite 4.6
Professionals (Edmonton, Alberta, Canada) software for
metabolite identification and quantitation.32
Validation
A higher field NMR spectrometer was used for the validation
experiment one year after the initial experiment on the urine of
7 male control rats and 8 male rats subjected to partial UTO.
Sample preparation and NMR experimental settings for these
data were similar to the description above except that the
sample was analyzed on a 700 MHz NMR spectrometer. 1D1H-NMR spectra were acquired at 2981K using a 5 mm Triple
Resonance Inverse (TCI) Cryoprobet with automatic tuning
and matching (ATMA) and a z-axis gradient amplifier and
digital lock on a BrukerAvance III spectrometer operating at
700 MHz proton resonance frequency (BrukerBiospin,
Fallanden, Switzerland). These spectra were acquired using a
9.5 ms (901) pulse calibrated with a 3601 pulse, 7.5 kHz spectral
width and a 2 s relaxation delay with water pre-saturation (PS)
using a calibrated CW irradiation attenuation, with 64
transients and 8 dummy scans collected into 32k data points
Publ
ishe
d on
06
May
201
1. D
ownl
oade
d by
Uni
vers
ity o
f B
irm
ingh
am o
n 31
/10/
2014
03:
59:4
9.
View Article Online
2188 Mol. BioSyst., 2011, 7, 2181–2188 This journal is c The Royal Society of Chemistry 2011
at 293.2 K. Analysis and processing of the data was identical
to the approach described for the initial experiments (see Data
reduction and exploratory analysis).
Conclusion
UTO in a rodent model demonstrates evidence of proximal
tubule and medullary injury. Specifically, the oxidative
metabolism of the mitochondria in renal proximal tubules is
impaired and the medulla excretes protective osmolytes to
counterbalance the resultant change in urine osmolality. The
pattern of proximal tubule and medullary injury is similar in
partial and complete UTO. However, decreased production of
osmolytes is noted in complete obstruction suggesting
irrecoverable loss of the ability to produce these renal
protective metabolites. The identified metabolite alterations
may serve as potential biomarkers for renal injury in UTO.
Correlation in other models and humans is required.
References
1 J. M. Belarmino and B. A. Kogan, Management of neonatalhydronephrosis, Early Hum. Dev., 2006, 82, 9.
2 M. G. Seikaly, P. L. Ho, L. Emmett, R. N. Fine and A. Tejani,Chronic renal insufficiency in children: the 2001 Annual Report ofthe NAPRTCS, Pediatr. Nephrol., 2003, 18, 796.
3 B. A. Thornhill, L. E. Burt, C. Chen, M. S. Forbes andR. L. Chevalier, Variable chronic partial ureteral obstruction inthe neonatal rat: a new model of ureteropelvic junction obstruction,Kidney Int., 2005, 67, 42.
4 K. H. Yoo, V. F. Norwood, S. S. el-Dahr, I. Yosipiv andR. L. Chevalier, Regulation of angiotensin II AT1 and AT2 receptorsin neonatal ureteral obstruction, Am. J. Physiol., 1997, 273, R503.
5 R. L. Chevalier and R. A. Gomez, Response of the renin-angiotensin system to relief of neonatal ureteral obstruction, Am.J. Physiol., 1988, 255, F1070.
6 R. L. Chevalier, Molecular and cellular pathophysiology ofobstructive nephropathy, Pediatr. Nephrol., 1999, 13, 612.
7 P. L. Zhang, C. A. Peters and S. Rosen, Ureteropelvic junctionobstruction: morphological and clinical studies, Pediatr. Nephrol.,2000, 14, 820.
8 J. K. Nicholson, J. C. Lindon and E. Holmes, ‘Metabonomics’:understanding the metabolic responses of living systems topathophysiological stimuli via multivariate statistical analysis ofbiological NMR spectroscopic data, Xenobiotica, 1999, 29, 1181.
9 J. K. Nicholson, J. Connelly, J. C. Lindon and E. Holmes,Metabonomics: a platform for studying drug toxicity and genefunction, Nat. Rev. Drug Discovery, 2002, 1, 153.
10 E. Holmes, I. D. Wilson and J. K. Nicholson, Metabolic pheno-typing in health and disease, Cell, 2008, 134, 714.
11 Y. Yoshioka, T. Tsutsumi, M. Adachi and A. Tokumura, Alteredphospolipid profile in urine of rats with unilateral ureteralobstruction, Metabolomics, epub May: 2009.
12 N. Serkova, T. F. Fuller, J. Klawitter, C. E. Freise andC. U. Niemann, H-NMR-based metabolic signatures of mild andsevere ischemia/reperfusion injury in rat kidney transplants,Kidney Int., 2005, 67, 1142.
13 E. M. Lenz, J. Bright, R. Knight, I. D. Wilson and H. Major, Ametabonomic investigation of the biochemical effects of mercuricchloride in the rat using 1H NMR and HPLC-TOF/MS: timedependent changes in the urinary profile of endogenous metabo-lites as a result of nephrotoxicity, Analyst, 2004, 129, 535.
