June 2018

Medical Mass Spectrometry Vol. 2 No. 1

Review

Pathogenesis of uremic sarcopenia based on metabolic alteration
1,2
Emiko Sato *1

Division of Clinical Pharmacology and Therapeutics, Tohoku University Graduate School of Pharmaceutical Sciences,
Sendai, Miyagi 980‒8578, Japan
2
Division of Nephrology, Endocrinology and Vascular Medicine, Tohoku University Graduate School of Medicine,
Sendai, Miyagi 980‒8574, Japan

Abstract

Sarcopenia, the degenerative loss of skeletal muscle mass, is associated with increased morbidity and mortality

for patients with chronic kidney disease (CKD), where it is speciﬁcally referred to as uremic sarcopenia. However, the details of the abnormal metabolic processes induced by uremic toxins remain unclear. Recently, we clariﬁed the pathogenic
mechanisms of uremic sarcopenia using liquid chromatography-mass spectrometry (MS) and MS imaging, which revealed
that uremic toxin indoxyl sulfate accumulates in the muscle tissue of CKD model mice. Moreover, capillary electrophoresis
MS-metabolomics of a muscle cell line suggested that indoxyl sulfate induces metabolic alterations such as upregulationof glycolysis, including the pentose phosphate pathway, for protection against oxidative stress. This altered metabolic ﬂow
leads to downregulation of the tricarboxylic acid cycle resulting in an ATP shortage. In a clinical study, plasma indoxyl
sulfate levels were associated with skeletal muscle mass reduction in CKD patients. In this review, I discuss the known
pathogenic mechanisms of uremic sarcopenia induced by the uremic toxin indoxyl sulfate with a focus on the consequent
metabolic alteration and mitochondrial dysfunction.
Key words: Uremic toxin, mass spectrometry imaging, mass spectrometry, metabolomics

Introduction

Patients with CKD typically suffer from uremic sarcopenia,

Chronic kidney disease (CKD), which is a consequenceof lifestyle-related diseases such as diabetes and hyperten-which is the progressive loss of skeletal muscle mass andsion, is a major health problem worldwide associated withthe risk of end-stage renal disease and cardiovascular dis-the reduction of muscle strength is associated with a poor
5)
prognosis . Thus, uremic sarcopenia is a serious complica-

ease. Although cardiovascular disease is the primary cause

strength

3,4). Sarcopenia is observed in all CKD stages, and, other complications also

tion of CKD that requires medical treatment. Althoughinﬂammation, hormonal and immunological changes, meta-

substantially contribute to the high mortality rate. Skeletal

bolic acidosis, and reduction of protein intake are all known

muscle is the major tissue of energy consumption in theto be involved in the pathogenesis of sarcopenia in CKD,

body, and is also the primary site of glucose disposal.

the speciﬁc pathogenic mechanisms of uremic sarcopenia
remain unclear. Accordingly, there is currently no efﬁca-

of death for patients with CKD

1,2)*Corresponding author

cious therapy for uremic sarcopenia.

Emiko SatoCKD progression results in the accumulation of uremic
toxins in the circulation, some of which cannot be ade-

Division of Clinical Pharmacology and Therapeutics, Tohoku
University Graduate School of Pharmaceutical Sciences,
Sendai, Miyagi 980‒8578, Japan

quately removed by dialysis therapy, including pro6)
tein-bound uremic toxins . These accumulated uremic tox-

Tel: +81‒22‒795‒6807, Fax: +81‒22‒795‒6839
E-mail: emiko@med.tohoku.ac.jp

ins are involved in the various complications of CKD such

Received December 28, 2017. Accepted April 13, 2018.

as hypertension, cardiovascular disease, neurological

Epub June 25, 2018.

impairment, and bone disorders

DOI: 10.24508/mms.2018.06.005

the pathogenic mechanisms of uremic sarcopenia using
2

7‒11)

. Recently, we revealedJune 2018

Medical Mass Spectrometry Vol. 2 No. 1

mass spectrometry (MS) . In this review, I summarize theUremic Toxins and Sarcopenia

insight gained into these pathogenic mechanisms with afocus on the effects of metabolic alterations and mitochon-

Several waste products accumulate in the circulation due
6)
to the reduction of renal function , and those with a nega-

dria dysfunction occurring in the muscle cells due to thetive impact on biological functions are known as uremic

impact of the accumulation of uremic toxins.

:
toxins . Uremic toxins are classiﬁed into three groups
low-molecular-weight solutes (<500 Da), protein-boundUremic Sarcopenia
Sarcopenia refers to the reduction in muscle mass andlow-molecular-weight solutes (<500 Da), and intermediate-sized molecules (>500 Da). These uremic toxins con-function, which is associated with the physiological aging.

tribute to the pathogenesis of CKD-related complications

Uremic sarcopenia, which represents muscle wasting insuch as hypertension, cardiovascular disease, neurological

CKD patients, is associated with increased morbidity and. Although uremic toxins
have been generally considered to be linked to the patho-

12)

21)

7‒10)

disorders, and bone disorders

4)

mortality . Uremic sarcopenia is common with an overall
4,13)

prevalence of 〜50％ in patients undergoing dialysis

22,23).

genesis of uremic sarcopenia, the details remain unclear.

Multiple factors such as inﬂammation, hormonal changes,

Our preliminary examination of this association suggested

myocellular changes, immunological changes, protein
energy wasting, renin-angiotensin system changes, muscle

that plasma indoxyl sulfate (IS) and methylguanidine levels

protein balance changes, and uremic changes are known to4)contribute to uremic sarcopenia . The ATP-dependent ubiq-

level of CKD patients. Based on these results, we further

uitin-proteasome system (UPS) pathway is recognized to bemyoblast cell line C2C12 using MTT assays, and foundinvolved in one of the most important forms of muscle loss

that only IS had a toxic effect on the muscle by restricting

and is characterized as the primary cause of muscle masscell growth . Recently, Enoki et al. examined the effect ofwere negatively correlated with the skeletal muscle mass
examined the toxic effects of these two uremic toxins on

12)

14)

24)

degradation in CKD patients . Inﬂammation, metabolic

uremic toxins on the C2C12 cells . They speciﬁcallyfocused on six protein-bound uremic toxins, IS, indole ace-

acidosis and insulin resistance, which are complications of
15)
CKD, activate the UPS to degrade muscle protein . Insu-

tic acid, p-cresyl sulfate, hippuric acid, kynurenic acid, and3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, which

lin/insulin-like growth factor I (IGF-I) signaling plays animportant role in the regulation of cell growth, cell prolifer-

are known to accumulate in the circulation under a condi25)

tion of CKD due to a high afﬁnity for serum albumin .
They found that IS had the strongest anti-proliferative

ation, development, and maintenance of several tissues
within the human body.
Satellite cells, located under the basal lamina of myoﬁbers, maintain muscle mass. They are activated by muscle

effect for C2C12 cells and induced skeletal muscle atrophy
by increasing the production of atrogin-1 and myostatin

injury and they express the transcription factors MyoD andmyogenin, which trigger cell proliferation and differentia-

through inducing muscular oxidative stress-mediated24)
inﬂammation . IS is an end-product of tryptophan metabo-tion during repair. Impairment of insulin/IGF-I signaling

lism and has been associated with CKD progression .

and satellite cell function leads to muscle wasting. In a

Nishikawa et al. found that IS might be involved in therodent CKD model, metabolic acidosis, excess angiotensin

impairment of exercise capacity through oxidative stress,

II, and inﬂammation were found to be the factors causing
muscle wasting via impairment of insulin/IGF-I signal-and that the removal of uremic toxins by using an oral
adsorbent AST-120 improved exercise capacity in CKD. In addition, satellite cell function was found to bemouse models . However, the speciﬁc effects of IS toxicity on muscle cells remain unclear. Thus, we further exam-

ing

26)

5,16‒19)27)

20)

impaired in a rodent CKD model . Although the activationof UPS, impairment of the insulin/IGF-I pathway, andined the association between IS and sarcopenia using an
12)adenine-induced CKD mouse model that is characterized

impairment of satellite cell function are considered patho-

by a signiﬁcant increase in the plasma IS level. To evaluate

genic mechanisms of uremic sarcopenia, no potential agent
for therapy or prevention is yet available for muscle wast-

the relationship between IS and skeletal muscle changes ina CKD condition, we compared the cross-sectional area ofing in CKD. Therefore, novel strategies with regard to thethe anterior tibia muscle and intramuscular IS between control and CKD mice. The cross-sectional area of the anterior

therapy or prevention of uremic sarcopenia are required.