14 E. M. Lenz, J. Bright, R. Knight, F. R. Westwood, D. Davies,H. Major and I. D. Wilson, Metabonomics with 1H-NMR spectro-scopy and liquid chromatography-mass spectrometry applied to theinvestigation of metabolic changes caused by gentamicin-inducednephrotoxicity in the rat, Biomarkers, 2005, 10, 173.
15 C. U. Niemann and N. J. Serkova, Biochemical mechanisms ofnephrotoxicity: application for metabolomics, Expert Opin. DrugMetab. Toxicol., 2007, 3, 527.
16 E. Bairaktari, K. Katopodis, K. C. Siamopoulos and O. Tsolas,Paraquat-induced renal injury studied by 1H nuclear magneticresonance spectroscopy of urine, Clin. Chem., 1998, 44, 1256.
17 R. E. Williams, M. Jacobsen and E. A. Lock, 1H NMR patternrecognition and 31P NMR studies with d-Serine in rat urine andkidney, time- and dose-related metabolic effects, Chem. Res.Toxicol., 2003, 16, 1207.
18 T. Hauet, H. Baumert, H. Gibelin, C. Godart, M. Carretier andM. Eugene, Citrate, acetate and renal medullary osmolyte excre-tion in urine as predictor of renal changes after cold ischaemia andtransplantation, Clin. Chem. Lab. Med., 2000, 38, 1093.
19 L. Wei, P. Liao, H. Wu, X. Li, F. Pei, W. Li and Y. Wu,Toxicological effects of cinnabar in rats by NMR-based metabolicprofiling of urine and serum, Toxicol. Appl. Pharmacol., 2008, 227,417.
20 E. M. Lenz, J. Bright, R. Knight, I. D. Wilson and H. Major,Cyclosporin A-induced changes in endogenous metabolites in raturine: a metabonomic investigation using high field 1H NMRspectroscopy, HPLC-TOF/MS and chemometrics, J. Pharm.Biomed. Anal., 2004, 35, 599.
21 H. H. Hagar, E. El Etter and M. Arafa, Taurine attenuateshypertension and renal dysfunction induced by cyclosporine A inrats, Clin. Exp. Pharmacol. Physiol., 2006, 33, 189.
22 T. Deguchi, M. Takemoto, N. Uehara, W. E. Lindup, A. Suenagaand M. Otagiri, Renal clearance of endogenous hippurate corre-lates with expression levels of renal organic anion transporters inuremic rats, J. Pharmacol. Exp. Ther., 2005, 314, 932.
23 M. Malyusz, W. Kahler and G. Gronow, Hippurate metabolism asa hydroxyl radical trapping mechanism in the rat kidney, KidneyBlood Pressure Res., 2001, 24, 149.
24 W. Y. Huang, C. A. Peters, D. Zurakowski, J. G. Borer,D. A. Diamond, S. B. Bauer, D. L. McLellan and S. Rosen, Renalbiopsy in congenital ureteropelvic junction obstruction: evidencefor parenchymal maldevelopment, Kidney Int., 2006, 69, 137.
25 J. S. Elder, R. Stansbrey, B. B. Dahms and A. A. Selzman, Renalhistological changes secondary to ureteropelvic junction obstruc-tion, J. Urol., 1995, 154, 719.
26 R. Dzurik, V. Spustova, Z. Krivosikova and K. Gazdikova,Hippurate participates in the correction of metabolic acidosis,Kidney Int., 2001, 59(s78), 278.
27 D. O. McGregor, W. J. Dellow, M. Lever, P. M. George,R. A. Robson and S. T. Chambers, Dimethylglycine accumulatesin uremia and predicts elevated plasma homocysteine concentra-tions, Kidney Int., 2001, 59, 2267.
28 T. K. Karakach, R. M. Flight and P. D. Wentzell, Bootstrapmethod for the estimation of measurement uncertainty in spotteddual-colorDNA microarrays, Anal. Bioanal. Chem., 2007, 389,2125.
29 A. H. Ulm and F. Miller, An operation to produce experimentalreversible hydronephrosis in dogs, J. Urol., 1962, 88, 337.
30 S. Macho, F. Sales, M. P. Callao, M. S. Larrechi and F. X. Rius,Outlier detection in the ethylene content determination inpropylene copolymer by near-infrared spectroscopy and multi-variate calibration, Appl. Spectrosc., 2001, 55, 1532.
31 R. K. Beebe, J. R. Pell and M. B. Seasholtz, Chemometrics:A practical guide, 1989.
32 A. M. Weljie, J. Newton, P. Mercier, E. Carlson and C. M. Slupsky,Targeted profiling: quantitative analysis of 1H NMR metabolomicsdata, Anal. Chem., 2006, 78, 4430.
Publ
ishe
d on
06
May
201
1. D
ownl
oade
d by
Uni
vers
ity o
f B
irm
ingh
am o
n 31
/10/
2014
03:
59:4
9.
View Article Online