3

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Medical Mass Spectrometry Vol. 2 No. 1

fused before harvesting to ﬂush out blood. Fifty-milligramof muscle tissue was mixed with 800 μL of 0.1％ formic
acid methanol containing 2.5 μg/mL IS-d4, and homogenized for 30 s at 6000 rpm at room temperature using a Percellys 24 lysing and homogenization system (M&S Instruments Inc., Osaka, Japan). After homogenization, sample
was sonicated for 15 min and then centrifugated at
16,400×g for 20 min at 4°
C. An equal volume of 0.1％ formic acid was added to the supernatant, and the mixture wasanalyzed by LC-MS/MS. For plasma, 150 μL of 0.1％ formic acid methanol containing 2.5 μg/mL IS-d4, and vortex
for 1 s. After vortexing, sample was sonicated for 15 min
and then centrifugated at 16,400×g for 20 min at 4°
C. An
equal volume of 0.1％ formic acid was added to the supernatant, and the mixture was analyzed by LC-MS/MS. Quantitative analysis of IS was performed using a Nanospace
SI-II HPLC platform (Shiseido, Tokyo, Japan) coupled to a
TSQ Quantiva mass spectrometer (Thermo Fisher Scientiﬁc, Waltham, MA, USA), and operated in the negativemode. 100×2.0 mm Capcell Pak C18 MG-III 3 μm column(Shiseido) was used. 10 mM ammonium acetic acid andacetonitrile were used as mobile phase for gradient elution.
Quantiﬁcation analyses by MS/MS were performed by
selected reaction monitoring (SRM) mode. SRM conditionand collision energy were as follow: m/z 212 → 80, 21 eV
for IS and m/z 216 → 80, 30 eV for IS-d4. Plasma IS levelwas 20-fold higher in the CKD group than in the control
group, and muscle IS level was 6.2-fold higher in the CKDgroup than in the control group (Fig. 1C). In addition to
LC-MS/MS analysis, we visualized IS on muscle tissue by
Fig. 1.

MS imaging (Fig. 1D). Methods of sample preparation andmeasurement for mass spectrometry imaging were as fol-Comparison of cross-sectional area and indoxyl
sulfate levels in muscle tissues between healthy
and chronic kidney disease (CKD) mice.

lows: Muscle tissues were sectioned at 10-μm thickness
with a cryostat and thaw-mounted onto ITO-coated glass(A) Hematoxylin and eosin staining of the anterior tibia
muscle. (B) Comparison of the cross-sectional area between control and CKD mice. (C) Indoxyl sulfate accumulation in plasma and muscle. Modiﬁed from Ref. 28.
(D) Mass spectrometry imaging-based cellular uptake ofindoxyl sulfate into CKD mice. Reproduced from Ref.
12 with permission.

slide. 9-aminoacridine (9-AA) was used as a matrix. Matrix
assisted laser desorption/ionization time-of-ﬂight mass®
spectrometry (MALDI-TOFMS, AXIMA Conﬁdence, Shimadzu) equipped with a 377 nm N2 laser was used for spec-

trometry analysis. Mass spectra were acquired with thelaser frequency in the negative and scanning mass range
from m/z 50 to m/z 1000 at high-resolution mode. Metabo-tibia muscle was signiﬁcantly smaller in CKD mousemodel than that in control mouse (Fig. 1A, 1B). IS accumu-

lites were identiﬁed with the MS/MS spectrum from results

lation in muscle tissues was examined using liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Fig.

of IS chemical standard analysis. IS signal was detected
only on muscle tissue of CKD group (Fig. 1D). Our results

28)1C) . Methods of sample preparation and measurementfor LC-MS/MS were as follows: Muscle tissue was per-indicated that the skeletal muscle was atrophied and IS
accumulated in the muscle of CKD mice.
4

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Fig. 2.

Medical Mass Spectrometry Vol. 2 No. 1

Comparison of metabolites involved in glycolysis, pentose phosphate pathway, tricarboxylic acid cycle, glutathione
metabolism, and purine metabolism in C2C12 cells.

The relative changes of metabolite levels between non-treated (control) and indoxyl sulfate-treated C2C12 cells extracts are shown.
Data are mean±standard deviation, *p<0.05, **p<0.01, ***p<0.0001 compared to the control based on Welch t-test. The vertical
6
axis shows the absolute value (pmol/10 cells). G6P: glucose 6-phosphate; F6P: fructose 6-phosphate; F1,6P: fructose 1,6-diphosphate; GA3P: glyceraldehyde 3 phosphate; 3-PG: 3-phosphoglyceriate; 2-PG: 2-phosphoglycerate; PEP: phosphoenolpyruvate;
6-PG: 6-phosphogluconate; Ru5P: ribulose 5-phosphate; R5P: ribose 5-phosphate; PRPP: phosphoribosyl pyrophosphate; X5P:
xylulose 5-phosphate; S7P: sedoheptulose 7-phosphate; E4P: erythrose 4-phosphate; IMP: inosine monophosphate; HXA: hypoxanthine; XA: xanthine. Reproduced from Ref. 12 with permission.

CKD and Metabolic Alteration

A common feature of CKD is alteration of metabolic pro-

(UUO) rat renal failure model using MALDI-MS imaging.
They identiﬁed 21 metabolites involved in glycolysis, tri-

cesses and pathways in the body. In particular, CKD is

carboxylic acid (TCA) cycle, ATP metabolism, fatty acids

associated with a negative nitrogen balance, and metabolic

metabolism, as well as antioxidants and metal ions that

acidosis is observed in CKD patients when the glomerular
ﬁltration rate decreases to below 20‒25％ of the normalchanged in the UUO rat kidney. In addition, hippuric acid,
which is a protein-bound uremic toxin, accumulated in the

29)

12)

level . In CKD metabolic acidosis, muscle proteolysis is
stimulated by activating the ATP-dependent UPS pathway.

UUO rat kidney. In our study , we investigated the effectsof IS on intracellular metabolic alterations in skeletal mus-Impairment of carbohydrate oxidation caused by protein

cle using C2C12 cells and capillary electrophoresis-mass
spectrometry (CE-MS). CE-MS based target quantitative

degradation signaling in muscle cells ultimately contributes
30)
to sarcopenia . Therefore, metabolic alteration in the tis-analysis was performed at Human Metabolome Technolo-sues plays an important role in CKD progression. Recently,
31)
Liu et al. revealed metabolic changes of the kidney occur-

gies, Inc. (Yamagata, Japan). To measure the levels of
metabolites in the C2C12 cells, an aliquot of methanol con-ring during renal ﬁbrosis in a unilateral ureteral obstruction

taining internal standards was added to 1×10 cells to65June 2018

Fig. 3.

Medical Mass Spectrometry Vol. 2 No. 1

Comparison of parameters of oxidative stress, pentose phosphate pathway activity, and tricarboxylic acid cycle metabolites in C2C12 cells.

The GSH/GSSG ratio, total glutathione (GSH + 2GSSG) level, glucose 6-phosphate (G6P) /ribose 5-phosphate (R5P), total oxaloacetate-related amino acid, total Glu-related amino acid content, and malate/Asp ratio for the non-treated (control) and indoxyl sulfate-treated C2C12 cells extracts are shown. Data are mean±standard deviation; * p<0.05, ** p<0.01, compared with the control by
Welch t-test. Reproduced from Ref. 12 with permission.

Oxidative Stress-Induced Metabolic Alteration

extract cellular metabolites. Resultant cellular extract wascentrifuged, and upper aqueous layer was centrifugally ﬁl-

As described above, the MS-metabolomics study identi-

tered through a Millipore 5-kDa cutoff ﬁlter to remove proteins. The ﬁltrate was used for CE-MS analysis. Cationicﬁed upregulation of the glutathione metabolism pathway

compounds were measured in the positive mode of
CE-TOFMS (Agilent CE-TOFMS system, Fused silica cap-suggested that IS might induce oxidative stress. To test thisand PPP in C2C12 cells following IS stimulation, which
hypothesis, we performed electron spin resonance (ESR)
analysis using the spin-trap reagent 5,5-dimethyl-1-pyrro-

illary, i.d. 50 μm×80 cm), and anionic compounds were
measured in the positive and negative mode of CE-MS/MS

line-N-oxide (DMPO). The ESR spectrum of the IS-stimulated C2C12 cells culture medium showed a typical spec-(Agilent CE system and Agilent 6400 TripleQuad LC/MS,
Fused silica capillary, i.d. 50 μm×80 cm). CE-MS analysis

trum of hydroxyl radicals trapped by DMPO, conﬁrming

could be induced by IS stimulation to C2C12 cells (Fig. 2),that IS induces the generation of reactive oxygen species inC2C12 cells. We further examined the molecular mecha-

which resulted in an increase in the metabolites involved inglycolysis, the pentose phosphate pathway (PPP), and glu-

nisms driving this IS-induced upregulation of PPP in
C2C12 cells. In an oxidative condition, glutathione disul-tathione metabolism and a decrease of the metabolites

ﬁde (GSSG) is reduced to glutathione by GSSG reductase

involved in the TCA cycle, glutamine, and glutamate.

at the expense of NADPH. The major functions of PPP

These metabolic alterations were further conﬁrmed withinclude the production of NADPH for protection against
32)
oxidative damage . Nuclear factor (erythroid-2-related fac-

revealed that similar metabolic alterations found in CKDparametric analysis (Fig. 3). The results of metabolomics
analyses in C2C12 cells suggested that IS upregulated gly-tor) -2 (Nrf2) is one of the main transcriptional activators incolysis, PPP, and glutathione metabolism and downregu-

33)

response to oxidative stress , and additionally regulates
the expression of genes in the PPP such as glucose-6-phos-

lated the TCA cycle and glutamine anabolism.
6June 2018

Medical Mass Spectrometry Vol. 2 No. 1

indicated low levels of mitochondrial enzymes in the muscle biopsy of patients undergoing dialysis, and decreased
levels of mitochondrial oxidative enzymes, synthesis ofmuscle contractile mixed muscle proteins, myosin heavy
chain, and mitochondrial proteins were observed in the4)muscle biopsy of CKD patients . Thus, our results were inline with these previous studies. IS induced mitochondrial
dysfunction such as a reduction of ATP production andinduction of mitochondrial network disintegration in
12)
C2C12 cells . Overall, these results indicate that the mitochondrial dysfunction induced by IS plays an importantrole in the pathogenesis of uremic sarcopenia.

Toward a Better Understanding of Uremic
Sarcopenia in CKD Patients
Fig. 4.

Several factors are considered to be involved in the

Hypothesized mechanisms of metabolic alteration
caused by oxidative stress induced by indoxyl sulfate in muscle cells.

development of uremic sarcopenia, such as inﬂammation,
metabolic acidosis, reduced protein intake, physical inactiv-

The cellular requirement for NADPH increase due toindoxyl sulfate-induced oxidative stress in muscle cells.
The cells change the metabolic ﬂow to adjust the cellularrequirements via pentose phosphate pathway activation
to produce NADPH.

ity, excess angiotensin II, abnormalities in insulin/IGF-I,
myostatin expression, reduction in satellite cell function,
4)and hormonal, immunological, and myocellular changes .
As outlined in this review, we and others have demonstrated the development of sarcopenia in CKD mice orphate dehydrogenase (G6PD) and phosphogluconate dehy-

24,27,35)

rats

34)

, including the reduction of body and skeletal

drogenase (PGD) . Indeed, we found that IS stimulation

muscle weight, mitochondria dysfunction, and exercise

induced Nrf2 protein expression and increased the levels of

capacity reduction. However, animal CKD conditions are

enzymes (G6PD and PGD) related to NADPH production

not necessarily equivalent to human CKD conditions. In
our clinical study, plasma IS levels were found to be signiﬁ-

in the PPP in C2C12 cells. These results indicated that thecellular requirement for NADPH increased along with theaccumulation of IS-induced oxidative damage in muscle

cantly increased and associated with the skeletal muscle
12)

mass reduction in CKD patients . Although these results
suggest that IS is involved in skeletal muscle mass reduc-cells, and thus the cells altered the metabolic ﬂow to makethese adjustments for NADPH through PPP activation (Fig. 4).

tion, further clinical studies are needed to validate whether
similar metabolic changes are observed in the muscle tis-

Sarcopenia and Mitochondrial Dysfunction

sues of CKD patients.

mics also revealed that the TCA cycle was downregulated

In conclusion, in CKD conditions, accumulated IS
induces oxidative stress in muscle cells and alters the meta-

In addition to the effects of IS on the PPP, MS-metabolo-in C2C12 cells following IS stimulation, which suggests
mitochondrial dysfunction accompanied by oxidative dam-

bolic ﬂow toward an excess anti-oxidative response of theage. The skeletal muscle is highly metabolic and requires a

production leading to sarcopenia (Fig. 5). Targeting thesevast amount of mitochondria for sufﬁcient ATP production.

metabolic

PPP through Nrf2 activation, resulting in a decrease of ATP

Thus, mitochondrial abnormalities result in a decrease ofdysfunction
induced by IS in muscle tissues may be useful for the pre-ATP production, leading to an ATP shortage in muscle
cells. A study in CKD rats showed that the expression lev-

vention or treatment of uremic sarcopenia. A recent clinicalstudy found that AST-120 continuously reduced circulating27)

alterations

andmitochondrialels of mitochondrial biogenesis genes were decreased . In
addition, the numbers of mitochondria-rich type I ﬁbers

IS levels and improved oxidative stress in maintenance

35)

that the oral administration of probiotics reduced uremic

36)

hemodialysis patients . Another clinical study reported

were decreased in CKD mice . Moreover, a clinical study7June 2018

Medical Mass Spectrometry Vol. 2 No. 1

Conﬂict of Interest
The author declares no competing interest.
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